INCT INBEQMeDI

1. São Carlos - SP Institute of Physics of São Carlos (IFSC) University of São Paulo (USP)

INBEQMeDI SUMMARY REPORT 2009-2013

Coordinator Vice-Coordinator Technology Transfer Outreach Finance

Richard C. Garratt Adriano D. Andricopulo Otavio H. Thiemann Leila M. Beltramini Rejane N. Brasil

Cristallography

Biophysics

Glaucius Oliva Richard C. Garratt Otavio H. Thiemann Adriano D. Andricopulo Ilana Lopes B.C. Camargo Eduardo Horjales Reboredo Rafael V.C. Guido

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

Department of Chemistry (DQ) Federal University of São Carlos (UFSCar) Arlene Gonçalves Correa Dulce Helena F. de Souza Paulo Cezar Vieira

Headquarter Institute of Physics of São Carlos University of São Paulo Avenida Trabalhador Sãocarlense 400 CEP 13566-590 São Carlos - SP

2. São Paulo - SP Institute of Chemistry (IQ) University of São Paulo (USP) Shacker Chuck Farah

Institute of Biosciences (IB) University of São Paulo (USP)

Associated Laboratories Department of Chemistry - DQ/UFSCar Institute of Chemistry - IQ/USP Institute of Biosciences - IB/USP Institute of Biomedical Sciences - ICB/USP Medical Faculty of Ribeirão Preto - FMRP/USP Faculty of Pharmaceutical Sciences of Ribeirão Preto - FCFRP/USP Department of Biochemistry and Molecular Biology - DBB/UFV Department of Chemistry - DQ/UEPG

instituto b oc ê c s de biociências

Célia R. da Silva Garcia Institute of Biomedical Sciences (ICB) University of São Paulo (USP) Ariel Mariano Silber

3. Ribeirão Preto - SP instituto de biociências

Medical Faculty of Ribeirão Preto (FMRP) University of São Paulo (USP) Angela Kaysel Cruz

Faculty of Pharmaceutical Sciences of Rib. Preto (FCFRP) University of São Paulo (USP) Monica Talarico Pupo Production

4. Viçosa - MG Department of Biochemistry and Molecular Biology (DBB) Federal University of Viçosa (UFV) Juliana Lopes Rangel Fietto

5. Ponta Grossa - PR Department of Chemistry (DQ) State University of Ponta Grossa (UEPG) Jorge Iulek

Summary

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Research report. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Innovation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Outreach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Facts and Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Internationalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Associate laboratories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

INTRODUCTION

WHO AND WHAT IS INBEQMeDI

The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases INBEQMeDI The National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases (INBEQMeDI) came into existence at the beginning of 2009. After a positive evaluation during the period from 2010-2011, it subsequently received additional funding guaranteeing its existence until the end of 2013. INBEQMeDI has as its principal research focus molecular targets from microorganisms associated with infectious diseases, mostly tropical parasites such as trypanosomes, schistosomes, Plasmodium and Leishmania. Its mission is to perform both basic and developmental research within this field to the highest possible standard which it is able to achieve.

We are also active in outreach programs which aim to raise awareness about the importance of these topics, and of biotechnology in general, to 21st century society.

Introduction 2

The Institute arose naturally as a result of previous

Many of our research projects ultimately aim towards

collaborative projects between its constituent

the study of novel targets for therapeutic intervention.

laboratories, most significantly the CEPID program

Nevertheless, much effort is still concentrated on the

funded by FAPESP. Currently we are jointly financed

acquisition of solid basic knowledge of the molecular

by CNPq and FAPESP (research councils representing

biology of the organisms of interest. It is the view of

the federal government and the State of Sao Paulo

INBEQMeDI scientists that there is no effective way to

respectively) as well as by the Ministry of Health. Our

separate these two endeavors. Furthermore, the Institute

headquarters are based at the Institute of Physics of

is well aware of its limitations as to how far along the

São Carlos (IFSC), University of São Paulo (USP), which

drug development pipeline it can reasonable expect to

received brand new facilities at the beginning of 2013

go. Beyond that point it is essential to seek partnerships

permitting the expansion and modernization of our

with industry and governmental organizations in order

research laboratories. The remainder of INBEQMeDI

for subsequent project development and technology

is spread over eight other institutions within the States

transfer. INBEQMeDI aims to bridge this gap wherever

of São Paulo, Minas Gerais and Paraná. More details of

possible.

each participant laboratory can be found elsewhere in this summary report.

In this summary report you will see an overview of the activities undertaken by INBEQMeDI since its inception

Hopefully the research we report here will leave

in 2009 to the present day. We believe that we have made

a clear impression of the diversity of experimental

significant strides in the direction of generating high

approaches used by the Institute in order to fulfill its

quality scientific knowledge about important infectious

mission. We aim to use this diversity in an integrated

agents which cause diseases that lead to considerable

fashion by which, for example, novel compounds

human suffering worldwide. We are an integrated

identified and/or synthesized by our chemists can be

group of scientists working towards common goals and

fed into in vitro and in vivo studies of the pathogenic

part of the wider international scientific community.

organisms of interest. For this we count on the expertise

We strongly believe that it is essential that such work

of molecular biologists, biochemists and parasitologists

continues. Hopefully this message comes across in the

as well as physicists, crystallographers and chemists.

pages which follow.

Introduction 3

AN INTEGRATED APPROACH Drug discovery is a multidisciplinary subject driven by innovation that integrates knowledge from a variety of fields of science, including, but not limited to, chemistry, biology and physics. The area of drug design involves the cooperative work of scientists with a diverse range of backgrounds and technical skills, trying to tackle complex problems using a combination of approaches and technologies. Innovative strategies, methods and tools have benefited from several scientific and technological advances to enable scientists to gain an exceptional understanding of the basic principles

their efforts towards the effective integration of structural biology and medicinal chemistry approaches, with emphasis in the area of neglected tropical diseases (NTDs). NTDs are a major global cause of illness, morbidity, long-term disability, and death, with severe medical and psychological consequences for millions of men, women and children. Despite the high prevalence of these diseases worldwide, in most cases their treatment is inadequate, generating an urgent demand for new antiparasitic drugs. However, in addition to the traditional challenges involved in the complex process of drug discovery and development, there is the hurdle of the lack of investments in this field. Therefore, strategies

and mechanisms underlying the processes of protein-

that allow the identification of promising compounds

ligand binding and biological activity. The integration

as well as the reduction in drug discovery costs and

of available knowledge of several 3D protein structures

time are extremely useful in this field. The biology of

with hundreds of thousands of small-molecules have

parasitic organisms has been continuously studied in

attracted the attention of scientists from all over the

detail, providing a solid base for the selection of relevant

world for the application of structure- (SBDD) and

molecular targets for drug discovery. Following this, one

ligand-based drug design (LBDD) approaches, which

of the most important challenges in drug design is the

have been successfully applied in hit identification and

development of innovative new chemical entities (NCEs)

lead optimization across a range of key therapeutic

from an incredibly large reservoir of real and virtual

targets. In this context, INBEQMeDI scientists directed

possible compounds, considering that NCEs expected to

Figure 1. Drug design strategies employed in INBEQMeDI.

4 Summary Report 2009-2013 | INBEQMeDI

Figure 2. General workflow of the drug discovery process: from hit identification to NCE discovery.

advance into clinical trials should have an ideal balance

research and development (R&D) is evident across all

of pharmacodynamic and pharmacokinetic properties,

areas of research, providing new opportunities and

as well as in vivo efficacy and safety. In line with this,

ideas. The challenges lie in the continuous improvement

some of our projects have focused on the identification

of the incorporation of these and other approaches at

and optimization of lead candidates using modern

different stages in order to improve pharmacodynamic

strategies based on our increasing understanding of

and pharmacokinetic properties of lead compounds at

the fundamental principles of medicinal chemistry,

multiple levels of complexity, leading to the discovery

molecular and structural biology, and parasitology

of high-quality NCEs for use in humans.

(Figure 1).

To face these challenges, a multidisciplinary team

In general, in the early stages of the drug discovery

of leading scientists has been working in collaboration

process, chemical libraries varying widely in size and

in the INBEQMeDI projects, developing and applying

complexity are screened for the identification of new

drug discovery approaches and technologies that rely

hits (i.e., ligands, bioactive compounds). The selection of

on state-of-the-art methods in medicinal chemistry,

a small fraction of compounds with sufficient promise

natural products chemistry, synthetic organic chemistry,

for further optimization is a major challenge at this

and molecular and structural biology, providing not

stage of this lengthy and risky process. Traditional

only solid scientific skills and expertise in all relevant

strategies in structural biology and medicinal chemistry

subfields, but also a well-organized structure for the

are often combined with SBDD and LBDD approaches

integration of modern approaches to drug discovery.

to explore the vast chemical and biological space as a

Furthermore, our INCT has established a number of

key component in the process of hit-to-lead generation,

collaborations with other laboratories, either within

lead optimization and NCE discovery (Figure 2). The

the country or abroad, to more effectively fulfill its

integration of fundamental fields driving pharmaceutical

interdisciplinary objectives.

Introduction 5

1

The relevance of proline metabolism in Trypanosoma cruzi

RESEARCH REPORTS

Lisvane S. Paes1, Brian A. S. Mantilla1, Anahí Magdaleno1, Ariel M. Silber1 Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil

1

Introduction Since the early ´70s, the involvement of proline, glutamate, aspartate, glutamine and asparagine, leucine and isoleucine as oxidable energy sources in Trypanosoma cruzi is well known. In the ´80s, it was also shown that proline, aspartate and glutamate were also involved in differentiation processes occurring inside the vector. Some years later we characterized the proline uptake by this parasite and we established its relationship with differentiation among intracellular stages of T. cruzi. The quantity of biological processes in which proline, glutamate and other metabolically related amino acids are involved in this parasite led us to revisit this topic.

Results Proline, glucose and the energy metabolism along the infection We proposed that proline is a relevant energy source for the intracellular stages.

The trypomastigotes (living in the extracellular medium where glucose is available), have the highest glucose and minimum proline transport, being dependant mainly on glucose metabolism for energy requirements. However, amastigotes have the highest intracellular free proline concentration but proline transport is low and no transport of glucose was evidenced. The amino acid pool of the amastigotes is consumed during replication and/or differentiation to the intracellular epimastigote stage, which has the lowest levels of intracellular free proline concentration, and

Figure 1. The role of proline in the resistance to oxidative stress in T. cruzi: A) Thiozilidina-4-carboxylate (T4C) acts as a specific inhibitor of the proline uptake, leading the cells to reduced levels of intracellular free proline.

Cells treated with an IC20 of T4C and challenged with an IC20 of H2O2 showed a synergistic effect suggesting that decreased intracellular free proline makes the cells more sensitive to H2O2. B) Knock out yeasts for ProDH, which had also their intracellular free proline increased (C) with respect to those complemented with T. cruzi ProDH, were more resistant to the treatment with H2O2, confirming this idea. D) Finally, the general oxidation state of the yeasts was evaluated through the measurement of the GSSG/GSH ratio after the H2O2 challenge.

The obtained results show that yeasts bearing a functional ProDH (T. cruzi ProDH) and having lower proline levels presented a more oxidized state than knock-outs and control.

6 Summary Report 2009-2013 | INBEQMeDI

The relevance of proline metabolism in Trypanosoma cruzi

Proline: the energy support of the host-cell invasion The invasion process by trypomastigotes is energy dependant. However, the energy sources supporting it had not been identified. We evaluated the participation of proline as a fuel for parasite invasion. Significant diminution of intracellular ATP correlated with parasite ability to attach and to invade the mammalian hostcells. After starvation, proline (but neither glucose nor glutamate) was able to restore the intracellular ATP levels, and attachment and invasion abilities, showing that proline energetically support these early steps in the mammalian infection process [2].

Proline dehydrogenase is able to transfer electrons to the respiratory chain Proline is initially oxidized by a FAD dependant proline dehydrogenase (ProDH). This fact led us to hypothesize that this enzyme could be transferring electrons directly to the respiratory chain at the level of Complex II. This hypothesis was confirmed by the fact that mitochondrial vesicles are able to consume O2 and produce ATP at the same level when stimulated with proline or succinate.

Proline is involved in the resistance to oxidative and nutritional stresses As ProDHs in other organisms are involved in the regulation of the generation of oxygen reactive species (ROS) and apoptosis, we investigated if this could be the case for the T. cruzi enzyme. Surprisingly, T. cruzi cells treated with a proline analogue which inhibits proline uptake and causes a diminution in the intracellular proline content, showed that resistance to oxidative stress increased with intracellular free proline accumulation [3]. In addition, intracellular free proline is related to their resistance to metabolic stress.

These functions are fundamental for the parasite in all

100

Relative quantity

depends upon external proline supplement to proceed with the differentiation to trypomastigote. Accordingly, this stage shows the highest proline transport. Proline then plays an important role in the metabolism of the intracellular forms [1].

