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Coordinator Vice-Coordinator Technology Transfer Outreach Finance

Glaucius Oliva Richard C. Garratt Otavio H. Thiemann Leila M. Beltramini Francisco Fernando Falvo Rejane Nogueira Brasil


Ligia Rafaela Prado


Rui Cintra

Headquarters Center for Structural Molecular Biotechnology Institute of Physics of São Carlos University of São Paulo Avenida Trabalhador Sãocarlense 400 CEP 13566-590 São Carlos - SP

Associated Laboratories Department of Chemistry - UFSCar Institute of Chemistry - USP Institute of Biosciences - USP Institute of Biomedical Sciences -USP Medical Faculty of Ribeirao Preto - USP Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP Department of Biochemistry and Molecular Biology - UFV Department of Chemistry - UEPG

instituto de biociências


SUMMARY Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Science Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Outreach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Innovation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Infrastructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Facts and Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Papers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Emails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases (INBEQMeDI) is a joint initiative focused on the development of structural and biological studies of specific molecular targets from microorganisms associated principally with neglected infectious diseases. Its mission is to perform both basic and developmental research within the field to the highest standard it is able to achieve. It aims towards contributing to the development of new drug candidates and associated technologies for the treatment or prevention of endemic diseases such as, but not limited to, Chagas’ disease, malaria, leishmaniasis and schistosomiasis. INBEQMeDI formally began operation on the 1st of January 2009, as part of the INCT initiative of the Brazilian Ministry of Science and Technology. It is managed by an internal committee of five members and overseen by an independent international board of evaluation. It is jointly funded by two research councils, CNPq and FAPESP together with the Ministry of Health, representing contributions from both federal government and the state of São Paulo. Much of the research undertaken began well before the official inauguration and is bolstered by previous successful collaborations between many of the participants who have conducted frontline research in structural biotechnology and medicinal chemistry over recent years. Besides the fundamental research which underpins these initiatives, our past efforts have also included patent registration,

INBEQMeDI is a virtual institute spread over the 10 different laboratories which comprise it. More than anything, INBEQMeDI is the people that work within those laboratories.

technology transfer and the dissemination of science through several outreach programs aimed principally towards high-school students and their teachers. INBEQMeDI represents an opportunity to continue and expand these activities. The headquarters of the Institute are based at the Center for Structural Molecular Biotechnology of the Institute of Physics of São Carlos (IFSC), University of São Paulo (USP). This is the largest and most traditional research group in Protein Crystallography and Structure-Based Drug Design in Brazil. However, the most significant aspect of INBEQMeDI is not one particular group of researchers but rather the way in which all of the associated laboratories attempt to work in a coordinated and cohesive manner in order to undertake international standard research. These laboratories include (i) the Laboratory of Natural Products and Organic Synthesis of the Department of Chemistry, Federal University of São Carlos (DQ-UFSCar); (ii) the Laboratory for the Study of the Molecular Bases of Signal Transduction in the Malaria Parasite, Institute of Biosciences (IB-USP); (iii) The Laboratory of Biochemistry of the Department of Parasitology, Institute of Biomedical Sciences (ICB-USP); (iv) The Laboratory of Molecular Parasitology, of the Medical Faculty of Ribeirão Preto (FMRP-USP); (v) The Laboratory of Protein Structure and Function, Institute of Chemistry (IQ-USP) and (vi) The Laboratory of Chemical and Biological Prospection in Fungi and Actinobacter, of the Faculty of Pharmaceutical Sciences of Ribeirão Preto (FCFRP-USP). Furthermore, INBEQMeDI includes two young research groups as Associate Laboratories, based at the State University of Ponta Grossa in the State of Paraná (DQ-UEPG), and at the Federal University of Viçosa in Minas Gerais (DBB-UFV).




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2. São Paulo - SP Inst In stititut st ittut ueo off C Che hemistry (IQ Q) Un niver ivver e si sity ity of Sã São o Pa Paul u o (U (USP SP P) Shac Sh acke ac kerr C ke Ch huc uckk Fa Fara rah h

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3. Ribeirão Preto - SP Medi Me d ca cal F Faacu cultltltyy off R Rib ibei ib e rã rão o Pr P et eto o (F ( MR R P) Un U nivver ersi s ty of Sã ão P Paaul ulo o (U USP P) An ng ge ela Kay ayse se el Cr Cruz uzz

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4. Viçosa - MG Depa De paarttme p ment n o nt off Bi B oc oche hemi mist stry ry and Mollec e ul u ar a Bio iollogy log (DBB) ( F de Fe d ra al Univ Un niv iver ersi sity t o ty off Vi Viço çosa sa ((UF UF FV) J liiaan Ju na Lo Lope pess Ra Rang ng gell Fie iett tto o

5. Ponta Grossa - PR De D epa art rtme me ent n of Ch C em mis istr try (DQ) S at St ate e Un nivver ersi sity ty of Ponta Grossa (UEPG) J rg Jo ge Iu Iule lekk le



Diversity is undoubtedly one of the cornerstones of the Institute and the principal investigators not surprisingly come from a wide spectrum of scientific backgrounds. These include biology, biochemistry, chemistry, structural biology, parasitology, molecular biology and physics. This diversity and complementarity reflects well the inter-disciplinary research necessary in order to achieve the Institute’s major goals. Very often a given research project will involve the use of a wide range of different techniques including Protein Crystallography, Multidimensional NMR, Spectroscopy, Molecular Modeling, Bioinformatics, Medicinal Chemistry, Cheminformatics, Organic Synthesis, Natural Products Chemistry, Molecular Immunology, Cell Biology, Pharmacology and Molecular Biology. We aim to make this a synergistic process in which research into the fundamental biology of the etiological agents supports efforts into the development of novel compounds as potential new drugs for the treatment of the diseases they cause. For this reason the majority of research projects are selected on the basis of their potential as targets for chemotherapeutic intervention. However, this is not done at the expense of neglecting curiosity-based research into the fundamental cellular processes of the fascinating organisms under study. Furthermore, the Institute is well aware of its limitations as to how far along the drug development pipeline it can reasonably expect to go. Beyond that point it is essential to seek partnerships with industry and governmental organizations in order for subsequent project development and technology transfer. It is for this reason that the WHO World Reference Center for Medicinal Chemistry in Chagas’ Disease, which is housed at INBEQMeDI and coordinated by one of its members, represents such a critical component of our activities. Wherever possible, INBEQMeDI is committed to advancing the public understanding of science and many of its members have accumulated considerable experience in this noble cause over recent years. The gap between the current advances in biotechnology and the difficulty with which the public assimilates such ideas is perceived to be widening in an alarming fashion. There is an urgent demand for adequate educational programs to disseminate the basic concepts of molecular biology such as what genes actually are and how they codify for proteins. Myths and misconceptions in the minds of the general public have raised unnecessary and sometimes irrational over-reactions against science and scientists involved in genetic research and biotechnology. It is INBEQMeDI’s responsibility to be part of attempts to reverse this process. Although INBEQMeDI has only recently come into existence, considerable effort has been made during its first year of operation towards progressing on all of the fronts described above. It is the intention of this first Annual Activity Report to describe some of the principal advances which have been made. We hope that it will serve as a backdrop to subsequent reports in order that the evolution of our research into the structural biology of infectious agents and the medicinal chemistry necessary to combat their effects can be more readily appreciated.




01. Drug Design Approaches for Neglected Tropical Diseases: Integration of Structural Biology and Medicinal Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 02. Inf uence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 03. Structure and Calcium Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 04. The Relevance of Proline Metabolism in Trypanosoma cruzi. . . . . . . . . . . . . . . . 26 05. Anacardic Acid Derivatives as Inhibitors of Glyceraldehyde-3-Phosphate Dehydrogenase from Trypanosoma cruzi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 06. The Schistosome Purinome Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Drug Design Approaches for Neglected Tropical Diseases: Integration of Structural Biology and Medicinal Chemistry Rafael V.C. Guido, Glaucius Oliva, Adriano D. Andricopulo Laboratório de Química Medicinal e Computacional, Instituto de Física de São Carlos Universidade de São Paulo, São Carlos, SP, Brasil

Abstract Drug discovery is a highly complex and costly process, which demands integrated efforts in several relevant aspects involving innovation, knowledge, information, technologies, expertise, R&D investments and management skills. The shift from traditional to genomics- and proteomics-based drug research has fundamentally transformed key research and development (R&D) strategies addressed to the design of new chemical entities (NCEs) as drug candidates against a variety of biological targets. The combination of available knowledge of several 3D protein structures with hundreds of thousands of small-molecules have attracted the attention of scientists from all over the world for the application of structure- (SBDD) and ligand-based drug design (LBDD) approaches in medicinal chemistry.1-3 This research highlight summarizes some of our continuing efforts in INBEQMeDI towards the effective integration of structural biology and medicinal chemistry approaches, with emphasis in the use of SBDD and LBDD strategies in the area of neglected tropical diseases. Keywords structural biology, medicinal chemistry, drug design, neglected tropical diseases

Original publications: see complete reference list.






the integrated medicinal chemistry approach employed


toward the discovery of new inhibitors of Schistosoma

experimental and computational methods. In line

mansoni purine nucleoside phosphorylase (SmPNP),

with this, some of our projects in INBEQMeDI

a key enzyme involved in the purine salvage pathway

have focused on the identification and optimization

of S. mansoni, one of the causative agents of human

of lead candidates for a variety of infectious parasitic diseases, integrating modern strategies based on our increasing understanding of the fundamental principles of medicinal chemistry, structural biology and parasitology (Figure 1). One of the most important challenges in drug design is the development of innovative new chemical entities (NCEs) from an incredibly large reservoir of real and virtual possible compounds. In INBEQMeDI,

schistosomiasis.4 In this work, we have described the

Science Highlights

Drug discovery is currently driven by innovation

development of a structure-based pharmacophore model for ligands of SmPNP, which was subsequently employed in virtual screening studies leading to the identification of three thioxothiazolidinones derivatives with substantial in vitro inhibitory activity against the parasite enzyme (Figure 2). Synthesis, biochemical evaluation and structure-activity relationship (SAR)

experimental and computational strategies are integrated

investigations led to the successful development

in order to facilitate the identification and optimization

of a small set of thioxothiazolidinone derivatives

of new biologically active compounds that possess the

harboring a novel chemical scaffold as new competitive

ability to modulate specific molecular targets.

inhibitors of SmPNP at the low micromolar range.

Several steps of the drug discovery process (e.g.; hit

Seven compounds were identified with IC50 values

identification, lead optimization, NCE development)

below 100 ¾M. The most potent inhibitors 1–3

can be improved in a rational way with the application

represent new potential lead compounds for further

of computational methods. An example can be seen in

development for the therapy of schistosomiasis.

Figure 1. Drug design strategies employed in INBEQMeDI.



Drug Design Approaches for Neglected Tropical Diseases

Structure-Based Drug Design

New Lead Compounds

Figure 2. Integrated approach carried out toward the identification of novel inhibitors of SmPNP.

Medicinal chemistry studies aimed at elucidating

(Figure 3). Statistically significant models, with high

fundamental aspects of the relationships between

predictive power were obtained. The final QSAR

structural (or property) descriptors and biological

models and the information gathered from the

activity are important in the understanding of the

3D CoMFA and CoMSIA contour maps provided

activity of interest and may enable the prediction of

important molecular insights into the structural basis

the biological property for new compounds. The

involved in the molecular recognition process of this

integration of modern molecular modeling techniques

family of cruzain inhibitors, which are useful for

and advanced two– and three–dimensional quantitative

the design of new structurally related analogs with

structure-activity relationship (2D and 3D QSAR,

improved potency.

respectively) methods has been successfully used to provide important insights into the chemical and structural determinants of ligand binding affinity and selectivity. For instance, the combination of molecular docking and QSAR methods is advantageous because it allows direct visualization and interpretation of modeling results within the protein binding site, revealing essential ligand-receptor interactions. In this regard, comparative molecular field analysis (CoMFA)

QSAR approach used to explore the chemical and biological space of data sets of compounds, is another example of the importance of QSAR methods in drug design.6,7 HQSAR is a modern 2D QSAR approach based on specialized molecular fragments (molecular holograms) that implicitly encode 3D structural information, such as hybridization and

and comparative molecular similarity indices analysis

chirality. With the transformation of the chemical

(CoMSIA) studies were conducted on a series of

representation of a molecule into its corresponding

thiosemicarbazone and semicarbazone derivatives

molecular hologram, this method requires no explicit

as small-molecule inhibitors of cruzain, the major

3D information (e.g., determination of 3D structures,

cysteine protease of the parasite Trypanosoma cruzi,

putative binding conformations, and molecular

which has been considered an important target for the

structural alignment). HQSAR explains differences

development of new antitrypanosomal agents. In this

in the observed activity (e.g., Ki, IC50, EC50) in a

work, molecular docking studies were performed in

series of molecules by quantifying variations within

order to identify the preferred binding mode of the

their calculated molecular holograms. It is important

inhibitors into the enzyme active site, and to generate

to note that besides predicting accurately property

structural alignments for the 3D QSAR investigations

values of untested compounds (e.g., potency, affinity),



Hologram QSAR (HQSAR), a fragment-based

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

Guido et al.

Figure 3. Combination of molecular modeling and 3D QSAR methods in medicinal chemistry.

HQSAR models can also provide useful insights

concentrations of IAA. Subsequently, the X–ray

into the relationships between structural fragments

crystallographic data of the complex of T. cruzi GAPDH

(micromolecule) and biological activity. Thus, it

and IAA were used to investigate the structural basis

is possible to identify specific individual atomic

underlying the mechanism of enzyme inactivation.

contributions as determinants of increased biological

The analysis of the complex confirms that the modified

activity in the absence of the 3D crystal structure.

catalytic cysteine (carbocysteine, CCS166) is hydrogen

On the other hand, when 3D structural information

bonded to the O2D of the NAD+ molecule mediated

is available, this allows a comparison of the HQSAR

by a water molecule (W119) (Figure 5). Additionally,

results to determine if they are in agreement with the

the delocalized –electrons from the carboxylate group

3D chemical environment of the target protein.

of IAA form a – interaction with the nicotinamide and

moiety. Accordingly, the crystallographic data shows

computational tools have significantly evolved and

that the cofactor molecule plays a central role in the

transformed structure- (SBDD) and ligand-based

binding of the inhibitor within the GAPDH active

drug design (LBDD) methods in tools of large impact

site. In addition to providing invaluable information

in modern medicinal chemistry. These methods

about the mechanism of action, the X–ray complex

allow the investigation of the structural and chemical

structure also revealed important insights into

basis involved in molecular recognition mechanisms,

the binding conformation of IAA into the T. cruzi

selectivity/specificity and biological activity. In this

GAPDH active site. Specific knowledge about ligand

context, the integration of kinetic and structure–

binding mode and mechanism of action was of great

based methods is useful in inhibitor drug design. An

importance to guide structure–based inhibitor design

example of this approach was the application of state

studies towards the development of novel GAPDH

of the art kinetic and crystallographic methods in

inhibitors with enhanced potency and affinity.






order to improve our understanding of the binding

Selectivity plays a crucial role in the design of en-

mode and intermolecular interactions between the

zyme inhibitors as novel antiparasitic agents, particu-

enzyme glyceraldehyde–3–phosphate dehydrogenase

larly in cases where the target enzyme is also present

(GAPDH) from Trypanosoma cruzi and the iodoacetate

in the human host. In addition to its important role in


inhibitor (IAA). The time–dependent irreversible

the design of potent enzyme inhibitors, SBDD meth-

inactivation of T. cruzi GAPDH was determined by

ods are especially attractive for the design of small-

kinetic measurements in the presence of increasing

molecule modulators that selectively interact with



Drug Design Approaches for Neglected Tropical Diseases

Figure 4. Hologram QSAR as an attractive method to study the chemical and biological space of data sets of compounds.

Kinetic Measurements

X-ray Crystallography

Mode of Action

Figure 5. State of the art kinetic and crystallographic methods revealed important molecular interactions involved in ligand-binding and enzyme inhibition (PDB ID, 3DMT). Hydrogen bonds and – interaction are indicated as blue and yellow dashed lines.


distinct receptors. In this context, kinetic studies were

H-bonding, volume and hydrophobicity properties

carried out in order to determine the inhibitory po-

correlate with the inhibitory potency of these com-

tency, mode of action and enzyme selectivity of a se-

pounds. In order to allow the development of mecha-

ries of 9–substituted–9–deazaguanines and other pu-

nism-based SSRs, the type of inhibition of this series

rine analogs as inhibitors of SmPNP and human PNP

of compounds with respect to the physiological sub-

(HsPNP).9 In order to better explore the molecular

strate for SmPNP was evaluated. The results indicated

aspects involved in enzyme selectivity, values of IC50

that the inhibition of SmPNP was found to be com-

(concentration of compound required for 50% inhibi-

petitive, thereby the inhibitors exclusively binds to the

tion of PNP) were determined for both SmPNP and

free enzyme, in direct competition with the substrate.

HsPNP. The nucleoside analogs (4-6) showed good

Crystallographic studies were undertaken to investi-

selectivity towards the parasite enzyme (from about

gate the binding mode of this series of selective inhib-

2 to 6.4-fold, Figure 6). The SARs and structure–se-

itors into the substrate binding cavity of SmPNP. The

lectivity relationships (SSRs) indicated that electronic,

crystallographic structure of SmPNP in complex with

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

Guido et al.

Inhibitory Potency and Selectivity

Mode of Action

X-ray Crystallography

Figure 6. SBDD studies employed to explore the molecular basis underlying inhibitor affinity and enzyme selectivity (PDB ID, 3IEX).

guanosine (compound 4 analog) revealed that a more

In sum, the combination of kinetic investigation and

extensive H-bonding network is present in the purine

crystallographic studies provided important structural

binding site of the parasite enzyme, highlighting the

insights for rational inhibitor design, revealing consis-

importance of Ser247 (Val245 in HsPNP) and Tyr202

tent structural differences in the binding mode of the

(Phe200 in HsPNP) as key residues to be explore for

inhibitors in the active sites of the parasite and human

the development of selective inhibitors of SmPNP.


