evotec DDup Special Edition #2

DDup SERVICES

DRUG DISCOVERY SERVICES HIGHLY EXPERIENCED SCIENTISTS, STATE-OF-THE-ART TECHNOLOGIES & BROAD RANGE OF THERAPEUTIC AREAS EXPERTISE

CAPABILITIES INCLUDE Integrated Services Target Identification & Validation Hit Identification Compound Management Research Informatics Chemistry DMPK Proteomics & Metabolomics Biomarker Discovery Cell & Protein production In vivo & in vitro pharmacology

SECTIONS

1

2

3a

3b

INTEGRATED SERVICES

TARGET IDENTIFICATION & VALIDATION

HIT IDENTIFICATION – HTS

HIT IDENTIFICATION – HCS

3c

4

5

6

RESEARCH INFORMATICS & IN-SILICO DRUG DESIGN

CHEMISTRY

HIT IDENTIFICATION – SBDD COMPOUND MANAGEMENT

Bulk Cell Production

7

8

9

10

DRUG METABOLISM & PHARMACOKINETICS („DMPK“)

PROTEOMICS METABOLOMICS & BIOMARKER DISCOVERY

CELL & PROTEIN PRODUCTION

IN VITRO PHARMACOLOGY, MICROBIOLOGY & TRANSLATIONAL SCIENCE

11

12

13

14

ION CHANNELS

IN VIVO PHARMACOLOGY

EVOMAB

nt RECORD & CLIENTS eTRACK m p o l Deve leads

INNOVATION EFFICIENCY WITH EVOTEC

A message from Evotec Chief Operating Officer, Dr Mario Polywka

Drug development is a high-reward but high-risk process, with as few as one in 10,000 new molecular entities making the successful but costly and lengthy journey from discovery to market. The pharmaceutical industry is struggling with the fall in R&D productivity and increasing demands from the regulatory authorities who demand first- and best-in-class therapies rather than me-toos.

Evotec’s drug discovery platform was established to deliver an industrialised, cutting-edge, comprehensive and unbiased infrastructure to meet the industry’s need for innovation in drug discovery. Using a strategic outsourcing partner like Evotec to drive Innovation Efficiency, reduces the risk and increases the chance of success in drug discovery without the need for the industry to invest in fixed cost structures.

Evotec’s portfolio of capabilities includes target identification and validation, compound management, screening, hit-to-lead and lead optimisation medicinal chemistry, in vivo and in vitro pharmacology, proteo­ mics and biomarker science, protein production and early CMC activities.

Working with Evotec, our partners gain access to an industry-leading team of scientists with years of expe-

rience in the Pharma and biotech industry. This experience effectively marries target and disease biology expertise with a world-class technology infrastructure. These scientists have successfully delivered clinical candidates in their past careers but more importantly they continue to do so at Evotec for our clients. The intellectual input of Evotec’s scientists is one of the pillar’s supporting Evotec’s business model. The most visible sign of our scientist’s ability to deliver innovative solutions is the number of customer patents on which Evotec scientists are named inventors. This number grows yearly and currently exceeds 200 and covers many therapeutic areas and target classes. Evotec is the partner of choice for standalone services which are charged on a purely fee-for-service basis. Alternatively, we can offer holistic, fully integrated drug discovery solutions through a variety of commercial structures. Our aim is to meet your requirements for the drug discovery solution you are seeking both scientifically and commercially. Our track record is second to none as testified by the number of projects that have moved through to the clinic and the strong portfolio of satisfied partners we have worked with through the years.

Evotec has been involved in more than 250 partnerships since its inception in 1993 and has delivered more than 30 pre-clinical candidates and 20 clinical candidates both in partnerships and within its own proprietary drug discovery efforts.

This document showcases the stateof-the-art offerings of Evotec to address the challenges of Innovation Efficiency within our industry. In particular with this Drug Discovery Services update we introduce you to our new and expanded capabilities since the strategic alliance with Sanofi and the acquisition of an integrated drug discovery operation in Toulouse, France. This transaction gives us access to much needed capacity and some new capabilities including our state-of-the-art compound management facility – a first for us in Europe – as well as a ground breaking, innovation led access to the Sanofi compound library made available to our partners for screening. Of course the key addition is almost 200 first class scientists with significant experience in drug discovery bursting to collaborate with new and existing partners. This addition cements our place as the leading drug discovery partner in the world.

Please use this document to define how we can help you and contact us for the most innovation- and costefficient solution to your drug discovery requirements.

Yours sincerely Mario Polywka

SECTION 1

INTEGRATED SERVICES

Craig Johnstone Executive Vice President, Drug Discovery and Site Head, Toulouse

TARGET ID & VALIDATION

Molecular biology and cloning Bioinformatics In vitro target validation In vivo target validation Target deconvolution

In the next sections of this document, we have laid out a selection of the key technical capabilities and technologies which we have on offer at Evotec, and these capabilities can be accessed as stand-alone services on demand. In addition, through our extensive drug discovery know-how and experience and expert project management, we bring seamlessly integrated drug discovery capabilities to bear on our collaborations. We understand that each partner has different needs, internal capabilities and capacities, so through in-depth understanding of the project goals and your specific needs, we develop a coherent plan which enables you to leverage the benefits of our large, flexible, high-quality organisation as a one-stop, cost-effective solution, no SCREENING

Assay development and screening (u)HTS High-content screening Biophysical methods Electrophysiology In silico screening technologies Fragment-based drug discovery Compound management

matter where the project lies on the gene-to candidate continuum.

Each collaboration has its own individual challenges so at Evotec “we start with the end in mind”, meaning that we consider the intended indication, frequency and route of administration, safety and efficacy demands, early development strategy etc as part of the product profile which in turn impacts directly on the project plan. Our scientists work with our partner’s scientists to select and execute the most promising strategy and technologies to deliver on the project goals. We also build into our processes key decision points that will guide us through the project and continually measure our progress against these. Using this

HIT-TO-LEAD

LEAD OPTIMISATION

Disease biology and target class expertise Medicinal, synthetic & computational chemistry Library design, procurement and synthesis High-throughput chemistry Protein-ligand crystallography and structure-based drug design In vitro & in vivo pharmacology In vitro ADME, rodent PK & bioanalysis Cellular selectivity analysis Cellular MoA analysis In silico ADMET

DISEASE BIOLOGY EXPERTISE PROVEN PROGRAMME MANAGEMENT

approach, we provide innovative and efficient solutions that are always focused on the needs of our partners whilst minimising waste.

Furthermore, by using Evotec as a single provider of integrated services, you can spend more time thinking about the science and less time managing the interfaces between multiple service providers.

Every single day, Evotec scientists are striving to solve drug discovery problems for their clients. Our scientists have made significant contributions to cutting-edge science throughout their careers and are drawn from a variety of backgrounds. They have been successful in all major therapeutic areas and target classes.

Their ideas, inspiration, creativity, innovation and insightful analysis are all key elements of Evotec’s value proposition, and are critical contributions to successful and productive integrated collaborations. In addition, the conduct of such science is heavily dependent on being able to carry out the practical work smoothly, quickly, and without delays, problems, and frustration. Our client’s satisfaction, and our performance, is dependent on the effective unification of slick, productive, practical execution with good ideas, problem solving and creativity: we call it “Innovation Efficiency”.

As a result of this unique combination of depth, breadth, knowledge and experience of drug discovery with operational excellence, Evotec has established a successful track record in assisting academic institutions, not-for-profit foundations, biotech and pharmaceutical companies in developing novel therapeutics.

Powerful combination of experience and analysis to extract learning and drive next cycle

Excellent understanding of molecular interactions, structural insight and product properties to enable simultaneous multi-parameter optimisation

ANALYSIS

DESIGN

CYCLE TIME

TEST Rapid, relevant, high-capacity efficacy and non-efficacy testing

MAKE MAKE

Rapid and efficient synthetic execution.

Bespoke, complex molecules and designed libraries / sparse arrays

WHY EVOTEC? Industry-leading capabilities in integrated drug discovery World-class suite of compound screening and profiling technologies Proven ability to progress targets to pre-clinical development within 3 years Consistent delivery and innovation for our partners Internal processes optimised to focus on quality and speed allowing for reduced cycle times and thereby accelerating our partner’s discovery efforts Single contracting partner reduces complexity Experienced and proven senior scientists act as project leaders and provide a single point of contact to our partner organisations Expert advice on overall project strategy

Securing IP protection and supporting grant applications

SECTION 2

TARGET IDENTIFICATION AND VALIDATION

Gene knock-out and over-expression studies both in vitro and in vivo as well as access to relevant in vivo disease models World-class ex vivo imaging techno­ logy platform using tissue sections to study cellular and molecular events Phenotypic screening of complex cellular systems for hit identifi­cation Target deconvolution using proteomics

Mark Slack Vice President In vitro Pharmacology

OFFERING Evotec has established a world-leading platform to support the discovery and validation of innovative diseasemodifying targets across different therapeutic areas as well as aid target deconvolution following a phenotypic screen. Our offering comprises: Differential expression studies followed by bioinformatics-driven data mining and hypothesis building

IN VITRO TARGET MODULATION CAPABILITIES

The powerful combination of CRISPR and disease-related cellular models coupled with a variety of read-outs including high-content imaging are designed to provide an accelerated solution to target identification, validation and target deconvolution following a phenotypic screen. Our offering includes: Screening of shRNA and CRISPR libraries for target identification GENE EXPRESSION PROFILING Validation of specific targets in various cellular models using Changes in gene expression, as disease-relevant read-outs result of a disease state or as a consequence of drug action, give invaluable insights into biological systems. Additionally, expertise in working From early target iden­ tification with non­traditional cellular models to mode of action studies and such as co-cultures and primary cell biomarker identification the analysis culture is available. of gene expression data supports the drug discovery processes. Our capaIN VIVO bilities include: TARGET VALIDATION Design and execution of transcrip- Evotec has disease models in a tomics studies in vitro or in vivo number of areas such as dia­ betes, including rigorous quality control diabetic complications, kidney diseases, neurodegeneration, oncol Bioinformatics analysis to deterogy, inflammation and pain that mine differential expression and can be used for target validation. generate annotated gene lists Cluster analysis to identify groups Approaches utilised include: of regulated genes Application of target-selective small molecules or biologics Data interpretation turning raw in acute or sub-chronic in vivo data into meaningful biological studies in rodents and pharmacological information

Virus-based target in vivo knockdown and over-expression techniques A variety of read-outs have been established that allow the investigation of target modulation. Such studies are run on a routine basis and additional read-outs are developed on a continuous basis.