% (Proline transport) % (Glucose transport) % (Intracellular proline)

80 60 40 20 0 Trypomastigotes Amastigotes

Intracellular Trypomastigotes epimastigotes

Figure 2. Scheme illustrating the variations of relative quantities of glucose or proline uptake, or free intracellular proline concentrations were represented for each life cycle stage along the mammalian cell infection cycle.

the environments it goes through along its complex life cycle.

Concluding remarks T. cruzi is exposed to different environments (some of them somehow extreme, like the host-cell cytoplasm) along its complex life cycle. Most of these environments could be considered dangerous for most of organisms with a biochemical logics such as a bioenergetics based mainly on glucose oxidation. The fact that this parasite alternates from a bioenergetics based on glucose consumption to one based on proline consumption, or the fact that proline and not glucose fuels the host cell invasion constitute interesting examples. The same applies to some strategies of resistance to stress conditions: we 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 metabolism, which is leading to proposal that its transporters and enzymes related to its metabolism are relevant targets for therapeutic drug design. Further work is being undertaken to validate these targets and to rationally design inhibitors against key proteins participating in this interesting metabolic pathway.

References [1] Silber AM, Tonelli R, Lopes C, Cunha-e-Silva N, Torrecilhas A, Schumacher RI, Colli W, Alves M (2009) Mol Biochem Parasitol 168:102-108.

[2] Martins RM, Covarrubias C, Rojas RG, Silber AM, Yoshida N (2009) Infect Immun 77: 3023-3032.

[3] Magdaleno A, Ahn IY, Paes LS, Silber AM (2009) PLoS One 4:e4534.

Research Reports 7

2 RESEARCH REPORTS

Structure and interactions of the major surface antigen of pathogenic Leptospira sp Pricila Hauk1,2, Cristiane R. Guzzo1,3, Angela Barbosa2, Henrique R. Ramos2, Paulo L. Ho2, Chuck S. Farah1 Biochemistry Department, University of São Paulo, São Paulo, SP, Brazil Center for Biotechnology, Butantan Institute, São Paulo, SP, Brazil 3 Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil 1 2

Leptospirosis, caused by the spirochaete Leptospira, is an important emerging infectious disease. The disease is more prevalent in tropical countries, due to climatic and environmental conditions. The main symptoms of the disease include fever, jaundice, and tubule-interstitial nephritis. Despite its prevalence and importance, many fundamental aspects of Leptospira interrogans biology are poorly understood.

Structural and Functional Studies of LipL32 The most abundant antigen found in the leptospiral total protein profile is LipL32. This highly immunogenic outer-membrane lipoprotein is conserved among pathogenic Leptospira species but not observed in the non-pathogenic saprophytic L. biflexa. It is therefore considered a promising target for vaccine development and diagnosis of leptospirosis. Recently, studies using a L eptospira interrogans lipL32 mutant showed that LipL32 does not play a essential role in either acute or chronic models of animal infection. While these data did not identify an indispensible role in pathogenesis, it does not exclude an important function for LipL32 in mediating the host-pathogen interaction.

We determined the X-ray structure of LipL32 [1,2] (Figure 1). Its tertiary structure showed a jelly roll fold similar to those presented by some calcium-binding and ECM-

Figure 1. LipL3221-272 structure. (a) Cartoon model of the LipL3221-272 monomer. The N- and C-termini are indicated. β-strands, α-helices and 310 helices are numbered in the order in which they appear in the sequence.

Helices are colored red, the eight β-strands that form the core jelly-roll topology are shown in blue, while the other β-strands are show in yellow.

8 Summary Report 2009-2013 | INBEQMeDI

Structure and interactions of the major surface antigen of pathogenic Leptospira sp

binding proteins. 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.

Subsequent to the termination of this work, another group resolved the crystal structure of Ca2+-LipL32 complex. These crystal structures have provided a wealth of information from which to raise hypothesis regarding the molecular mechanisms by which LipL32 interacts with Ca2+ and with proteins of the extracellular matrix.

We therefore decided to characterize the LipL32Q67A, LipL32 S247A, and LipL32 D163–168A mutants in terms of their abilities to interact with host proteins previously characterized as LipL32 targets: full-length fibronectin and its 30-kDa fragment (F30), type IV collagen, and plasminogen [3]. Binding studies based on an ELISA were performed in the presence or absence of Ca2+ using

Figure 2. Structure of the bacterial second messenger bis(3´ 5´) cyclic di-GMP (c-di-GMP). GGDEF domains are diguanylate cyclases (DGCs) that synthesize c-di-GMP from two GTP molecules. EAL e HD-GYP are phosphodiesterases, responsible for c-di-GMP degradation to pGpG and GMP, respectively.

wild-type and LipL32 mutants as well as a fragment derived from the LipL32 C terminus (LipL32185–272_His tag) as a positive control and the unrelated leptospiral protein rLIC11030 as a negative control. The K Dapp values obtained for wild-type LipL32 binding with different substrates (F30, full-length fibronectin, collagen type IV and plasminogen) changed very little or not at all upon addition of Ca2+. The same was observed for the Q67A and S247A mutants, both of which retain the ability to bind Ca2+. Furthermore, the D163–168A mutant and LipL32 C-terminal fragment, both of which lack an intact Ca2+-binding site, interacted with target molecules with affinities equal or greater (in the case of the C-terminal fragment) than that observed for the wild-type protein.

Future Work – c-di-GMP signaling in Leptospira The molecule bis(3´ 5´) cyclic diGMP (c-di-GMP) is a universal second messenger molecule that controls global lifestyle behaviors (motility, biofilm formation, virulence, etc) in Gram negative bacteria (Figure 2). In previous work, we characterized a set of interactions among proteins that control c-di-GMP synthesis, degradation and signaling in Xanthomonas species [4-8].

Some of these c-di-GMP signaling pathways have been shown to regulate macromolecular secretion systems [4-

Our results lead us to conclude that whereas Ca2+

6]. We have therefore embarked on a new project aimed

binding contributes to the conformational stability of

at studying Leptospira c-di-GMP signaling pathways.

LipL32, metal binding does not play a crucial role in

Proteins involved in c-di-GMP metabolism and signaling

mediating or modulating its interactions with tested

have been cloned into expression vectors, expressed,

host extracellular matrix proteins. As Ca2+is abundant in

purified and crystallization trials initiated. We plan to

extracellular host fluids at concentrations well above the

construct a Leptospira interrogans two-hybrid library to

Ca2+ binding constants measured in this study, it is most

identify protein-protein interactions involved in these

likely that Ca2+ plays a structural role in LipL32 function

signaling pathways. Finally, Leptospira gene knockout

or contributes to other uncharacterized biological

strains will be produced to study c-di-GMP signaling in

LipL32 activities.

vivo and animal disease models.

References [1] Hauk P, Guzzo CR, Ho PL, Farah CS (2009) Acta Crystallogr Sect F Struct Biol Cryst Commun 65:307-309.

[2] Hauk P, Guzzo CR, Roman Ramos H, Ho PL, Farah CS (2009) J Mol Biol 390:722-736.

[3] Hauk P, Barbosa AS, Ho PL, Farah CS (2012) J Biol Chem 287:4826-4834.

[4] Guzzo CR, Dunger G, Salinas RK., Farah CS (2013) J Mol Biol 425:2174-2197.

[5] Guzzo CR, Salinas RK, Andrade MO, Farah CS (2009) J Mol Biol 393:848-866.

[6] Ryan RP, McCarthy Y, Kiely PA, O’Connor R, Farah CS, Armitage JP, Dow JM (2012) Mol Microbiol 86:557-567.

[7] Ryan RP, McCarthy Y, Andrade M, Farah CS, Armitage JP, Dow JM (2010) Proc Natl Acad Sci USA 107:5989-5994.

[8] Andrade MO, Alegria MC, Guzzo CR, Docena C, Rosa MCP, Ramos CHI, Farah CS (2006) Molecular Microbiology 62:537-551.

Research Reports 9

3 research reports

Structural studies of enzymes involved in nucleotide metabolism from blood fluke Schistosoma mansoni Juliana R. T. de Souza1, Larissa Romanelo1, Richard C. Garratt1, Ricardo DeMarco1, Jose Brandão-Neto2, Humberto D.M. Pereira1 Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, England

1 2

Schistosomiasis is a severely debilitating disease caused by parasites of the genus Schistosoma, among which Schistosoma mansoni is the most prevalent species in the world and is estimated to infect approximately 200 million people. S. mansoni lacks the de novo purine pathway and depends on its host for purine requirements. In the case of pyrimidine metabolism, the parasite possesses both de novo and salvage pathways. Furthermore, the thymidylate cycle is also functional. These pathways have been used in the past as targets for chemotherapeutic intervention. However, little attention has been devoted due to their importance for worm metabolism.

The current project aims to solve crystallographic structures of all enzymes involved in the purine salvage pathway and the thymidylate cycle as well as some specifically selected from the pyrimidine salvage and de novo pathways. This is being done together with the determination of their kinetic constants. Three rounds of cloning and expression were performed. In the first, genes were amplified from an enriched cDNA library and were cloned and expressed using standard procedures at IFSC-USP. In the remaining two, synthetic genes were cloned in multiple vectors and parallel expression was performed using the OPPF-UK facilities. Using these approaches 31 genes were cloned and expressed, 28 of the resulting proteins were soluble, 27 of which were purified and 23 submitted to robotic crystallization trials.

Crystals for 18 enzymes were successfully obtained, 16 of which were subject to diffraction measurements resulting in nine crystal structures. Kinetic assays for 7 of these enzymes were also performed.

Figure 1 shows a summary of the resulting structures. Adenosine kinase (AK) was the first structure solved and catalyzes the reaction: adenosine + ATP ↔ AMP +ADP. Two complexes were solved to 2.25Å: the ternary complex AK-adenosine-AMP and the complex between AK and tubercidin. Several differences were found in the adenosine binding site that explains the preference for some adenosine analogues in contrast to the human enzyme [1]. Uridine phosphorylase (UP) catalyzes the phosphorolysis of uridine/thymidine to uracil/thymine plus ribose-1-phosphate, participating in the pyrimidine salvage pathway. The parasite possess two isoforms for this enzyme, one with the active site totally conserved in comparison with its human counterpart (UP-GQ) and other with several differences (UP-DL). We cloned the two isoforms and solved several structures for both isoforms up to 1.65Å resolution. The differences in the active site between the two isoforms suggest different roles in nucleotide metabolism. Adenylate kinase (ADK) is involved in the conversion of 2 ADP into AMP +ATP, and also has a role in energy homeostasis. The structure of ADK in its apo form was obtained to 2.05Å resolution. Despite many efforts we were unable to obtain complex structures due to the crystallization conditions [2]. Dihydrofolate reductase (DHFR) is the enzyme that converts dihydrofolate into tetrahydrofolate in the presence of NADPH, and is extensively used as a drug target. The structure for the schistosome enzyme was solved to 1.95Å resolution. The structure obtained is in its apo form and the most prominent result was the conformation of the W loop that enters into the folate site. Adenylosuccinate lyase cleaves adenylosuccinate into

10 Summary Report 2009-2013 | INBEQMeDI

Structural studies of enzymes involved in nucleotide metabolism from blood fluke Schistosoma mansoni

adenosine 5’-monophosphate and fumarate. Datasets were obtained up to 2.3Å resolution and refinement is currently under way. The SmNDPK enzyme (EC 2.7.4.6) is a component of the purine salvage pathway and catalyses the γ-phosphoryl transfer from nucleoside triphosphate to a nucleoside diphosphate. Dozens of datasets were obtained from co-crystallization experiments and several structures were solved and refined including the apo form at 1.7Å and its complex with ATP. The second isoform for purine nucleoside phosphorylase was also crystallized and several datasets collected. The crystal diffracts to 1.5Å resolution and two structures were solved. Studies of PNP2 specificity

will be performed to determinate the function of this enzyme. The methylthioadenosine phosphorylase (MTAP) uses 5’-deoxy-5’-methylthioadenosine (MTA) as one possible substrate and also catalyzes the reaction: S-methyl-5’-thioadenosine + phosphate ↔ adenine + S-methyl-5-thio-α-D-ribose 1-phosphate. In this case four complexes were obtained, as well as dozens of datasets for three MTAP mutants for investigation their differential specificity in comparison to their human homologue.

The structural data together with kinetic data will be invaluable in the understanding of purine metabolism in this important human parasite.

Figure 1. Structures Solved. 1 - Methylthioadenosine phosphorylase; 2 - Adenosine kinase; 3 - Adenylate kinase; 4 - Nucleoside diphosphate kinase; 5 - Purine nucleoside phosphorylase isoform 2; 6 - Uridine phosphorylase isoform DL; 7 - Adenylosuccinate lyase; 8 - Purine nucleoside phosphorylase isoform 1; 9 - Uridine phosphorylase isoform CQ; 10 - Dihydrofolate reductase.