References 1. Guido RVC, Oliva G, Andricopulo AD. Virtual screening and its integration with modern drug design technologies. Curr. Med. Chem., 2008, 15, 37–46. 2. Andricopulo AD, Salum LB, Abraham DJ. Structurebased drug design strategies in medicinal chemistry. Curr. Top. Med. Chem., 2009, 9, 771-790. 3. Guido RVC, Oliva G. Structure-Based Drug Discovery for Tropical Diseases. Curr. Top. Med. Chem., 2009, 9, 824-843. 4. Postigo MP, Guido RVC, Oliva G, Castilho MS, Pitta IR, Albuquerque JFC, Andricopulo AD. Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening. J. Chem. Inf. Model., 2010, in press. 5. Trossini GH, Guido RVC, Oliva G, Ferreira EI, Andricopulo AD. Quantitative structure activity


relationships for a series of inhibitors of cruzain from Trypanosoma cruzi: molecular modeling, CoMFA and CoMSIA studies. J. Mol. Graph. Model., 2009, 28, 3–11. 6. Salum LB, Andricopulo AD. Fragment-based QSAR: perspectives in drug design. Mol. Divers., 2009, 13, 277–285. 7. Salum LB, Andricopulo AD. Fragment-based QSAR strategies in drug design. Expert Opin. Drug Discov., 2010, 5, 405-412. 8. Guido RVC, Balliano TL, Andricopulo AD, Oliva G. Kinetic and crystallographic studies on glyceraldehyde–3–phosphate dehydrogenase from Trypanosoma cruzi in complex with iodoacetate. Lett. Drug Discov. Des., 2009, 6, 210–214. 9. Castilho MS, Postigo MP, Pereira HM, Oliva G, Andricopulo AD. Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies. Bioorg. Med. Chem., 2010, 18, 1421–1427.


Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence Ramon F. Santos1, Marcela A.S. Pôssa1, Matheus S. Bastos1, Paulo M.M. Guedes1, Marcia R. Almeida2, Ricardo DeMarco3, Sergio Verjovski-Almeida4, Maria T. Bahia1, Juliana L.R. Fietto1,2 1

Núcleo de Pesquisa em Ciências Biológicas Universidade Federal de Ouro Preto, Ouro Preto, MG, Brasil Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brasil 3 Instituto de Física de São Carlos, Universidade de São Paulo, São Paulo, SP, Brasil 4 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil 2

Abstract Trypanosoma cruzi is the etiological agent of Chagas’ disease. Presently there are no vaccines or effective treatment, making the development of new therapies to control Chagas’ disease very desirable. In this study, we evaluated the influence of Ecto-Nucleoside-TriphosphateDiphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. We verified that trypomastigotes resulting from sequential sub-cultivation exhibit lower Ecto-NTPDases activity and infectivity. Exposure of parasites to NTPDase inhibitors or anti-NTPDase-1 polyclonal antiserum also resulted in reduced infectivity. Mice infections with Ecto-NTPDaseinhibited trypomastigotes exhibited lower parasitemia levels and higher survival rates in relation to control experiments. Taken together our data suggest that Ecto-NTPDases might play an important role in the parasite infection process and might be a good candidate target for development of therapies. Keywords apyrase, host-parasite interaction, neglected diseases, Chagas’ disease

Original publications: PLoS Negl Trop Dis 3(3): e387, 2009. DOI:10.1371/journal.pntd.0000387

Nucleoside Triphosphate Diphosphohydrolases (NTPDases) are enzymes that hydrolyze nucleoside triand diphosphates with the release of orthophosphate and are dependent on the presence of divalent cations, such as calcium and magnesium. NTPDases perform different functions in a variety of organisms, including neurotransmission, nucleotide recycling and protein

of synthesizing purines thorough a De Novo route and therefore must acquire either nucleosides or nucleobases directly from the host, which will serve as a purine source for the parasite2. Several transporters of Therefore, the breakage of tri- and di-phosphate

Science Highlights

In addition, protozoan parasites are incapable


nucleotides to mono-phosphate nucleotide promoted by NTPDases may represent a first step to obtain the nucleotides from the host circulation.


Ecto-NTPDase activity have been detected at

Nucleotides play important roles as extracellular signaling molecules for blood cells1. Extracellular

the surface of a variety of human parasites, including Trypanosoma3,




ADP in the bloodstream plays an important role as

Toxoplasma6, suggesting that such proteins could play

an activator of platelet aggregation, while ATP is a

a important role in the interface between parasite and

potent inhibitor of this process. Nucleotides also

host. In fact, it has been proposed that Schistosoma

play important roles in modulation of the immune

mansoni ecto-NTPDase may play an important role

system, with ATP as a key molecule in process such as

in immune evasion by lowering ATP and ADP levels

the inhibition of Fc receptor-mediated phagocytosis

thus inhibiting platelet aggregation and cytotoxic

and proliferation of lymphocytes1. Considering that

responses4. Secretion of one of the two Toxoplasma gondii

several human parasites resides in the bloodstream,

NTPDase isoforms in the host cell vacuole was

it is not unexpected that such parasites may display

associated with the parasite virulence6. Higher

systems interfering with nucleotide signaling with the

ATPase activity was detected in Leishmania amazonensis

aim of subvert host blood cells signaling and prevent

virulent promastigotes when compared to the activity

the mobilization of an effective immune response.

of promastigotes avirulent strains5. Considering their

CD39-H.Sapiens CD39-M.musculus CD39L1-H.sapiens ATPDase1-S.mansoni CD39-L3-H.sapiens NTPase-N.caninum apyrase-lysosomal-H.sapiens 1000




Ynd1p-S.cerevisiae 966

NTPDase-1 T.brucei NTPDase-1 T.cruzi GDPase-L.major NTPDase-2 T.brucei

960 698


NTPASE-II-T.gondii NTPASE-I-T.gondii

868 968 981


502 934 788 979 969


Apyrase-C.elegans ATPDase2-S.mansoni CD39-L4-H.sapiens CD39-L2-H.sapiens NTPDase L.major

Phylogenetic tree constructed with the Neighbour Joining method from the multiple alignment ofseveral apyrases from different organisms using the ClustalX program. The numbers represent the statistical confidence of the branches, assigned by a bootstrap analysis (in 1000 samplings). The T. cruzi NTPDase-1, kinetoplastid apyrases and other human parasites’ apyrases are represented in blue, red and green, respectively. Figure 1. Evolution of NTPDases from parasites.



Science Highlights

Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity in Trypanosoma cruzi

ecto-location and the importance of these enzymes

forms of the parasite3, thus likely to be the responsible

to the parasite survival, it is natural to consider these

for the ecto-NTPDase activity detected in the parasite.

proteins as potential targets for therapy.

Considering the location and the demonstrated

Trypanosoma cruzi is the etiological agent of Chagas’

importance of NTPDases for blood dwelling parasites, it

disease being an obligate intracellular parasite. Three

is tempting to propose therapies based on the inhibition

distinct forms are compose the T. cruzi life cycle:

of its activity. The work described in this report aimed

amastigotes, a dividing form found intracellularly

to obtain data on importance of NTPDase activity in

in mammalian hosts; epimastigotes, a non-infecting

the process of infection to permit a better evaluation of

form found in the insect vector’s digestive tract, and

the value of these enzymes as targets for development

trypomastigotes that occur in the lumen of the rectum

of therapies.

of the insect and in the mammalian host. The infection in the mammalian host affects mainly muscle tissues

Results and discussion

in the heart and digestive tract. There are currently no








effective vaccines or drugs for treatment of Chagas’

trypomastigotes in VERO cell cultures was performed

disease, especially in the chronic phase.

and both ecto-nucleotidase and infectivity capacity

T. cruzi has only one described NTPDase gene

were measured (Figure 2), in order to verify if any

(named NTPDase-1), which was discovered by

correlation between those two parameters could

computational searches of the T. cruzi genome3.

be established. Ecto-nuclease activity assays were

Interestingly, Trypanosoma brucei and Leishmania major,

performed with whole live parasites in a, appropriated

other kinetoplastids with described genomes display two

medium. Each global exit of parasites to the culture

different NTPDase genes (Figure 1). This NTPDase

supernatant after completion of the intracellular life

was immunolocalized at the external membrane in all

cycle was named as ‘‘one passage (P)’’. Infectivity and


d b e



Blood trypomastigotes (infective form) were obtained from infected mice (a) and used to infect VERO cell (b). Extra cellular parasites collected from culture medium (c) were used in the following experiments: measurement of whole parasite ecto-ATPDase activities (d); evaluation of the in vitro infectivity (e) and evaluation of the in vivo infectivity and virulence using murine model (f). Figure 2. Summary of the methodologies used in this work.


The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

ecto-ATPase/ADPase ratios were measured for four

adenosine-5-triphosphate); Gadolinium, a lanthanide

passages, after which the infectivity levels were too


low to allow the recovery of sufficient parasites to

compound. After pre-incubation of parasites with

perform enzymatic assays (Figure 3). It was verified

different concentrations of inhibitors, assays of

that parasites going to multiple passages tended to be

nucleotide hydrolysis activity in whole parasites and

less infective and also to display lower ecto-ATPase/

infection of VERO cells were performed. Incubation

ADPase ratios, suggesting a possible correlation

with any of the three inhibitors resulted in decrease

between those two factors2.

of both ecto-ATPase and ADPase activity over a





Aiming to obtain more data on this possible

range of concentrations. Parasites pre-treated with

correlation, experiments of treatment of live parasites

either Suramin or Gadolinium showed significantly

with different known NTPDase inhibitors were

diminished infection levels (Figure 4A). These

performed. The inhibitors tested were: ARL67156

results provided further evidence for a link between

(6-N,N-Diethyl-b-c-dibromomethylene-D -

NTPDase activity and infectivity of T. cruzi.

Science Highlights

Santos et al.

4000 A Ecto-nucleotidase activity (nmol Pi.10 8 parasites-1.h-1)

3500 3000 2500 2000 Passage P1-1 P1-2 P3-1 P3-2 P4-1 Ama-like-P3

1500 1000 500 0





ATPase/ADPase 12.32 35.68 3.34 1.84 5.6 2.02

P4-1 Ama-like-P3

Cellular Passage Ecto-ATPDase (solid bars) and Ecto-ADPDase (open bars) activities from live trypomastigotes from different cellular passages (P1, P3 and P4). P1-1 and P1-2 are the first and second massive exits of parasites from the 1st passage; in the same way P3-1 and P3-2 are the first and second massive exits of parasites for the 3rd passage. Data are mean Âą SE of triplicate assays from one experiment. The inset shows the ATPase/ADPase hydrolytic activities ratio.

Microphotograph of infected VERO cells culture after 24 h of parasite-cell interaction at the 3rd to 4th passage. Spherical bodies are non-internalized amastigote-like parasites. Zoom from box section shown in B. Black arrow exemplifies a non-internalized amastigote-like and white arrow a noninternalized trypomastigote parasite. Reproduced with permission from Santos R.F. et al., PLoS Negl Trop Dis 3(3): e387. Š Santos R.F. et al.

Figure 3. T. cruzi infectivity and ecto-ATPase/ADPase ratio decrease during in vitro cultivation.



Science Highlights

Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity in Trypanosoma cruzi

Suramin (at 0.1 mM concentration) was also

still unknown.

capable of inhibit ATPDase activity of recombinant T.

Finally, in vivo assays in mice were performed using

cruzi NTPDase-1 to 40% of its original activity, which

P1 Y trypomastigotes pre-treated with the optimal

was very similar to the extent of Suramin inhibition of

concentration of Ecto-NTPDase inhibitors. After

hydrolytic activity on live parasites. This indicates that

treatment the parasites were used for infection of

the effects of this drug on intact parasites may be a result

mice. Micro were monitored daily during 40 days in

the action of this inhibitor to the Ecto-NTPDase-1

relation to the number of parasites in collected blood

on their surface. Nonetheless, Gadolinium and ARL

samples (parasitemia) and the number of surviving

67156 were not able to inhibit of T. cruzi NTPDase-1,

mice as a measure of virulence. It was possible to note

suggesting that additional enzymes with NTPDase

that parasitemia and mortality of mice infected with

activity may be present in the parasite surface to

treated parasites were lower than the negative parallel

account for the effects of these inhibitors in the assays

control experiment (Figure 4B). ARL 67156 was the

performed with the whole parasite.

most effective drug, resulting in approximately 60%

A polyclonal serum against T. cruzi NTPDase-1 was raised by immunization of rabbits with the

higher mice survival when compared to the control experiment.

recombinant protein. Incubation of whole parasites

Taken together these experiments showed the

with this serum also induced a decrease of parasite

importance of NTPDase activity for the infection

infectivity (Figure 4A). This reinforce the notion

process of the parasite and indicate that therapies

that T. cruzi NTPDase-1 is important to infection

targeting the proteins responsible for such activity

process, although the details of how this happen are

are feasible. The successful use of known ATPDase

Mammalian cell infection

Infectivity analysis

Effect of apyrase inhibitors and anti-apyrase antibody on in vitro infectivity of trypomastigotes.

Infectivity Ecto-NTPDase Inhibitors Inhibition (%) ARL 67156 (300 M) 42 ± 1.17 Gadolinium (300 M) 65 ± 2.90* Suramin (100 M) 71 ± 0.85* Policlonal anti-NTPDase-1 (1:50) 53 ± 0.58*

Ecto-ATPDase inhibited parasites

Virulence analysis

Mortality in Swiss mice infected with 5,000 parasites/0.1 mL blood (Y strain P1 trypomastigotes).

Surviving mice (%)

Ecto-ATPDase inhibited parasites

Mouse infection

100 80 60 Control ARL 67156 300 PM Gadolinium 300 PM Suramin 300 PM Suramin 1 mM

40 20 0 0









Days post infection Figure 4. Influence of Apyrase inhibitors on parasite infectivity and virulence. Parasites were pre-treated with ARL67156, Gadolinium or Suramin as indicated; a negative control assay is included, omitting the parasites’ drug pre-treatment. Data are from one experiment using the mean value of a group of 10 mice in each treatment. Modified with permission from Santos R.F. et al., PLoS Negl Trop Dis 3(3): e387. © Santos R.F. et al.


The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

inhibitors to block infection process of trypanosomes

for inhibition experiments with different nucleotide

provides a proof-of-principle for development of

analogs as well as studying its structure to obtain

future drugs based on this concept. Direct inhibition

further insights on the development of inhibitors.

of NTPDase-1 activity by the inhibitor Suramin


together with its ecto-localization suggests that this

This work was supported by Fundaçãoo de Amparo

protein is one of the possible molecular targets for an

a Pesquisa do Estado de Minas Gerais (FAPEMIG),

eventual therapy based on blockage of infection by

Conselho Nacional de Desenvolvimento Científico

inhibition of ecto-ATPDase activity. We are currently

e Tecnológico (CNPq) and Coordenação de Aper-

utilizing the recombinant form of T. cruzi NTPDase-1

feiçoamento de Pessoal de Nível Superior (CAPES).

References 1. Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli A, Torboli M, Bolognesi G, Baricordi OR. Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood. 2001 Feb 1;97(3):587-600. 2. Landfear SM, Ullman B, Carter NS, Sanchez MA. Nucleoside and nucleobase transporters in parasitic protozoa. Eukaryot Cell. 2004 Apr;3(2):245-54. 3. Fietto JL, DeMarco R, Nascimento IP, Castro IM, Carvalho TM, de Souza W, Bahia MT, Alves MJ, Verjovski-Almeida S. Characterization and immunolocalization of an NTP diphosphohydrolase of Trypanosoma cruzi. Biochem Biophys Res Commun. 2004 316(2):45460. 4. Vasconcelos EG, Ferreira ST, Carvalho TM, Souza W, Kettlun AM, Mancilla M, Valenzuela


Science Highlights

Santos et al.

MA, Verjovski-Almeida S. Partial purification and immunohistochemical localization of ATP diphosphohydrolase from Schistosoma mansoni. Immunological cross-reactivities with potato apyrase and Toxoplasma gondii nucleoside triphosphate hydrolase. J Biol Chem. 1996 271(36):22139-45. 5. Berrêdo-Pinho M, Peres-Sampaio CE, Chrispim PP, Belmont-Firpo R, Lemos AP, Martiny A, Vannier-Santos MA, Meyer-Fernandes JR. A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence. Arch Biochem Biophys. 2001 Jul 1;391(1):16-24 6. Asai T, Miura S, Sibley LD, Okabayashi H, Takeuchi T. Biochemical and molecular characterization of nucleoside triphosphate hydrolase isozymes from the parasitic protozoan Toxoplasma gondii. J Biol Chem. 1995 May 12;270(19):11391-7.


Structure and Calcium Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp. Pricila Hauk1,2,3, Cristiane R. Guzzo3, Henrique Roman-Ramos1,3, Paulo Lee Ho1,2,3, Chuck S. Farah3 Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brasil Programa de Pós-Graduação Interunidades em Biotecnologia USP/IPT/Instituto Butantan, Instituto de Ciências Biomédicas da USP, São Paulo, SP, Brasil 3 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil 1 2

Abstract Leptospirosis, caused by the spirochaete Leptospira, is an important emerging infectious disease. LipL32 is the major exposed outer membrane protein (OMP) found exclusively in pathogenic leptospires. It is highly immunogenic and has been shown to bind to host extracellular matrix (ECM) components, including collagens, fibronectin and laminin. In this work we crystallized recombinant LipL32 protein and determined its structure to 2.25 Å resolution. Initial phases were determined using the multiwavelength anomalous dispersion technique with data collected from selenomethionine-containing crystals at the MX2 beamline at the LNLS. The LipL32 monomer is made of a jelly-roll fold core from which protrude several peripheral secondary structures. Some structural features suggested that LipL32 could bind Ca2+ ions and indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium binding properties of LipL32. Keywords Leptospira, LipL32, MAD-phasing, jelly-roll fold, calcium-binding

Original publications: J Mol Biol 390, 722-736, 2009. Acta Crystallogr Sect F Struct Biol Cryst Commun 65, 307-309 , 2009.

Many fundamental aspects of Leptospira interrogans biology are poorly understood. The most abundant antigen found in the leptospiral total protein profile is LipL323. This highly immunogenic outer-membrane lipoprotein is conserved among pathogenic Leptospira

Science Highlights

the crystal structure of the Ca2+-LipL32 complex.