Classical pharmacodynamic and efficacy read-outs such as blood glucose and HbA1c for diabetes, behavioural read-outs for pain and neurodegeneration A world-class ex vivo histology and immunohistochemistry platform investigating cellular and molecular markers in tissue sections prepared from treated animals USE OF PHENOTYPIC ASSAYS TO IDENTIFY NEW DISEASE RELEVANT TARGETS AND PATHWAYS

The ability to study compound effects in a disease-relevant cell type in a target-unbiased way offers enormous potential and has thus become increasingly popular in recent years, with more complex and diseaserelevant cellular systems becoming available for higher throughput assays.

Identification of new mechanisms to treat kidney disease: Phenotypic screening of conditionally immortalised human podocytes

Evotec has in-depth expertise in the pounds) and Sanofi collection (800K area of phenotypic screening and compounds) target deconvolution.

Proteomics-based target High-Content Screening deconvolution Evotec has a world-class high-content Evotec has established a mass screening platform that is increasingly spectrometry-based approach to used for hit identification through ex­ perimentally determine the binphenotypic screening ding partner(s) of a lead compound in a relevant cellular context: >15 years know-how in the design and implementation of phenotypic Metabolic or chemical labelling assays using cell lines but also includ Optimised linker chemistry capaing complex cellular systems utilising bilities primary cells and stem cells in the Determination of Kd values for area of neurodegeneration, inflamma- each putative binding partner tion, metabolic disease and oncology Combine with post-translational events to understand global cellular Small molecule compound librareffects ies: Evotec collection (400K com-

Overview of Evotec capabilities and expertise in target identification and validation across different disease areas

METABOLIC DISEASE

ONCOLOGY

CNS, NEURODEGENERATION, PAIN

+++

n.a.

+++

shRNA / CRISPR/ overexpression

++

++

++

Cellular / ex vivo tissue imaging

+++

++

+++

In vivo efficacy studies

+++

+++

+++

Differential gene expression

SECTION 3A

HIT IDENTIFICATION HIGH-THROUGHPUT SCREENING OFFERING Evotec has a long history in highthroughput screening (“HTS”) utilising large client compound collections and also our own 400,000 compound collection together with the exclusive access to the Sanofi library of 700,000 molecules. Evotec’s screening platform is differentiated through integrating a variety of detection technologies with high-throughput capabilities.

This technology platform allows for assay miniaturisation and use of multiple read-out parameters to enable the reduction of false positives and negatives. Evotec’s technology platform

Dirk Ullmann Executive Vice President Lead Discovery

includes nuclear magnetic resonance spectrometry (“NMR”), surface plasmon resonance spectrometry (“SPR”), high-content screening (“HCS”), highthroughput mass spectrometry based screening (“HTMS”) and high-throughput RT-qPCR. OVERVIEW SCREENING CAPABILITIES AT EVOTEC

Evotec selects the most suitable screening strategy for your target – our approach is to let the target dictate the screening strategy and not the other way around.

Screening platforms at Evotec

*Available RapidFire MS capacity allows for up to 10,000 data points per day

EVOSCREEN MARK II

EVOSCREEN MARK III

384 HTS SYSTEM

RADIOMETRIC PLATFORM

1536 HTS SYSTEM II

1536 HTS SYSTEM III

384 MTS SYSTEM

SEC-LC/MS PLATFORM

No. of Systems

1

1

3

semi-automated benchtop devices

1

1

3

1

Assay Volume

1µl/well

5-10µl/well

20-50µl/well

20-50µl/well

5-20µl/well

5-20µl/well

20-50µl/well

7.5µlwell

Plates

2080-well

1536-well

384-well

96 and 384-well

384/1536-well

384/1536-well

384-well

96

Formats

Biochemical

Cellular + Biochemical

Cellular + Biochemical

Cellular + Biochemical

Cellular+ Biochemical+ Microorganism

Cellular + Biochemical

Cellular+ Biochemical+ Microorganism

Biochemical

Readers

Insight™ FCS+plus reader

PE EnVision

FLIPRTetra PE EnVision Tecan Safire RapidFire MS

PE TopCount (2) PE MicroBeta2 (3)

Tecan Infinite Arrayscan off line RapidFireMS off line

Tecan Infinite Roche Lightcycler Arrayscan off line RapidFireMS off line

PE Envision Beckmann DTX 880 Arrayscan off line

SEC-LC/MS

Assay principles

Binding Enzyme activity

Binding Enzyme activity Reporter gene

Binding Enzyme activity Ca2+ flux Second messenger Membrane potential

Radioligand binding FlashPlate SPA Filtration

Phenotypic screen mRNA detection Enzyme activity Reporter gene Second messenger Cell infection Reporter gene Enzyme activity Phenotypic

Enzyme activity Second messenger Reporter gene Phenotypic

Affinity binding

FCS+plus

Fluorescence Luminescence Absorption Alpha screen

Fluorescence Luminescence Absorption Alpha screen Mass spectrometry*

up to 100,000 / day

up to 100,000 / day

up to 40,000 / day

Detection

Throughput

Isotopes: H, 35S, 14C, 32P, 33 59 P, Fe, 125I

3

Fluorescence Fluorescence Fluorescence Luminescence HTRF HTRF Absorption Luminescence Luminescence Alpha screen Absorption Absorption Mass spectrometry Mass spectrometry Mass spectrometry*

up to 25,000 / day

up to 100,000/day

up to 100,000/day

up to 15,000/day

Mass spectrometry

up to 25,000/day (in mixture)

Assay development and HTS track record SCREENING

ASSAY DEVELOPMENT Other targets 121

GPCR 107

PPI 11

NEu

Others 64 Protein Protein 34 ADMET 5

Nuclear Hormone Receptor 9 ADME/Tox assays 18

Ion Channel 19 Protease 16 Kinase 19

Ion Channel 68

GPCR 78 Enzyme other 93

Kinease, protease, phosphatase & other enzymes 145

>475 assays developed Success rate >95% High percentage of cellular assays

Evotec has twenty years of experience in assay development; in particular in HTS, but also to support hit-to-lead & lead optimisation (H2L/LO) programmes. Our experience includes: A broad portfolio of assays covering the major target classes (see chart)

>320 uHTS screens Specific hits with EC50<25μM identified in most screens

New target classes in the field of epigenetics, transporter biology and protein-protein interaction

Anti-infective screens ran on several pathogenic strains

High numbers of unique assays developed as no literature precedence was available

Assay read-outs covering a wide range of technologies including biochemical, molecular and cellular assays

Assays using label-free and biophysical methods

SECTION 3A

For structure-based approaches to drug discovery, Evotec has a 20,000 fragment set available for biochemical screening and a 3,000 fragment set specifically selected for NMR and SPR screening. After signing the multi-component strategic alliance with Sanofi in March 2015, the Sanofi compound library is also made available to our partners for screening. This creates one of the largest and most valuable sources of starting points for drug discovery with approx. 700,000 compounds physically available to screen which ultimately represent 2,000,000 molecules.

Evotec screens customer libraries of any size, virtual screening derived hits and also smaller, more targetdirected libraries.

SCREENING PROCESS

THE OPTIMUM SCREENING COLLECTION

Our library contains 400,000 compounds consisting of 70,000 proprietary Evotec compounds, a set of 330,000 maximally diverse “islands” and 20,000 natural products from Analyticon.

Screening process

Diverse islands 330,000

Evotec’s screening library is differentiated through its quality, diversity and novelty and has a successful track record of delivering potent and attractive compounds as star­ting points for medicinal chemistry optimisation.

The integration and connection of the different stages of the screening process from assay development to automation to compound management/tracking and finally data processing are essential. The Evotec screening team manages this complexity on a daily basis to deliver high-quality data on time to our clients.

In Evotec’s view the success of a screening project is determined by: Target selection Right strategy for target expression Evotec fragment 20,000 to address specific sites of interest Robust and sensitive assay system Evotec proprietary 70,000 Well-selected compound collection for screening Reliable screening process Analyticon NPs 20,000 Careful characterisation of screening hits in orthogonal test systems

Distribution of calculated properties in Evotec’s discovery screening library Molecular weight distribution

0

CLogP distribution

100 200 300 400 500 600 700 800 900 Molecular weight

-5.0

Rotatable bond count

2

4

6

0

2.5 CLogP

5.0

7.5

10

H-Bond acceptor distribution

8 10 12 14 16 18 20 Rotable bonds

1

DATA HANDLING

Evotec’s data analysis platform, Aplus, is suited to support all screening processes from HTS including multivariate analysis for HCS to SPR

Reagent production and assay development

-2.5

TPSA (Topological polar surface area)

MINIATURISATION

2

3

4

0

20 40 60 80 100 120 140 160 180 TPSA

H-Bond donor distribution

5 6 7 8 9 10 11 12 H-bond acceptors

1

2

3 4 5 H-bond donors

6

7

and also HTMS. This software facili- flexible to meet customer needs in tates the integration of all tech- data formats for upload in different niques into one analysis platform environments. and streamlines the process flows.