References [1] Romanello L, Bachega JFR, Cassago A, Brandao-Neto J, Garratt RC, DeMarco R, Pereira HDM (2013) Acta Cryst D 69:126-136.

[2] Marques IA, Romanello L, DeMarco R, Pereira HDM (2012) Mol Biochem Parasitol 185:157-160.

Research Reports 11

4 research reports

NTPDases (Apirases) of Trypanosoma cruzi and Leishmania Juliana L. R. Fietto1, Raphael S. Vasconcellos1, Christiane M. Moura1, Matheus S. Bastos1, Ramon F. Santos1, Márcia R. de Almeida1, Luís C. C. Afonso2, Maria T. Bahia2 Department of Biochemistry and Molecular Biology, Federal University of Viçosa, Viçosa, MG, Brazil Institute of Exact and Biological Sciences, Federal University of Ouro Preto, Ouro Preto, MG, Brazil

1 2

In this work we foccused on the NTPDases from T. cruzi and Leishmania, causative agents of Chagas Disease and Leishmaniases respectively. The first step in this work was to clone and heterologously express the NTPDases to be used in biochemical characterization, target validation and drug screening assays (search for inhibitors to be tested both in vitro and in vivo). We have been successful in the expression and characterization of 03 different NTPDases, one from T. cruzi (NTPDase-1) and two from Leishmania infantum (NTPDase-1 and 2). The second step was to validate the targets. We have performed different assays for in vitro and in vivo infections in situations where the NTPDases from parasites were inhibited (by general NTPDases inhibitors) or blocked by specific antibodies. The results showed lower levels of infection or lower virulence when NTPDases were inhibited or blocked. The next step was the search of specific inhibitors. We designed two different approaches: the rational drug design (crystallizations, structure determinations and in silico search of putative inhibitors) and a “blind” search using synthetic and natural products from the INBEQMeDI library of compounds. We were unsuccessful in the first approach because we were unable to obtain the recombinant proteins in conditions for crystallization. The second approach came up with three different compounds and two different plant extracts able to inhibit the recombinant enzymes. We are currently characterizing the inhibitors biochemically and determining their IC50 values and inhibitory mechanism. Furthermore we are testing the effect of these compounds and extracts directly on parasites (trypanosomicidal and Leishmanicidal effects). The next step will be the evaluation of inhibition of in vitro infections.

Furthermore, we have used the Leishmania recombinant proteins in immunodiagnostic tests and applied for a national and international patent for the use of Leishmania NTPDases in biotechnological applications including diagnosis, prognosis, chemotherapy, drug design, antibody production and vaccine compositions to be used in any type of Leishmaniasis.

12 Summary Report 2009-2013 | INBEQMeDI

NTPDases (Apirases) of Trypanosoma cruzi and Leishmania

Figure 1. Potential biotechnological applications of NTPDases.

References [1] Leite PM, Gomes RS, Figueiredo AB, Serafim TD, Tafuri WL, de Souza CC, Moura SA, Fietto JL, Melo MN, Ribeiro-Dias F, Oliveira MA, Rabello A, Afonso LC (2012) PLoS Negl Trop Dis 6:e1850.

[2] de Souza RF, dos Santos YL, de Souza Vasconcellos R, Borges-Pereira L, Caldas IS, de Almeida MR, Bahia MT, Fietto JL (2012) Acta Trop 125:60-66.

[3] de Souza MC, de Assis EA, Gomes RS, Marques da Silva Ede A, Melo MN, Fietto JL, Afonso LC (2010) Acta Trop 115:262-269.

[4] Santos RF, Pôssa MA, Bastos MS, Guedes PM, Almeida MR, Demarco R, Verjovski-Almeida S, Bahia MT, Fietto JL (2009) PLoS Negl Trop Dis 3:e387.

Research Reports 13

5 RESEARCH REPORTS

Leishmanicidal Activity of Aphidicolin, Deoxy-aphidicolin and their Semisynthetic Derivatives Gabriela B. dos Santos1, Marília O. de Almeida1, Iara A. Cardoso1, Viviane Manfrin1, Fernanda O. Chagas1, Adriana A. Lopes1, Juliano S. Toledo2, Camila F. Pinzan2, Alexandre S. de Araujo3, Flavio S. Emery1, Angela K. Cruz2, Mônica T. Pupo1 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil 2 School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil 3 Institute of Biosciences, Letters and Exact Sciences of São José do Rio Preto, State University of São Paulo “Julio de Mesquita Filho”, São José do Rio Preto, SP, Brazil 1

The investigation of natural products has provided an important source of hits and leads for drug discovery, and the structure-activity studies of active natural leads appears to provide an alternative approach for developing new safe and selective leishmanicidal compounds against each Leishmania species. Among natural products, diterpenes have shown high activity against different Leishmania species.

We have found that the endophytic fungus Nigrospora sphaerica, isolated from Smallanthus sonchifolius (Asteraceae), is a prolific producer of the anticancer aphidicolane-type diterpene aphidicolin (1), while 3-deoxyaphidicolin (2) is also produced in lower yields [1]. The mevalonate biosynthetic pathway of 1 has been established by 13C-1-glucose feeding experiments using N. sphaerica liquid cultures [2].

One previous literature report has described a high activity for aphidicolin against the protozoan Leishmania major. However, despite its promising leishmanicidal potential, aphidicolin presents therapeutically unsuitable physico-chemical and pharmacokinetic profiles. The semi-synthetic approach is widely used to develop new treatments for neglected diseases and has led to new methodologies for functional group transformation, such as alkylation and acylation.

Figure 1. Chemical structures of natural products 1-2 and semi-synthesis of compounds 3-8 from 1.

14 Summary Report 2009-2013 | INBEQMeDI

Leishmanicidal Activity of Aphidicolin, Deoxy-aphidicolin and their Semisynthetic Derivatives

A series of aphidicolin derivatives were synthesised via modification of the primary and secondary hydroxyl groups [3]. We have proposed a preliminary evaluation of the structural requirements for the leishmanicidal activity of aphidicolin and its semisynthetic derivatives based on biological assays. This work presents the use of a semi-synthetic approach to create derivatives from the natural lead aphidicolin for biological assays against Leishmania spp (Figure 1). Eight compounds have been synthesised and tested against different species of the Leishmania protozoa (Table 1). The preliminary evaluation demonstrated high leishmanicidal activity for aphidicolin (1), while the oxime derivative (8) shows moderate selectivity for the L. braziliensis species, which is commonly found in several South American countries (Table 1). None of the compounds showed cytotoxicity against mammalian cells (Figure 2).

Since compounds 1 and 8 demonstrated higher potential for the inhibition of Leishmania growth, they were selected for cytotoxic activity studies in mammalian cells. Compounds 1 and 8 had no effect on the growth of normal cells. Because 1 and 8 demonstrated the IC50 values on promastigote cells and exhibited no cytotoxic effects on mammalian cells, they were tested against amastigotes, the stage of the parasite adapted to living in vertebrate host cells.

These compounds exhibited potent activity against the amastigotes (Figure 2), the evolutionary

Table 1. IC 50 values of compounds 1, 2, 7 and 8 against different promastigote Leishmania species.

Compound

L. braziliensis (avg ± SD)

L. major (avg ± SD)

1

0.37 ± 0.11

0.17 ± 0.05

2

2.28 ± 0.33

0.95 ± 0.16

7

2.7 ± 1.17

inactive

8

0.85 ± 0.23

inactive

geneticin*

5.7 × 10-6

3.3 × 10-6

*positive control; avg, average; SD, standard deviation

form responsible for the pathological development.

Compound 1 reduced the infection index of the Leishmania amastigotes by 90% at concentrations of 0.1 µM, and compound 8 reduced the infection index by approximately 70% at 0.5 µM (Figure 2) [3].

Aphidicolin (1) was found to be more effective as a leishmanicidal hit than 8. However, clinical studies previously reported have shown that 1 undergoes rapid in vivo oxidative metabolism, producing 3-ketoaphidicolin (2). We found that 2 is less active than 1 and 8 as a leishmanicidal. Compound 8 is metabolically stable and active against promastigotes and amastigotes of L. braziliensis (Table 1 and Figure 2), therefore it has been considered for further in vivo studies as an antiparasitic agent.

Figure 2. Biological evaluation of compounds 1 (left) and 8 (right) against amastigotes of Leishmania braziliensis. Negative control, non-treated infected macrophages. Positive control, infected macrophages treated with geneticin [10 µg/mL].

References [1] Gallo MBC, Chagas FO, Almeida MO, Macedo CC, Cavalcanti BC, Barros FWA, Moraes MO, Costa-Lotuffo LV, Pessoa CO, Bastos JK, Pupo MT (2009) J Basic Microbiol 49:142-151.

[2] Lopes AA, Pupo MT (2011) J Braz Chem Soc 22:80-85.

[3] Santos GB, Almeida MO, Cardoso IA, Manfrin V, Chagas FO, Toledo JS, Pinzan CF, Araujo AS, Cruz AK, Pupo MT, Emery FS (2013) Bioorg Med Chem Lett (submitted).

Research Reports 15

6 RESEARCH REPORTS

Biochemical and structural studies of the arginase from Leishmania amazonensis Monica R. C. Iemma1, Lorena R. F. de Souza1, Caroindes J. Correa1, Paulo C. Vieira1, Dulce H. F. de Souza1 Department of Chemistry, Federal University of São Carlos, São Carlos, SP, Brazil

1

Investigation of the arginase from L. amazonensis was undertaken using two different approaches: search for compounds isolated from bushland plants with potential inhibitory activity against the enzyme and crystallization experiments for structural studies using X-ray diffraction. Different parts of bushland plants (Aegiphila lhotskiana; Anadenanthera falcata; Bauhinia holophylla; Byrsonima coccolobifolia; Ouratea spectabilis; Tocoyena formosa; Qualea grandiflora; Cochlospermum regium) were used in screening against the arginase enzyme (Figure 1). Among the plants tested, the crude extracts from stem (IC50 = 39.7 mcg/mL) and leaves (IC50 = 25.4 mcg/mL) from B. coccolobifolia proved to be the most effective. These extracts exhibited also good inhibitory activity on the growth of promastigotes of L. (L.) braziliensis. The extracts were fractionated and again tested against the enzyme. Compounds of the class of lignans presented moderate inhibition with IC50 values between 13.7 and 35.1 µM. Furthermore, we describe lignans and antraquinone derivative as arginase inhibitors for the first time. (–)-Epicatechin, (+)-catechin and derivatives of aglycones showed strong enzymatic inhibition with IC50 values in the range of 0.9-3.7 µM, suggesting that the subtype flavan-3-ol has important structural features that allowed these substances to bind to the enzyme. Although flavonoids have been recently characterized as inhibitors of recombinant arginase from L. (L.) amazonensis, there are still few studies exploiting their inhibitory effects against the enzyme.

The present study in the search for new inhibitors of arginase, differs from

Figure 1. Screening of crude extracts against arginase enzyme from L. amazonensis. E, ethanolic extract; F, leaves; C, stem; Fr, fruits; R, roots; Al, Aegiphila lhotskiana; Af, Anadenanthera falcata; Bh, Bauhinia holophylla; Bc, Byrsonima coccolobifolia; Os, Ouratea spectabilis; Tf, Tocoyena formosa; Qg, Qualea grandiflora; Cr, Cochlospermum regium.

16 Summary Report 2009-2013 | INBEQMeDI

Biochemical and structural studies of the arginase from Leishmania amazonensis

R1

R2

R3

R4

R5

R6

R7

OH

OH

H

O-α-L-rhamnopyranose

OH

H

OH

OH

OH

H

β-D-glucopyranose

OH

H

OH

OH

OCH3

H

O-α-L-rhamnopyranose

OH

H

OH

OH

OCH3

OH

O-α-L-rhamnopyranose

OH

H

OH

OH

OH

OH

O-α-L-rhamnopyranose

OH

H

OH

OH

OH

OH

H

OH

H

OH

OH

OH

H

H

OH

H

OH

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

R1

R2

R3

R4

R5

OH

OH

(+) OH

OH

OH

OH

OH

(–) OH

OH

OH

H

OH

O-α-L-rhamnopyranose

OH

OH

H

OH

O-(3”-O-trans-cinnamoyl)-αrhamnopyranose

OH

OH

OH

OH

(+) OAc

OH

OH

OH

OH

(+) OAc

OAc

OAc

Compound 1 2

R1

Flavonols

R2

7 8 9 10 11

O

R7 R6

R4 R5

12

R3

O

Compound 3 4

R1

Flavan-3-ols

R2

13 O

R4

14 15

R3

R5

16 Compound

Compound

Compound

OCH3 OH

OH

5

H

H H3CO

OCH3

OH

H 3C

O

CH3

6

17

CH3

H3CO

O

O

HO

OCH3

OH

OCH3 Figure 2. Chemical structures and IC50 values of several flavonoids as inhibitors of arginase.

previous reports in the literature since these active

In the crystallization experiments, 500 conditions

flavonoids were found through a bioguided study. To

were tested but proved to be unsuccessful. The

exploit further the arginase inhibitory activity of this

oligomerization state of the enzyme was studied

series of naturally occurring compounds several other

using dynamic light scattering which showed that the

flavonoids were evaluated. In Figure 2 we report the IC50

sample was present as a non-functional oligomer in

values for the compounds analysed (1-17) together with

spite of its relatively high purity. DLS measurements

their corresponding structures. Due to the inhibitory

with the enzyme in different conditions (buffer, salt

activity of flavonoids we also evaluated the simple

and additives) were performed in order to establish

molecule 17 (hydroquinone). Among all compounds

a condition in which the formation of agglomerates

tested hydroquinone was the most active with an IC50

was minimized. Based on this new knowledge, the

of 53.2 ± 5.0 nM. Further studies are required in order

purification procedure was reassessed and adapted

to fully understand structure-activity relationships

including the addition of a third purification step

for this system. These studies were performed in

allowing for the separation of possible oligomeric forms

collaboration with Prof. Paulo Cesar Vieira, from the

of the enzyme. This new procedure is being used to

Federal University of São Carlos, and Prof. Izabela

purify the enzyme, which will be tested in crystallization

Golden, from the University of Brasília.

trials in the next stage of the project.