The crystal structures of this important leptospiral protein1,7,8 have provided a wealth of information from which to raise hypothesis regarding the molecular mechanisms by which LipL32 interacts with Ca2+ and with ECM proteins.

species but not observed in the non-pathogenic

Results and discussion

saprophytic L. biflexa4,5. It is therefore considered a promising target for vaccine development and

Resolution of the LipL32 Structure

diagnosis of leptospirosis. Recently, studies using

Recombinant LipL3221-272 containing selenome-

a Leptospira interrogans LipL32 mutant showed that

tionine was crystallized in space group P3221. This

LipL32 does not play a essential role in either acute or

fragment corresponds to the full-length mature

chronic models of animal infection6. While these data

protein minus the N-terminal lipid-anchored cysteine

did not identify an indispensible role in pathogenesis,

residue. The X ray diffraction datasets (Table 1) used to

it does not exclude an important function for LipL32

resolve the structure were collected from two crystals

in mediating the host-pathogen interaction.

at the MX2 beamline at the LNLS1,2. Initial phases

Here, we describe the resolution of the X-ray

were estimated using a MAD data set collected from

structure of LipL32, which represents a fundamental

one crystal at two wavelengths, 0.97814 and 0.978308

step towards understanding its structure-function

Å, corresponding to the peak and inflection points of

relationships. The resolved tertiary structure showed

the fluorescence spectrum, respectively. The f ’ and f ’’

a jelly roll fold similar to those presented by some

anomalous scattering factors were estimated from the



fluorescence spectrum of the SeMet-labelled protein

Additional biochemical characterizations confirmed

crystal using the program CHOOCH. The program

the capacity of LipL32 to bind calcium ions. Subsequent

SHELXD was used to find the selenium sites in the

to the termination of this work, anoher group resolved

asymmetric unit and these positions were refined and




119 2

1 122

126 72

113 7

62 225

98 182 173 134 215 6


111 47






184 170

2 1





N 35


54 233



139 208





3 153 145 3101

193 198 201 203

Cartoon model of the LipL3221-272 monomer. The N- and C-termini are indicated. -strands, -helices and 310 helices are numbered in the order in which they appear in the sequence. Helices are colored red, the eight -strands that form the core jelly-roll topology are shown in blue, while the other -strands are show in yellow.



263 C

Topology diagram for LipL3221-272 in which helices are represented as red rectangles and -strands as arrows.

Figure 1. LipL3221-272 structure and crystal contacts. Figures reproduced from Hauk et al.1.



Science Highlights

Structure and Calcium Binding Activity of LipL32

phases calculated up to 2.6 Å resolution using the

interesting feature is the loop between 3 and 9

program SHARP (note that while the dataset for this

(residues 154-169), that contains a large proportion

crystal was complete only up to 2.93 Å, incomplete

of acidic amino acids (AKPVQKLDDDDDGDD)

data was collected up to 2.6 Å). Phases were refined by

of which those underlined are disordered with no

density modification and the electron-density map was

significant electron density in both monomers). This

used to construct a preliminary polyalanine model using

loop has been subsequently shown to be involved in

the program ARP/wARP that contained 303 residues


distributed over 27 peptide segments. Interpretation residues was performed using the program COOT.

Comparison of LipL3221-272 with other protein structures

Subsequently, a second dataset was collected from a

LipL3221-272 has no significant sequence similarity

new crystal that diffracted to 2.25 Å resolution using

with proteins of known structure. We therefore used

of electron-density maps and construction of missing

1.459 Å radiation . The initial model was used to

the Dali program9 to search for protein structures

calculate phases for the 2.25 Å resolution dataset using

with topologies similar to that of LipL3221-272. This

the Phaser program. Structural refinement of the

analysis found a small group of proteins with Z-scores

LipL32 model was done using ARP/wARP, REFMAC,

above 5.0. All these proteins share a common jelly-

CNS, and COOT. TLS was used in the final cycles

roll fold topology at their core (shown in blue in Figs

of refinement with each chain in the asymmetric unit

1a and 1f) but all differ in the nature and number of

divided into 15 independent group segments.

secondary structure elements inserted in the loops


Even though Matthews coefficient analysis

between these strands. The structure with the highest

suggested 3 or 4 molecules in the asymmetric unit, the

Dali z-score (8.8) was that of the isolated domain III

final model contained only two monomers (Mathews

from human calpain 7 (pdb 2qfe; unpublished) which

coefficient = 4.03) and 69.5% solvent. The refined

is distinguished from the others by the presence of a

structure converged to Rwork and Rfree values of 0.184

C-terminal -strand that is topologically analogous to

and 0.227, respectively. The two monomers in the

13 of LipL3221-272.

asymmetric unit are related by a non-crystallographic two-fold axis and are very similar in structure, with a RMSD of 0.6 Å for all atoms. The LipL32 structure has been deposited in the Protein Data Bank (pdb) under code 3FRL.


Many of the proteins with topologies most similar to LipL32 have been shown to bind calcium ions. For example, domain III from calpain, collagen-binding domain (CBD) from ColG, and many icosahedral RNA virus coat proteins, undergo significant structural

LipL32 structure

rearrangements upon binding calcium10-13. Analysis of

The monomer structure is built around a central

the superposition of LipL3221-272 with the Ca2+-ColG

jelly-fold -sandwich topology of 8 -strands (strands

collagen binding domain structure revealed that some

3, 5, 6, 8, 9, 10, 11, 12) from which protrude loops that

of the secondary structure elements that contribute to

carry the other secondary structure elements (Fig. 1a,b).

the ColG Ca2+-binding site are absent in LipL3221-272.

Some of these peripheral structures are -strands (4,

Another possible Ca2+-binding site was identified

7 and 13) that associate with the edges of the central

as the loop between 3 and 9 that contains seven

-sandwich. Four alpha helices and two 310 helices are

aspartate residues within an eight amino acid stretch

also found in these loops (Fig. 1a,b). The two 310 helices

(residues 161-168). A portion of this loop containing

are both found in a stretch between 10 and 11. 3101

the first three aspartates (residues 161-168) are absent

is two turns in length (residues 193-198) while 3102

in our crystal structure due to lack of electron density.

is made up of only one turn (residues 201-203). One

We predicted that this loop could adopt a more ordered

conspicuous feature of the LipL3221-272 structure is an

structure upon ligand binding. This prediction was

N-terminal -hairpin (1-2) that protrudes from

born out by the subsequent determination of the

the more compact portion of the molecule. Another

Ca2+-LipL32 crystal structure7 (also see Fig. 3 below).

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

Hauk et al.

LipL3221-272 specifically binds calcium ions LipL3221-272 is capable of interacting with calcium ions. We therefore used circular dichroism and fluorescence spectroscopy to detect structural

Molar Ellipticity x 10-6 (deg.cm2.dmol-1)

The above observations led us to test whether

0.6 0.4 0.2 0.0 -0.2

changes in LipL3221-272 induced by the addition


of calcium ions1. Circular dichroism spectra

-0.6 200 210 220 230 240 250 260

of LipL3221-272 clearly demonstrate a change in secondary structure content upon the addition of Ca2+ but not upon the addition of Mg2+ (Fig. 2a). LipL3221-272 possesses three tryptophan residues,

Wavelength (nm)

Circular dichroism (CD) spectra of LipL3221-272 in the absence or presence of Ca2+ or Mg2+. CD experiments were performed with 10 µM protein in 10 mM Tris-Cl pH 8.0, 50 mM KCl in the presence or absence of either 1 mM CaCl2 or 1 mM MgCl2.

Trp85, Trp130 and Trp134 all of which are


and a significant blue-shift in the LipL3221-272 tryptophan emission was observed upon the addition of 1 mM CaCl2 but not upon addition of 1 mM MgCl2 (Fig. 2b). Furthermore, the fluorescence of 1-amino-2-naphthol-4-sulfonic acid (ANS) was

Relative Fluorescence

significantly buried within the LipL3221-272 structure

observed to increase upon the addition of Ca2+ (data





with an increase in the surface hydrophobicity of the LipL3221-272 molecule. None of these changes were observed when the experiments were repeated using Mg2+, Zn2+ or Cu2+ ions (data not shown)1.


or absence of Ca2+ 1. In the CD experiments, the midpoint of the conformational transition (Tm) was observed to increase from 50 °C in the absence of Ca2+ to 56 °C in the presence of Ca2+ while only a very small change in the Tm (+1 °C) was observed upon addition of MgCl2 (Fig. 2c). A similar result was obtained by monitoring ANS fluorescence: in the presence or absence of Mg2+ the Tm was 51 °C while in the presence of Ca2+ it increased to 58 °C (data not shown)1. No changes in thermal stability were observed in the presence of 1 mM ZnCl2 or CuCl2 (data not shown).


Fraction Folded

We then tested whether the addition of Ca2+

thermal denaturation of the protein in the presence



LipL3221-272 tryptophan fluorescence in the absence of divalent metals or in the presence of Ca2+ or Mg2+. A blue-shift in Trp emission is observed in the presence of Ca2+. Intrinsic fluorescence experiments were performed with 2 µM protein in 10 mM Tris-Cl pH 8.0, 50 mM KCl in the presence or absence of either 1 mM CaCl2 or MgCl2. Excitation wavelength was 285 nm. 1.0

CD and ANS fluorescence were monitored during


Wavelength (nm)

Calcium binding by LipL3221-272 increases protein stability affected the conformational stability of LipL3221-272.



not shown), indicating an overall structural change

0.6 0.4 0.2 0.0 30





Temperature (°C) LipL32

LipL32 + MgCl2

LipL32 + CaCl2

Tm LipL32 = 50 °C Tm LipL32 + MgCl2 = 51 °C Tm LipL32 + CaCl2 = 56 °C

Thermal stability of LipL3221-272 in different conditions. Circular dichroism was used to detect temperature-induced changes in LipL3221-272 secondary structure (see Materials and Methods). Figure 2. LipL3221-272 binds Ca2+ ions. Conditions were the same as in part (a). Figures reproduced from Hauk et al.1.


Structure and Calcium Binding Activity of LipL32

motility and chemotaxis, host-pathogen interactions, stability and integrity of the outer lipopolysaccharide layer and bacterial cell wall and specific enzyme activity14-17 and calcium ions are absolutely required for Leptospira spp to grow and survive18,19. We demonstrated for the first time that LipL32 binds Ca2+. The data also suggest that LipL32 does not bind Mg2+, Zn2+ or Cu2+ ions, at least not in a manner similar to that observed for Ca2+, since no significant structural changes or increases in thermal stability were observed in these cases. Analysis of the LipL32 structure pointed to two putative Ca2+binding sites. One of these, the loop between 3 and 9 that contains seven aspartate residues within an eight amino acid stretch (residues 161-168) has Superposinion of the apo-LipL32 structure (this work, in blue and green)1 and the Ca2+-LipL32 structure determined by Tung et al. (in red and yellow)7. Residues directly involved in Ca2+-binding are shown as stick models in green (apo) and yellow (Ca2+-bound form). Arrows indicate distances between equivalent atoms in the two structures (residues D164, E173, Y178 and F193). The Ca2+ ion is shown as a transparent yellow sphere. Figure 3. Comparison of LipL32 apo and Ca2+ structures.

recently been shown to contribute significantly to the Ca2+-binding site in the Ca2+-LipL32 structure7. As shown in Figure 3, Ca2+-binding induced large structural rearrangements in several peripheral secondary structure elements and loops (for example, a 23A shift for residue D164). The crystal structures of this important leptospiral protein1,7,8 have provided a wealth of information from which to raise hypothesis regarding the molecular mechanisms by which LipL32 interacts with Ca2+


and with ECM proteins. These hypotheses will be

The importance of calcium as a key regulator in

addressed in future experimental studies using site-

several fundamental aspects of eukaryote biology is well established. An increasing importance for a role for calcium in prokaryotes is also being implicated in several processes, including signal transduction, cell cycle and division control, competence, pathogenesis,


directed LipL32 mutants.

Acknowledgements The authors would like to thank CNPq, Fapesp and the Fundação Butantan for financial support.

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

References 1. Hauk, P., Guzzo, C. R., Roman Ramos, H., Ho, P. L., and Farah, C. S. (2009) Structure and calciumbinding activity of LipL32, the major surface antigen of pathogenic Leptospira sp, J Mol Biol 390, 722-736. 2. Hauk, P., Guzzo, C. R., Ho, P. L., and Farah, C. S. (2009) Crystallization and preliminary X-ray analysis of LipL32 from Leptospira interrogans serovar Copenhageni, Acta Crystallogr Sect F Struct Biol Cryst Commun 65, 307-309. 3. Zuerner, R. L., Knudtson, W., Bolin, C. A., and Trueba, G. (1991) Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona, Microb Pathog 10, 311322. 4. Haake, D. A., Chao, G., Zuerner, R. L., Barnett, J. K., Barnett, D., Mazel, M., Matsunaga, J., Levett, P. N., and Bolin, C. A. (2000) The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection, Infect Immun 68, 2276-2285. 5. Picardeau, M., Bulach, D. M., Bouchier, C., Zuerner, R. L., Zidane, N., Wilson, P. J., Creno, S., Kuczek, E. S., Bommezzadri, S., Davis, J. C., McGrath, A., Johnson, M. J., Boursaux-Eude, C., Seemann, T., Rouy, Z., Coppel, R. L., Rood, J. I., Lajus, A., Davies, J. K., Medigue, C., and Adler, B. (2008) Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis, PLoS ONE 3, e1607. 6. Murray, G. L., Srikram, A., Hoke, D. E., Wunder, E. A., Jr., Henry, R., Lo, M., Zhang, K., Sermswan, R. W., Ko, A. I., and Adler, B. (2008) The major surface protein LipL32 is not required for either acute or chronic infection with Leptospira interrogans, Infect Immun.

B., and Rossjohn, J. (2009) Crystal structure of LipL32, the most abundant surface protein of pathogenic Leptospira spp, J Mol Biol 387, 12291238. 9. Holm, L., Kaariainen, S., Rosenstrom, P., and Schenkel, A. (2008) Searching protein structure databases with DaliLite v.3, Bioinformatics 24, 27802781. 10. Johnson, J. E. (2003) Virus particle dynamics, Adv Protein Chem 64, 197-218. 11. Speir, J. A., Bothner, B., Qu, C., Willits, D. A., Young, M. J., and Johnson, J. E. (2006) Enhanced local symmetry interactions globally stabilize a mutant virus capsid that maintains infectivity and capsid dynamics, J Virol 80, 3582-3591. 12. Tompa, P., Emori, Y., Sorimachi, H., Suzuki, K., and Friedrich, P. (2001) Domain III of calpain is a ca2+-regulated phospholipid-binding domain, Biochem Biophys Res Commun 280, 1333-1339. 13. Wilson, J. J., Matsushita, O., Okabe, A., and Sakon, J. (2003) A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation, EMBO J 22, 1743-1752. 14. Michiels, J., Xi, C., Verhaert, J., and Vanderleyden, J. (2002) The functions of Ca(2+) in bacteria: a role for EF-hand proteins?, Trends Microbiol 10, 8793. 15. Norris, V., Chen, M., Goldberg, M., Voskuil, J., McGurk, G., and Holland, I. B. (1991) Calcium in bacteria: a solution to which problem?, Mol Microbiol 5, 775-778. 16. Norris, V., Grant, S., Freestone, P., Canvin, J., Sheikh, F. N., Toth, I., Trinei, M., Modha, K., and Norman, R. I. (1996) Calcium signalling in bacteria, J Bacteriol 178, 3677-3682. 17. Onek, L. A., and Smith, R. J. (1992) Calmodulin and calcium mediated regulation in prokaryotes, J Gen Microbiol 138, 1039-1049.

7. Tung, J. Y., Yang, C. W., Chou, S. W., Lin, C. C., and Sun, Y. J. Calcium binds to LipL32, a lipoprotein from pathogenic Leptospira, and modulates fibronectin binding, J Biol Chem 285, 3245-3252.

18. Johnson, R. C., and Gary, N. D. (1963) Nutrition of Leptospira Pomona. Iii. Calcium, Magnesium, and Potassium Requirements, J Bacteriol 85, 983985.

8. Vivian, J. P., Beddoe, T., McAlister, A. D., Wilce, M. C., Zaker-Tabrizi, L., Troy, S., Byres, E., Hoke, D. E., Cullen, P. A., Lo, M., Murray, G. L., Adler,

19. Shenberg, E. (1967) Growth of pathogenic Leptospira in chemically defined media, J Bacteriol 93, 1598-1606.


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Hauk et al.


The Relevance of Proline Metabolism in Trypanosoma cruzi Ariel M. Silber Departamento de Parasitologia, Instituto de Ciências Biomédicas Universidade de São Paulo, São Paulo, SP, Brasil

Abstract The flagellated parasite Trypanosoma cruzi, the etiological agent for Chagas’ disease, has a complex life cycle in mammals and insect vectors. During its life cycle, the parasite alternates between infective non-replicative and replicative non-infective stages. T. cruzi uses proline and glutamate, among other amino acids, as energy sources. Biochemical evidence supports the hypothesis that T. cruzi oxidizes L-proline into glutamate, this being a central pathway for T. cruzi metabolism. Furthermore, proline has been previously implicated in metacyclogenesis, the parasite differentiation process occurring in the insect vector. In the present work, we investigated the role of proline and proline metabolism in several biological processes which are essential for the parasite life cycle. During this research, we discovered that a metabolic switch occurs from a metabolism based on the consumption of glucose to one based on the consumption of proline, where the latter constitutes a major energy supply for growth and differentiation of the intracellular stages. In addition, proline is also a major energy source for host-cell invasion. Our results also showed that the hexose transporter TcHT, the only glucose transporter found in T. cruzi up to now, has a dual function: it is responsible for the uptake of glucose from the extracellular medium into the cytosol, and it is also responsible for the glucose uptake from the cytosol into the intracellular organelle where glycolysis begin: the glycosome. Finally, we learned that the inhibition of proline transport by using structural analogues of proline led to a diminished intracellular free proline concentration, which correlated with an increased sensitivity to oxidative, thermal and nutritional stress. The diversity of processes in which proline is involved in T. cruzi, sugest this metabolite to be particularly relevant for the survival of these organisms, allowing us to propose its transporters and the enzymes involved in its metabolism as interesting targets for the treatment of T. cruzi infection. Keywords amino acid metabolism, proline, glucose transport, host-cell invasion

Original publications: PLoS ONE 4, e4534, 2009. Mol Biochem Parasitol In press, 2009.

metacyclic trypomastigotes, which are eliminated

Trypanosoma cruzi, the etiological agent responsible

in feces and deposited on the mammals’ skin while

for Chagas’ disease, affects some 10 million people

the triatomine bug bites and feeds. Trypomastigotes

in the Americas. This organism has a complex life

enter the body and invade host cells where they

cycle involving mammals and reduviid insects,

differentiate into dividing amastigotes and after

which serve as vectors for the infection. These

proliferating, differentiate into the trypomastigote

become infected when they bite an infected mammal

form after passing through a transient epimastigote-

carrying trypomastigote forms of the parasite

like stage. Finally, the trypomastigotes lyse host

circulating in its bloodstream. Trypomastigotes,

cells and are released into the extracellular medium,

infective non-dividing forms of the parasite are

where they can invade other cells or the bloodstream,

ingested with the blood and in the insect’s digestive

becoming capable of invading other tissues or a non-

tube, they differentiate into the dividing and non-

infected reduviid insect, thus completing the cycle

infective epimastigote form. In the terminal portion

(Boscardin et al.) (Figure 1).