For customer reporting, Evotec is

Adaptation on screening platform

MARKER LIBRARY SCREEN

Primary HTS screen n=1

DATA STATISTICS A+

Primary hits (3sigma threshold)

HIT CONFIRMATION SCREEN N=3

Identification of clusters & singletons

PRELIMINARY SAR

Specific hits with confirmed conc. dependent activity

TEST IN ORTHOGONAL ASSAY

Hits with conc. dependent activity

11PT DRCS N=2 + LC/MS CHECK

Confirmed hits

SECTION 3B

HIT IDENTIFICATION HIGH-CONTENT SCREENING primary cells from appropriate hosts.

This approach is a core part of Evotec’s efforts to identify novel targets and drugs with disease-modifying potential.

Ina Sternberger Group Leader In vitro Pharmacology

OFFERING Evotec specialises in the development and implementation of high-content based assays for high-throughput screening and compound validation to probe the pathophysiological environment. High-content screening (“HCS”) is routinely used throughout the hit identification cascade from target validation to mechanistic studies. The assays developed at Evotec encompass both a target centric as well as phenotypic approach with emphasis on tissue samples, stem cell derived or

Evotec’s HCS expertise is built around the OPERA® platform where an essential understanding of the mechanics and image analy­ sis tools, gathered over a period of more than 15 years in the develop­ment of the platform, flows into the hit identification process. Additional platforms include the Operetta® and ArrayScan™ to fulfill different requirements in resolution and throughput. The platform is completed with a power­ ful data management system and powerful data analysis tools includ­ing multifactorial analysis. Based on this expertise, Evotec has performed many high-throughput screens of up to 400,000 compounds and devel­ oped over 30 assays to study mode of action of compounds and targets in a multitude of cellular backgrounds.

PHENOTYPES Proliferation Cell cycle analysis Cell death/necrosis/apoptosis Differentiation markers DNA damage Cytoskeleton rearrangements Neurite outgrowth Mitochondrial mass/-toxicity Endocytosis Synapse density Neurodegeneration/-protection Intracellular aggregation Post-translational modification

new realm of drug discovery opportunities that otherwise could not be addressed. This includes experienced multi-disciplinary teams that build strategies for hit follow up towards identifying the molecular mechanism of action of a potential drug.

Accessing Evotec’s HCS platform offers a road to novel target space achieved via an array of disease-related secondary models post-HTS coupled PHENOTYPIC DRUG DISCOVERY The nature of HCS describes primarily to target deconvolution via the applia target agnostic approach measuring cation of bioinformatics, genetic tools phenotypic/morpho­logical changes and finally mass spectrometry based proteomic approaches. associated with target engagement.

The use of phenotypic screening Other assay formats that are routinely provides Evotec’s clients with a whole devel­oped on Evotec’s HCS platform

programmes across a variety of ther apeutic areas. At present, the main focus of HCS is in the area of oncology, kidney disease, diabetes, pain, inflammation and neurodegenera­tion.

Here the strengths of HCS are combined with regenerative medi­ DISEASE AREA EXPERTISE HCS at Evotec is now a common cine concepts to probe and discover element in many drug discovery new treatment paradigms such as are designed to investigate subtle or rare subcellular events. The table shows examples of cellular events that Evotec has experience with on the OPERA® platform.

beta cell regeneration for diabetes and podocyte protection for diabetic nephropathy. Applying stem cell technology in phenotypic and highcontent screening is also an area of active research at Evotec where we are committed to delivering new treatment paradigms for ALS and other degenerative diseases.

Examples of track record

TARGET/PHENOTYPE

READ-OUT

DRUG ID PHASE

Transporter

Internalisation

HTS 250K

Fibrosis

αSma expression/actin alignment

HTS 50K

Beta cell proliferation

Insulin expression coupled to BrdU stain

HTS 10K Biologics

Anchorage independent growth

Target validation

siRNA screen

Diabetic Neuropathy

Actin rearrangement

Known drug collection

Striatal neuron development

DARP32 positive cells

Protocol development

Neurodegeneration/ALS

Neurite outgrowth in co-culture system

10K HTS + known drugs

Transporter

Binding of fluorescent ligand

HTS of 100K

Huntington’s protein aggregation

Number of aggregates

Known drug collection

NFκB translocation

Nuclear accumulation in HUVECs

Lead optimisation

PTM enzyme in AID

NET formation in primary human neutrophils

Compound validation

DNA repair/oncology

DNA damage response in tumour cell line

HTS 50K

Autophagy

LC3 accumulation in autophagosomes

Pilot screen

Epigenetics

Protein methylation

Hit follow-up

Mucosal immunity

Reporter gene expression

HTS 400k

SECTION 3C

HIT IDENTIFICATION

STRUCTURE-BASED DRUG DESIGN (SBDD) Guided by high-quality data, SBDD at Evotec distils information into hypothesis-driven medicinal chemistry. By minimising unnecessary and wasteful compound synthesis, productivity is increased and chemistry teams are able to focus on an efficient path to the final drug.

NMR SCREENING AND HIT QUALIFICATION

NMR screening as part of the Evotec FBDD platform is an alternative option for the identification of new starting points for SBDD programmes. Evotec offers both protein-observed NMR assays monitoring chemical shift perturbations (SAR-by-NMR) as well Evotec has a comprehensive SBDD as ligand-detected NMR assays (Satuplatform comprising state-of-the-art ration Transfer Difference (“STD”) NMR and Water LOGSY) for hit idencapabilities in: tification. Hence, a broad range of Computational chemistry target proteins amenable to NMR Protein engineering screening technique can be covered.

X-ray crystallography Biophysical screening tools such 1 H, 15N-Trosy Spectra (37kDa kinase, as NMR, SPR, MS and ITC 2 H, 15N-labelled) John Barker Vice President Structural Biology

OFFERING The SBDD platform at Evotec includes a comprehensive fragment-based drug discovery (“FBDD”) platform. Sensitive screening techniques, such as the FCS+plus (proprietary to Evotec) or NMR, SPR or mass spectrometry (MS), combined with specifically selected fragment compound collections, identify diverse, active low molecular weight fragments providing multiple highly efficient starting points for chemical optimisation.

FRAGMENT COLLECTIONS

We employ our collection of 20,000 fragments for biochemical, high concentration and SPR screening, a 3,000 fragment set for NMR screening and a 3d enriched 340 fragment set for crystal soaking. All sets are optimally designed for quality, diversity and solubility at high concentrations to provide tractable starting points for optimisation within SBDD projects. The collection integrity is maintained following validated processes used in our HTS collection to maintain quality, diversity and novelty. apo-protein Protein + fragment

complex crystallography. Delivery of protein complexes is fine-tuned across a project’s lifetime to maintain the highest possible resolution while meeting the demanding cycle times of modern dis­covery. Experience covers the major target families with recent focus on crystallo­graphy of membrane proteins.

Novel Hsp90 fragment hits, identified in under one month from crystals entering the new High-Throughput Diamond Light Source process

Evotec has employed its NMR screening platform on more than 30 protein targets covering a whole range of target classes from frequently studied enzymes (like kinases or proteases) over nucleotide binding proteins or epi­genetic targets, to protein-protein inter­ action targets, even receptor domains. NMR also adds con­siderable value to biochemical screening campaigns by validating hit compounds for direct binding to the target protein and ultimately correlating this information to structure data, thereby enabling or facilitating SBDD programmes.

to screen both fragment sets thus increasing the chance to find bona fide binders of the target.

Independent of the primary screening strategy applied the assay techniques available at Evotec allow qualification of hits using alternative read-outs. Here we typically combine the above mentioned approaches with orthogonal assays and technologies like Isothermal Titration Calorimetry (“ITC”), Differential Scanning Fluorimetry (“DSF”) and Microscale Thermophoresis (“MST”). MST is applied in two modes available, the label free as well as the labelled SURFACE PLASMON RESONANCE version of the technology. This allows for maximal flexibility on a (“SPR”) SCREENING AND HIT broad variety of targets in conjuncQUALIFICATION Another powerful tool for study- tion with low affinity fragment like ing biomolecular interactions in a compounds. sensitive and label-free manner in real-time is SPR. While commonly STRUCTURAL BIOLOGY used at later stages of drug discovery Evotec is located within a few miles projects to qualify hit compounds of the UK’s third generation synchrowith regards to binding kinetics, the tron and coupled with our own instrumentation available at Evotec in-house X-ray source offers the also allows for primary screening ideal infrastructure for rapid cycle of fragments. Direct binding assays times in X-ray crystallography. The run on the Biacore™ 4000 screening Structural biology team provides device enable the identification of solutions to novel structure elucidafragments interacting with immobi- tion of drug targets and optimisalised target proteins. As the through- tion of crystal systems to support put is sufficiently high, we are able industrialisation of protein:ligand

The team is co-located with the medicinal chemistry and computational chemistry teams to maxi­ mise interaction across integrated discovery programmes. The use of 3D virtual reality has provided the chemistry team with the ideal environment to understand and effectively mine structural information to focus synthetic strategies and rapidly progress optimisation. Working with innovative computational scoring techniques, Evotec is experienced in maximising the value of structural information, providing foundation for rational design. The team is actively engaged with a newly established crystal soaking facility for medium-throughput fragment screening by X-ray crystallography, hosted at Diamond Light Source.