Research Reports 17

7 RESEARCH REPORTS

Identification of Polyether Antibiotics Produced by the Endophyte Streptomyces platensis RTd22 Larissa Varella1, Eduardo J. Crevelin1, Luiz A. B. de Moraes2, Andrei N. G. Dabul3, Ilana L. B. C. Camargo3, Mônica T. Pupo1 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil 2 School of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil 3 Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil 1

The endophytic actinobacteria Streptomyces platensis RTd22 was isolated from the roots of Tithonia diversifolia (Asteraceae) and cultured on rice solid medium and ISP-2 liquid medium. Extracts and sub-fractions obtained after the chromatographic procedures showed remarkable antibiotic activity against Staphylococcus aureus ATCC 6538, S. saprophyticus ATCC 15305 and S. aureus ATCC 29213. The subfraction FAL-5 also showed high activity against three antibiotic-resistant bacteria: vancomycin-intermediate S. aureus Mu50 (VISA) (0.781µg/mL), methicillin-resistant S. aureus SA16 [1] (MRSA) (0.781 µg/mL) and vancomycin-resistant Enterococcus faecium VRE16 (0.781 µg/mL) (Table 1). This sub-fraction was found to be more

Figure 1. S. platensis RTd22 culture in ISP-2 agar and chemical structures of polyether antibiotics grisorixin and nigericin.

18 Summary Report 2009-2013 | INBEQMeDI

Identification of Polyether Antibiotics Produced by the Endophyte Streptomyces platensis RTd22

Table 1. Minimal inhibitory concentration (MIC) value (µg.mL-1) of extracts, subfraction and commercial antibiotics determined by microdilution assay.

Gram-positive bacteria

FHS

FAS

FBS

FAL

FBL

FAL-5

Van

Dap

Pen

S. aureus ATCC 6538

0.781

0.781

200

0.195

400

0.195

-

-

0.115

S. aureus ATCC 15305

0.781

0.781

400

1.562

>400

0.195

-

-

1.475

S. aureus ATCC 29213

0.39

3.125

6.25

0.39

100

0.39

1.00

1.00

-

S. aureus Mu50 ATCC 700699

0.781

6.25

6.25

0.781

100

0.781

4.00

1.50

-

S. aureus SA16

1.562

6.25

12.5

1.562

200

0.781

2.00

0.5

-

E. faecium VRE 16

0.781

6.25

12.5

0.781

200

0.781

>256

3.00

>256

FHS: Hexane extract from rice solid medium; FAS: Ethyl acetate extract from rice solid medium; FBS: n-butanol extract from rice solid medium FAL: Ethyl acetate fraction from ISP-2 liquid medium; FBL: n-butanol extract from ISP-2 liquid medium FAL-5: subfraction from FAL extract, Van: vancomycin, Dap: daptomycin and Pen: penicillin.

potent than the commercial antibiotics vancomycin and

in the literature. These compounds are able to form

daptomycin, used as positive controls. Analyses by ESI-

complexes with metal cations and transport them

MS and ESI-MS/MS (Xevo® TQ-S, Waters Corporation)

across the lipid bilayer. As a consequence, the whole

and H NMR of the subfraction allowed the detection

process leads to changes in osmotic pressure inside

of ions m/z 726 [M+NH4]+ and m/z 731 [M+Na]+ which

the cell, causing death of the bacterial cell. Despite

correspond the ionophore antibiotic grisorixin (1),

the high in vitro effectiveness of carboxyl polyethers,

a nigericin derivative, structurally related polyether

the main obstacle for their use as drugs to control

polyketides [2] (Figure 1). The antibacterial activities

human diseases seems to be their potential toxicity.

of the carboxyl ionophore to Gram-positive bacteria,

The reduction of this toxicity by medicinal chemistry

including multidrug-resistant strains of pathogenic

approaches might improve their potential as candidates

bacteria such as MRSA and VRE are already reported

for use clinical.

1

References [1] Dabul ANG, Camargo ILBC (2013) Epidemiol Infect 29:1-5 [Epub ahead of print].

[2] Varella L, Crevelin EJ, De Moraes LAB, Dabul ANG, Camargo ILBC, Pupo MT (2012) Planta Medica 78:1156.

Research Reports 19

8

Selenocysteine synthesis pathway in Protozoa

RESEARCH REPORTS

Marco T. A. da Silva1, Izaltina S. J. Cavalli1, Otavio H. Thiemann1 Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil

1

Biological trace elements are chemical elements required in minute quantities by an organism, that otherwise would be toxic, and function in different ways as essential components of enzymes. Among these elements, selenium (Se), the major metalloid micronutrient, occurs naturally in foods almost exclusively in organic compounds.

The primary forms of Se are selenomethionine, Se-methylselenomethionine, selenocystine and selenocysteine. Selenocysteine (Sec – U), the 21st naturally occurring amino acid, is co-translationally incorporated into selenoproteins encoded by an in-frame UGA codon. The human selenoproteome includes 25 selenoproteins containing at least one selenocysteine. Selenoproteins are essential for mammals.

Although the function of most selenoproteins remains unknown, the few which have been characterized are oxidoreductases, protecting the cell against oxidative stress.

The selenocysteine synthetic pathway was first described in Leishmania in 2006 [1] and later the existence of three selenoproteins in Kinetoplastida was proposed, two with mammalian homologs, SelK and SelT, and a third, SelTryp exclusive of Kinetoplastida. Additionally, auranofin, a selective inhibitor of selenoproteins, strongly inhibits the growth of Trypanosoma brucei cultures (Figure 1). The identification of selenoprotein genes in Kinetoplastida and the characterization of the selenocysteine synthesis and insertion pathways are also relevant from an evolutionary perspective, since selenoproteomes have been previously identified in the three domains of life, however they are absent in fungi and plants. What would be the selective pressure that maintained such a pathway in several organisms and lacking in others?

Among the proteins involved in selenocysteine biosynthesis and insertion machinery in Kinetoplastidae, the best characterized is the Selenophosphate synthase (SPS) [2]. The insertion of selenium into Sec-dependent enzymes requires the formation of a highly reactive reduced selenium donor compound monoselenophosphate. Monoselenophosphate synthesis is catalyzed by SPS in a 1:1:1 ratio from selenide and ATP.

Figure 1. The selenocysteine synthesis pathway from Bacteria, Archea, Eukaryote and Trypanosomes are comparable. SelA route is exclusive for the bacterial pathway. The PSTK/SepSecS pathway is found in archaea and eukaryotes. Trypanosomes posses an Archea-like pathway.

20 Summary Report 2009-2013 | INBEQMeDI

Selenocysteine synthesis pathway in Protozoa

RNAi mediated ablation of the T.brucei SPS mRNA severely impairs selenoprotein synthesis. Costa et al.

(2011) [3] demonstrated that under stress conditions T. brucei cells lacking SPS presented growth deficiency in both the procyclic and bloodstream forms indicating the relevance of senelocysteine metabolism for stress protection of the parasite cell, a condition it encounters during its life cycle and therefore may even be a relevant drug target.

The process of Sec incorporation into selenoproteins differs from bacteria to archaea and eukarya although not all species within these kingdoms have the capacity for selenoprotein synthesis (Figure 2). However, in all organisms that have the selenocysteine pathway Sec is formed in a tRNA-dependent conversion of serine bound to tRNASec by seryl-tRNA synthetase. Kinetoplastida tRNA[Sec] present common features observed in other known tRNASec but it has its own peculiarities. The presence of the anticodon UCA has been identified and required for the recognition of the UGA codon.

Furthermore, the predicted folding of tRNASec revealed the characteristic long variable loop arm consisting of a six base-pair long stem and presence of an usual pseudopirimidine site. Another important feature observed in T. brucei tRNASec is the long D-loop with 7 base pairs, contrasting with the tRNASer short D-loop of 3-4 base pairs. The length and secondary structure of the D-loop, but not its sequence composition, are the major components by which archaea and human PSTKs discriminate between tRNASec and tRNASer. The mutated human tRNASec with a 4 base pair D-loop has shown decreased PSTK phosphorylation when compared with the wild type with a 6 base pair D-loop and recent data demonstrated that identification of tRNASec in archea is dependent on the D-loop structure.

Moreover, the Kinetoplastidae tRNA Sec has a 7/5 structure in the acceptor-TψC stems with 7 nucleotides in the acceptor stem and five nucleotides in the TψC stem, while the human and the archea tRNASec harbor a 9/4 structure. These structural differences in the tRNA Sec of Kinetoplastidae suggest an equivalent adaptive modification in the enzymes that interact and recognize such tRNA, including the ribosome A-site that differentiates the parasite selenocysteine.

Little is known about selenium metabolism in trypanosomatids cells but the effect of auranofin (AF), a specific inhibitor of selenoproteins, in T. brucei brucei cells has been demonstrated. In various organisms

Figure 2. Sec incorporation pathway in Bacteria, Archea, Eukaryote and Trypanosomes comparing the known mediators of the interaction between Sec-tRNA, SECIS element and the ribosome.

selenoproteins have oxireductase functions, preventing or repairing damage to cellular components, besides the regular oxi-reduction reactions. AF showed high activity against these proteins, essential to the parasite survival, reducing T. cruzi viability in vitro and in vivo.

It is not known if Kinetoplastidae possess a selenium detoxification mechanism, as observed in other organisms such as mammals. In these cells, inorganic forms of selenium such as selenite and selenate are reduced to selenide (H 2Se), reactions catalyzed by glutathione reductase. It was demonstrated that the methylation of selenide by methyltransferase, using S-adenosylmethionine as methyl donor identified (CH3)2Se (dimethyl selenide) as a major product. The methylated Se compounds are the major forms in which it is excreted in mammals. The volatile dimethyl selenide is lost in the lungs and the soluble trimethylselenonium cation is excreted in the urine. The mechanism of trypanosome selenium detoxification remains unclear.

It is possible that selenium is essential for kinetoplastid cell viability in various growth stages, not only during the stationary phase or under oxidative stress. However, further experiments are needed to better understand the role of selenium in the trypanosomatids development.

References [1] Cassago A,, Rodrigues EM, Prieto EL, Gaston KW, Alfonzo JD, Iribar MP, Berry MJ, Cruz AK, Thiemann OH (2006) Mol Biochem Parasitol 149:128-134.

[2] Sculaccio SA, Rodrigues EM, Cordeiro AT, Magalhaes A, Braga AL, Alberto EE, Thiemann OH (2008) Mol Biochem Parasitol 162:165-171.

[3] Costa FC, Oliva MAV, de Jesus TCL, Schenkman S, Thiemann OH (2011) Mol Biochem Parasitol 180:47- 50.

Research Reports 21

9 RESEARCH REPORTS

Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening Matheus P. Postigo1, Rafael V. C. Guido1, Glaucius Oliva1, Marcelo S. Castilho2, Ivan R. Pitta3, Julianna F. C. de Albuquerque3, Adriano D. Andricopulo1 Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil Faculty of Farmacy, Federal University of Bahia, Salvador, BA, Brazil 3 Department of Antibiotics, Federal University of Pernambuco, Recife, PE, Brazil 1 2

Drug discovery is currently driven by innovation and knowledge employing a combination of experimental and computational methods [1,2]. In line with this, some of our projects in INBEQMeDI have focused on the identification and optimization 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.

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. Several steps of the drug discovery process (e.g.; hit identification, lead optimization, NCE development) can be improved in a rational way with the application of computational methods [2].

Figure 1. Integrated approach carried out toward the identification of novel inhibitors of SmPNP.

22 Summary Report 2009-2013 | INBEQMeDI

Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening

Figure 2. Thioxothiazolidinone derivatives as new lead compounds for schistosomiasis.