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of the digestive tube, epimastigotes differentiate into


Vertebrate host Trypomastigote

Intracellular epimastigote



Metacyclic trypomastigote

Invertebrate host

Figure 1. Schematic representation of the T. cruzi life cycle: In the insect vector, epimastigotes replicate and differentiate into infective non-dividing metacyclic trypomastigotes. These forms invade the host-cells and achieves the cytoplasm, where divide. After replication, the amastigotes differentiate into trypomastigotes passing through a transient stage called intracellular epimastigote. Trypomastigotes lyse the host-cells and are released to the extracellular medium, where they can invade neighbor cells or can fall into the blood stream. When a non-infected insect vector bites an infected mammalian host having circulating trypomastigotes, this insect vector is infected, being able to transmit the infection by T. cruzi to another mammalian host, perpetuating the transmission of the infection.



The Relevance of Proline Metabolism in Trypanosoma cruzi

Mainly two phases are recognized in the clinical evolution of the disease: the acute phase, which is asymptomatic in most of cases, and the chronic phase, which can be asymptomatic and last for the entire life of the infected patient (indeterminate form affecting some 70% of the chronic individuals) or symptomatic (affecting some 30% of the individuals chronically infected), which itself can be divided in both cardiac and the digestive forms. Although occurring at a lower frequency, the chronic phase can also consist of alterations affecting the peripheral or central nervous system. Despite the fact that Chagas’ disease was described for the first time a century ago, only two therapeutic compounds have been shown to be useful against the human infection by T. cruzi: Benznidazole (BZL) and Nifurtimox (NF). The treatment is effective under several circumstances including during the acute phase as well as in congenital infections, reactivation of an infection or in early chronic disease. However, its efficacy during the chronic phase is still controversial. In cases involving either drug-resistant or partially resistant strains, neither BZL nor NF therapies work well. All these observations make the search for new drugs a mandatory task. Figure 2. Examples of amastigote infected tissues: arrows show amastigote nests in an infected heart muscle (A) and intestinal epithelial tissue (B).

Amino acids are metabolites that have a myriad of biological roles in several organisms, beyond their participation as the fundamental building blocks for proteins. Since the early ‘70s, several amino acids have been proposed to be relevant to the bioenergetics of T. cruzi. At that time the involvement of proline, glutamate, aspartate, glutamine, asparagine, leucine

Glucose transport

Intracellular proline

Proline transport

and isoleucine as oxidable energy sources was well demonstrated. In the early ‘80s, it was also shown


Relative quantity

that the amino acids proline, aspartate and glutamate 80

were involved in differentiation processes occurring inside the vector, while the amino acids leucine and


isoleucine negatively regulated this process. These observations called our attention to the fact that some


major cellular processes such as cell remodeling


prior to parasite infection of the mammalian host are probably regulated by the kind of amino acid present in

0 Trypomastigotes Amastigotes Intracellular Trypomastigotes Epimastigotes

Figure 3. Representation of the variation of the proline and glucose transport activity (red and green lines) and the intracellular free proline content (blue line) along the T. cruzi host-cell infection.


the medium (reviewed by (Silber et al., 2005)). Finally, some years ago we characterized proline uptake by the parasite (Silber et al., 2002), and established it’s relationship with differentiation among intracellular stages of (Tonelli et al., 2004).

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Silber A.M.

Figure 4. Subcellular localization of the T. cruzi glucose transporter. Epimastigotes were labeled with a specific antibody recognizing the glucose transporter (A), a typical reservosome protein (cruzipain) (B), a specific dye for DNA (nuclear and kinetoplastid DNA) (C), and processed for immunofluorescence microscopy. The merged image is also shown (D). The epimastigotes were also submitted to transmission electron microscopy (E and F). Arrows, glycosomes; small dots correspond to a glycosomal specific marker (glyceraldehydes 3 phosphate dehydrogenase); large dots correspond to the glucose transporter.

The particularly large number of biological

evaluated. The mRNA and protein levels varied with

processes in which proline and glutamate are involved

the specific activity over the course of the infection,

in this parasite led us to revisit this topic. We are

being particularly relevant in trypomastigotes in

currently investigating this metabolic pathway with

contrast to the intracellular stages which showed little

particular emphasis on its relevance for the intracellular

(if any) glucose transport activity. Furthermore, when

cycle of the parasite, as well as some yet to be described

expressed, the glucose transporter was located not only

roles for proline, glutamate, or the enzymes involved

in the plasma membrane of the parasite (as expected)

in their inter-conversion in T. cruzi.

and in reservosomes (vesicles participating of the endocytic pathway) as previously reported (Sant’Anna

What did we learn about the role of proline in T. cruzi?

et al., 2009), but also in a unique organelle called the glycosome, in which the first part of glycolysis occurs

1. Proline, glucose and energy metabolism over the time course of the infection

(Figure 3). This fact led us to attribute two functions

As mentioned above, we were able to establish a

glucose from the external medium into the cytosol

relationship between proline uptake and differentiation

and the uptake of glucose from the cytosol into the

during the intracellular cycle, which raised the question:

glycosome (Silber et al., 2009). Glucose uptake activity

is proline a relevant energy source for the intracellular stages of

of the different stages of the parasite during the

T. cruzi? To address this problem, we first evaluated

infection cycle was also compared with previous data on

how glucose was being used over the time course of

proline uptake and on the intracellular proline reserves

the mammalian infection cycle. The expression and

in order to create a broader overall picture (Fig. 4).

activity levels, as well as the location of the single

Trypomastigotes, which live in the extracellular medium

known glucose transporter (Tetaud et al., 1994) were

where glucose is available, have the highest glucose and


to the single glucose transporter of T. cruzi: to transport


The Relevance of Proline Metabolism in Trypanosoma cruzi

the lowest proline transport activities, being probably

first oxidation step is catalyzed by a FAD dependent

dependant mainly on glucose metabolism for energy

proline dehydrogenase (ProDH) which uses proline

requirements. Amastigotes in turn have the highest

as a source of electrons to be feed into the respiratory

intracellular free proline concentration but their proline

chain (unpublished data). As it has been proposed

transport activity is low and there was no evidence of

that the activity of orthologs of this enzyme in other

glucose transport whatsoever, being more dependent

organisms are involved in the regulation of the

on endogenous amino acids. Apparently, the amino acid

generation of reactive oxygen species (ROS) and

pool of the amastigotes is consumed during replication

apoptosis, we investigated if this could also be the

and/or differentiation to the intracellular epimastigote

case for the T. cruzi enzyme. Surprisingly, experiments

stage, which has the lowest levels of intracellular free

performed on T. cruzi cells treated with a proline

proline concentration and depends upon an external supplement of this metabolite in order to differentiate into the trypomastigote stage. Accordingly, this stage shows the highest proline transport activity and the amino acid is likely to be provided by the proline pool of the host cell. Proline (and probably other amino acids) therefore plays an important role in the metabolism of the intracellular forms where glucose transport activity is severely diminished (Silber et al., 2009).

analogue which competitively inhibits proline uptake (thereby causing a reduction in intracellular proline content) showed that resistance to oxidative stress was related to intracellular free proline accumulation and not to the regulation of the enzyme activity (Magdaleno et al., 2009). In addition, it was observed that the ability to accumulate intracellular free proline is related to the cells’ resistance to metabolic stresses. All of these functions of the amino acid proline are

2. Proline: the energy support of the host-cell invasion

fundamental for the parasite in all the environments it

It has previously been established that the invasion

encounters during its complex life cycle.

process by trypomastigotes is energy dependant (Schenkman et al.). However, the energy source supporting this process had not been identified. As

T. cruzi is exposed to many different environments

proline is a relevant energy source in trypanosomatids

(some of them extreme, such as the host-cell

its participation as an energy source during parasite

cytoplasm) along the course of its complex life cycle.

invasion was deemed worthy of evaluation. Firstly, the

Most of these environments could be considered

parasites were depleted of ATP by starving them for

dangerous for most organisms employing standard

different periods of time. We observed that starvation

biochemistry such as that based on glucose dependent

for 16 hours caused a significant reduction in

bioenergetics. The fact that this parasite alternates

intracellular ATP, which correlated with the parasite’s

from a bioenergetics based on glucose consumption

ability to attach to and invade mammalian host-cells.

to one based on proline consumption, or the fact that

It was also observed that after starvation, proline (but

proline and not glucose fuels the host cell invasion,

neither glucose nor glutamate) was able to restore

constitute interesting examples. The same applies to

the intracellular ATP levels as well as the capacity to attach and invade showing that proline is able to energetically support these early steps of the infection process (Martins et al., 2009).


Concluding remarks

some strategies for resistance to stress conditions: we have learned that T. cruzi uses proline accumulation as a defense against oxidative and nutritional stress. Proline seems to be a nodal point in the parasite’s metabolism,

3. Proline is involved in the resistance to oxidative and nutritional stress

which tends to suggest that its transporters, as well as

Proline, as an energy source, is involved in redox

relevant targets for therapeutic drug design. Further

metabolic reactions. It is oxidized to glutamate,

work is being undertaken to validate these targets and

which in turn is deaminated to -ketoglutarate, an

to rationally design inhibitors against key proteins

intermediate in the tricarboxylic acid cycle. The

participating in this interesting metabolic pathway.

the enzymes related to its metabolism, may well be

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References 1. Boscardin, S.B., Torrecilhas, A.C., Manarin, R., Revelli, S., Rey, E.G., Tonelli, R.R., Silber, A.M., Chagas’ disease: an update on immune mechanisms and therapeutic strategies. J Cell Mol Med. 2. Magdaleno, A., Ahn, I.Y., Paes, L.S., Silber, A.M., 2009. Actions of a proline analogue, L-thiazolidine4-carboxylic acid (T4C), on Trypanosoma cruzi. PLoS ONE 4, e4534. 3. Martins, R.M., Covarrubias, C., Rojas, R.G., Silber, A.M., Yoshida, N., 2009. Use of L-proline and ATP production by Trypanosoma cruzi metacyclic forms as requirements for host cell invasion. Infect Immun 77, 3023-3032. 4. Sant’Anna, C., Nakayasu, E.S., Pereira, M.G., Lourenco, D., de Souza, W., Almeida, I.C., Cunha, E.S.N.L., 2009. Subcellular proteomics of Trypanosoma cruzi reservosomes. Proteomics 9, 1782-1794. 5. Silber, A., Tonelli, R., Martinelli, M., Colli, W., Alves, M., 2002. Active transport of L-proline in


Trypanosoma cruzi. J. Eukaryot. Microbiol. 49, 441446. 6. Silber, A., Tonelli, R., Lopes, C., Cunha-e-Silva, N., Torrecilhas, A., Schumacher, R.I., Colli, W., Alves, M., 2009. Glucose uptake in the mammalian stages of Trypanosoma cruzi. Mol Biochem Parasitol In press.

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Silber A.M.

7. Silber, A.M., Colli, W., Ulrich, H., Alves, M.J., Pereira, C.A., 2005. Amino acid metabolic routes in Trypanosoma cruzi: possible therapeutic targets against Chagas’ disease. Curr. Drug. Targets. Infect. Disord. 5, 53-64. 8. Tetaud, E., Bringaud, F., Chabas, S., Barrett, M.P., Baltz, T., 1994. Characterization of glucose transport and cloning of a hexose transporter gene in Trypanosoma cruzi. Proc. Natl. Acad. Sci. U S A 91, 8278-8282. 9. Tonelli, R.R., Silber, A.M., Almeida-de-Faria, M., Hirata, I.Y., Colli, W., Alves, M.J., 2004. L-proline is essential for the intracellular differentiation of Trypanosoma cruzi. Cell. Microbiol. 6, 733-741.


Anacardic Acid Derivatives as Inhibitors of Glyceraldehyde-3Phosphate Dehydrogenase from Trypanosoma cruzi Junia M. Pereira1, Richele P. Severino1, Paulo C. Vieira1, João B. Fernandes1, M. Fátima G.F. da Silva1, Aderson Zottis2, Rafael V.C. Guido2, Adriano D. Andricopulo2, Glaucius Oliva2, Arlene G. Corrêa1 1 2

Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brasil Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brasil

Abstract Chagas’ disease, a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi, is a major public health problem affecting millions of individuals in Latin America. On the basis of its essential role in the life cycle of T. cruzi, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered an attractive target for the development of novel antitrypanosomatid agents. In the present work, we describe the inhibitory effects of a small library of natural and synthetic anacardic acid derivatives against the target enzyme. The most potent inhibitors, 6-n-pentadecyl- (1) and 6-n-dodecylsalicilic acids (10e), have IC50 values of 28 and 55 M, respectively. The inhibition was not reversed or prevented by the addition of Triton X-100, indicating that aggregate-based inhibition did not occur. Detailed mechanistic characterization of the effects of these compounds on the T. cruzi GAPDH-catalyzed reaction showed clear noncompetitive inhibition with respect to both substrate and cofactor. X-ray crystallography indicated the structural determinants underlying the new binding site as well as provided important insights for further studies aimed at the development of the inhibitors as novel antitrypanosomatid agents. Keywords anacardic acid, GAPDH, inhibitor, natural products

Original publications: Bioorg Med Chem 16, 8889–8895, 2008.







parasite Trypanosoma cruzi, is a major cause of illness, morbidity, long-term disability, and death in Latin America. Currently, there are over 9 million people infected with T. cruzi, resulting in a variety of adverse health events such as heart failure, with more than 50,000 deaths each year. It is thought that another 100 million people are at risk of infection. In spite of the alarming health, economic, and social consequences of this parasitic infection, the limited existing drug therapy (nifurtimox and benznidazole) suffers from a combination of drawbacks including poor efficacy, and serious side effects. Therefore, there is an urgent need for new safe and effective therapy against Chagas’ disease.1

hundreds of bioactive compounds have been isolated from plants, animal or microorganisms with different architectures, presenting high chemical diversity. However, we have few examples of “natural drugs” (templates) which are formulated for ready availability

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the 19th century were the alkaloids and since then,


as medicine. In the majority of cases the “natural drugs” must be chemically modified in order to improve their pharmacological profile. The medicinal chemist synthesizes structural analogs which are more appropriate for the target, for biological, chemical or even physico-chemical reasons. Several natural products and synthetic compounds have been evaluated against T. cruzi GAPDH using a standard biochemical assay.4,5 Among these, a mixture of anacardic acids, isolated from the Brazilian cashew-nut shell liquid, presented promising results.



Chemically, anacardic acids feature a convenient

(GAPDH, EC is a key enzyme involved

salicylic acid system and a long side chain at the

in the parasite’s glycolytic pathway (Figure 1). This

6-position, in which a double bond is found at C-8

homotetrameric enzyme catalyzes the oxidative

in the monoene, diene and triene components

phosphorylation of D-glyceraldehyde-3-phosphate

(Figure 3).6 These compounds exhibit a wide range



of biological activities (e.g.; antimicrobial, antitumoral,

and inorganic phosphate

molluscicide, antifungal, insecticide), stimulating the

(Figure 2).2 GAPDH plays a central role in controlling

search for new derivatives with improved properties.

ATP production in pathogenic parasites such as T. cruzi,

A variety of synthetic methods for the preparation

T. brucei, and Leishmania sp., the causative agents of

of anacardic acids, as well as for converting these

Chagas’ disease, sleeping sickness, and leishmaniasis,

materials to other useful compounds, has been


1,3-diphosphoglycerate +

in presence of NAD

respectively. On the basis of the essential role in the life cycle of trypanosomes, GAPDH has been considered an important target for drug development. Considering that intracellular amastigotes depend on glycolysis for ATP production, the inhibition of the glycosomal GAPDH would prevent T. cruzi from being infective. In addition, the enzyme GAPDH from the pathogenic parasites T. cruzi, T. brucei, and Leishmania mexicana are closely related (about 90% sequence identity) and possess important structural differences for drug design when compared to the homologue protein of the mammalian host (about 45% sequence identity). Natural Products are very important for human therapy due to their ability to modulate many pharmacological events.3 The use of natural products in medicine is as old as human civilization. The first natural products isolated from natural sources in


Figure 1. T. cruzi GAPDH enzyme.


Anacardic Acid Derivatives as GAPDH Inhibitors

reported. As part of our research program aimed at discovering novel T. cruzi GAPDH inhibitors and in order to identify the mode of action of new inhibitors, we have synthesized and evaluated a small library of anacardic acid analogues.

Results and discussion In the present study, we have investigated the activity of Anacardium occidentale (cashew, a tree in the flowering plant family Anacardiaceae) extracts against T. cruzi GAPDH. Cashew nut shell liquid is a rich source of anacardic acids, and the access to these compounds was possible through the utilization of rotation locular counter-current chromatography (RLCC). A mixture of anacardic acids containing different degrees of unsaturation in the alkenyl side chain was obtained, and produced about 80% GAPDH inhibition at a dose of 1 mg/mL. Next, we have reduced by hydrogenation the double bonds of the different components of the mixture, obtaining a single derivative. This strategy allowed the evaluation of the pure anacardic acid derivative 1 (Figure 3) which has an IC50 value of 28 ď ­M, and can be considered one of the most potent inhibitor of T. cruzi GAPDH yet described. Based on these preliminary results, we have employed the methodology described by Yamagiwa et al.7 in order to prepare a series of anacardic acid derivatives starting from a common intermediate (6) (Scheme 1). Among the synthesized anacardic acids are compounds containing 6-12 carbons in the alkyl side chain, with the presence or absence of a polar group at the end, and also substituted at the carboxyl or methoxyl groups. Twenty-one derivatives were prepared






evaluation against T. cruzi GAPDH. The two most potent inhibitors, among the 21 derivatives evaluated against the parasite enzyme, are compounds 1 and 10e (with IC50 values of 28 and 55 ď ­M, respectively), which were then selected for further enzyme kinetic studies. Firstly, these compounds were confirmed to be authentic inhibitors of the target enzyme, since in all cases enzyme inhibition was not affected by the Figure 2. Proposed mechanism for GAPDH enzymatic reaction. Source: H. R. Horton, Principles of biochemistry. Prentice Hall: New Jersey, 1996.


presence of 0.01% Triton X-100 (results not shown). If aggregates formed by a promiscuous compound

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

Pereira et al.