The system brings together a host of advanced automation technologies centered on acoustic liquid handling and high-efficiency synchrotron data collection. As mentioned above, Evotec has developed a 3d-enriched library with the option to run in a cocktail mode designed by genetic algorithm methods for maximum cocktail diversity and enhanced experimental efficiency. The capability of screening many hundreds of diverse fragments is achievable within a matter of weeks, delivering immediate structures to guide hypothesis-driven medicinal chemistry optimisation.

SECTION 4

COMPOUND MANAGEMENT

Scott Snyder Executive Vice President Compound Management

OFFERING Evotec is uniquely qualified in delivering its customers small and large molecule library management needs through over a decade of being the world’s leading compound management operation. Evotec expanded its operational presence in 2013 and again in 2015 by adding capacity and capabilities in Branford Connecticut and Toulouse France. These facilities are strategically positioned to support the strong presence of Evotec’s drug discovery services along the east coast of the U.S. and across Europe.

Our service offering includes: Cost effective compound identifi­ cation, selection, and procurement High-throughput compound analysis Multi-format plating and reformatting Inert or dry atmosphere and low temperature storage and processing Fast order fulfilment and worldwide delivery Multi-site disaster recovery and business continuity “In-sourcing” of Evotec sample management staff to client sites Access to compound collections of some of its partners through an open innovation strategy

and governmental organisations; which results in a deep understanding of its customers’ needs and deadlines. Each client has a dedicated Project Manager who provides a single point of contact to insure an open line of communication is always maintained.

COMPOUND IDENTIFICATION, SELECTION, AND PROCUREMENT

Evotec has extensive experience in the identification, selection and procurement of small molecule compounds possessing very specific (including commercial, physicochemical or bio­ activity-related) properties. Evotec has the capa­bility to apply compound identification tools as required by the client.

Once compounds are identified for purchase, our Project Management group consults with selected suppliers to negotiate terms on behalf of our client requirements, e.g. price, purity and format. Evotec purchases tens of thousands of compounds on a regular basis for a number of clients; therefore, the client can benefit from our vendor relations and volume Evotec has a broad range of discounts. compound management expertise accross many sectors including COMPOUND QUALITY CONTROL large Pharma, biotechs, foundations Evotec typically uses a three-part

incoming Quality Control (“QC”) process comprised of weight, solubility and LC-MS purity and identity to ensure that all collections start with high-quality compounds. Systems include a Waters 8-channel LC-MS system, with digital UV, ELSD and an LCT Premier mass spectrometer, and two Waters LCT Premier XE uPLC systems featuring positive and negative ESI and UV and ELS detection. Evotec also utilises a Xevo G2 Q-TOF system for quantitative determination and high resolution of compounds in DMSO solution.

COMPOUND STORAGE Evotec has invested in industryleading automated storage systems and supporting informatics. Storage capabilities support vials, tubes, plates and bulk materials at controlled temperatures of -80°C, -20°C (inert or humidity controlled atmosphere) and ambient. Actual storage conditions are dictated by sample type, containers and length of storage. Automated tube, vial and plate storage systems are equipped with high-speed pickers for fast arraying of tubes, plates and vials to support large plating, weighing and screening campaigns and rapid follow-up. Evotec can also prepare a disaster recovery set of the client’s collection for storage at one of our other compound management facili-

ties. This set can be readily accessed and processed for distribution at the client’s request.

COMPOUND DISPENSATION AND REFORMATTING DMSO solutions

Evotec has the capability for automated arraying of large numbers of compounds in DMSO or in water between a large variety of containers suitable for distribution or storage. Our extensive liquid handling resources cover the widest range of possible volumes.

High-volume systems include the eight channel pipettors, Tecan, Hamilton and Sias systems that can dispense and transfer 1 uL to multi-millilit­ers in vials and plates Mid-volume systems include the Beckman and Agilent plate replicators (formerly VPrep and Bravo) that can dispense from 1 uL to 2 mL to and from 96, 384 and 1536 SBS format arrays Low-volume systems include the LabCyte Echo 550 and 555 that acous­tically dispenses volumes below 1 uL using multiples of 2.5 nL drops of solution, and the TTP Mosquito, which dispense volumes between 50 nl and 1.2 ml

Neat compound dispensation

Evotec has the capability to transfer neat samples via manual weighing, Volatile Solvent Trans­fer (“VST”) or Solvent Transfer methods, and using automation (Flexiweigh and XPert dose technologies). Range for automatic distribution goes from submilligram (0.2mg to 30mg).

SECTION 4

COMPOUND SHIPMENT

Our team has extensive experience in shipping chemicals, research compounds, controlled substances and reagents. Evotec delivers and receives compounds in all formats, both domestically and overseas (including, but not limited to, North America, Europe and Asia). We are well-versed in docu-

mentation requirements, tariff classification and import/export customs requirements. We have the ability to facilitate the import/export processes through the use of brokers as required by the have client.

and Tracking (“ComIT”) software system and the industry leading commercial application (Titian Mosaic) to manage, record and report all manipulations of client samples. These systems operate independent of each other at different sites but both deliver DATA MANAGEMENT functionality and Evotec currently uses both a end-to-end traceability. proprietary Compound Inventory

Experts in Sample Logistics

> 84 million samples delivered to 450+ recipients in 18 countries

In support of our disaster reco­ very and business continuity plans Evotec employs i365 Data Backup and Recovery Solution to store incremental backups on a nightly basis and full server backups on a weekly basis. Data is transferred to two offsite locations automatically.

All data transmissions are encrypted and password protected at all times for security and confidentiality.

QUALITY MANAGEMENT

Evotec operates under the ISO 9001:2008 Quality Management and internal auditing principles but is not formally ISO 9000 certified. We have well-defined and documented procedures to monitor and ensure the consistency of our output. Our process enables us to find defects early in the process and our procedures ensure corrective action is taken whenever a defect occurs, with the goal being to implement correc-

tions with minimal impact to our instances, Evotec reviews requirements with our clients and design final deliverables. solutions including need identification, process design, facility IN-SOURCING AND layout, process workflow, vendor CONSULTING SERVICES If a prospective client decides to qualification and selection, and even retain compound management complete staffing and management services within their operation then of onsite services.

Evotec also offers both in-sourcing and/or consulting services. In these Our in-sourcing programme provides scientific staffing specifically designed to give clients the “flexible” workforce needed for anywhere from EVOTEC TRACK RECORD one year to multiyear partnerships.

Build and operate both private and public screening libraries We hire, train and manage our since 2004 employees to perform analytical Largest individual library consists of ~1.2 million compounds scientific programmes at client sites, Over 25 client collaborations working to individual specifications using their quality systems. This >84 million samples shipped to 450+ recipients in 18 countries advanced staffing model provides a Over 5 million samples acquired and processed for clients non-permanent, long-term and cost Experience with 223 compound global suppliers effective way to meet compound management staffing needs.

SECTION 5

RESEARCH INFORMATICS & IN-SILICO DRUG DESIGN

st em i

ut

e Knowledge Creat ists e l Ch m , Informat i i o na

Bioinformaticia Internal

Mike Bodkin,

NE W D

ATA

tatisticans

DATA

S t s, S

Organise M ana g e r s Da t a

on

is t s

ntis

ns, C

mp

olog

c ie

o

Ch

Scientists ogists Pharmac s, B i o l

External NE W DATA

Vice President Research Informatics

OFFERING The huge quantity of data that drug discovery generates both externally and internally requires that data informatics is at the heart of any modern research organisation. At Evotec the research informatics department smoothly integrates the disciplines of computational chemistry, bioinformatics, systems integration, data management and support informatics.

The team empowers the discovery scientist to deliver data-driven design hypothesis using commercial, propri-

etary and bespoke software offerings.

In short, the department delivers in-silico drug discovery and can be engaged in a standalone capacity or fully integrated with our discovery partners.

TARGET IDENTIFICATION

Evotec uses industry standard target interaction databases which combined with other ‘omics’ data can be used to construct target interaction maps. These networks can be significantly enhanced using our predictive

pharmacology tools. > 200M bioassay datapoints are currently sourced for model building. Pathway analysis can be used to identify new and novel targets proposed to modulate a disease hypothesis. Such approaches are fundamental in target deconvolution and drug re­purposing.

DRUGABILITY ASSESSMENT

Structure-based assessment for novel targets gives an additional read on drugability when no/few druglike compounds are known. New

2D sequence based “switchability” calculations projected onto 3D structures have successfully been used for predicting allosteric sites for modulation, surfaces for PPIs and mAB binding.

2D

HIT IDENTIFICATION

Fingerprint

For virtual screening Evotec uses multiple methods using the bestof-breed commercial software. A number of compound collections are available for screening: EvoSource (34M) Evotec Diversity (400K), Sanofi HTS (1.4M), Fragments (21K), Diverse Phenotype Library (20K) Annotated Druglike Library (6K) Chemgenomics and ortholog approaches also provide a good starting point for identifying novel actives. Evotec has licensed LeadIT and Spark for scaffold hopping but also has a number of in-house tools using structure merging and vector placement algorithms.

In-pocket

Shape

MULTI-OBJECTIVE DESIGN

Feauture

2.5D Fingerprint/ Feature

Docking

Field Virtual Screening Toolbox

Ideas generated can be scored across a range of objectives such as an ADMET profile (Evotec has its own proprietary ADMET models), target QSARs, off-target QSARs. The method can be used for both structure-based and ligand based design.