Structure-based enzyme inhibitor design approaches play a crucial role in the development of small-molecule drug candidates that act on specific enzymes, and have become a pivotal component of drug discovery programs [3]. An example can be seen in the integrated medicinal chemistry approach employed toward the discovery of new inhibitors of Schistosoma mansoni purine nucleoside phosphorylase (SmPNP), a key enzyme involved in the purine salvage pathway of S. mansoni, one of the causative agents of human schistosomiasis [4]. In this work, we have described the development of a structure-based pharmacophore model for ligands of SmPNP, which was subsequently employed in virtual screening studies leading to the identification of thioxothiazolidinones derivatives with substantial in vitro inhibitory activity against the parasite enzyme (Figure 1).

Synthesis, biochemical evaluation and structureactivity relationship (SAR) investigations led

to the successful development of a small set of thioxothiazolidinone derivatives harboring a novel chemical scaffold as inhibitors of SmPNP at the low micromolar range. To explore the mechanism of inhibition in more detail, we have determined K i values and the type of inhibition with respect to the physiological substrate of SmPNP (i.e., inosine). The results indicated that the inhibition of SmPNP was found to be competitive with respect to inosine, hence, this behavior is consistent with a mutually exclusive binding mode between inhibitor and substrate.

Accordingly, these results confirm the successful use of our structure-based virtual screening strategy, allowing the discovery of thioxothiazolidinone derivatives as a new class of competitive inhibitors of SmPNP, with affinity values in the low micromolar range. The most potent inhibitors 1–3 represent new potential lead compounds for further development for the therapy of schistosomiasis (Figure 2).

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. Structure-based 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, 50, 1693-1705.

Research Reports 23

10

Antileishmanial activity of natural products and synthetic analogs

RESEARCH REPORTS

Diógenes A. G. Cortez1, Celso V. Nakamura2, Arlene G. Corrêa3 Department of Pharmacy and Pharmacology, State University of Maringá, Maringá, PR, Brazil.

Department of Basic Health Sciences, State University of Maringá, Maringá, PR, Brazil.

3 Department of Chemistry, Federal University of São Carlos, São Carlos, SP, Brazil 1 2

The drugs utilized in the treatment of leishmaniasis (pentavalent antimonials, amphotericin B and pentamidine) are limited to some extent by their toxicity, requirement for intravenous administration, long-term treatment, lack of efficacy, and high cost, and are prone to stimulate drug resistance. A multidisciplinary approach to drug discovery, involving the generation of truly novel molecular diversity from natural product sources, combined with total and combinatorial synthetic methodologies, and including the manipulation of biosynthetic pathways, provides the best solution to the current productivity crisis facing the scientific community engaged in drug discovery and development.

Extensive studies have shown that plant extracts and chemically defined molecules of natural origin possess antileishmanial activity. Piper amalago L. has been used in folk medicine as an anti-inflammatory, analgesic, antipyretic, therapy for stomach problems, and vermifuge. Recently, we have reported that amides isolated from the leaves of P. amalago L., a derivative, and synthetic analogs (Figure 1) were evaluated against promastigote and amastigote forms of Leishmania amazonensis.

The derivative and synthetic analogs were synthesized with the aim of investigating whether they could improve the antileishmanial activity and contribute to the structure-activity relationship study. The compounds were also analyzed in terms of cytotoxicity toward macrophages, and the ability to induce nitric oxide production [1].

Natural compounds 1 and 2 showed the best antileishmanial activity with IC50 values of around 20 μM and 15 μM respectively, compound 2 being more active and selective than compound 1 with a selective index ratio (SI = CC50 J774G8 / IC50) of 12.89. The isolated amides inhibited significantly both promastigote and intracellular amastigote forms. It was possible to observe that the double bonds in the side chain having a (E,E) configuration in compound 2, led to an increase in the antileishmanial activity. The substituents of compounds 7-10 with deactivating electron-withdrawing groups (e.g., fluor, difluoromethoxy, trifluoromethoxy) have a negative effect on the activity.

Another plant, Calophyllum brasiliense Camb. (Clusiaceae), has been used in folk medicine for the treatment of rheumatism, varicose veins, hemorrhoids, and chronic ulcers. We have previously shown that extracts, fractions, and mainly coumarin

Figure 1. Amides tested against the promastigotes and intracellular amastigotes forms of L. amazonensis: natural compounds 1-2, derivative 3, and synthetic analogs 4-10.

24 Summary Report 2009-2013 | INBEQMeDI

Antileishmanial activity of natural products and synthetic analogs

Figure 2. Natural coumarins 11–12, (-)-mammea A/BB derivatives 13–16, and a synthetic coumarin 17.

(-)-mammea A/BB (11) isolated from C. brasiliense leaves and its derivatives show antileishmanial activity against L. amazonensis (Figure 2, Table 1) [2]. Compounds 11, 13, 14 and 16 were active not only against promastigote forms, but also against intracellular amastigote forms of L. amazonensis, being compound 13 the most potent with IC50 of 0.6 μM and a SI ratio of 144.2.

Further studies were carried out showing the morphological and ultrastructural alterations in L. amazonensis treated with natural, synthetic and derivative coumarins of (-)-mammea A/BB, observed by different microscopic techniques. In addition, we reported the drug target of (-) mammea A/BB and the derivative 13 in the parasite [3]. Ultrastructural analysis showed that compound 13 induced intense atypical cytoplasmic vacuolization and the appearance of autophagic vacuoles in L. amazonensis promastigotes.

The intense cytoplasmic vacuolization and autophagic like vacuoles suggest recycling of abnormal membrane structures, indicating a process of intracellular remodeling.

The results of the present study show that natural products represent an unparalleled source of molecular diversity in drug discovery and the development

Table 1. Antileishmanial activity of coumarins against promastigote forms of L. amazonensis

Compound

Promastigote IC50 (µM)

Amastigote IC50 (µM)

11

7.4 ± 0.30

14.3 ± 2.2

12

30.1 ± 3.5

n.d.a

13

0.9 ± 0.1

0.6 ± 0.0

14

2.4 ± 0.4

34.04 ± 2.6

15

15.1 ± 1.0

n.d.

16

1.9 ± 0.2

22.2 ± 3.9

17

60.2 ± 3.5

n.d.

Amphotericin B

0.063 ± 0.1

n.d.

Values represent the mean ± S.D. of at least three experiments performed in triplicate.a n.d.=not determined

of novel antiprotozoal agents. In this context, the compounds (-) mammea A/BB (11) and coumarin 13 are potential candidates for further research to develop new antiprotozoal drugs, considering their significant antileishmanial activity that may act in depolarization of the mitochondrial membrane potential cells, leading to death of the parasite.

References [1] Carrara VS, Cunha-Júnior EF, Torres-Santos EC, Correa AG, Monteiro JL, Demarchi IG, Lonardoni MVC, Cortez DAG (2013) Braz J Pharmacogn 23:447-454.

[2] Brenzan MA, Nakamura CV, Dias-Filho B, Ueda-Nakamura T, Young MCM, Alvim-Junior J, dos Santos AO, Cortez DAG, (2008) Biomed Pharmacother 62:651-658.

[3] Brenzan MA, Santos AO, Nakamura CV, Dias-Filho BP, Ueda-Nakamura T, Young MCM, Morgado DA, Cortez DAG (2012) J. Phytomedicine 19:223-230.

Research Reports 25

11

Host-Plasmodium signaling Fernanda Koyama1, Eduardo Alves1, Wania Rezende1, Dario Passos1, Célia R. S. Garcia1 Institute of Biosciences, University of São Paulo, São Paulo, SP, Brazil

research reports

1

The purpose of our collaborative work with the INBEQMeDI-INCT is to dissect the structure and function of the molecular players involved in signaling transduction pathways on Plasmodium – host interaction.

Malaria is the most devastating parasitic disease in humans, afflicting 300-500 million people worldwide and being responsible for 1.5-2.7 million deaths annually.

The rational development of a malaria vaccine and new therapies for the disease is urgently required, but its success is highly dependent of a better understanding of molecular details of parasite cell biology.

Recent contributions in the field from our laboratory as well as others have provided evidence that the parasite senses the environment and displays the handling machinery for controlling its own cell cycle by using second messengers such as calcium, cAMP and IP3 [1]. The parasite can sense its environment and adapt to benefit its survival, indeed this is essential for it to complete its life cycle (Figure 1). Melatonin (MLT) crosses the erythrocyte (RBC) surface membrane (EM) and the parasitophorous vacuolar membrane (PVM) into the parasitophorous vacuole (PV). Plasmodium senses MLT from the RBC and a cascade of signaling is initiated through an as yet unidentified melatonin receptor located in the parasite plasma membrane (PM). Melatonin signaling activates phospholipase C (PLC) that induces the production of inositol triphosphate (IP3). IP3 is able to mobilize intracellular Ca+2 from the endoplasmic reticulum (ER) leading to a rise in cytosolic Ca+2 concentration through open ER-localized IP3-sensitive Ca2+ channels. In addition, melatonin signaling activates adenylyl cyclase (AC) producing an increase in cAMP level and further activation of protein kinase A (PKA). PKA is involved in controlling the balance of gene expression in the nucleus (N). Melatonin is also implicated in the activation of gene transcription for the UPS machinery. Protein kinase 7 (PfPK7) seems to participate in the melatonin signaling pathway since parasites with a knockout of this kinase are not responsive to melatonin treatment, as measured by parasite intraerythrocytic stage distribution and activation of a subset of genes involved in UPS. However, how PfPK7 plays its role in melatonin signaling has not been identified yet. Most recently, it has been shown that melatonin increases ubiquination of PfNF-YB transcription factor and increases its expression.

So far few transcription factors have been described in P. falciparum and its potential signaling pathway and its role in the control of Plasmodium replication and development is poorly understood. The transcription factor NF-YB binds to the CCAAT motif of several gene promoters during the G2/M phase of the cell cycle and is thought to be present in several biological models. In Plasmodium, PfNF-YB is the CCAAT-box-binding subunit B of the NF-Y complex and is conserved from yeast to humans.

We recently reported that the controls that melatonin exerts upon the Plasmodium cell cycle involve the regulation of the transcription factor PfNF-YB expression as well as modulation of a subset of genes of the ubiquitin proteasome system. PfNF-YB (PF11_0477) expression is activated by melatonin signaling at the trophozoite stage of P. falciparum. In addition, the hormone affected the post-translational modification of PfNF-YB by ubiquitination [2].

26 Summary Report 2009-2013 | INBEQMeDI

Host-Plasmodium signaling

MLT

MLT (?)

AC (?)

(?) (?)

P

cAM

Ub B

F-Y

PfN

EM

PVM

Protease

(?)

IP3

7

PfPK

IP3 Ca2+

PKA

PM

IP3 N

e som

tea

Pro

RBC

PLC

PV

Cell cycle Kinases on TF expressi

IP3R

ER

Figure 1. Melatonin molecular signaling pathways in Plasmodium.

On top of that, our group reported the molecular identification of four serpentine receptors in malaria parasites and propose that these proteins, which are currently annotated as hypothetical, constitute novel members of the largest class of membrane receptors widespread in living organisms, the class of G protein-coupled receptors (GPCRs), more generally called serpentine or heptahelical receptors. Although serpentine receptors are present in such evolutionary

distant organisms as bacteria, fungi, plants and metazoans, up to now their presence in malaria parasites has not been demonstrated.

Finally, in collaboration with members of INBEQMeDI from the USP-SĂŁo Carlos crystallography group we have investigated the role of the enzyme heme oxygenase and its products on parasite heme metabolism. Our findings points to biliverdin as a new signaling molecule involved in the control of the P. falciparum cell cycle.

References [1] Alves E, Bartlett PJ, Garcia CR, Thomas AP (2011) J Biol Chem 286:5905-5912.

[2] Lima WR, Moraes M, Alves E, Azevedo MF, Passos DO, Garcia CRS (2013) J Pineal Res 54:145-153.

Research Reports 27

12 RESEARCH REPORTS

Structural studies of proteins from Trypanosoma cruzi involved in host invasion Renato A. Mortara1, Diana Bahia2, Claudio V. da Silva3; Claudia E. Munte4, Valeria Terkiel5, Eduardo Horjales4 Paulista School of Medicine, Federal University of São Paulo, São Paulo, SP, Brazil Department of General Biology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 3 Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, MG, Brazil 4 Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil 5 Institute for Research in Biotechnology, National University of General San Martín, San Martín, Argentina 1 2

Cell invasion by the different infective forms of Trypanosoma cruzi, the causative parasite of Chagas disease, involves several mechanisms which culminate in entry of T. cruzi into host cells. Numerous studies have been performed in order to identify surface and secreted proteins related to cell invasion, mostly from metacyclic and cultured trypomastigote forms. However, little attention has been given to the extracellular amastigote, also capable of invading mammalian cells via an alternative invasive mechanism. The molecules involved in this alternative process are still poorly known or studied and may be significant to T. cruzi survival under the highly cytotoxic conditions afforded by the host cells. In this context, a new protein named P21 has been recently identified. Its participation in the invasion mechanism cannot yet be explained at the molecular level, but our experiments suggest that P21 interacts with the CXCR4 chemokine receptor [1], activating phagocytosis in macrophages and promoting actin polymerization in mammalian cells.