6-n-pentadecylsalicilic acid (1) CO2H HO

6-[8(Z)-n-pentadecenyl]salicilic acid

Figure 3. Anacardium occidentale cashew nuts and examples of anacardic acids.




Cl SOCl2




94% OMe 3








OMe 4 1) LHMDS






OMe 5

OMe 6

PPh3+Cl - R(CH2)nCHO 18-51% 2) H2, Pd /C CO2Et 95-98%

( )n


CO2Et OMe 8a-c: n = 4,6,8/R = OTHP 8d,e: n = 6,9/R = CH3 O

AlCl3 (a-c) 42-64%

( )n

or BBr3 (d,e) 82%



( )n

NaOH 68-85%

OH 9a-c: n = 4,6,8/R = OH 9d,e: n = 6,9/R = CH3

CO2H OH 10a-c: n = 4,6,8/R = OH 10d,e: n = 6,9/R = CH3

R Cl O 2 2 DMSO 73%

( )6


CO2H OH 11

Scheme 1. Synthetic route for the preparation of anacardic acid derivatives.

are responsible for enzyme inhibition, removal of

inhibition with respect to the physiological substrates

the aggregates from a solution of the compound

G-3-P and NAD+, employing the two compounds

would decrease inhibition. Therefore, these results

(1 and 10e) with the lowest IC50 values for T. cruzi

eliminate possible mechanisms of inhibition through

GAPDH (Table 1 and Figure 4).

the interaction of aggregates of many compounds

The results shown in Table 1 and Figure 4 indicate

molecules with T. cruzi GAPDH, rather than the

that the inhibition of T. cruzi GAPDH was found to be

binding of the individual molecules. This so called

noncompetitive with respect to both substrates, where

promiscuous inhibition generally involves hits from

inhibitors have binding affinity for both free enzyme

virtual and high-throughput screening as well as some

and the enzyme-substrate complex. In this situation,

natural products and their synthetic small molecule

constants must be defined for the binary enzyme-



inhibitor complex (Ki) and ternary ESI (ď ĄKi). Data

To explore the mechanism of inhibition in more

for the most potent T. cruzi GAPDH inhibitors 1 and

detail, we have determined Ki values and the type of

10e are shown in the Lineweaver-Burk (or double-



Anacardic Acid Derivatives as GAPDH Inhibitors


[10e] - 0.0 μM [10e] - 5.0 μM [10e] - 6.0 μM [10e] - 7.5 μM

1/[NADH] x 10-4 P0.min-1

1/[NADH] x 10-4 P0.min-1




[1] - 0.0 μM [1] - 42.5 μM [1] - 45.0 μM [1] - 47.5 μM [1] - 50.0 μM




100 200

0 -20













1/[NAD+] x 10-4 P0.min-1

1/[NAD+] x 10-4 P0.min-1

Figure 4. Lineweaver-Burk plots showing that compounds 1(A) and 10e(B) inhibits T. cruzi GAPDH noncompetitively with respect to NAD+. Concentration of 10e: 0.0; 42.5; 45.0; 47.5 e 50.0 (M).

reciprocal plot were affected by the presence of the noncompetitive inhibitors. The pattern of lines seen when the plots for varying inhibitor concentrations are overlaid depends essentially on the value of . It can be seen in Figure 4 that the lines intersected at a value of 1/[S] less than zero and a value of 1/v greater than zero. Therefore, when  > 1, the inhibitor preferentially binds to the free enzyme, as confirmed by the dissociation constants depicted in Table 1. It is worth noting that noncompetitive inhibitors do not compete

Figure 5. Crystal structure of GAPDH in complex with the noncompetitive inhibitor 10e. The inhibitor is bound to a hydrophobic pocket located at the interfacial region among subunits A (orange), C (cyan) and A’ (green).

with substrate for binding to the free enzyme, they bind to the enzyme at a site distinct from the active site. When the inhibitor displays finite but unequal affinity for the two enzyme forms, they are also sometimes called mixed inhibitors, but this term

reciprocal) plots in Figure 4. Ki and Ki values obtained

should be avoided in inhibitors design.9

for these noncompetitive inhibitors are in the low

Prompted by the kinetic results, crystallization

micromolar range (Table 1), and were determined from

assays were carried out in order to identify the

the collected data employing the Sigma-Plot enzyme

new GAPDH binding site. In this context, co-

kinetics module. Lineweaver-Burk plots showed

crystallization assays were performed using a solution

intercepts of all lines (obtained at different inhibitor

of T. cruzi GAPDH at 10 mg/mL and NAD+ at

concentrations) in the upper left quadrant for the

saturating concentrations incubated with 10 mM


cofactor NAD (similar plots were observed for the

of 1 or 10e. Crystals of the GAPDH–10e complex

substrate G-3-P). Both slope and the y of the double-

were grown at 18°C by hanging drop vapor diffusion.

Table 1. Values Ki and Ki for the noncompetitive inhibitors of T. cruzi GAPDH. Inhibitor


NAD+ K i (mM)

G-3-P K i (mM)

K i (mM)

K i (mM)











The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

A single crystal of the GAPDH–10e complex was

a hydrophobic pocket formed by the side-chains of the

flash–cooled to 100 K with 15% polyethylene glycol

residues Leu102, Arg266, Ala265 and the main-chain

400 added to reservoir solution as a cryoprotectant

of Phe183 and Gly182 (Figure 5). Additionally, the

agent. Data collection was conducted in the Brazilian

crystallographic data shows that the NAD+ molecule

National Synchrotron Light Laboratory (LNLS). The

is also bound to GAPDH, which is consistent with the

crystal structure of the complex GAPDH–10e was

mechanism of action for this series of inhibitors. In

determined by molecular replacement and the native

summary, besides providing invaluable information

tetrameric GAPDH structure as a search model.

about the new binding site, the X–ray complex

The 10e inhibitor is bound to a hydrophobic pocket located at an interfacial region formed by

structure also revealed important insights into the binding conformation of 10e.

residues from A, C and A’ subunits. Subunits A and C

The inhibitors in the present study represent a new

are located in the dimeric interface within the tetramer,

chemical diversity for the target enzyme, and have

while the A’ subunit belongs to the next tetramer in

been shown to act noncompetitively in the presence

the crystal. The new binding cavity lies adjacent to

of both physiological substrates. The crystallographic


Science Highlights

Pereira et al.

the NAD binding site, which is in good agreement

structure revealed specific knowledge about ligand

with the mechanism of inhibition determined for

binding mode and mechanism of action, which is of

this series of inhibitors. The analysis of the complex

great importance to guide structure–based inhibitor

revealed several important interactions that orient the

design studies aimed at the development of the lead

inhibitor in the new binding site, specifically: (i) the

candidates as novel antitrypanosomatid agents. Taken

carboxylic substituent interacts via an ionic interaction

together our data indicate that potency and selectivity

with the charged NZ side-chain of the Lys101 residue;

can be achieved by inhibitors that bind differentially

(ii) the hydroxyl substituent is hydrogen bonded to

to the enzyme-substrate complex and free enzyme.

the NE2 side-chain of Gln91 mediated by a water molecule; (iii) the substituted phenyl moiety is in van


der Waals contact with the side-chain of Val88 and

The authors gratefully acknowledge the financial

Pro98; and (iv) the aliphatic alkyl substituent fits into

support from FAPESP, CAPES and CNPq.

References 1. Sanchez-Sancho, F.; Campillo, N.E., Paez, J.A. Chagas’ Disease: Progress and New Perspectives, Current Medicinal Chemistry, v. 17, n. 5, p. 423-452, 2010. 2. Souza, D.H.; Garratt, R.C.; Araujo, A.P.; Guimaraes, B.G.; Jesus, W.D.; Michels, P. A.; Hannaert, V.; Oliva. G. FEBS Lett. 1998, 424, 131. 3. Newman, D.J.; Cragg, G.M.; J. Nat. Prod. 2007, 70, 461. 4. Alvim-Jr., J.; Dias, R.L.A.; Castilho, M.S.; Oliva, G.; Corrêa, A.G. J. Braz. Chem. Soc. 2005, 16, 763. 5. de Macedo, E.M.S.; Wiggers, H.J.; Silva, M.G.V.; Braz-Filho, R.; Andricopulo, A.D.; Montanari, C.A., A New Bianthron Glycoside as Inhibitor


of Trypanosoma cruzi Glyceraldehyde 3-Phosphate Dehydrogenase Activity, Journal of the Brazilian Chemical Society, v. 20, n.5, p. 947, 2009. 6. Stasiuk, M.; Kozubek, A. Biological activity of phenolic lipids. Cellular and Molecular Life Sciences, v. 67, n. 6, p. 841-860, 2010. 7. Yamagiwa, Y.; Ohashi, K.; Sakamoto, Y.; Hirakawa, S.; Kamikawa, T.; Kubo, I. Tetrahedron 1987, 43, 3387. 8. Copeland, R.A. Evaluation of Enzyme Inhibitors in Drug Discovery. Wiley Interscience: New Jersey, 2005; p. 57. 9. Hannaert, V.; Opperdoes, F.R.; Michels, P.A.M. Protein Expr. Purif. 1995, 6, 244.


The Schistosome Purinome Project Humberto M. Pereira, Larissa Romanello, Juliana R.T. de Souza, Ivo Marques, Ricardo DeMarco, Alexandre Cassago, Victor Caldas, Angela Fala, Glaucius Oliva, Richard C. Garratt Centro de Biotecnologia Molecular Estrutural, Instituto de F鱈sica de S達o Carlos, Universidade de S達o Paulo, S達o Carlos, SP, Brasil

Abstract The schistosome purinome project adopts a parallel approach to solving many crystal structures of the enzymes involved in purine recovery in Schistosoma mansoni. Besides generating a fuller understanding of the biochemistry of the purine salvage pathway itself, one of the main goals of the project is to identify promising target enzymes for future inhibitor design. These may subsequently be of value in the treatment of the debilitating disease schistosomiasis. In this short report we highlight how a combination of teamwork together with automation wherever possible, have led to rapid progress during the first year of the existence of INBEQMeDI. Keywords purine metabolism, pyrimidine metabolism, salvage pathway, Schistosoma mansoni, crystal structure

Related publications: Journal of molecular biology 353, 584-599, 2005. Acta crystallographica D 59, 1096-1099, 2003. Acta crystallographica D 66, 73-79, 2010. Acta Tropica,114, 97-102 , 2010. Bioorganic & Medicinal Chemistry, 18, 1421-1427 , 2010.

relies in order to provide purine nucleotides essential

development of novel therapeutic agents against infec-

for DNA synthesis and other cellular functions (Senft

tious organisms. It therefore seems entirely appropriate

and Crabtree 1983; el Kouni and Naguib 1990). An

that a multidisciplinary center dedicated to research in

important difference between schistosomes and their

this area would adopt a series of different strategies in

human hosts is that the former lack an alternative

order to reach such a goal. Often the structure-based

pathway for the de novo synthesis of nucleotides

approach is considered an attractive choice as it allows

making their dependence on the salvage pathway all

for the rational use of knowledge about a validated tar-

the more critical.

get molecule or receptor (normally a protein essential

The schistosome purinome project aims to fully

for the metabolism of the infectious agent) in order to

characterize the component enzymes of the salvage

accelerate inhibitor design. This is based on a search

pathway from S. mansoni and is an integral part of

for ligand-receptor complimentarity.

the objectives of the INBEQMeDI. This project has

However, many such projects may fail due to

more recently been amplified to include also enzymes

several technical difficulties in obtaining the crystal

responsible for the recycling and de novo synthesis of

structure of the target protein. Firstly the protein may

pyrimidines. The starting point for the project was the

not readily crystallize or even if it does, the crystals may be of poor quality and diffract to low resolution. Even if good quality crystals are available the way in which the molecules pack within the crystal lattice may not be convenient for the subsequent stages of inhibitor development. This often requires the soaking of potential small molecule inhibitors through channels within the crystal until they bind specifically to the desired site in the protein (such as the active site of an enzyme for example). If this site is occluded as a result of unfortunate crystal packing, a new crystal form will probably need to be found. Even when all goes well up to this point, the target protein may turn out to be so similar to a human homologue in the region of the

Science Highlights

An ample variety of approaches exist for the

recently determined genome sequence for S. mansoni which permitted the initial design of primers for the amplification and cloning of all of the enzymes of interest. In some cases the bioinformatics analyses necessary prior to embarking on the design of primers for amplification can aid in correcting errors in the genome annotation as was the case for the enzyme methyl thioadenosine phosphorylase in the present study. All genes subsequently follow a standard pipeline whose principal steps include the expression, purification and crystallization of the corresponding protein (Figure 2). The accompanying table gives a good overall impression of the rate of progress since the inception

active site as to severely limit the chances of building specificity into the inhibitor. This may result in a potentially interesting and well-validated target falling by the wayside. All of these problems suggest that rather than placing all of ones ligand “eggs” in a single protein “basket”, it may be advantageous to start with a series of potential target proteins. This may particularly be the case if these are the component enzymes of an essential metabolic pathway, critical for the survival of the pathogenic agent. Schistosomes (Figure 1), parasites responsible for the severely debilitating disease schistosomiasis,

100  m

present several metabolic Achilles’ heals which might be exploitable by such an approach. One such pathway is the purine salvage pathway, on which the parasite


Figure 1. Schistosoma mansoni causes the debilitating diseases schistosomiasis, which affects around 200 million people worldwide.


The Schistosome Purinome Project

Figure 2. The pipeline used in the schistosome purinome project.

the conversion of adenosine to AMP as part of the purine salvage pathway, exemplifies an important point. Comparison of the active sites of the enzyme from schistosomes with that from the human host reveal some important and surprising differences. The substitution of threonine by alanine at position 136, isoleucine by glutamine at position 38, methionine by leucine at position 134 and valine by cysteine at position 123 significantly alter the landscape of residues around the substrate within the binding pocket. Such differences may be exploitable in future attempts Figure 3. Differences within the adenosine kinase 2 active site. The human enzyme is shown in green and that from schistosoma in white.

to develop schistosome specific ligands, starting compounds for drug development (Figure 3). However, this fortunate situation is very often not the case, as active sites of enzymes tend to be well

of INBEQMeDI (table 1). This clearly shows part

conserved during evolution. The structure of methyl

of the advantage of the parallel approach in that

thioadenosine phosphorylase is a good example where

some projects have advanced far more rapidly than

the human enzyme and that from schistosomes

others. Nevertheless overall progress on all fronts

present almost no differences at all. Clearly the choice

is quite impressive given the limited timeframe of

of this particular enzyme for specific inhibitor design

the project so far. The use of robotic crystallization

would be considerably more challenging.

can be identified as one of the factors which seem


An even more extreme example is a difference

to be important in improving our success rate at the


crystallization stage. Thus far, of the 24 enzymes

phosphorylase (UP) which catalyzes the phospho-

included in the study, four have had their three-

rolyitic cleavege of the nucleosides thymidine and

dimensional structures determined. These include

uridine to their correspondent pyrimidine bases and

adenosine kinase 2, uridine phosphorylase, methyl

ribose-1-phosphate. In this case the substitution of a

thioadenosine phosphorylase and adenylate kinase.

glycine in the human enzyme at position 126 by aspar-







Despite the fact that the project is still at a

tic acid would appear to completely impede substrate

prospective stage, the rapid progress made during

binding. A second critical substitution involves

this initial investment may save time in the future

residue 217 where a catalytically important glutamine

by being able to concentrate effort on target enzymes

has been replaced by a leucine. These alterations may

which are tractable to the structure-based approach.

render the enzyme completely inactive or limited to

However, the initial results already demonstrate some

a thus far unidentified alternative substrate . This is

interesting points. Adenosine kinase, which catalyzes

consistent with the difficulty we observe in soaking

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

Pereira et al.

Table 1. Progress made during 2009. Green indicates steps which have concluded and red indicates current bottlenecks. Crystal Puri-Pyrimidome

Primer Amplified Cloned


Soluble Purified screening Crystals Diffraction Structure Kinetics

Adenosine kinase 1 Adenosine kinase 2 Adenosine deaminase 1 Adenosine deaminase 2 APRT PNP2 HGPRT Thymidylate synthase UP_D UP_G UP_D/G NDPK DHFR Cytidine deaminase MTAP SHMT Citoplasmatic SHMT Mithocondrial Dihydroorotate dehydrogenase Cytidylate kinase Deoxycytidylate deaminase Uridine cytidine kinase 1 Uridine cytidine kinase 2 Adenylate kinase 1 Adenylate kinase 2 CAD Guanylate kinase

ligands into the crystals and is currently under investigation by direct enzyme assay (Figure 4). An important observation is that the schistosome genome appears to have two copies of the UP gene, the second of which has the expected glycine at position 126 and glutamine at position 217. This second isoenzyme may represent the catalytically active version responsible for phosphorolyitic cleavege of the pyrimidine 6-oxonucleosides. This once again raises questions concerning the real function of the D126/ L217 variant which represents approximately 90% of transcripts thus far sequenced. This picture is further complicated by trans-splicing events between the isoforms generating hybrid products. At present crystal-

Figure 4. The active site of D126 Uridine Phosphorylase showing the unexpected presence of Asp126 occluding part of the active site and substitution of glutamine by leucine at position 217.

lization trials are underway for all such enzymes. The schistosome purinome project is therefore progressing rapidly towards its initial aim of determining the maximum possible number of its component enzyme structures. From that point onwards we will be in a stronger position to establish efficient strategies in the search for novel inhibitors and their subsequent development.


References el Kouni, M. H. & Naguib, F. N. (1990). Int J Parasitol 20, 37-44. Senft, A. W. & Crabtree, G. W. (1983). Pharmacol Ther 20, 341-356.


INBEQMeDI devotes a considerable amount of its effort towards outreach programs which aim to connect to different sectors of the wider community. Considerable experience in such activities has been acquired by several INBEQMeDI members over recent years and the Institute is fully committed to its role in improving the public understanding of biomolecular sciences. Here we present some of the highlights from 2009. One of the principal outreach programs undertaken over the past year was a continuing education course carried out with teachers from elementary and secondary schools of the state of São Paulo (Brazil). The course, entitled “Structural Molecular Biology and its relationship with Biotechnology”, was given to 256 science and mathematics teachers. The participants were all coordinators of pedagogical workshops (PCOP) related to their fields of expertise. Together with the 91 Departments of Education of the São Paulo State Education Agency they are responsible for advising and training elementary and secondary school teachers registered at these departments. The goal was to update the participants on issues related to advances in science and technology. Particular emphasis was given to modern methodologies used in the fields of structural molecular biology and biotechnology and to propose appropriate ways in which to make the teaching and learning of these topics more enjoyable for students and teachers.