LIGAND OPTIMISATION

HIT EXPANSION The hits identified from screening require expansion to provide evidence of SAR. This is traditionally based upon chemical similarity and involves searching databases for ‘like’ molecules. Evotec also uses predictive pharmacology so that molecules that possess a similar target pathway signature can be identified. Library design, statistical analysis and virtual chemistry tools can be used to design the most information rich set of molecules for synthesis.

3D

3D

Evotec possesses significant expertise in QM guided SBDD using the fragment molecular orbital (FMO) approach. FMO calculates the strength of interaction between protein residues and ligand atoms to accurately pinpoint the drivers of molecular affinity. Molecular simulation (WaterScout) and ΔG (Nautilus) approaches can be applied to identify happy and unhappy waters.

Evotec’s large internal HPC (high performance computing) capacity, employing multiple Intel and GPU clusters, ensures that these approaches are routinely applied.

The Evotec ELN reaction transform library can be used to perform virtual Designing molecules for the clinic chemistry and grow fragments balances the multiple dimensions within the active site of protein. of medicinal chemistry and phar-

macology. Evotec has developed a number of statistical analysis tools for matched-molecular-pair analysis (MMPA) and Free-Wilson approaches. These can be used in combination with predictive modelling tools QSAR, QSPR to enable effective experimental design. PK/ PD modelling is done in collaboration with the ADMET group.

THE RESEARCH INFORMATICS TEAM

The infrastructure of research informatics at Evotec is supported by a strong team of data managers who organise and securely store data from across the globe. The systems integration group maintains the computing environment and includes the software developers who build and maintain the proprietary software tools.

The support informatics (training and education) and business operations teams provide the softer interface to the department and ensure the smooth operation of internal and external client relationships.

SECTION 6

CHEMISTRY We understand that each partner has different needs, internal capabilities and capacities. Evotec prides itself in being able to provide flexible, innovative and efficient solutions. Evotec’s experienced scientists can support your aspirations in a variety of ways.

The capabilities listed below can be accessed individually where required or brought together to form complete project solutions: Cutting-edge molecular design Rapid synthetic execution Expert advice on overall project strategy Developing strategy for and securing IP protection Scale-up chemistry Preparative chromatography Project management Dave Hallett

SYNTHESIS At the foundation of Evotec’s medicinal chemistry group is a talented and industry-experienced team of >175 synthetic organic chemists. Their skills are utilised during medicinal chemistry-driven projects, focusedOFFERING Small molecule drug discovery library preparation, scale-up synthesis requires a deep understanding of and when preparing chemical building biological context coupled to knowledge blocks or literature compounds. and experience of the property space required to produce safe, efficacious Synthetic execution is one of the drugs. Over the past 20 years, Evotec most cost- and time-consuming has developed expertise in numerous parts of the early discovery process. therapeutic areas including diseases We understand that where synthesis of the central nervous system (“CNS”), is concerned, speed is paramount. oncology, diabetes and metabolic The faster our teams can deliver diseases, pain, cardiovascular and molecules into bioassays, the faster thrombosis diseases, inflammation the project teams can obtain new knowledge. and anti-infectives.

Executive Vice President Chemistry Discovery Chemistry

Our approach is to employ, educate and retain talented chemists and to enable them with the latest technology and equipment.

More than 60% of our chemists are educated to PhD level >40% of our chemists have >8 years’ experience at major pharmaceutical and biotech companies prior to joining Evotec Our chemists utilise state-of-the art equipment known to accelerate productivity: microwave reactors, automated purification systems, parallel synthesisers These facets enable Evotec to provide superior synthetic chemistry and problem-solving capabilities to our partners and to efficiently deliver compounds in a shorter timeframe than our competitors.

Good molecular design often leads to complex syntheses (e.g. densely functionalised heterocycles, multiple chiral centres). Projects are successfully executed at Evotec that involve highly challenging syntheses and isolation procedures, for example: Natural products Steroids Macrocycles Nucleosides Carbohydrates Peptidomimetics The origin of the chemistry department in Abingdon was in asymmetric synthesis and Evotec routinely develops enantioselective synthetic

methods and has a full range of chiral separation technologies including SFC to deliver single isomers.

MEDICINAL CHEMISTRY

Evotec has a powerful core of experienced, industry-seasoned drug hunters who drive the molecular design process. Our teams in Abingdon, UK and Toulouse, France have co-located group of medicinal chemists, computational chemists, structural biologists and DMPK scientists that have made significant contributions to discovery projects throughout their careers: >100 development candidates Named inventors and authors on >860 patents and publications

plC50

Our scientists are drawn from a variety of backgrounds and have been successful in all major therapeutic areas and target classes. We have a particularly strong track record in prosecuting ion channel modulators, enzyme inhibitors and proteinprotein interaction inhibitors, with several compounds progressing to pre-clinical development and beyond.

Our teams are fluent and expert in both protein structure-guided and ligand-based design, utilising internal knowledge and experience as well as a wealth of supporting computational models.

Series A plc50 5.45 LLE 3.16 clogD 2.3

4

5

Series B plc50 5.58 LLE 2.9 clogD 2.7

Series A analogue plc50 6.8 LLE 5.3 clogD 1.5

6

Series C plc50 7.19 LLE 3.78 clogD 3.4

7 Series B analogue plc50 6.62 LLE 4.1 clogD 2.5

8

Series C analogue plc50 6.97 LLE 4.65 clogD 2.3

9

Series C plc50 7.29 LLE 4.02 clogD 3.3

LiPE6

0

1

2

3

LiPE3

LiPE5

4

LiPE2

An example of multiparameter optimisation.

Evotecâ&#x20AC;&#x2122;s design teams simultaneously improve physicochemical properties as well as biological activity. The plot above (-log10IC50 versus calculated LogD) highlights three different chemotypes from the same project. Over 2 iterations, the arrows indicate the improvements made to each series as seen from the improved Lipophilic Ligand Efficiency (LLE) scores

LiPE4

5

cLogD

SECTION 6

Selected templates investigated as Aurora kinase inhibitors, Phthalazinone Pyrazoles as Potent, Selective, and Orally Bioavailable Inhibitors of Aurora-A Kinase, J. Med. Chem. 2011, 54, 312–319

The focus of our medicinal chemistry teams is on high-quality design and crisp decision making. The process at Evotec is enabled by widespread availability of visualisation, analysis and design software tools. Our medicinal chemists target analogues within defined property space to answer specific structure-property and structure-activity questions – the emphasis is always on making the right compounds with optimisation of biological activity and properties in parallel. We firmly believe that the co-location and interaction of synthetic and medicinal chemistry, computational science, structural biology and DMPK enhances the overall design, analysis and communication process.

We continually review our internal processes (analysis, compound management, registration, dispatch etc.) to ensure that our discovery teams are working in the most efficient manner possible with the implementation of lean processes wherever possible. We have implemented a companywide electronic lab

notebook system and internal LIMS system for compound submissions.

In addition we have project specific databases and workflows to enable rapid data sharing within teams and with our clients.

Our focus is to ensure the shortest possible “design-make-test-analyse” cycle with typically one complete cycle per month. With this in place we can achieve; Key project decision points in a shorter timeframe Increased knowledge and experience due to more learning cycles per time unit Better quality output/product Less waste due to more informed decisions Effective, efficient teams

Large scale synthesis of > 140 compounds for advanced pharma cological profiling Their expertise allows them to cover areas such as: Large scale synthesis of API, inter mediates (up to 100-200g) Route scouting for IND process Optimisation of synthetic routes Crystalline Salt selections Full technical package for transfer to larger scales for GMP batches synthesis We ensure that synthetic processes match the safety requirements of large scale synthesis by using stateof-the-art equipments for calorimetric studies and in-process controls.

PREPARATIVE CHROMATOGRAPHY

SCALE-UP CHEMISTRY Evotec can supply large quantities of molecules of interest through a team of very experienced chemists originating from chemical development that has contributed to: Late development synthesis of > 20 pre-clinical and clinical candidates

Evotec has established a high quality preparative chromatography service catering from milligram to kilogram scales. The highly experienced group utilises complementary analytical and preparative HPLC and SFC separation techniques to provide:

DESIGN Excellent understanding of molecular interactions, structural insight and product properties to enable simultaneous multi-parameter optimisation

ANALYSE Powerful combination of experience and analysis to extract learning and drive next cycle

AN

GN

AL

SI

YS

E

DE

Design, synthesis and structureâ&#x20AC;&#x201C;activity relationships of a novel class of sulfonylpyridine inhibitors of Interleukin-2 inducible T-cell kinase (ITK), Bioorg. Med. Chem.

Lett. 2014, 24, 5818-5823

MA

ST

The group has also developed unique biomimetic oxidation models mimicking P450 monooxygenases for the synthesis, purification and structural identification of real and putative metabolites of active compounds.

TE

Separation of chiral compounds at the preparative scale (intermediates or final compounds) Successful purifications when higher resolution techniques are needed (peptides, vitamins, steroids, macrolides, amino-acid derivatives) Impurity isolation for structural identification Chiral analysis for asymmetric synthesis follow-up Evaluation of chiral stability of final molecules or development candidates

TEST Rapid, relevant, high-capacity efficacy and non-efficacy testing

KE

CYCLE TIME

MAKE Rapid and efficient synthethic execution.