As the first step towards P21 NMR structural studies, we developed a purification/ refolding protocol and performed the 1H, 13C and 15N sequential assignment using triple resonance NMR spectra (Bruker 600 MHz). The secondary structure prediction, based on chemical shifts (TALOS+), showed five α-helical regions and an unstructured N-terminal region (residues 1-42) (Figure 1). We also identified several parts of the protein which have multiple conformations, indicating great structural flexibility (Figure 2). Future structural and interaction 1H, 15N HSQC NMR experiments with

Figure 1. Secondary structure prediction based on CB, CA, CO, HN and N chemical shifts (TALOS+ ), showing five α-helix regions and an unstructured N-terminal region.

28 Summary Report 2009-2013 | INBEQMeDI

Structural studies of proteins from Trypanosoma cruzi involved in host invasion

Figure 2. Assigned 1H, 15N HSQC Spectrum of P21 from T. cruzi. Residues in an alternative conformation are indicated with an asterisk (*).

the N-terminal peptide of CXCR4, essential for protein activation, will be made, and the results may lead to a new therapy based on peptides, changing the current worldwide scenario of Chagas disease treatment.

Both parasitic and host cytoskeleton modifications essential to cell invasion are known to be triggered by different kinases, but little is known on the particularities and detailed function of the parasitic enzymes. We have determined de catalytic activity of mevalonate kinase from T. cruzi, an enzyme that is secreted by the parasite, a fact that is unique for this type of enzyme and we are developing crystallization

trials. TOR kinases are large enzymes (over 2500 amino acids) which are inhibited by rapamycin, thus inhibiting parasite cell growth. The structure of a large fragment of the human enzyme has recently been determined and we have cloned fragments of the three homologous enzymes present in T. cruzi (TOR1, TOR2 and TOR-like).

In a recent collaboration with Valeria Terkiel (U.San Martin, Argentina), we are studying the GPI anchored family of membrane proteins TASV also from T. cruzi.

They were selected for been overexpressed during host invasion in trypomastigote forms.

References [1] Rodrigues AA, Clemente TM, Santos MA, Machado FC, Gomes RGB, Moreira HHT, Cruz MC, BrĂ­gido PC, Santos PCF, Martins FA, Bahia D, Maricato JT, Janini LMR, Reboredo EH, Mortara RA, Silva CV (2012) PLoS One 7:e51384.

Research Reports 29

13

Septins from S. mansoni

research reports

Ana E. Zeraik1, Humberto D’M. Pereira1, Vitold E. Galkin2, Gabriel Rinaldi3,4, Yuri V. Santos1, Victoria H. Mann4, Anastas Popratiloff4 , José Brandão-Neto5, Richard C. Garratt1, Paul J. Brindley4, Ana P. U. Araújo1, Ricardo DeMarco1 Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, United States of America 3 Departmento de Genética, Universidad de la República (UDELAR), Montevideo, Uruguay 4 Department of Microbiology, Immunology & Tropical Medicine, George Washington University Medical Center, Washington, DC, United States of America 5 Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, England.

1 2

Septins are a family of eukaryote GTP binding proteins conserved from yeast to humans. Septins participate in diverse cellular functions including cytokinesis, organization of actin networks, cell polarity, vesicle trafficking and many others.

The septins assemble into hetero-oligomers to form filaments and rings.

Four septins of Schistosoma mansoni were found. These orthologues are more closely related to the SEPT5, SEPT10 and SEPT7 septins of humans, and hence we have termed the novel schistosome septins SmSEPT5, SmSEPT10, SmSEPT7.1 and SmSEPT7.2. Immunolocalization analyses undertaken with antibodies specific for SmSEPT5 and SmSEPT10 revealed a ubiquitous distribution of septins in the schistosomulum [1]. Septin and actin were co-localized in the muscular fibers of the sporocyst stage. Ciliated epidermal plates of the miracidium were found to be rich in septins. In the cercaria, septins colocalize with the protonephridial tubule system (Figure 1). Intriguingly, septins also localize at elevated levels in germ cells within the miracidium and sporocyst. This data indicates that septins are involved in several different functions in a large variety of tissues.

Figure 1. Protonephridial canals of the schistosome cercaria are septin rich structures. Optical section of cercariae labeled with phalloidin (green) and anti-SmSEPT10 (red). Arrow pointing the flame cells connected to the protonephridial canals. Scale bar, 20 µm.

30 Summary Report 2009-2013 | INBEQMeDI

Septins from S. mansoni

α6

α2 α3 β5 α4

β4 β1

α5’

β3

β6 α5 β7

α1

β8

β2

β10 β9 Figure 2. Tridimensional structure of the GTPase domain of Schistosoma mansoni Septin-10.

Crystal structures of a Schistosoma mansoni septin (SmSEPT10) GTPase domain bound either to GDP or GTP and of good resolution (~2 Å) were obtained [2] (Figure 2). These structures provided a reliable reference model for future septin structural studies since they represent the most detailed structures obtained so far for this class of proteins. For the first time, we were able to describe a mechanism of response of this class of proteins upon the binding of a magnesium ion coordinated with GTP. Understanding of this new mechanism opens new possibilities for the study of the dynamics of septins filaments. The description of a S. mansoni septin structure may help to the development of new specific drugs that may interfere with their function.

With the aim to investigate the function of septins in schistosomes, a recombinant heterofilament formed by

three of the schistosome septins were incubated with forchlorfenuron (FCF), which is a compound that exhibits reversible inhibition of septins dynamics in yeasts and mammalian cells [3]. It was observed that filaments from schistosome septins treated with this compound in vitro tended to display a more rapid polymerization of the monomers. Remarkably, treatment of several life cycle stages of schistosomes with FCF resulted in a complete paralysis and the effects were fully reversed after FCF was removed. We hypothesize that a direct effect on muscle contraction due to septin stabilization might be responsible for the reversible paralysis, since septins were localized by us in the muscles fibers of schistosomes by immunolocalization. This class of compounds may represent a promising new starting point for the development of therapeutics against schistosomiasis.

References [1] Zeraik AE, Rinaldi G, Mann VH, Popratiloff A, Araujo APU, DeMarco R, Brindley PJ (2013) Submitted.

[2] Zeraik AE, Pereira HDM, Santos YV, Brandão-Neto J, Garratt RC, Araújo APU, DeMarco R (2013) Submitted.

[3] Zeraik AE, Galkin VE, Rinaldi G, Garratt RC, Mann VH, Araujo APU, DeMarco R, Brindley PJ (2013) Submitted.

Research Reports 31

14 RESEARCH REPORTS

Identification of compounds that inhibits bacterial diguanylate cyclases involved in biofilm formation Helton J. Wiggers1, Éverton E. D. Silva1, Edson Crusca1, Marcos V. A. S. Navarro1 Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil

1

Recently, much emphasis has been given to the notion that bacterial cultures not only exist as a suspension of single cells, but also appear and function as multicellular communities, the so-called biofilms. In particular, biofilm formation plays a crucial role in nosocomial infections, pathologic colonization of organs, implants and catheters and infection by opportunistic pathogens. Such infections often escape from traditional treatments by presenting greater resistance to antibiotics, mainly due to the high concentration of bacteria embedded in a protective polymer matrix.

Thus, pharmacological interference in biofilm formation may represent a new potent and attractive strategy for the treatment of infectious diseases.

Occupying a central role in the process of biofilm formation, there is a messenger molecule found only in the microbial world, guanosine monophosphate (3’-5-)-cyclic dimeric (c-di-GMP), which controls secretion, adhesion, cell motility and increased cytotoxicity. Cyclic di-GMP signaling network is the most complex secondary signaling system discovered in bacteria and is especially prominent in Proteobacteria, including the major human pathogens P. aeruginosa, Salmonella typhimurium, E. coli and Vibrio cholerea, which possess numerous c-di-GMP-metabolizing proteins. The biosynthesis of c-di-GMP is controlled by oppose activities of diguanilate cyclases (DGC) harboring the GGDEF domain, which synthetizes c-di-GMP from two GTP molecules, and phosphodiesterases (PDE) (EAL and HD-GYP domains) that degrades c-di-GMP to pGpG or GMP. Given that depletion of DGC activity in many bacterial species completely abolishes their biofilm formation capacity, DGCs have emerged as potential targets for the development of novel anti-biofilm agents.

Aiming the identification of DGC inhibitors targeting the active site (A-site) of GGDEF domains (Figure 1), a virtual screening campaign was applied on the DrugBank database. Such database contains approximately 7000 entries that are used or are being tested in a wide variety of diseases. Molecules in this database have optimal pharmacokinetic properties and toxicological profiles, and the

a

b

Figure 1. Structure of C. crescentus DGC PleD (PDB ID 2VN0). A) Quaternary structure of PelD showing one of the protomers of the dimer as a cartoon and colored red and green, REC and GGDEF domains respectively.

The I-sites containing c-di-GMP and A-sites containing GTP-α-S are highlighted. B) Close up of the GGDEF A-site in complex with GTP-α-S, major conserved interactions are presented.

32 Summary Report 2009-2013 | INBEQMeDI

Identification of compounds that inhibits bacterial diguanylate cyclases involved in biofilm formation

a

b

c

Figure 2. Experimental results. A) A typical ESI mass spectrum obtained in positive ion mode for an aqueous solution containing WspRGGDEF domain 500 nM (control) and increasing concentrations of sulfadiazine. B) Typical phase contrast optical microscopy image presenting cell aggregates (top left) after 0.1 mM IPTG induction. The other microscopy images clearly show the absence of aggregates when 50 µM inhibitors are added to the cultures. Bottom graphs represents normalized cell aggregate area covered for each compound tested against YdeH (left) and WspR (right). C) Table summarizing the results for IC50 for YdeH and WspR enzymes and Kdapp values for WspRGGDEF domain. * Substrate of WspR enzyme. The WspRGGDEF domain has no enzyme activity. ND – not determined

repositioning of existing drugs for new indications can potentially avoid the expensive costs associated with early-stage testing of the hit compounds. An integrated in silico strategy was applied, employing ligand- and target-based virtual screening methods. This approach resulted in the selection of ten compounds, which were tested for enzyme inhibition against E. coli YdeH and P. aeruginosa WspR, binding and specificity to the A-site of WspR GGDEF domain and cellular anti-biofilm assays.

to measure apparent dissociation constants. (Figure 2C). Furthermore, competition experiments with GTP showed that the inhibitors specifically target the A-site of the GGDEF domain. It is interesting to note that two compounds mined in our virtual screening campaign (Sulfasalazine and Sulfadiazine) and a biofilm inhibitor previously reported (Sulfathiazole) share a common benzenesulfonamide moiety, indicating that this group could be important for molecular recognition.

A fluorescent method (PiPerTM Pyrophosphate Assay Kit) that detects inorganic pyrophosphate released during the conversion of GTP to c-di-GMP by the enzymes was employed to evaluate the inhibition capacity and IC50 of the selected compounds. Several of them exhibited inhibition properties to different degrees and dose-response curves were obtained for four compounds, with IC50 values fluctuating in the high micromolar range (Figure 2C). Given the intrinsic low enzymatic efficiency of DGCs and methodological difficulties of using the PiPer assay, we used electron spray ionization (ESI) mass spectrometry to direct measure the interaction of the inhibitors with the enzyme (Figure 1A). By using a construct of the isolated GGDEF domain of WspR, we observed interaction of compounds undetectable in the enzymatic assays, such as iodipamine and sulfadiazine, and were able

Finally, we developed an assay to access the antibiofilm activity of the identified DGC inhibitors. We used liquid cultures of E. coli overexpressing either P. aeruginosa WspR or E. coli YdeH and quantified cell aggregates by Contrast Phase Optical Microscopy. All compounds were able to inhibit cell aggregation at a concentration of 50 µM (Figure 2B).

In summary, the approach applied in this study lead to the discovery of DGC inhibitors capable to interfere in biofilm formation. Once the selected compounds are FDA-approved drugs, their scaffolds lie within a privileged chemical-biology space. Therefore, optimization of the compounds identified in this study could lead to more potent DGC inhibitors already harboring drug-like features and potentially resulting in the development of novel anti-biofilm agents.

Research Reports 33

INNOVATION

INBEQMeDI is aware of its responsibility to attempt to innovate and transfer technology to the productive sector wherever possible. We understand innovation to be any effective mechanism by which new technologies are made available to a wider public. Our research team is therefore always on the lookout for such possibilities.

The following are examples of the ways in which we attempt to turn this into reality.