Head-to-head meeting of the participants of the “Structural Molecular Biology and its relationship with Biotechnology” course offered to 256 Science and mathematics teachers.

Outreach Schedule of the course offered to 256 science and maths teachers. Activity Video conference

Content / Title Cells – a structure based on n a large assembly of mac acro romolecular in nte t ra r ct ctiions. Interaction with the teachers. Activity carried out with the “Virtual Cells ells” interactive so soft ftwa ft wa are re. e. Lecture: Genome organization in cells – transcr scription and translattion Workshop: DNA extraction – using fruit and veg. and home-made re reaagen nts t . Diisc scussion of the procedures used. Dynamics of timeline: History off the th main events and researchers related to the DNA revolution.

Head-to-head meeting

Ludic activity consisting of building three dimensional models using usin the “Building the molecules of life: DNA and RNA” kit: DNA composition, structure and replication; the transcription and translation of a gene. Lecture: Structure, folding and functions of proteins. Ludic activity consisting of building three dimensional models using the “Amino Acids Acid and Proteins” kit: Composition and structure of amino acids and polypeptide chains; constru truction of secondary structures.

Video conference Video conference

Genetic engineering, transgenic organisms and recombinant DNA technology. Interaction with the teachers. Biotechnology and the discovery of new medicines and vaccines: an arduous research challenge. Interaction with the teachers.

The course included three video conferences and one head-to-head meeting of all participants, comprising a total of 17 hours (see Table). Some of the activities of the head-to-head meeting were evaluated and the results presented at an international congress in the field of education and subsequently published (Bossolan et al. (2010). Direct contact with students, rather than their teachers, was addressed through a Science club for state school children in the São Carlos area. It takes place in the Interactive Museum of the CBME/ INBEQMeDI. In 2009, 26 weekly meetings were held for two groups of elementary school students (totaling 50 overall) and one group of 25 high school students. Besides their regular activities, the students participated in the following two events: “Knowledge Fayre” (as part of the activities of the National Week of Science and Technology), with an estimated 1,000 participants and “The Science Club Workshop”, with poster presentations referring to some of the “The Science Club Workshop”.

experiments undertaken by students of the club. The latter included approximately 100 attendees.




Head-to-head contact with students and teachers is always desirable but imposes limits on the size of the public which can be attended. In order to overcome this limitation INBEQMeDI is investing in a series of software based projects which aim to both inform and entertain. One such initiative is the development of interactive software on the topic of Chagas’ disease. The software provides a large amount of useful information about the disease itself, the insect vector, the lifecycle of the parasite, pathogenesis, the resulting socioeconomic losses and the treatment currently available. These issues are addressed in an interactive manner, which includes videos and audio clips, and are currently in the final stages of production. The user environment is a virtual carousel consisting of 10 frames which, when clicked, open new environments containing videos, interviews, text material and audio clips on the subject. In the future the same platform will provide the basis for alternative software on malaria, schistosomiasis, leishmaniasis and lepstospirosis. An alternative and entertaining way of reaching large numbers of students is to use board games as educational tools. INBEQMeDI has developed just such a board game. Once again it aims to aid in teaching concepts about the tropical diseases studied by the Institute’s researchers. The final version will be a card-based board game in which the pieces move according to the player’s ability to answer questions concerning the disease of interest. The intention is to reinforce correct concepts and eliminate erroneous ones. An extra element of excitement is added by joker cards and tips which influence the movement of pieces on the board. The game is currently in the final

Frames from the interactive software on Chagas’ disease.

stages of development as this report goes to press.

The interactive carousel.


The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

WHO Medicinal Chemistry Center for Chagas’ Disease Neglected tropical diseases: a major global problem More than 1 billion people, about one-sixth of the world’s population, suffer from one or more tropical diseases, causing severe illness, morbidity, long-term disability and death. Developing countries, and particularly the world’s poorest people, are the most vulnerable. These diseases have high medical, psychological, mental and economic consequences for millions of men, women, and children.

The urgent need for novel drugs In spite of the alarming health, economic, and social consequences of these debilitating infectious tropical diseases, the limited existing drug therapy suffers from a combination of drawbacks including poor efficacy, resistance, and serious side effects. Therefore, there is an urgent need for new drugs that can overcome resistance and are safe and effective for use in humans.

New medicinal chemistry center for Chagas’ disease in Brazil Lead discovery and optimization are key bottlenecks in the development of novel drugs for neglected tropical diseases such as malaria, tuberculosis, African sleeping sickness, leishmaniasis and Chagas’ disease. Recently, the medicinal chemistry group within INBEQMeDI has been selected by the World Health Organization (WHO) to join its Drug Discovery Networks, through its UNICEF/ UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), as a New TDR-WHO Medicinal Chemistry Center for Chagas’ disease. Our specific interests are in medicinal chemistry and drug design, organic synthesis, in vitro and in vivo biological evaluation and structural biology. The center is coordinated by INBEQMeDI scientist Adriano Andricopulo in colaboration with Glaucius Oliva, Otavio Thiemann and Luiz Carlos Dias (UNICAMP).


Innovation Drug Design Approach of the TDR-WHO Medicinal Chemistry Center for Chagas’ Disease.

Interfaces, monitoring and evaluation The significant progress made by the TDR screening programs over the past few years has allowed the identification of a series of promising lead candidates against various infectious diseases. Within this project, a Medicinal Chemistry Network was organized to effectively collaborate with TDR-WHO to take these compounds forward, enhancing their value, and providing new opportunities in drug discovery and development. In the TDR-WHO MedChem network, the different approaches to lead optimization and NCE discovery for tropical diseases are discussed, and a coordinated strategy that involves highly integrated partnerships between scientists in academic institutions and industry in both developed and developing countries is always emphasized. This strategy offers the promise of reducing the inherently high attrition rate of the early stages of discovery research, thereby increasing the chances of success and enhancing cost-effectiveness.


Source: Nwaka S, Hudson A. Innovative lead discovery strategies for tropical diseases. Nature Reviews. Drug Discovery, 2006, 5(11):941–955.


The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

the help of external consultants. The Expert Drug Discovery Advisory Committee provides strategic oversight and review. Industry partners provide in-kind support, allowing TDR to cope with the large volume


Network activities are managed and coordinated by TDR staff with

of work.

Vitro/Vivo Screening Network

Network Meetings

Drug Target/ in silico Screening Network

Database Management

Inter/intra Network Coordination

MedChem/ DMPK Network

Coordination of the integrated network for drug discovery for infectious tropical diseases by TDR source: management

The major goal of our TDR-WHO project is to identify new bioactive compounds with high in vitro and in vivo potency against Trypanosoma cruzi cultured in host cells. Initially, two TDR compounds were employed as lead candidates for medicinal chemistry optimization. Subsequently over 100 new small-molecule compounds have been synthesized and biologically evaluated followed by in vitro and in vivo pharmacokinetic studies on the most potent candidates. So far, we have selected a privileged set of compounds (equipotent or more potent than Benzonidazole, the only drug currently used for the treatment of Chagas’ disease) for further development. During the coming months, the synthetic work will be mainly guided by the outcome of the biological evaluation together with SAR and ADME studies. These results will generate valuable information in order to guide further studies employing ligand-based drug design (LBDD) strategies with a view to moving forward with the identification of promising candidates for clinical evaluation.



The ten laboratories which comprise INBEQMeDI are distributed amongst nine distinct institutes, each of them part of one of four different Brazilian universities. In total the Institute has laboratories based in five cities located in three different states in the south and southeast of the country. The laboratory infrastructure housed in each center is made available to all other researchers of the Institute with a view to optimizing resources and expertise. In the following pages we describe briefly the attributes of each group and the associated infrastructure. In some cases the latter has been significantly reinforced as a consequence of investment resulting from participation in INBEQMeDI.




1. and 2. Molecular Biology and Protein Expression. 3. and 4. Protein Purification and Biochemistry.


Crystallography and Structural Biology


SĂŁo Carlos Institute of Physics IFSC - USP

The major focus of the group is in the structural biology of the etiological agents responsible for endemic parasitic diseases in Brazil and in the


related medicinal chemistry necessary for encountering novel compounds of therapeutic interest. Members of the laboratory come from differing backgrounds and apply a wide range of both experimental and computational techniques varying from X-ray diffraction for the determination of the 3D structures of macromolecules to computational techniques for drug and vaccine design. The laboratories are well equipped and include the necessary infrastructure for most aspects of molecular biology, protein expression, protein purification and biochemistry, spectroscopic studies, protein crystallization, X-ray diffraction, bioassays (in vitro and in vivo), ligand- and structure-based approaches to drug design, computational analysis, microbiology and molecular epidemiology including Multilocus Sequencing Typing (MLST). Some of the major equipment installed within the laboratories of the crystallography group includes high and lowpressure liquid chromatography, Ă„kta FPLC protein purification systems, fluorimeters, 96-well plate spectrophotometers, a Phast electrophoresis system, DNA sequencers, a BIACore surface plasmonic resonance


system, automated image-plate diffractometers, thermostable chambers, a scintillator, a Biomek 3000 workstation, a crystallization robot and a series of workstations and clusters together with the associated software for medical chemistry applications.

Sergio Mascarenhas Molecular Biophysics Group The biophysics group of the Physics Institute is focused on interdisciplinary research in the fields of Biochemistry, Biophysics, and 7.

Molecular Biology. The group employs a broad range of biophysical and biochemical methods to investigate the structure and molecular mechanisms of proteins, peptides, and polymers as well as their interactions with biomimetics and other biological systems. In pursuing these research interests, several spectroscopic techniques are employed, most of which are available in our own laboratories. These include optical absorption, circular dichroism, fluorescence, and electron spin resonance, supported by traditional biochemistry and molecular biology. One of the research interests involves investigation of the genome, transcriptome and proteome of Schistosoma mansoni and particularly proteins involved in


host-parasite interaction. To this end sequencing and mass spectrometry

5. and 6. Protein Crystallography. 7. and 8. Medicinal Chemistry.

techniques are frequently employed. The group possesses an enviably infra-structure including spectropolarimeters, a fluorimeter equipped with pressure cell and pump, EPR spectrometers, a Magna-Nicolet ATRFT-IR spectrometer and microcalorimeters.




Bioscience Institute IB - USP The laboratory is primarily interested in understanding the molecular and cellular biology of the malaria parasites Plasmodium ssp. and particularly the molecular mechanisms used for cell signalling. This includes investigating how the malaria parasite uses second messengers to sense its environment and control its own cell cycle as well as studies of orphan receptors, members of the serpentine (or G-Protein Coupled Receptor) superfamily, whose presence was demonstrated by the group only recently. The purpose of the collaborative work within INBEQMeDI is to dissect the structure

9. 10.

and function of the molecular players involved in signal transduction pathways involved in Plasmodium – host interactions. Besides the 11.

basics, the laboratory is well equipped with a fluorimeter flexstation, real time PCR thermocyclers, FACSCalibur BD, confocal and inverted microscopes and thermo stable chambers.

Faculty of Medicine of Ribeirão Preto FMRP - USP At the FMRP comparative genomics and reverse genetics is used in the study of gene expression regulation in the protozoan parasite Leishmania. Elements and factors involved in mechanisms of posttranscriptional control of gene expression are under investigation as well as the occurrence and mode of action of ncRNAs in Leishmania. Studies are currently focused on a putative ncRNA, named ODD3, whose expression exerts a clear effect on the expression of other genes and results in drastic phenotypic changes. ODD3 studies

12. 13.

led to the identification of one of its targets, a putative Leucyl Phenylalanyl tRNA-protein transferase, the first enzyme to act in protein degradation via the N-end rule pathway. The objective is to study the structural and functional characteristics of this enzyme as a potential target for discovery of anti-leishmanial lead compounds. The laboratory is equipped to permit in vitro culturing and genetic manipulation of Leishmania, in vitro and in vivo infection with the parasite and to conduct a wide range of molecular biology techniques. Some of the major apparatus includes real time PCR, a DNA sequencer, pulsed field gel electrophoresis and an electroporator for bacterial and eukaryotic cells. A room approved for the manipulation of type II transgenic organisms is also available. 9. and 10. Spectroscopy at the Sergio Mascarenhas Molecular Biophysics Group. 11. FACSCalibur BD, Bioscience Institute, University of São Paulo. 12. and 13. Molecular Biology at the Faculty of Medicine of Ribeirão Preto, University of São Paulo.


The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

The Laboratory of Trypanosomatid Metabolism of the Institute of Biomedical Sciences performs fundamental research into the


Institute of Biomedical Sciences ICB - USP

metabolism of trypanosomatids. The laboratory is mainly oriented towards understanding: (i) which are the main metabolic resources these parasites use in each step of their life cycle; (ii) how the metabolism of these parasites interacts with the metabolism of their mammalian hosts; (iii) how the metabolism of these parasites provide 14.

them with a set of metabolic tools to deal with stress to which they are submitted during their life cycle; (iv) which metabolic pathways are


critical for parasite survival inside the mammalian host. The latter has led us to identify and validate metabolic targets for new therapeutic strategies. Our laboratory is equipped with most modern molecular biology and biochemistry equipment including thermocyclers, UV trans-illuminator with image-capture systems and software for band quantification, ELISA reader, double beam spectrophotometer with appropriate software for data treatment, laminar flow chambers, static incubators for microbiological manipulations etc. The laboratory has 16.

a P2 class culture room and the capacity to work with radiolabelled parasites as well as facilities for DNA sequencing, mass spectrometry and microarrays.

Chemistry Institute IQ - USP The principal focus of the laboratory is to understand the structural basis of biological processes important for bacterial pathogenicity. Model systems are the bacterial phytopathogen Xanthomonas axonopodis pv citri, responsible for citrus canker disease and the spirochaete Leptospira interrogans, responsible for Leptospirosis in humans. Interest is focused in understanding the following systems: (i) surface lipoproteins important for mediating pathogen-host interactions and evasion of host defense; (ii) signaling pathways involving the important second messenger cyclic diGMP, (iii) “quorum sensing” and (iv) type III and type IV secretion systems that mediate the transfer of bacterial virulence factors to the host cell. Studies combine a variety of approaches including protein crystallography, NMR, and other spectroscopic techniques, 17.

the production and characterization of bacterial knock-out strains and

14. 15. and 16. Molecular Biology at the Institute of Biomedical Sciences, University of São Paulo, USP. 17. Protein Purification at the Chemistry Institute, University of São Paulo.

the analysis of protein interaction networks. The laboratory is well equipped with FPLC systems for protein purification, a fluorimeter, a temperature-controlled room for the growth of protein crystals and access to a rotating anode X-ray source for the collection of X-ray diffraction data.




Faculty of Pharmaceutical Sciences FCFRP - USP Research centers on both microbial natural product chemistry and its biological/ecological significance. The laboratory is especially interested in all aspects of the discovery of novel natural products produced by symbiotic microorganisms, mainly endophytic fungi and actinobacteria. Most of the effort is directed towards the discovery 18.

of new biologically active microbial natural products, including antibiotics as well as antifungal, antiparasitic, and anticancer agents. These microorganisms are also studied for their capacity to induce the biotransformation of natural products and drugs. Recently, other approaches of particular interest include the utilization of mixed microbial cultures and chemical epigenetic modifiers to induce the expression of silent biosynthetic pathways as well as studies of the biosynthetic pathways themselves. The experimental procedures involve microbiological techniques for the isolation and cultivation of the microorganisms, chromatographic techniques for the isolation of


the natural products, and NMR and MS experiments are extensively 20.

used for structure elucidation. The laboratory is equipped with one HPLC-PDA-RID analytical system, one preparative recycling HPLC-UV system, one diode array spectrophotometer, three rotatory evaporators, among other equipment.

State University of Ponta Grossa UEPG The focus of the group is to solve the three dimensional structures of proteins and to study the relationships between the structure and function of these complex molecules, many of which are targets for the development of new drugs. To accomplish this, single crystal X-ray diffraction is used as well as molecular dynamics simulations. These studies include native proteins, mutants and protein-ligand complexes. The laboratory is equipped with an HPLC system as well as low pressure chromatography, a crystallization room and computers 21.

for crystallography/docking/molecular dynamics calculations. Being part of INBEQMeDI has enabled improvements in the laboratory infrastructure including the purchase of the HPLC system itself.

22. 18. and 19. Natual Products Chemistry at the Faculty of Pharmaceutical Sciences, University of São Paulo. 20. Protein Purification at the State University of Ponta Grossa. 21. and 22. Molecular Biology at the Federal University of Viçosa.


The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

The Laboratory of Animal Molecular Infectology (LIMA) is 23.

equipped to perform a whole range of cellular, biochemical and


Federal University of Viçosa LIMA - UFV

molecular biology techniques, such as the production of genetically modified organisms (bacteria and yeast) for the heterologous expression of foreign proteins, purification of recombinant proteins, biochemical and biological assays (in vitro and in vivo) as well as DNA, RNA and protein manipulation. Within INBEQMeDI the main goal of the LIMA-UFV group is to study a series of different proteins from protozoan parasites (Trypanosoma cruzi and Leishmania) as potential new targets for the development of drug candidates to treat Chagas’ disease and Leishmaniasis. The laboratory is equipped with analytical balances, a laminar air flow cabinet (biosafety levels 1 and 2), inverted microscope, horizontal and vertical electrophoresis systems, automated protein purification system (AKTA-purifier-GE), multiplate incubator and reader (absorbance, fluorescence and luminescence), real-time PCR and a Bioanalyzer besides a whole range of conventional apparatus.


Chemistry Department DQ - UFSCar The laboratories of the Organic Chemistry group are nationally respected in the field with state of the art facilities for wet chemistry and analysis (e.g. chromatography, mass spectrometry and NMR spectroscopy). One of the goals of our group is the development

25. 26.

of specific and potent inhibitors of enzymes from several parasites associated with neglected diseases. As such, natural products from plants are submitted to screening and in order to improve their activity and selectivity, new natural product derivatives are designed by molecular modeling and synthesized employing combinatorial chemistry. Perform protein expression and purification of recombinant proteins that will be used in these assays as well as crystallization experiments for the determination of their three-dimensional structures. The Laboratory of Synthesis of Natural Products is

23. and 24. Organic and Natural Products Chemistry, Federal University of São Carlos. 25. and 26. Biochemistry and Molecular Biology at the Chemistry Department, Federal University of São Carlos.

equipped with gas chromatography including FID, mass spectrometry, electroantenngraphy and infrared detectors as well as a semi-automatic synthesizer Syncore and a single mode microwave reactor Discover. The Laboratory of Biochemistry and Molecular Biology is equipped with High Performance Liquid Chromatography (HPLC), a Thermal Cycler for PCR, a spectrophotometer, Gel electrophoresis equipment and a whole host of basic equipment for standard molecular biology procedures.