Bespoke, complex molecules and designed libraries/ sparse arrays

EVOTEC TRACK RECORD Over the last 15 years, Evotec chemists have supported >125 hit-to-lead and/or lead optimisation projects Evotec scientists are named inventors on >200 client patents and have helped identify >30 pre-clinical candidates Our project teams have made major contributions to the identification of >20 compounds that have been approved for clinical trials

SECTION 7

DRUG METABOLISM

& PHARMACOKINETICS („DMPK“)

Eric Erdociain Head of the ADME-PK unit, Evotec France

OFFERING Over the past 20 years, Evotec has developed expertise in numerous therapeutic areas including diseases of the central nervous system (“CNS”), pain, oncology, diabetes and metabolic diseases, inflammation and anti-infectives.

synthetic and medicinal chemistry, computational science and structural biology) enhances the overall design, analysis and communication process: More learning cycles per unit time = more knowledge and experience = better quality output. More, betterinformed decisions = less waste.

This collective experience has consolidated the view that successful small molecule drug discovery requires a deep understanding of biological context coupled with knowledge and experience of the property space required to deliver safe, efficacious drugs. DMPK continues to be a key player in this success since the major sources of attrition – clinical efficacy and safety – are clearly linked to exposure in key tissues either on- or off-target.

Collectively, this group of experienced scientists has made significant contributions to discovery projects throughout their careers: > 90 development candidates Named inventors and authors on > 750 patents and publications

Evotec offers a comprehensive and flexible range of in vitro and in vivo DMPK studies designed to cover the majority of activities in the screen-tocandidate phase. The scientific experWe understand that each partner has tise and processes are accessed in a different needs, internal capabilities variety of ways by our partners and and capacities and Evotec prides project teams. itself in being able to provide flexible, innovative and efficient solutions.

IN VITRO COMPOUND SCREENING AND PROFILING Evotec’s experienced scientists can Routine compound testing support your aspirations in a variety (e.g. weekly cycle times) of ways, providing mechanistic Evotec’s combination of routine in interpretation as well as timely vitro assays and our flexibility to delivery of quality data. We firmly address your individual requirements believe that the co-location and enables us to offer a truly bespoke, interaction of disciplines (e.g. DMPK, differentiated service: Experienced

SO

T RP

ION

R

AB

RE

TIO

N

TION Metabolic stability Microsomes Hepatocytes Plasma issues Metabolite identification in vitro, in vivo Drug-drug interactions CYP inhibition, induction

ME TA

For integrated projects, a range of physico-chemical assays are availa足 ble together with analysis of permeability (PAMPA, Caco-2, MDCK II), metabolic stability (plasma, microsomes and hepatocytes for a range of species), plasma protein bindMetabowl studies ing (multiple species) and drugBile duct cannulation drug interaction (DDI) potential Transport (cytochrome P450, CYP, inhibition).

Recent additions to our portfolio include human transporter assays (e.g. OATP1B1) and CYP induction analysis (PXR receptor, HepaRG cells or cryopreserved human hepatocytes). Additional bespoke studies Evotec with unique insight in several are often accommodated to trouble- areas. shoot emerging challenges within projects.

STAND-ALONE SCREENING Automated, high-throughput Estimation of human pharmaco足 assays allows for profiling of several kinetic parameters, dose and exposure hundreds of compounds per week provides a contemporary framework against a broad panel of DMPK assays for multi-parameter optimisation.

Tailored packages designed to address a specific problem associWorking with multiple partners ated with a compound, or more across a variety of sectors provides frequently, a series of compounds

DIS T

Plasma protein binding Blood: plasma partitioning Tissue binding Transport PK

U IB M LIS

Aqueous solubility LogD PAMPA Caco-2 MDCKII Transport HIF stability PK

BO

DMPK scientists delivering data of excellent quality coupled with validated, cost efficient and highly automated processes. Interpretation of data and consultation is provided by our DMPK experts within integrated project teams.

In the pharmaceutical industry, early assessment of liabilities of potential drug candidates is an important element to decrease the high attrition rate in the drug discovery and development process. One of the key challenges is optimising the balance between drug efficacy and potential adverse effects at the earliest possible point in time for de-risking costintensive activities especially during late-stage clinical development.

EX C

SECTION 7

Once key liabilities are identified, Examples of capacity for our higher throughput assays DMPK assays can be applied to rapidly design out such properties, ASSAY CAPACITY (WEEKLY) sometimes making use of quantitative structure-activity (QSAR) analysis.

Plasma protein binding (multiple 100 To meet this demand, Evotec has species) established an industrialised high144 throughput profiling platform for Permeability (e.g. MDCKII) routine compound screening against Microsomal stability (multiple species) 200 a panel of the most critical targets: Hepatocyte stability (multiple species) 100 Potential DDIs can be determined by using our panel of cytochrome P450 (“CYP”) reversible inhibition (“RI”) assays for the major CYP isoforms.

- Assays for the four most highly relevant CYP isoforms are established on our high-throughput RapidFire/MS platform.

- Medium-throughput LC/MS/MS based assays are provided for CYP1A2 and 2C19.

For CYP3A4, analysis of timedependent inhibition (“TDI”) and induction are also provided.

In support of discovery chemistry programmes, externalisation of such activities is providing a unique business opportunity for some partners to reserve internal capacity for core research activities, expand their assay portfolio and to mitigate risk.

Pharmacological sensitivity and statistical robustness of the DMPK assays is routinely monitored by a range of reference compound DMPK assays are performed according to validated SOPs and provide 8-point IC50/EC50 for test compounds.

Turnaround time from compound reception to data reporting is 7 working days. Assays are scaleable towards higher throughput.

CYP inhibition (reversible)

200 – 800 (CYP-dependent)

CYP inhibition (time-dependent, CYP3A4 IC50 shift)

700

CYP Induction (hPXR)

800

TECHNOLOGY PLATFORM AND EXPERT KNOWLEDGE

Within many collaborations with major pharmaceutical and biotech companies, Evotec has successfully provided expert knowledge in development and integration of complex processes for our collaborator’s drug discovery programmes including: Support logistics of compound shipments in close interaction with global carriers.

Management of large libraries and regularly shipped smaller compound batches for DMPK or SAR profiling.

Broad expertise in development and validation of > 500 biochemical, biophysical and cell-based assays.

Compound profiling using benchtop and fully automated screening platforms.

State-of-the-art bioanalytical readout technologies including e.g.

RapidFire/MS, FLIPR, SPR, fluorescence, radiometric, high-content imaging.

Flexible data analysis to provide customised formats enabling rapid

data uploads to internal or external data bases Professional project management with experienced leads IN VIVO Pharmacokinetics & PKPD support

Obtaining pharmacokinetic data is a key requirement in the evaluation of new chemical entities providing validation of any in vitro-in vivo extra­polations and an understanding of ADME processes in pre-clinical species which can provide confidence in translation to Man and/or highlight key risks. As part of Evotec’s integrated drug discovery platform, in vivo pharma­cology studies are also further supported by DMPK and histology.

Evotec offers:

Various administration routes: intravenous (incl. infusion), per os (PO), intraperitoneal, subcutaneous, intra-cerebrospinal and intra­ muscular.

Cassette PK screening of up to 5 compounds simultaneously.

Different sampling routes:

jugular vein cannulation, cardiac puncture, tail vein microsampling, retro-orbital.

Different matrices: blood, plasma, cerebrospinal fluid, tissues, bile, urine and faeces.

Data generated with the latest version of Phoenix® WinNonlin®6.3 software, analysis performed using rigorous acceptance criteria.

Solid state evaluation, salt screening and enhanced formulation for problematic compounds.

Biomarker development and application within projects.

Simulations from early PK data assist study design and relationships between response, concentration and time based on exposure (often in target tissues) support translation to Man and early assessments of clinical dose and associated exposure.

Analysis of biomarkers can greatly enhance this process. Integration of such information in static or ideally dynamic (physiologically-based pharmacodynamic; PBPK) models forms the foundation for DDI and safety risk assessment.

Response

Parameter

Time

Concentration

1

Response

Parameter

B

2

2

Schematic illustrating PKPD for rapid (A) and slower (B) equilibrating compounds

1

Time

Concentration

1000 IV PO

Concentration (ng/ml)

100

10

1 Representative PK profile from an oral bioavailability study

0.1 0

6

12 Time (h)

18

24

100

80

% Maximum Effect

DMPK also provide analysis and interpretation of pharmacokineticpharmacodynamic (PKPD) relationships in support of PD and/or disease models conducted by our colleagues in Biolo­gy. It is important to emphasise here the need to study concentration–time and response– time profiles since the temporal relationship between these varies with different targets and the position of a target in a biological pathway. Hence the time to equilibrium between the plasma and target tissue can be very rapid (e.g. anti-thrombotic agent) or delayed (e.g. anti-psychotic).

A

60

40

PK-PD plot for range of compounds illustrating value of considering unbound concentration in relation to affinity for the target

20

0

0.01

0.11

1 10 Unbound [Plasma]: Target IC50

100

SECTION 8

PROTEOMICS METABOLOMICS AND BIOMARKER DISCOVERY OFFERING Evotec offers unique proteomics and metabolomics services to address key issues in drug and biomarker discovery. We continuously advance our capabilities in mass spectrometrybased proteomics and metabolomics to ensure unrivalled comprehensiveness and data quality when analysing cells, animal models and patient samples.