PK/DB – Database for Pharmacokinetic Properties PK/DB is a database of pharmacokinetic properties of compound designed as a tool to aid in pharmacokinetic studies and in silico ADME (Absorption, Distribution, Metabolism, and Excretion) prediction. The database is freely available on the internet for ready access by the medicinal chemistry community worldwide and has received more than 55,000 visits so far.

NuBBEdb – a Natural Products database NuBBEdb is the result of a collaborative initiative between INBEQMeDI and INOFAR. It is a database of Brazilian natural products which is currently based on a collection of compounds from UNESP – Araraquara. It has the potential to be expanded and thereby provide a valuable resource for medicinal chemists worldwide. It has attracted the interest of the curators of the Zinc database in the USA.

LAM Educational LAM Educacional is a spinoff company which commercializes products derived initially from the activities of the CBME/CEPID project which have been continued within the auspices of INBEQMeDI. LAM Educacional specializes in the production of educational tools such as model building kits, software etc.

Innovation 34

Sm14 Patent: Synthetic gene for expressing Sm14 in Pichia pastoris, methods for producing and purifying Sm14 and the use thereof as a vaccine and diagnostic medium”.

A patent for the use of peptides derived from the Sm14 antigen for vaccination against helminthes was granted in 2012. This is a joint invention involving researchers from CBME (now INBEQMeDI) and the Oswaldo Cruz Institute (RJ). The use of this technology is currently being exploited by the Brazilian company Ourofino.

Malaria Patent: “Pharmaceutical Composition, Drug Screening Method and Method for Treating Malaria”.

This invention relates to compounds that bind to serpentine receptors present in Plasmodium as well as to means for screening promising potential drugs and to a method for treating malaria.

Pharmaceutical formulations and applications Patent: “Process for the preparation and pharmaceutical formulations for 4-quinolinones and quinolines and use thereof”.

This invention describes the pharmaceutical applications of 4-quinolinones and quinoline derivatives for the treatment of diseases such as inflammatory and autoimmune diseases, as well as their use as anti-coagulants, antivenins, analgesics and anti-thrombotics.

Insecticides Patent: “Processo de Preparação de Complexos Metálicos de Hesperidina e esperitina, Complexos Metálicos e Composições Inseticidas para o Controle de Insetos pragas Urbanos, da Agricultura e da Silvicultura”.

Applications of novel compounds in the control of insect plagues.

Innovation 35

Collaborative project with GlaxoSmithKline via “Trust in Science” One of only eight laboratories in Brazil chosen to participate in GSK’s “Trust in Science” program (and the only one to involve organic synthesis), our effort aim towards the identification of novel compounds for the treatment of Chagas disease.

A joint patent application with GSK has been filed - “Compostos de quinoxalina e composições farmacêuticas contendo os mesmos”.

Protein folder “Protein Folder” is a teaching aid developed under the auspices of the CBME/CEPID. Its use and application in schools and workshops continues as part of the activities of INBEQMeDI. A public call for proposals has been made and a company from the UK has shown great interest in acquiring the technology. It is expected to be commercialized in the near future.

Novel enzyme inhibitors Patent: “Compostos, uso, composição farmacêutica inibidora de redutase em microorganismos, ligante de InhA, e, método para obtenção de ligantes de InhA a partir de derivados de acetamida, ureia, fenol e carboxamida”.

This innovation refers to the development of a pharmacophore model for ligands of 2-trans-enoyl-ACP (CoA) reductase from M. tuberculosis (MtInhA), as well as a pharmacophore-based virtual screening approach. The strategy resulted in the identification of four compounds from different chemical classes with in vitro inhibitory activity against MtInhA in the micromolar range.

Diagnosis of leishmaniasis Patent: “Recombinant e-ntpdases, use for producing a diagnostic kit for detecting antibodies in various types of leishmaniasis caused by species of the Leishmania genus”.

A prototype for an ELISA-based diagnostic kit for Leishmaniosis in dogs has been developed based on the NTPDase-2 antigen. Early diagnosis is critical for the control of the infection and if successful its socio-economic impact is undeniable. A patent has been filed protecting the invention.

36 Summary Report 2009-2013 | INBEQMeDI

WHO – World Health Organization From 2009 to 2012 INBEQMeDI was part of the World Reference Center for Medicinal Chemistry in Chagas Disease. As such part of the only research center of the World Health Organization (WHO) in Latin America. Besides other research centers throughout the world, this initiative involved Big Pharm companies such as Pfizer and Merck and resulted in the identification of dozens of new molecular hits. The project will continue under the auspices of the DNDi (Drugs for Neglected Diseases initiative) who will coordinate subsequent developmental stages.

Industrial collaboration INBEQMeDI scientists interact with the pharmaceutical and biotech industries via different mechanisms. These include collaborative projects which are co-financed by industry as well as consultancy, in which our expertise is used for advancing technology.

Innovation 37

OUTREACH

Since its inception, the outreach division of INBEQMeDI has concerned itself with the development of novel teaching aids and in devising educational strategies for increasing awareness about neglected infectious diseases and the need for drugs for their treatment. Our team attempts to innovate in the production of educational software, games, science clubs and teacher training programs. In the following pages we hope to give an overview of the progress which has been made over the last four and a half years.

An Interactive Science Museum has been running for many years at the headquarters of the Outreach division of INBEQMeD. It has permanent displays and interactive exhibits.

Within this building INBEQMeDI is also able to offer courses for both teachers and students alike.

INBEQMeDI is also active outside the State of São Paulo. Teacher training courses have been run in the cities of Ponta Grossa in the State of Paraná and Viçosa in Minas Gerais. These courses counted on the invaluable help of local INBEQMeDI scientists for their successful implementation and execution.

A teacher training course during the winter holidays is run for local science teachers. The course runs for a week and addresses modern techniques in molecular biology including heterologous expression of proteins.

Outreach 38

In 2009 and 2010 INBEQMeDI ran courses for ~600 pedagogical coordinators from 95 different regions of the State of São Paulo.

The event, held in the small town of Serra Negra, was widely considered a success and inspired several further activities subsequently organized by INBEQMeDI’s outreach division

The above initiative led to an agreement with the State Secretariat for Education (SEE) of the State of São Paulo, by which a series of teacher training courses were run with the aim of updating science teachers in the fields of molecular biology, biotechnology and drug design. Among others places, these courses have been run in São Carlos, Ribeirão Preto, Catanduva, São João da Boa Vista, José Bonifácio, Tupã etc.

Teachers who have participated in our training program subsequently pass on their newly acquired expertise to their students in the classroom.

When requested by local school teachers, INBEQMeDI runs special workshops for students from São Carlos and surrounding areas. Teaching aids, such as molecular models and interactive games, which have been developed by INBEQMeDI over the years are used to motivate the students and stimulate their sense of criticism.

Outreach 39

A Science Club is run for interested students from local public schools. The club meets weekly to perform practical experiments on many scientific subjects in groups of 20-30 students. INBEQMeDI also organizes field trips for the students and encourages them to participate in outreach events such as National Science and Technology week.

An interactive educational resource on Chagas´ disease has been developed which uses different computational tools either in terms of hardware or software. The resource contains 13 videos that range from 30 seconds to 4 minutes, all containing useful information about Chagas disease.

A similar resource has been developed for Malaria. An opening scenario shows a typical wetland with native residents, health workers and tourists arriving by boat to the site. On clicking on these elements, animations, videos and audio clips are activated.

These deal with topics such as the symptoms of the disease, the parasite life cycle, prevention, diagnosis and treatment.

Wordfinder: A web application which is integrated within social media such as Facebook, deals with tropical infectious diseases such as Chagas´ disease, Schistosomiasis and Leishmaniosis. The game is divided into two levels of difficulty and keeps track of both the time spent by the user and his/her success rate. Its flexible architecture allows the ready introduction of new content.

40 Summary Report 2009-2013 | INBEQMeDI

Other computational resources include a Jmol application for displaying the 3D structure of cruzain (a potential drug target for Chagas’ disease) and a web application for crossword puzzles of different levels of difficulty.

Explanatory videos about neglected diseases have been made available via YouTube.

A board game of Protein Synthesis (also available as an electronic version). Each player is required to synthesize a given protein using the eukaryotic intracellular machinery. The game aims to aid in assimilating and clarify fundamental concepts in molecular and cell biology in the mind of the student.

The book “Química Verde: Fundamentos e Aplicações” was published in 2009 with a view to increasing awareness of the theme with in Brazil and neighbouring countries.

In 2010 it won the Jabuti prize (bronze) and was subsequently translated into Spanish and re-launched during the 4th International Conference on Green Chemistry.

Under construction: A Touch Screen application about infectious diseases and a corresponding web application for reinforcing students’ knowledge are currently under development.

A Magic Book employing enhanced reality techniques and an electronic game will be added in the near future.

Outreach 41

FACTS AND FIGURES

The way we interact

The way we interact

instituto de biociências instituto

CRYSTALLOGRAPHY CRYSTALLOGRAPHY BIOPHYSICS BIOPHYSICS

INBEQMeDI interacts with other INCTs in a synergistic manner

Facts and Figures 42

Research Team

INBEQMeDI has a good balance of both experienced and young researchers as demonstrated by the distribution of CNPq research scholarships.

MSc PhD

10

11

10

11

4

5

5

7

12

14

Once established INBEQMeDI has maintained the level of theses and dissertations finalized per year.

2009

2010

2011

2012

2013*

25

28

30

67

* From January to June.

Undergraduate students

MSc

PhD

INBEQMeDI relies upon the dedication of a large number of undergraduate and post-graduate students, as well as post-docs. The impact of INBEQMeDI is therefore expected to continue for many years to come as more theses and dissertations are finalized.

Post Doc

2013

Facts and Figures 43

Publications

INBEQMeDI’s publications are roughly equally distributed amongst its main fields of interest.

INBEQMeDI productivity has been roughly stable over the course of its existence and the majority of its publications appear in international journals with recognized impact factors.

Several INBEQMeDI papers were chosen to illustrate the front cover of both international and brazilian journals

44 Summary Report 2009-2013 | INBEQMeDI

Selected papers

Santos RF, Pôssa MAS, Bastos MS, Guedes PMM, Almeida MR, DeMarco R, Verjovski-Almeida S, Bahia MT, Fietto JLR (2009) Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence. PLoS Neglected Tropical Diseases (Online) 3:e387.

DeMarco R, Mathieson W, Manuel SJ, Dillon GP, Curwen RS, Ashton PD, Ivens AC, Berriman M, Verjovski-Almeida S, Wilson RA (2010) Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts. Genome Research 20:1112-1121.

Cruz LN, Alves E, Leal MT, Juliano MA, Rosenthal PJ, Juliano L, Garcia CR (2010) FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii. International Journal for Parasitology 41:363-372.

Alves E, Bartlett PJ, Garcia CR, Thomas AP (2011) Melatonin and IP3-induced Ca2+ release from intracellular stores in the malaria parasite Plasmodium falciparum within infected red blood cells. Journal of Biological Chemistry 13:1-11.

Chiaradia LD, Martins PG, Cordeiro MN, Guido RV, Ecco G, Andricopulo AD, Yunes RA, Vernal J, Nunes RJ, Terenzi H (2012) Synthesis, Biological Evaluation, and Molecular Modeling of Chalcone Derivatives as Potent Inhibitors of Mycobacterium tuberculosis Protein Tyrosine Phosphatases (PtpA and PtpB). Journal of Medicinal Chemistry 55:390-402.

Gazarini ML, Beraldo FH, Almeida FM, Bootman M, Da Silva AM, Garcia CR (2011) Melatonin triggers PKA activation in the rodent malaria parasite Plasmodium chabaudi. Journal of Pineal Research 50:64-70.

Koyama FC, Ribeiro RY, Garcia JL, Azevedo MF, Chakrabarti D, Garcia CR (2012) Ubiquitin Proteasome System and the atypical kinase PfPK7 are involved in melatonin signaling in Plasmodium falciparum. Journal of Pineal Research 53:147-153.

Garcia CR (2009) Molecular and cellular approaches to understanding pathogen-host interactions in neglected diseases. Current Opinion in Microbiology 12:392-393.

DeMarco R, Verjovski-Almeida S (2009) Schistosomes - proteomics studies for potential novel vaccines and drug targets. Drug Discovery Today 14:472-478.

Hauk P, Guzzo CR, Roman Ramos H, Ho PL, Farah CS (2009) Structure and calcium-binding activity of LipL32, the major surface antigen of pathogenic Leptospira sp.. Journal of Molecular Biology 390:722-736.

Guido RV, Oliva G (2009) Structure-based drug discovery for tropical diseases. Current Topics in Medicinal Chemistry 9:824-843.

Pereira HM, Rezende MM, Castilho MS, Oliva G, Garratt RC (2010) Adenosine binding to low-molecular-weight purine nucleoside phosphorylase: the structural basis for recognition based on its complex with the enzyme from Schistosoma mansoni. Acta Crystallographica D Biological Crystallography 66:73-79.