Facts and Figures

The way we interact

instituto b oc ências c as de biociências biociê iinstit tit t tituto


Previously existing collaborations.

New collaborations.

INBEQMeDI maintains many collaborations with other institution both within Brazil and abroad and has undertaken joint initiatives together with other INCTs. These include INOFAR and INBEB based in the city of Rio de Janeiro and INDI in Curitiba.







IFSC - USP (Crystallography)


IFSC - USP (Biophysics)












Facts and Figures



The current INBEQMeDI research team includes 24 principal investigators spread over the different intitutions.








4 The research team is well balanced in terms of experience including both young investigators and a large number of research council (CNPq) scholarship holders.



2 1D

3 CNPq scholars. 1C






15 11





7 4

IFSC USP Biophysics


7 4

5 3






IFSC USP Crystallography

14 12









INBEQMeDI relies upon the dedication of a large number of post-docs, students and technical staff distributed over all of its laboratories.










Beginning of INBEQMeDI´s activities.


Impact Factor 2009

Number of publications


Facts and Figures


In 2009 the members of INBEMeDI published a total of 86 full papers, the majority in international journals with recognized impact factors.





These 86 publications cover a wide range of topics which often span traditional disciplines.

During 2009 several INBEQMeDI papers were chosen to illustrate the front cover of both international and Brazilian journals. 56

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases




Facts and Figures


80 National

Participation in national and international events is an important aspect of INBEQMeDI. A total of 101 presentations in 2009 were distribuited as shown.



CNPq / Ministério da Saúde





Investiment in thousands of R$



The researchers of INBEQMeDI are well financed and in 2009 raised counterpart funding from their home institutions and research councils (at home and abroad) as well as industry. Not all of these resources are necessarily invested in INBEQMeDI projects.



In 2009 members of INBEQMeDI were involved in the organization of a series of scientific events. These included both international and national conferences as well as internal meetings of the Institute itself. The latter serve as important forums for discussion and exchange of ideas whilst the former represent one of the many ways in which the Institute integrates within a wider community.

Internal Events 1st INBEQMeDI Workshop In July 2009 representatives of the research groups which comprise INBEQMeDI met in São Carlos for their first inhouse research seminar, after an inaugural meeting held the previous January. The principal investigators reported on their latest findings and there were plenty of opportunities for establishing new collaborations within the group.

Joint INCT Workshop In December 2009 a two-day event was held in the city of Rio de Janeiro together with two other Institutes of Science and Technology. The Institute of Drugs and Medicines and the Institute of Structural Biology and Bioimaging are headed by Prof. Eliezer Barreiro and Prof. Jerson Lima da Silva respectively and undertake research in related fields to those of INBEQMeDI. This joint meeting was a first attempt to stimulate collaborative projects of mutual interest.

External Events 2nd Brazilian Conference on Natural Products Mônica Pupo and Paulo Vieira were members of the Organizing Committee of the “2nd Brazilian Conference on Natural Products (BCNP) and XXVIII Annual Meeting on Micromolecular Evolution, Systematics and Ecology (RESEM)”, from November 9 to 12, held in São Pedro, SP. The event involved the participation of approximately 400 attendees, including 20 speakers (14 from abroad). The scientific program included seven plenary lectures, 13 short lectures and 14 oral presentations of selected abstracts.


1st Workshop on Epidemiological and Molecular Approaches to Understand Malaria Infection Celia Garcia of INBEQMeDI organized the 1st Workshop on Epidemiological and Molecular Approaches to Understand Malaria Infection during the month of May 2009 at the Institute of Biosciences, USP. There were a total of approximately 100 participants including post-graduate students, post-doctoral fellows and researchers.

The Brazilian Meeting on Organic Synthesis The Brazilian Meeting on Organic Synthesis (BMOS) is a biannual 1.

scientific conference that has become one of the most important international events of the Brazilian Chemical Society. The 13th edition of the BMOS was held in S達o Pedro, Brazil, from August 31st to September 6th, at Hotel Colina Verde. The program included 11 plenary lectures, 5 invited lectures, and 2 short courses, by distinguished chemists from universities and pharmaceutical companies. Nine short presentations were selected by the organizing committee from

2. 3.

the submitted abstracts for oral communication. In total, the event had 341 participants from 17 different countries and members of INBEQMeDI were part of the organizing committee.

XXXIX Summer School in Chemistry 4.

Dulce Helena Ferreira of INBEQMeDI was part of the organizaing committee of the XXXIX Summer School in Chemistry which took place at the Federal University of S達o Carlos during February 2009. Plenary lectures were given by speakers from different countries including Canada, Isreal, Austria, USA and Spain.


1. and 2. 2nd Brazilian Conference on Natural Products (BCNP) 3. XVIII Annual Meeting of the International Network of Protein Engineering Centers (INPEC). 4. and 5. 1st INBEQMeDI Workshop.

XVIII Annual Meeting of the International Network of Protein Engineering Centers In 2009 members of INBEQMeDI were responsible for the organization of the XVIII Annual Meeting of the International Network of Protein Engineering Centers (INPEC), held in Ubatuba, SP. Members of the S達o Carlos Physics Institute represent the Brazilian node of INPEC which has been organizing international events since 1991. On this occasion there were approximately 100 participants including representatives from 18 different countries. Both scientific and social events were judged a great success.



The complete list of 86 publications resulting from INBEQMeDI activities in 2009 is given below. The majority of papers were published in international journals with recognized impact factors and the associated numbers and related data are given in the Facts & Figures section of this document. Many of these papers are the result of collaborations with groups outside INBEQMeDI.

1. Freitas, Renato F.; Prokopczyk, Igor M.; Zottis, Aderson; Oliva, Glaucius; Andricopulo, Adriano D.; Trevisan, Maria Teresa S.; Vilegas, Wagner; Silva, Maria Goretti V.; Montanari, Carlos A. Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors. Bioorganic & Medicinal Chemistry, v. 17, p. 2476-2482, 2009. 2. Trossini, Gustavo H.G.; Guido, Rafael V.C.; Oliva, Glaucius; Ferreira, Elizabeth I.; Andricopulo, Adriano D. Quantitative structure activity relationships for a series of inhibitors of cruzain from Trypanosoma cruzi: Molecular modeling, CoMFA and CoMSIA studies. Journal of Molecular Graphics & Modelling, v. 28, p. 3-11, 2009. 3. Leite, Ana C.; Ambrozin, Alessandra R.P.; Castilho, Marcelo S.; Vieira, Paulo C.; Fernandes, João B.; Oliva, Glaucius; Silva, Maria Fátima das G.F. da; Thiemann, Otávio H.; Lima, M. Inês S.; Pirani, José R. Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3phosphate dehydrogenase enzyme inhibitors. Revista Brasileira de Farmacognosia (Impresso), v. 19, p. 1-6, 2009. 4. Leite, Ney Ribeiro; Krogh, Renata; Xu, Wei; Ishida, Yuko; Iulek, Jorge; Leal, Walter S.; Oliva, Glaucius; Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive LidPlos One, v. 4, p. e8006, 2009. 5. Oliva, G; Guido, Rafael V.C. Structure-Based Drug Discovery for Tropical Diseases. Current Topics in Medicinal Chemistry (Print), v. 9, p. 824-843, 2009. 6. Oliva, G; Guido, Rafael V.C.; Balliano, T.L.; Andricopulo, Adriano D. Kinetic and Crystallographic Studies on Glyceraldehyde-3-Phosphate Dehydrogenase from Trypanosoma cruzi in Complex with Iodoacetate. Letters in Drug Design & Discovery, v. 2009, p. 210-214, 2009. 7. Salum, L.B.; Andricopulo, A.D. Fragment-Based QSAR: Perspectives in Drug Design. Molecular Diversity, v. 13, p. 277-285, 2009. 8. Borchhardt, D.M.; Andricopulo, A.D. CoMFA and CoMSIA 3D QSAR Models for a Series of Cyclic Imides with Analgesic Activity. Medicinal Chemistry, v. 5, p. 6673, 2009. 9. Salum, L.B.; Dias, L.C.; Andricopulo, A.D. FragmentBased QSAR and Molecular Modeling Studies on a Series of Discodermolide Analogs as MicrotubuleStabilizing Anticancer Agents. QSAR & Combinatorial Science, v. 28, p. 325-337, 2009.

10. Guido, R.V.C.; Andricopulo, A.D. Modelagem Molecular de Fármacos. Revista processos químicos, v. 3, p. 24-36, 2009. 11. Andricopulo, A.D. Structure- and Ligand-Based Drug Design: Advances and Perspectives. Current Topics in Medicinal Chemistry (Print), v. 9, p. 754-755, 2009. 12. Salum, L.B.; Dias, L.C.; Andricopulo, A.D. Structural and Chemical Basis for Anticancer Activity of a Series of -Tubulin Ligands: Molecular Modeling and 3D QSAR Studies. Journal of the Brazilian Chemical Society, v. 20, p. 693-703, 2009. 13. Dias, L.C.; Lima, D.J.P.; Goncalves, C.C.S.; Andricopulo, A.D. Synthesis of the C(11)-C(23) Fragment of the Potent Antitumor Agent Dictyostatin. European Journal of Organic Chemistry, v. 2009, p. 1491-1494, 2009. 14. Macedo, E.M.S.; Wiggers, H.J.; Silva, M.G.V.; BrazFilho, R.; Andricopulo, A.D.; Montanari, C.A.A New Bianthron Glycoside as Inhibitor of Trypanosoma cruzi Glyceraldehyde 3-Phosphate Dehydrogenase Activity. Journal of the Brazilian Chemical Society, v. 20, p. 947953, 2009. 15. Andricopulo, A.D.; Salum, L.B.; Abraham, D.J. Structure-Based Drug Design Strategies In Medicinal Chemistry. Current Topics in Medicinal Chemistry (Print), v. 9, p. 771-790, 2009. 16. Dias, L.C.; Dessoy, M.A.; Silva, J.J.N.; Thiemann, O.; Oliva, G.; Andricopulo, A.D. Quimioterapia da Doença de Chagas: Estado da Arte e Perspectivas no Desenvolvimento de Novos Fármacos. Química Nova (Impresso), v. 32, p. 2444-2457, 2009. 17. Valadares, N.F.; Salum, L.B.; Polikarpov, I.; Andricopulo, A.D.; Garratt, R. Role of Halogen Bonds in Thyroid Hormone Receptor Selectivity: Pharmacophore-Based 3D-QSSR Studies. Journal of Chemical Information and Modeling, v. 49, p. 2606-2616, 2009. 18. Andricopulo, A.D. Avanços e Desafios no Desenvolvimento de Novos Fármacos para o Tratamento da Doença de Chagas. Revista de Patologia Tropical (Impresso), v. 38, p. 1409-14010, 2009. 19. Cuff, A.L.; Sillitoe, I.; Lewis, T.; Redfern, O.C.; Garratt, R.; Thornton, J.; Orengo, C.A. The CATH classification revisited--architectures reviewed and new ways to characterize structural divergence in superfamilies. Nucleic Acids Research, v. 37, p. D310-D314, 2009.

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases

21. Cuff, Alison; Redfern, Oliver C.; Greene, Lesley; Sillitoe, Ian; Lewis, Tony; Dibley, Mark; Reid, Adam; Pearl, Frances; Dallman, Tim; Todd, Annabel; Garratt, R.C. The CATH Hierarchy Revisited Structural Divergence in Domain Superfamilies and the Continuity of Fold Space. Structure (London), v. 17, p. 1051-1062, 2009. 22. Damalio, J.C.P.; Nobre, T.M.; Garcia, W; Garratt, R; Araújo, A.P.U. Aggregation studies of human septin 2 and its binding to phosphatidylinositol-4,5-biphosphate. The FEBS Journal (Print), v. 276, p. 160-161, 2009. 23. Cordeiro, Artur T.; Thiemann, Otavio H.; Michels, Paul A.M. Inhibition of Trypanosoma brucei glucose-6phosphate dehydrogenase by human steroids and their effects on the viability of cultured parasites. Bioorganic & Medicinal Chemistry, v. 17, p. 2483-2489, 2009. 24. Silva, C.H.T.P.; Silva, M.; Iulek, J.; Thiemann, O.H. Structural Complexes of Human Adenine Phosphoribosyltransferase reveal novel features of the APRT Catalytic Mechanism. Journal of Biomolecular Structure and Dynamics, v. 25, p. 589-598, 2009. 25. Lopes, J.L.; Valadares, N; Moraes, D.I.; Rosa, J.C.; Araújo, H.S.; Beltramini, L.M. Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa. Phytochemistry, v. 70, p. 871-879, 2009. 26. Lopes, J L S; Nobre, T M; Siano, A; Humpola, V; Bossolan, N R S; Zaniquelli, M; Tonarelli, G; Beltramini, L.M. Disruption of Saccharomyces cerevisiae by Plantaricin149 and its mechanism of action using biomembrane model systems. Biochimica et Biophysica Acta. Biomembranes, v. XX, p. in press-xx, 2009. 27. Pinho, R.T.; Beltramini, L.M.; Alves, C.R.; De-Simone, S.G. Trypanosoma cruzi: isolation and characterization of aspartyl proteases. Experimental Parasitology, v. 122, p. 128-133, 2009. 28. Talhari, D.T.; Moraes, M.L.; Castilho, P.V.; Oliveira Junior, O.N.; Beltramini, L.M.; Araújo, A.P.U. Interaction of a C-terminal peptide of Bos taurus diacylglycerol acyltransferase 1 with model membranes (in press). Biochimica et Biophysica Acta. Biomembranes, v. x, p. xi-xii, 2009. 29. Célia Sulzbacher Caruso; Travensolo, R.F.; Bicudo, R.C.; Lemos, E.G.M.; Araujo, A P U; Carrilho, E. -Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization 3 4 57 58. Microbial Pathogenesis, v. 47, p. 118-127, 2009. 30. Gamarra, L.F.; Pontuschka, W.M.; Mamani, J.B.; Cornejo, D.R.; Oliveira, T.R.; Vieira, E D.; Costa-Filho, A.J.; Amaro Jr, E. Magnetic characterization by SQUID and FMR of a biocompatible ferrofluid based on Fe O. Journal of Physics. Condensed Matter, v. 21, p. 115104, 2009. 31. Facchin, Gianella; Kremer, Eduardo; Barrio, Daniel A.; Etcheverry, Susana B.; Costa-Filho, A.J.; Torre, María H. Interaction of Cu-dipeptide complexes with Calf Thymus DNA and antiproliferative activity of [Cu(alaphe)] in osteosarcoma-derived cells. Polyhedron, v. 28, p. 2329-2334, 2009.


32. Tarallo, M.B.; Costa-Filho, A.J.; Vieira, E.D.; Monge, A.; Leite, C.Q.; Borthagaray, G.; Gambino, D.; Torre, M.H. Research of new Mixed-Chelate Copper Complexes with Quinoxaline N1,N4-Dioxide Derivatives and Alanine as Ligands, Potential Antimycobacterial Agents. Anales de la Asociación Química Argentina, v. 97, p. 8089, 2009. 33. Viera, I.; Gómez, M.A.; Ellena, J.; Costa-Filho, A.J.; Migliaro, E.R.; Domínguez, L.; Torre, M.H. Synthesis, structural characterization and ex vivo biological properties of a new complex [Cu(propranolol)2] 2H2O, a potential beta-blocker. Polyhedron, v. 28, p. 3647-3653, 2009. 34. DeMarco, Ricardo; Verjovski-Almeida, Sergio Schistosomes - proteomics studies for potential novel vaccines and drug targets. Drug Discovery Today, v. 14, p. 472-478, 2009. 35. Santos, Ramon F.; Pôssa, Marcela A.S.; Bastos, Matheus S.; Guedes, Paulo M.M.; Almeida, Márcia R.; Demarco, Ricardo; Verjovski-Almeida, Sergio; Bahia, Maria T.; Fietto, Juliana L.R.; Meyer-Fernandes, J.R. Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence. Plos neglected tropical diseases, v. 3, p. e387, 2009. 36. Berriman, Matthew; Haas, Brian J.; Loverde, Philip T.; Wilson, R. Alan; Dillon, Gary P.; Cerqueira, Gustavo C.; Mashiyama, Susan T.; Al-Lazikani, Bissan; Andrade, Luiza F.; Ashton, Peter D.; Aslett, Martin A.; Bartholomeu, Daniella C.; Blandin, Gaelle; Caffrey, Conor R.; Coghlan, Avril; Coulson, Richard; Day, Tim A.; Delcher, Art; Demarco, Ricardo; Djikeng, Appolinaire; Eyre, Tina; Gamble, John A.; Ghedin, Elodie; Gu, Yong; HertzFowler, Christiane; Hirai, Hirohisha; Hirai, Yuriko; Houston, Robin; Ivens, AlAsdair; Johnston, David A.; Lacerda, Daniela; Macedo, Camila D.; Mcveigh, Paul; Ning, Zemin; Oliveira, Guilherme; Overington, John P.; Parkhill, Julian; Pertea, Mihaela; Pierce, Raymond J.; Protasio, Anna V. The genome of the blood fluke Schistosoma mansoni. Nature (London), v. 460, p. 352358, 2009. 37. Oliveira, Katia C.; Carvalho, Mariana L.P.; Venancio, Thiago M.; Miyasato, Patricia A.; Kawano, Toshie; Demarco, Ricardo; Verjovski-Almeida, Sergio Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile. Plos Neglected Tropical Diseases, v. 3, p. e556, 2009. 38. Guzzo, Cristiane R.; FARAH, S.C. Expression, crystallization and preliminary crystallographic analysis of PilZ from pv.. Acta Crystallographica. Series F, v. 65, p. 304-306, 2009. 39. Hauk, Pricila; Guzzo, Cristiane R.; Ho, Paulo L.; Farah, Chuck S. Crystallization and preliminary X-ray analysis of LipL32 from serovar Copenhageni. Acta Crystallographica. Series F, v. 65, p. 307-309, 2009. 40. Correa, F.; Farah, Chuck S.; Salinas, R.K. Mg2+ ions bind at the C-terminal region of skeletal muscle αtropomyosin. Biopolymers (New York), v. 91, p. 583590, 2009. 41. Aguilar-Ramirez, P.; Reis, E.S.; Florido, M.P.C.; Barbosa, A.S.; Farah, Chuck S.; Costa-Carvalho, B.T.; Isaac, L. Skipping of Exon 30 in C5 gene Results in Complete Human C5 Deficiency and demonstrates the importance of C5d and CUB domains for stability. Molecular Immunology, v. 46, p. 2116-2123, 2009. 42. Hauk, Pricila; Guzzo, Cristiane Rodrigues; Ramos, Henrique Roman; Ho, Paulo Lee; Farah, Chuck S. Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.. Journal of Molecular Biology, v. 390, p. 722-736, 2009.