PROTEOMICS

Evotec`s proteomics platform encompasses: Highly optimised experimental strategies tailored to different proteomics applications and project needs

Henrik Daub Senior Vice President Chemical Biology

TARGET DECONVOLUTION

TARGET DISCOVERY

CELLULAR MODE-OF-ACTION ANALYSIS TO SUPPORT LO

HT SCREENS/ SCAFFOLD SELECTION

Target ID & validation

Screening

ON/OFF TARGET PROFILING

MODE OF ACTION ANALYSIS

H2L

LO

EVOTEC CELLULAR TARGET PROFILING™ TO SUPPORT H2L Evotec Cellular Target Profiling™

Industry-leading capabilities in high-end quantitative mass spectrometry Experience and infrastructure to analyse the enormous amounts of data generated in large-scale proteomics projects Advanced statistics and bio-informatics for systems-wide data analysis In-depth data interpretation to extract relevant drug target and mode-of-action information from proteomics experiments Extensive track record to deliver high-quality results within agreed timelines

Subproteomic Evotec Cellular Target Profiling™

RESPONSE PREDICTION

Pre-clinic

DRUG REPOSITIONING

BIOMARKER ASSAYS

PhI/II

PROTEOMIC SIGNATURES FOR BIOMARKER DEVELOPMENT Omics Technologies (Phosphoproteomics, Acetylomics, Proteomics)

technology captures target specificity Evotec’s chemical proteomics and affinity information by methods such as Evotec Cellular quantifying target binding across Target Profiling™ reveal cellular defined sets of affinity purification drug targets and selectivity in an and drug competition experiments. unbiased manner. Other Evotec proteomics technologies record Evotec Cellular Target Profiling™ protein modification and expression Reveals and verifies specific on a proteome-wide scale for cellular targets of a drug by drug mode-of-action analysis or quantitative mass spectrometry biomarker discovery efforts. Thus, Determines target-specific dissociation our platform offers highly innovative constants for the compound studied, solutions that can be leveraged ranking targets according to their across the entire drug discovery and likely physiological relevance development pipeline.

Performs selectivity analysis on a proteome-wide scale against native, post-translationally modified QUANTITATIVE proteins in the pre-sence of cellular CHEMICAL PROTEOMICS co-factors and complex partners Evotec scientists have pioneered chemical proteomics applications for Has an extensive, non-target the identification of cellular small class restricted track record in molecule targets. The basic concept successful profiling of diverse involves functional immobilisation small molecule compounds of an appropriate linker derivative of a drug, followed by affinity Selectivity data about cellular onpurification of target proteins from and off-target liabilities is particucell or tissue extracts and finally their larly useful to inform decisions at identification by MS analysis. Unlike various stages of drug development, other competitor technologies, our for example in the lead optimisation Evotec Cellular Target Profiling™ phase or in the pre-clinical candidate

selection process. Moreover, chemical proteomics enables target deconvolution of bioactive compounds identified in phenotypic screens. Cellular target identification is the crucial step to enable further drug optimisation and development. Evotec Cellular Target Profiling™ perfectly complements Evotec’s medicinal chemistry and high-content screening capabilities to deliver new drugs and targets across many therapeutic areas.

Numerous small molecule kinase inhibitors are currently being developed for treatment of cancer, inflammation or autoimmune diseases.

However, drug discovery faces significant challenges, particularly due to unacceptable safety pharmacology profiles caused by an inhibitor’s lack of selectivity. Undesirable off-target kinase profiles frequently induce toxicity that might halt the development of candidate drugs. To decrease the high attrition rate the design of selective as well as multi-specific inhibitors is becoming increasingly important.

Evotec Cellular Target Profiling™ key elements Test compound

1 Cell or tissue sample

2 Enrichment of target proteins by compound affinity chromatography

3 Competition of target proteins by test compound

4 LC-MS analysis

(1) A lysate of the cell line or tissue of interest is prepared. (2) A linker derivative of the test compound is immobilised on sepharose beads and used to enrich compound target proteins. (3) Competition assays with the free test compound and (4) quantitative mass spectrometry analysis reveal the compound’s affinity profile, (5) ranking all protein targets according to their Kd values for free compound.

5 List of target proteins ranked according to their K d values Sepharose resin Linker derivative of test cpd test cpd Target proteins

SECTION 8

KinAffinity® is Evotec’s hit-to-lead compatible approach for rapid target profiling of kinase inhibitors in cell and tissue samples. Unlike traditional biochemical kinase panel screening, the inhibitor’s target affinities are determined simultaneously for a large number of native kinases within their physiological cellular environment, thereby complementing traditional biochemical assays that use only recombinant proteins.

KinAffinity® employs a ready-touse affinity matrix comprising wellcharacterised broad-spectrum kinase inhibitors to enrich the subproteome of endogenously expressed kinases of cells or tissues.

This enables rapid determination of kinase target Kd values for free

kinase inhibitors without needing to immobilise the inhibitor.

Evotec’s third chemical proteomics platform makes use of reactive drug conjugates for covalent target capture, enabling applications such activity-based protein profiling (ABPP) of a wide range of enzyme classes including serine hydrolases, metalloproteases, oxidoreductases, histone deacetylases, and glutathione S-transferases. ABPP visualises only the active forms of particular enzymes using reactive chemical probes directed to the active site of a particular target protein (or protein family).

Besides the monitoring specific enzymatic activities, ABPP can also be used to identify and characterise (unknown) protein functions, to study up- and down- regulation of enzymatic

activity in various disease states, to discover and evaluate putative new enzyme inhibitors, and to identify the protein targets of covalently binding natural products.

Evotec has also developed Photo­ Affinity Labeling combined with Mass Spectrometry (PALMS), a powerful technology to identify the target(s) of a drug, to determine the affinity and selectivity of drugtarget interaction, to determine drugtarget stoichiometry, and to identify ligand binding sites. PALMS uses an analog of a biologically active ligand (small-molecule, peptide), known as a photoaffinity probe, that bears photo-reactive and reporter functional groups, to identify protein binding partners. During PALMS, the photoaffinity probe binds proteins through reversible affinity interaction.

PhosphoScout® Workflow

Isotope labelling

UV-irradiation of reversible complexes generates a covalent bond between the photoaffinity probe and target proteins. Photo-crosslinked protein targets can be visualised by the reporter group using conventional imaging and identified using affinity purification combined with quantitative mass spectrometry.

GLOBAL ANALYSIS OF PROTEIN MODIFICATIONS

Proteins

Peptides

Global phosphoproteome enrichment

LC-MS

Evotec’s proteomics platform for global protein modification analysis permits the identification and quantification of about 15,000 phosphorylation sites, 1,000 lysine acetylation sites, more than 5,000 ubiquit­ ination sites, or several hundred arginine methylation sites in a single experiment.

Moreover, Evotec has established bioorthogonal chemical reporter strategies based on non-native, non-perturbing chemical handles that can be modified in living systems through highly selective reactions with exogenously delivered probes.

These approaches allow specific profiling of modification-specific sub-proteomes including N- and O-linked glycoproteins, O-GlcNAcmodified glycoproteins, sialic acidmodified proteins, fucosylated glycoproteins, palmitoylated ­or myristoylated proteins and iso­­ prenylated proteins.

Such studies may be performed in cells, tissues or patient samples.

Accurate quantification is enabled by differential isotope labelling and label-free approaches, while highly optimised peptide enrichment and analysis on fast, sensitive and accurate mass spectrometers ensure highest coverage. Evotec’s platform provides highly reliable results,

both for in-depth bioinformatics involving enrichment, cluster, network andmotif analysis, as well as for expert data interpretation on the level of site-specific changes.

Our quantitative phosphoproteomics ­(PhosphoScout®) ­enables ­the un­biased and ­comprehensive ­ investigation of signalling pathways, to delineate the cellular modes of action for kinase inhibitors. Typical applications include: Identification of specific pharmacodynamic read-outs for therapeutic kinase inhibition Selection of lead compounds or pre-clinical candidates with maximal on- and minimal off-target activity in cellular conditions, either as single agents or in combination with other drugs Monitoring phosphoproteome regula­ tion in different biological models to shed light on factors underlying differential biological activity of lead compounds or pre-clinical candidates In addition to PhosphoScout®, Evotec’s acetylomics and ubiquitinomics platforms offer similar applications for HDAC and E3 ligase and methyltransferase inhibitors using highly optimised workflows with regard to sensitivity and selectivity. As a result, these technology platforms enable comprehensive mode of action studies in the biologically relevant context.

GLOBAL PROTEOME PROFILING AND BIOMARKER DISCOVERY

Evotec offers industry-leading plat­ forms for comprehensive protein expression profiling allowing unbiased, proteome-wide target and

SECTION 8

signatures in lung cancer cell lines, mouse xenograft models and leukemia patients.

In conclusion, Evotec offers an integrated and unique platform of innovative proteomics technologies for drug and biomarker discovery.

Moreover, Evotec is constantly investing in R&D activities dedicated to advance its high-end proteomics capabilities and maintain industry leadership in the years to come.

METABOLOMICS

biomarker discovery on the functional protein level. Depending on the application and required throughput, tailored proteome analysis workflows are available: Deep proteome profiling for de­tection of up to 10,000 proteins from cell or tissue samples upon efficient peptide pre-fractionation Single-shot proteome profiling to a depth of 5,000 proteins in cell or tissue lysate, enabling comparative proteome analysis across 100+ samples Comprehensive analysis of any type of biological material, including formalin-fixed paraffin-­ embedded (“FFPE”) solid tumour samples and body fluids such as plasma and CSF.

Targeted mass spectromety analysis by Multiple Reaction Monitoring (“MRM”) and Parallel Reaction Monitoring (“PRM”) fully established at Evotec for follow-up validation experiments

Both the expertise and the data processing infrastructure are in place for accurate quantification across many different samples, such as whole panels of cell lines, xenograft models or patient tissue and body fluid samples. Such data, together with drug response information, provides a unique basis for the discovery of predictive protein biomarkers, arguably one of the most exciting proteomics applications in future medicine.