Boscardin SB, Torrecilhas AC, Manarin R, Revelli S, Rey EG, Tonelli RR, Silber AM (2010) Chaga’s disease: an update on immune mechanisms and therapeutic strategies. Journal of Cellular and Molecular Medicine 14:1373-1384.

Postigo MP, Guido RV, Oliva G, Castilho MS, da R Pitta I, de Albuquerque JF, Andricopulo AD (2010) Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening. Journal of Chemical Information and Modeling 50:1693-1705.

Facts and Figures 45

internationalization

International collaboration

Internationalization 46

Our talks worldwide Our courses worldwide Foreign students visiting us Our students travelling abroad Our international collaborations

Internationalization 47

Talks in foreign institutions and conferences

36

10 2009

15 2010

Courses and workshops outside of Brazil “Química verde, base de la tecnología sustentable”. XVIII Jornadas de Jóvenes Investigadores (19th – 21th Oct. 2010). Asociación de Universidades Grupo Montevideo (AUGM). Universidad Nacional del Litoral. Santa Fe, Argentina

“Introduction to Drug Design Computer Programs I”. Workshop on Drug Design and Neglected Tropical Diseases (5th – 16th Nov. 2012).

United Nations University (UNU-BIOLAC) Universidad de Los Andes Merida, Venezuela

“Biophysical Techniques for the Study of Intermolecular Interactions in Structural Biology”. Fundación Instituto Leloir and Facultad de Farmacia y Bioquímica (19th – 27th Nov. 2012).

Universidad de Buenos Aires. Buenos Aires, Argentina

“Molecular Genetic Analyses of Leishmania Biology” (Fall semester 2012). Yale School of Public Health and the International Center for Medical Training and Research (CIDEIM). Internet-based post graduated course (Colombia, Argentina, Ecuador, Guatemala and Honduras)

20 08 2011

2012

2013*

* From January to June.

“Drug Target Selection from a Structural Perspective”. Workshop on Drug Design and Neglected Tropical Diseases (5th – 16th Nov. 2012).

United Nations University (UNU-BIOLAC) Universidad de Los Andes Merida, Venezuela “Introduction to Drug Design Computer Programs II”. Workshop on Drug Design and Neglected Tropical Diseases (5th – 16th Nov. 2012).

United Nations University (UNU-BIOLAC) Universidad de Los Andes Merida, Venezuela “Aplicaciones Biológicas de la Resonancia de Espín Electrónico: Teoría y Práctica” (29th – 30th Aug. 2012). Facultad de Medicina. Universidad de La República. Montevideo, Uruguay “ P l a nt a s M e d i c i n a l e s y M e d i c a m e nto s Fitoterápicos, Situacion Actual en el Mercosur” (5th – 9th Mar. 2012). Universidad de La República.

Montevideo, Uruguay “ P l a nt a s M e d i c i n a l e s y M e d i c a m e nto s Fitoterápicos, Situacion Actual en el Mercosur” (27th Feb. – 9th Mar. 2012). Universidad Nacional de Asunción. Asunción, Paraguay

Cutting edge research facilities

INBEQMeDI uses frontier research facilities worldwide. Diamond is a 3rd generation Synchrotron Radiation facility providing highly collimated high-brightness X-ray beams for crystallography. Via international collaboration INBEQMeDI also has access to high resolution NMR spectrometry (900 MHz) and high throughput protein expression facilities housed respectively at the technical University of Munich and at the Oxford Protein Production Facility – UK.

48 Summary Report 2009-2013 | INBEQMeDI

Protein folder courses

INBEQMeDI teaching tools are used worldwide Students in Ica, Peru, teach their colleagues using INBEQMeDI DNA Models.

SGC (the Structural Genomic Consortium, Oxford, UK) and the Wellcome Trust (the largest private research foundation in Europe) have both used INBEQMeDI model building kits in their outreach programs

Internationalization 49

Organization of international events

“13th Brazilian Meeting on Organic Synthesis (BMOS)” 31st August – 4th November 2009 São Pedro, SP, Brazil

“4 th International IUPAC Conference on Green Chemistry (ICGC)” 28th – 31th August 2012 Foz do Iguaçu, PR, Brazil

“2 nd Brazilian Conference on Natural Products (BCNP)” and “XXVIII Annual Meeting on Micromolecular Evolution, Systematics and Ecology (RESEM)” 9th – 12th November 2009 São Pedro, SP, Brazil

“XVIII International Network of Protein Engineering Centers (INPEC) Meeting” 25th – 28th October 2009 Ubatuba, SP, Brazil

50 Summary Report 2009-2013 | INBEQMeDI

“3rd International Workshop on Spectroscopy for Biology (IWSB)” 18th – 22th October 2010 - Maresias, SP, Brazil

Brazilian Purine Club

3rd Meeting

Purinergic Signaling: Biological and Therapeutic Implications September 22-24, 2012

Ouro Preto, Minas Gerais, Brazil

Confirmed Speakers: Francesco Di Virgilio - Italy Pedro Muanis Persechini - Brazil György Haskó - USA Fritz Markwardt – Germany Detlev Boison - USA Lisiane Porciúncula - Brazil Rodrigo Cunha - Portugal Tiana Tasca - Brazil Shucui Jiang – USA Mark J. Wall – UK Vickram Ramkumar - USA

Roberto Paes de Carvalho – Brazil Herbert Zimmermann - Germany Jean Sévigny - Canada Diogo Onofre G. de Souza – Brazil Henning Ulrich – Brazil Maria Paula Faillace – Argentine Eliana Scemes – USA Juan Pablo Huidobro Toro – Chile Imogen Coe – Canada Miguel Diaz-Hernandez - Spain David Ojcius - USA

Join us at Ouro Preto, an UNESCO World Heritage Site! www.purinas2012.com.br

Support:

Organizing Committee: Ana Lúcia Marques Ventura Ana Maria Oliveira Battastini Henning Ulrich Jose Roberto Meyer Fernandes Juliana Lopes Rangel Fietto Luis Carlos Crocco Afonso Robson Coutinho Silva

Secretariat: Visa Congressos e Eventos Contact: Marta Staico or Deborah Zandona E-mail: martaeventos@visaturismo.com.br Phone: +55-31-3291-9819 / +55-31-7815-1526 Fax: +55-31-3224-3066

“3rd Meeting of the Brazilian Purine Club”

“USP Conference: Modern Topics in Magnetic Resonance”

22 – 24 September 2012

15th – 17th May 2013

Ouro Preto, MG, Brazil

São Paulo, SP, Brazil

th

th

“International Meeting on Cell Biology of Pathogens” 7th – 10th August 2011 Guarujá, SP, Brazil

Internationalization 51

ASSOCIATED LABORATORIES

IFSC - USP – Cristallography • Structural and Molecular Biology • Protein expression and Purification • Crystallography and Homology Modelling • Theoretical Medicinal Chemistry • In vitro and In vivo assays • Enzyme assays • Pathogenic micro-organisms

IFSC - USP – Biophysics • Structural and Molecular Biology • NMR and EPR spectroscopy • Heterologous protein expression and purification • Spectroscopic and biophysical characterization of proteins • Protein crystallography • Mass spectrometry

DQ - UFSCar • Synthetic chemistry • Combinatorial chemistry • Natural Products Chemistry • Mass spectrometry • NMR spectroscopy • Chromatography • Molecular and Structural Biology

FMRP - USP • Parasitology of Leishmania • Gene expression • Molecular and cell biology • Cell culture and genetic manipulation of Leishmania

Associated Laboratories 52

FCFRP - USP • Natural product chemistry • Symbiotic organisms • Enzyme assays • Microbial cell culture • Chromatography

IQ - USP • Structural and Molecular Biology • Target molecules from Leptospira • Protein Crystallography • NMR spectroscopy • Signalling pathways

ICB - USP • Trypanosome metabolism • Life cycle maintenance in trypanosomes • Cell culture • Molecular and cell biology

IB - USP • Molecular and Cell Biology • Plasmodium falciparum • Signal transduction • Parasite-host interactions • Confocal microscopy

Associated Laboratories 53

DBB - UFV • Molecular and cell biology • Biochemistry • Protozoan parasites • Target identification • Enzyme assays

DQ - UEPG • Protein expression and purification • Protein Crystallization • Molecular Dynamics Simulation • Bioinformatics

“90 % of the equipment we have in the laboratory was financed by INBEQMeDI. This has allowed us to over-express proteins for crystallization purposes, thereby increasing our perspectives for effectively interacting with other members of the team” (Jorge Iulek)

“Our participation in the INBEQMeDI has allowed us to aquire an automated protein purification system and to provide regular supplies of consumables necessary to inprove the quality of the research we undertake. Furthermode we have strengthened both our research collaborations and student training” (Juliana Fietto)

54 Summary Report 2009-2013 | INBEQMeDI

E-mails Institute of Physics of São Carlos - USP Crystallography Glaucius Oliva - oliva@ifsc.usp.br Richard C. Garratt - richard@ifsc.usp.br Otavio H. Thiemann - theimann@ifsc.usp.br Adriano D. Andricopulo - aandrico@ifsc.usp.br Eduardo Horjales Reboredo - horjales@ifsc.usp.br Ilana Lopes B.C. Camargo - ilanacamargo@ifsc.usp.br Rafael V.C. Guido - rvcguido@ifsc.usp.br Biophysics Antonio José da Costa Filho - ajcosta@ifsc.usp.br Claudia Elisabeth Munte - claudia.munte@ifsc.usp.br Ricardo de Marco - rdemarco@ifsc.usp.br Leila M. Beltramini - leila@ifsc.usp.br Ana Paula U. Araújo - anapaula@ifsc.usp.br Marcos Vicente A.S. Navarro - mvasnavarro@ifsc.usp.br Nelma R.S. Bossolan - nelma@ifsc.usp.br Department of Chemistry - UFSCar Arlene Gonçalves Correa - agcorrea@ufscar.br Dulce Helena F. de Souza - dulce@ufscar.br Paulo Cezar Vieira - paulo@dq.ufscar.br Institute of Chemistry - USP Shaker Chuck Farah - chsfarah@iq.usp.br Institute of Biosciences - USP Célia R. da Silva Garcia - cgarcia@usp.br Institute of Biomedical Sciences - USP Ariel Mariano Silber - asilber@usp.br Medical Faculty of Ribeirao Preto - USP Angela Kaysel Cruz - akcruz@fmrp.usp.br Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP Monica Talarico Pupo - mtpupo@fcfrp.usp.br Department of Biochemistry and Molecular Biology - UFV Juliana Lopes Rangel Fietto - jufietto@ufv.br Department of Chemistry - UEPG Jorge Iulek - iulek@uepg.br

E-mails 55

Notes:

1. São Carlos - SP Institute of Physics of São Carlos (IFSC) University of São Paulo (USP)

INBEQMeDI SUMMARY REPORT 2009-2013

Coordinator Vice-Coordinator Technology Transfer Outreach Finance

Richard C. Garratt Adriano D. Andricopulo Otavio H. Thiemann Leila M. Beltramini Rejane N. Brasil

Cristallography

Biophysics

Glaucius Oliva Richard C. Garratt Otavio H. Thiemann Adriano D. Andricopulo Ilana Lopes B.C. Camargo Eduardo Horjales Reboredo Rafael V.C. Guido

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

Department of Chemistry (DQ) Federal University of São Carlos (UFSCar) Arlene Gonçalves Correa Dulce Helena F. de Souza Paulo Cezar Vieira

Headquarter Institute of Physics of São Carlos University of São Paulo Avenida Trabalhador Sãocarlense 400 CEP 13566-590 São Carlos - SP

2. São Paulo - SP Institute of Chemistry (IQ) University of São Paulo (USP) Shacker Chuck Farah

Institute of Biosciences (IB) University of São Paulo (USP)

Associated Laboratories Department of Chemistry - DQ/UFSCar Institute of Chemistry - IQ/USP Institute of Biosciences - IB/USP Institute of Biomedical Sciences - ICB/USP Medical Faculty of Ribeirão Preto - FMRP/USP Faculty of Pharmaceutical Sciences of Ribeirão Preto - FCFRP/USP Department of Biochemistry and Molecular Biology - DBB/UFV Department of Chemistry - DQ/UEPG

instituto b oc ê c s de biociências

Célia R. da Silva Garcia Institute of Biomedical Sciences (ICB) University of São Paulo (USP) Ariel Mariano Silber

3. Ribeirão Preto - SP instituto de biociências

Medical Faculty of Ribeirão Preto (FMRP) University of São Paulo (USP) Angela Kaysel Cruz

Faculty of Pharmaceutical Sciences of Rib. Preto (FCFRP) University of São Paulo (USP) Monica Talarico Pupo Production

4. Viçosa - MG Department of Biochemistry and Molecular Biology (DBB) Federal University of Viçosa (UFV) Juliana Lopes Rangel Fietto

5. Ponta Grossa - PR Department of Chemistry (DQ) State University of Ponta Grossa (UEPG) Jorge Iulek