20. Bachega, Jose Fernando Ruggiero; Navarro, Marcos Vicente Albuquerque Salles; Bleicher, Lucas; BortoletoBugs, Raquel Kely; Dive, Daniel; Hoffmann, Pascal; Viscogliosi, Eric; Garratt, Richard Charles; Garratt, R.C. Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity. Proteins, v. 77, p. 26-37, 2009.


Papers 62

43. Guzzo, Cristiane R.; Salinas, Roberto K.; Andrade, Maxuel O.; Farah, Chuck S. PILZ Protein Structure and Interactions with PILB and the FIMX EAL Domain: Implications for Control of Type IV Pilus Biogenesis. Journal of Molecular Biology, v. 393, p. 848-866, 2009. 44. Bagnaresi, P; Alves, E; Silva, H.B.; Epiphanio, S.; Mota, M.M.; Garcia, C.R.S. Unlike the synchronous Plasmodium falciparum and P. chabaudi infection, the P. berghei and P. yoelli asynchronous infections are not affected by melatonin.. International Journal of General Medicine, v. 2, p. 47-55, 2009. 45. Koyama, F; Chakrabarti, D; Garcia, C.R.S. Molecular machinery of signal transduction and cell cycle regulation in Plasmodium.. Molecular and Biochemical Parasitology (Print), v. 165, p. 2-7, 2009. 46. Aranha Camargo, Lm; Oliveira, S; Basano, S; Garcia, C.R.S. Antimalarials and the fight against malaria in Brazil. Therapeutics and Clinical Risk Management, v. 5, p. 311-317, 2009. 47. Garcia, C.R.S. Molecular and cellular approaches to understanding pathogen host interactions in neglected diseases. Current Opinion in Microbiology, v. 12, p. 392393, 2009. 48. Sartorello, R; Amaya, Mj; Nathanson, Mh; Garcia, C.R.S. The Plasmodium receptor for activated c kinase protein inhibits Ca2+ signaling in mammalian cells. Biochemical and Biophysical Research Communications (Print), v. 389, p. 586-592, 2009. 49. Villanova, G.V.; Nardelli, S.C.; Cribb, P.; Magdaleno, A.; Silber, A.M.; Motta, M.C.M.; Schenkman, S.; Serra, E.C. Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation. International Journal for Parasitology, v. 39, p. 665-673, 2009. 50. Santos, M.G.; Paes, L.S.; Zampierin R.A.; Laranjeira da Silva, M.F.; Silber, A.M.; Floeter-Winter, M.L. Biochemical Characterization of Serine Transport in Leishmania (Leishmania) amazonensis. Molecular and Biochemical Parasitology, v. 163, p. 107-113, 2009. 51. Stolic; Miskovic; Magdaleno, A.; Silber, A.M.; Piantanida, I.; Bajic; Glava -Obrovac, L. Effect of 3,4-EthylenedioxyExtension of Thiophene Core on the DNA/RNA Binding Properties and Biological Activity of Bis-Benzimidazole Amidines. Bioorganic & Medicinal Chemistry, v. 15, p. 2544-2554, 2009. 52. Magdaleno, A.; Ahn, I.Y.; Paes, L.S.; Silber, A.M. Actions of a Proline Analogue, L-Thiazolidine-4-Carboxylic Acid, on Trypanosoma cruzi.. PLoS ONE, v. 4, p. 4534, 2009. 53. Martins, R.M.; Covarruvias, C.; Rojas Galvez, L.R.; Silber, A.M.; Yoshida, N. Use of L-proline and ATP production by Trypanosoma cruzi metacyclic forms as 4 requirement for host cell invasion. Infection and Immunity, v. 77, p. 3023-3032, 2009. 54. Silber, A.M.; Tonelli, Renata Rosito; Lopes, Camila Galvão; Cunha-E-Silva, N.L.; Torrecilhas, A.C.; Schumacher, R.I.; Colli, Walter; Alves, M.J.M. Glucose uptake in the mammalian stages of Trypanosoma cruzi. Molecular and Biochemical Parasitology (Print), v. 168, p. 102-108, 2009. 55. Godoy, P.D. D. M.; Nogueira-Junior, L.A.; Paes, L.S.; Cornejo, A.; Martins, R.M.; Silber, A.M.; Schenkman, S.; Elias, M.C. Trypanosome Prereplication Machinery Contains a Single Functional Orc1/Cdc6 Protein, Which Is Typical of Archaea. Eukaryotic Cell, v. 8, p. 1592-1603, 2009.

56. Gallo, M.B.C.; Chagas, F.O.; Almeida, M.O.; Macedo, C.C.; Cavalcanti, B.C.; Barros, F.W.A.; Moraes, M.O.; Costa-Lotuffo, L.V.; Pessoa, C.O.; Bastos, Jairo Kenupp; Pupo, M.T. Endophytic fungi found in association with Smallanthus sonchifolius (Asteraceae) as resourceful producers of cytotoxic bioactive natural products. Journal of Basic Microbiology, v. 49, p. 142-151, 2009. 57. Guimarães, Denise Oliveira; Borges, K.B.; Bonato, P.S.; Pupo, M.T. A simple method for the quantitative analysis of tyrosol by HPLC in liquid Czapek cultures from endophytic fungi. Journal of the Brazilian Chemical Society, v. 20, p. 188-194, 2009. 58. Verza, M.; Arakawa, N.S.; Lopes, N.P.; Kato, M.J.; Pupo, M.T.; Said, S.; Carvalho, Ivone Biotransformation of a tetrahydrofuran lignan by the endophytic fungus Phomopsis sp.. Journal of the Brazilian Chemical Society, v. 20, p. 195-200, 2009. 59. Borges, K.B.; Pupo, M.T.; Freitas, L.A.P.; Bonato, P.S. Box-Behnken design for the optimization of an enantioselective method for the simultaneous analysis of propranolol and 4-hydroxypropranolol by capillary electrophoresis. Electrophoresis, v. 30, p. 2874-2881, 2009. 60. Borges, K.B.; Borges, W.S.; Duran-Patron, R.; Pupo, M.T.; Bonato, P.S.; Collado, I.G. Stereoselective biotransformations using fungi as biocatalysts. Tetrahedron. Asymmetry, v. 20, p. 385-397, 2009. 61. Yuan, H.; Pupo, M.T.; Blois, J.; Smith, A.; Weissleder, R.; Clardy, J.; Josephson, L. A stabilized demethoxyviridin derivative inhibits PI3 kinase. Bioorganic & Medicinal Chemistry Letters, v. 19, p. 4223-4227, 2009. 62. Borges, W.S.; Borges, K.B.; Bonato, P.S.; Said, S.; Pupo, M.T. Endophytic Fungi: Natural Products, Enzymes and Biotransformation. Current Organic Chemistry, v. 13, p. 1137-1163, 2009. 63. Borges, K.B.; Pupo, M.T.; Bonato, P.S. Enantioselective analysis of propranolol and 4-hydroxypropranolol by capillary electrophoresis with application to biotransformation studies employing endophytic fungi. Electrophoresis (Weinheim. Print), v. 30, p. 3910-3917, 2009. 64. Borges, K.B.; Okano, L.T.; Pupo, M.T.; Bonato, P.S. Enantioselective analysis of fluoxetine and norfluoxetine by HPLC in culture medium for application in biotransformation studies employing fungi. Chromatographia (Wiesbaden), v. 70, p. 1335-1342, 2009. 65. de Toledo, Juliano Simões; Junqueira dos Santos, André F.; Rodrigues de Moura, Tatiana; Antoniazi, Simone Aparecida; Brodskyn, Cláudia; Indiani de Oliveira, Camila; Barral, Aldina; Cruz, Angela Kaysel, Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo. Parasitology International, v. 58, p. 45-50, 2009. 66. Cruz, A.K.; de Toledo, Juliano Simões; Mofolusho Falade; Mônica Cristina Terrão; Sumalee Kamchonwongpaisan; Dennis E. Kyle; Chairat Uthaipibull Current Treatment and Drug Discovery Against Leishmania spp and Plasmodium spp: a review. Current Drug Targets, v. 10, p. 1-15, 2009. 67. Nogueira, C.M.; Parmanhan, B.R.; Farias, P.P.; Correa, Arlene A importância crescente dos carboidratos em química medicinal. revista virtual de química, v. 1, p. 149-159, 2009.

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78. Fernandes, Carromberth C.; Vieira, Paulo C.; Silva, Virgínia C. da; Dall’Oglio, Evandro L.; Silva, Luiz E. da; Sousa Jr., Paulo T. de 6-acetonyl-N-methyldihydrodecarine, a new alkaloid from Zanthoxylum riedelianum. Journal of the Brazilian Chemical Society (Impresso), v. 20, p. 379-382, 2009.

69. Leite, Ana Cristina; Pereira, Luciene G.B.; Vieira, Paulo C.; Fernandes, João B.; Silva, M. Fatima das G.F. da Effects of Limonoids from Cipadessa fruticosa on Fall Armyworm. Zeitschrift für Naturforschung. C, A Journal of Biosciences, v. 64, p. 441-446, 2009.

79. dos Santos, Djalma A.P.; de C. Braga, Patricia A.; Fernandes, João B.; Vieira, Paulo C.; Magalhães, Aderbal F.; Magalhães, Eva G.; Marsaioli, Anita J.; de S. Moraes, Valéria R.; Rattray, Lauren; Croft, Simon L.; da Silva, M. Fàtima das G.F. Anti-African trypanocidal and antimalarial activity of natural flavonoids, dibenzoylmethanes and synthetic analogues. Journal of Pharmacy and Pharmacology, v. 61, p. 257-266, 2009.

70. A. Garcia Cortez, Diógenes; E. Ranieri Cortez, Lucia; B. Fernandes, João; C. Vieira, Paulo; G. Ferreira, Antonio; Fátima das G.F. da Silva, M. New Alkaloids from Conchocarpus gaudichaudianus. Heterocycles (Sendai), v. 78, p. 2053-2059, 2009. 71. Sousa Júnior, Paulo T.; Dall’oglio, Evandro L.; Silva, Luiz Everson da; Figueiredo, Uir S.; Vieira, Paulo C.; Machado, Helen V.; Santos, Luciane G. dos Gênero Acosmium: composição química e potencial farmacológico. Revista Brasileira de Farmacognosia (Impresso), v. 19, p. 150157, 2009. 72. Santos, Djalma A.P. dos; Vieira, Paulo C.; Silva, M. Fátima das G.F. da; Fernandes, João B.; Rattray, Lauren; Croft, Simon L. Antiparasitic activities of acridone alkaloids from Swinglea glutinosa (Bl.) Merr.. Journal of the Brazilian Chemical Society (Impresso), v. 20, p. 644-651, 2009. 73. Pinto, Angelo C.; Zucco, Cesar; Andrade, Jailson B. de; Vieira, Paulo C. Recursos humanos para novos cenários. Química Nova (Impresso), v. 32, p. 567-570, 2009. 74. Severino, Vanessa Gisele Pasqualotto; Cazal, Cristiane de Melo; Forim, Moacir Rossi; da Silva, Maria Fátima das Graças Fernandes; Rodrigues-Filho, Edson; Fernandes, João Batista; Vieira, Paulo Cezar Isolation of secondary metabolites from Hortia oreadica (Rutaceae) leaves through high-speed counter-current chromatography. Journal of Chromatography (Print), v. 1216, p. 42754281, 2009. 75. Cazal, Cristiane de Melo; Batalhão, Jaqueline Raquel; Domingues, Vanessa de Cássia; Bueno, Odair Corrêa; Filho, Edson Rodrigues; Forim, Moacir R.; da Silva, Maria Fátima G. Fernandes; Vieira, Paulo Cezar; Fernandes, João Batista High-speed counter-current chromatographic isolation of ricinine, an insecticide from Ricinus communis. Journal of Chromatography (Print), v. 1216, p. 4290-4294, 2009. 76. Cazal, Cristiane de Melo; Domingues, Vanessa de Cássia; Batalhão, Jaqueline Raquel; Bueno, Odair Corrêa; Filho, Edson Rodrigues; da Silva, Maria Fátima G. Fernandes; Vieira, Paulo Cezar; Fernandes, João Batista Isolation of xanthyletin, an inhibitor of ants symbiotic fungus, by high-speed counter-current chromatography. Journal of Chromatography (Print), v. 1216, p. 4307-4312, 2009. 77. Moccelini, Sally Katiuce; Silva, Virgínia Claudia da; Ndiaye, Eliane Augusto; Sousa Jr., Paulo Teixeira de; Vieira, Paulo Cezar Estudo fitoquímico das cascas das raízes de Zanthoxylum rigidum Humb. & Bonpl. ex Willd (Rutaceae). Química Nova (Impresso), v. 32, p. 131-133, 2009.



68. Matos, Andréia Pereira; Nebo, Liliane; Vieira, Paulo Cezar; Fernandes, João Batista; Silva, Maria Fátima das Graças Fernandes D; Rodrigues, Ricardo Ribeiro Constituintes químicos e atividade inseticida dos extratos de frutos de Trichilia elegans E T. catigua (Meliaceae). Química Nova (Impresso), v. 32, p. 1553-1556, 2009.

80. Januário, Ana Helena; Vieira, Paulo Cezar; Silva, Maria Fátima das Graças Fernandes D; Fernandes, João Batista; Silva, Jorge José de Brito; Conserva, Lucia Maria Alcaloides -indolopiridoquinazolínicos de Esenbeckia grandiflora mart. (Rutaceae). Química Nova (Impresso), v. 32, p. 2034-2038, 2009. 81. Rother, Débora C.; Souza, Tiago F.; Malaspina, Osmar; Bueno, Odair C.; Silva, Maria de Fátima das G.F. da; Vieira, Paulo C.; Fernandes, João B. Suscetibilidade de operárias e larvas de abelhas sociais em relação à ricinina. Iheringia. Série Zoologia (Impresso), v. 99, p. 61-65, 2009. 82. Reche, Karine V.G.; de Souza, Gezimar D.; Trapp, Marília A.; Rodrigues-Filho, Edson; Silva, Sebastião C.; Fernandes, João B.; Vieira, Paulo C.; Muller, Manfred W.; da Silva, M. Fátima das G.F. Methyl angolensate changes in Khaya ivorensis after fungal infection. Phytochemistry, v. 70, p. 2027-2033, 2009. 83. Cameron, S.; Martini, Viviane Paula; Iulek, J.; Hunter, William Nigel Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase complexed with 6-phosphogluconate. Acta Crystallographica. Series F, v. 65, p. 450-454, 2009. 84. de Oliveira, L.S.; Mourão, L.C.; Oliveira, K.A.; da Matta Agostini, M.; de Oliveira, A.C.; de Almeida, M.R.; Fietto, J.L.R.; Conceição, L.G.; Filho, J.D.R.; Galvão, M.A.M.; Mafra, C. Molecular detection of in cats in Brazil. Clinical Microbiology and Infection, p. 1-2, 2009. 85. Santos, Ramon F.; Pôssa, Marcela A.S.; Bastos, Matheus S.; Guedes, Paulo M.M.; Almeida, Márcia R.; Demarco, Ricardo; Verjovski-Almeida, Sergio; Bahia, Maria T.; Fietto, Juliana L.R. Influence of EctoNucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence. PLoS Neglected Tropical Diseases, v. 3, p. e387, 2009. 86. dos Santos Sant&Amp; da Silva Paes, Lisvane; Vieira Paiva, Argentino Filogônio; Fietto, Luciano Gomes; Totola, Antônio Helvécio; Magalhães Trópia, Maria José; Lemos Giunchetti, Denise Silveira; Lucas, Cândida; Fietto, Juliana Lopes Rangel; Brandão, Rogelio Lopes; Castro, Ieso Miranda Protective effect of ions against cell death induced by acid stress in. FEMS Yeast Research, v. 9, p. 701-712, 2009.


Institute of Physics of São Carlos - USP Crystallography Glaucius Oliva - Richard C. Garratt - Otavio H. Thiemann - Adriano D. Andricopulo - Eduardo Horjales Reboredo - Ilana Lopes B.C. Camargo - Rafael V.C. Guido -

Biophysics Antonio José da Costa Filho - Claudia Elisabeth Munte - Ricardo de Marco - Leila M. Beltramini - Ana Paula U. Araújo - Marcos Vicente A.S. Navarro - Nelma R.S. Bossolan -

Department of Chemistry - UFSCar Arlene Gonçalves Correa - Dulce Helena F. de Souza - Paulo Cezar Vieira -

Institute of Chemistry - USP Shaker Chuck Farah -

Institute of Biosciences - USP Célia R. da Silva Garcia -

Institute of Biomedical Sciences - USP Ariel Mariano Silber -

Medical Faculty of Ribeirao Preto - USP Angela Kaysel Cruz -

Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP Monica Talarico Pupo -

Department of Biochemistry and Molecular Biology - UFV Juliana Lopes Rangel Fietto -

Department of Chemistry - UEPG Jorge Iulek -

Coordinator Vice-Coordinator Technology Transfer Outreach Finance

Glaucius Oliva Richard C. Garratt Otavio H. Thiemann Leila M. Beltramini Francisco Fernando Falvo Rejane Nogueira Brasil


Ligia Rafaela Prado


Rui Cintra

Headquarters Center for Structural Molecular Biotechnology Institute of Physics of São Carlos University of São Paulo Avenida Trabalhador Sãocarlense 400 CEP 13566-590 São Carlos - SP

Associated Laboratories Department of Chemistry - UFSCar Institute of Chemistry - USP Institute of Biosciences - USP Institute of Biomedical Sciences -USP Medical Faculty of Ribeirao Preto - USP Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP Department of Biochemistry and Molecular Biology - UFV Department of Chemistry - UEPG

instituto de biociências



Activity Report 2009

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