EVOTEC’S THREE-TIER STRATEGY TO SUPPORT ONCOLOGY BIOMARKER DISCOVERY

Evotec technologies cover the entire biomarker discovery phase including elaborate bioinformatics for the identification and verification of predictive multivariate markers, so-called protein signatures. These capabilities have been demonstrated in various cancer-­ related projects, delivering response prediction

Metabolomics can be defined as the quantitative assessment of the metabolic responses of a biological system (cell, tissue, organ or biological fluids) at a particular time and to measure the changes in the metabolic response when exposed to any pathophysiological stimuli and/or genetic alterations. Metabolomics have a potential role in the pharmaceutical research and development (R&D), starting from drug discovery to clinical development and even beyond.

Evotec has established expertise and state-of-the-art methodologies in targeted-metabolomics for the functional analysis of metabolic networks with applications in health, biotechnology and microbiology.

Evotec offers unique metabolomics services to address key issues in biomarker discovery and/or validation in various therapeutic areas including cancer, metabolic syndrome, diabete, CNS and cardiovascular diseases. Applications of metabolomics include pre-clinical biomarker identification, disease mechanism and validation of animal models for disease, and the discovery of biomarkers for patient stratification as well as clinical safety and efficacy assessment.

Our targeted metabolomics approach focuses on the analysis of specific group of metabolites related to certain metabolic pathways or a class of compounds i­ncluding ­endo­ c­annabinoids, eicosanoids, ­cortico­ steroids, neurosteroids, oxysterols, nucleotides free fatty acids, and nicotinamide metabolome. ­ ­A detailed list of all compounds which have been measured and quantified so far is available upon request. Due to its sensitivity and specificity, our approach allows absolute quantitation

of metabolites using selected reaction monitoring (SRM), with detection limit of ng/mL, in sample matrices (e.g., biofluids, cells, organs) and also in-vivo on awake or anesthetised animals using microdialysis coupled wit h XLC-MS/MS. IN-VITRO/EX-VIVO TARGETED METABOLOMICS (CHRONIC STUDIES)

Absolute quantification of metabolites is performed in complex matrices including animal body fluids and

1

Pre-clinical biomarker discovery in cultured or primary cells

Early identification of predictive biomarker candidates by proteome profiling of cell line panels

2

Pre-clinical biomarker discovery in animal models

Unbiased biomarker identification from mouse xenograft models by global and targeted proteome analysis

3

Clinical biomarker in patient samples

Proteome-wide biomarker identification from patient-derived leukemia cells or FFPE tumour tissue

tissues (brain, adipose tissue, liver, intestine, muscle, tumours, plasma, blood, serum, urine, CSF), human body fluids and tissues (plasma, serum, blood, CSF, adipose tissue, brain), and cell lines (cells and culture medium), enabling comparative metabolome analysis across 100+ samples.

Measurements in further matrices can be established upon request.

IN VIVO TARGETED METABOLOMICS (ACUTE STUDIES)

Relative quantification of metabolites is performed directly in awake (1 probe) or anesthetised (2 probes) animals.

Microdialysis can be performed in many tissues including brain skeletal muscles, adipose tissues, liver, tumours.

In kinetic studies, animals can be ­monitored continuously for 24 to 48 hours with a time-point generally every 30 min.

SECTION 9

CELL & PROTEIN PRODUCTION

Evotec’s wealth of expertise covers all principles including cell line generation, small- or large-scale transient trans­ fection, frozen cell technologies and protein expression from small-scale proof-of-concept studies through batch production for HTS, counter screening activities as well as structural biology applications. For clients looking to outsource the management and supply of their own cell collections, Evotec offers a cell bank management scheme, establishing, propagating and distributing thousands of cell lines between various locations.

Joachim Kraemer Senior Vice President Bioreagents, Biophysics and Assay Development

OFFERING Evotec provides access to a comprehensive base of services and capabilities around cell and protein production. These can be accessed as a stand-alone function or as an integral part of a wider collaborative programme accessing our drug discovery expertise. Combining expertise in cell culture and protein production methods with expertise in functional assays, assay development, HTS and compound profiling offers clients the best-possible platform for cost-efficient and timely delivery of highest quality data for their drug discovery programmes.

100L wave system for mammalian or insect cells and all the related separation techniques in a controlled environment.

Relevant in-process and final controls are elaborated and run to document quality prior to batch release.

CELL LINE GENERATION AND BANKING

From recombinant cells which have been either generated by stable transfection or viral transduction, monoclonal cell lines are isolated and screened for their functional expression of the target protein. After a first broad screening on expression level In 2015 the offering for reagents has using immuno­ fluorescence labelling, undergone a rapid expansion with Western blot, or RT-PCR, usually a new facilities opening in Princeton, secondary screening of selected clones NJ, on the East Coast of the US, to by a cell-based assay is applied. service the North American market, and the acquisition of the Sanofi site There are numerous reasons why a in Toulouse, France. In Princeton target may not be stably expressed the team offers rapid cycle times of in mammalian cells and a transient the highest quality in cell and protein approach may be more appropriate for production with a focus on membrane cell-based screening. To address this proteins.

In Toulouse, the team problem, Evotec routinely uses a variety brings new capabilities in large scale of transfection technologies from lipid ­microorganism or cell culture processes based to viral mediated delivery to as well as the relevant purification achieve transient expression. steps to provide larger amounts of high grade quality proteins batches. It also Particularly the MaxCyte e­lectropo­ incorporates scientists with research, ration platform offers access to large development and industrial expertise batches of up to 10 billion cells, a costin the biotherapeutics field (from effective and efficient transfection recombinant hormones, enzymes to process. Transiently transfected or antibody production). The team is expert virus transduced cells can be provided in adapting laboratory scale results to as assay-ready frozen instant cells, large scale equipment including 20L to which are functionally expressing the 450L bioreactors for bacteria or yeast, target instantly after thawing.

Cell line generation

(n=81 projects)

The availability of cells is a critical bottleneck in screening campaigns.

Frozen instant cells, which can be Others 13 assayed without prior cultivation have become a valid and frequently used Reporter cell lines 3 alternative to cells from a continuous Enzymes 6 growing culture.

Evotec’s cell culture unit was one of the first suppliers of frozen instant cells for global pharmaceutical research.

Cells are: Expanded under controlled culture conditions to large bulk quantities Harvested applying a gentle method which does not affect surface receptors Prepared to a density which corresponds to the requirements of your assay, the cells are dispensed into vials using an automated filling device. This fast processing of the cells significantly decreases the time from harvest to freezing, which is critical for maintaining the quality of frozen instant cells Frozen in a controlled rate freezer and stored in the vapour phase of a N2 tank until they are shipped.

For your ligand binding assays or in vitro transporter activity assays, we offer cell membranes in bulk in two different purity grades. All batches of cells and cell-based products are carefully controlled for their quality before they are shipped to you. Frozen instant cells are checked not only for viability but also for their ability to adhere shortly after thawing. A good “fitness” of the cells is often correlated with fully functional response.

Finally, the assay performance of the frozen cells is compared to cells from a continuous growing culture considering Z’, S/B and EC50.

Kinases 4

GPCR 28

Ion channel 11 Transporters 9 NHRs 2 Cytokine receptors 5

Transient transfections (n=61 projects)

GPCR 13 Others 20

Enzymes 3 Proteases 13

Ion channel 10 Transporters 2

Membranes (n=57 projects) Others 16

Transporters 9

GPCR 19

Ion channel 13

Assay ready frozen cells (n=300 projects)

GPCR 118

Others 51 Ion channel 76 Primary cells 10 Kinases 15 NHRs 11

Transporters 19

SECTION 9

DNA Engineering

Stably Transfected Cells

Transient transfection

Non Transfected Cells

Bulk Cell Production

Frozen Instant Cells

Membrane Preparation

Custom Cell & Reagent Supply

Evotec offers professional management and maintenance of client cell banks encompassing the addition of new cell lines and shipment of assay ready cells and vials to the clientâ&#x20AC;&#x2122;s research centres. A dedicated team with access to the necessary bioinformatics, quality control processes and infrastructure can additionally provide support in the sourcing and maintenance of disease relevant cell banks such as oncology. The databases covering all aspects of the respective cell line such as growth rates, morphology and genetic confirmation are available over a web-based client portal providing

Membrane Preparation

Evotecs Screening Services

clients with the essential access to all protein production facilities provide necessary information. comprehensive coverage of expression hosts, purification techniques and analysis tools and delivers a unique RECOMBINANT solution for the efficient delivery PROTEIN PRODUCTION Protein production lies at the heart of of proteins in a timely fashion. Our drug discovery. Evotecâ&#x20AC;&#x2122;s recombinant capabilities can be accessed flexibly, either as standalone units or as part of an integrated drug discovery programme.

Construct Cloning & design

Quality

expression

Protein purification

All proteins are prepared with end use in mind, including the delivery of high-quality proteins for bio-assay and biophysical applications.

Labelled proteins for NMR and Mass Spectroscopy

Biotinylated proteins for Surface Plasmon Resonance Yields and quality suitable for Isothermal Calorimetry, Differen­tial Scanning Fluorimetry and Microscale Thermophoresis High through-put protein purification and construct triage to deliver crystal grade material for macromolecular X-ray crystallo­graphy

Specific expertise in the team covers Expression hosts in 2014, the common protein families and also >550 production runs completed extends into the more demanding areas of macromolecular complexes (protein:protein and protein:DNA) and membrane proteins.

E. coli Insect cells 68,3% The team has unparalleled experience 27,5% in labelling of proteins for NMR work including ubiquitous 15N, 13C and 2H in E. coli and yeast and side chain The production platform successfully specific labelling in insect expression Pichia pastoris Mammalian deliv­ers on hundreds of recombinant hosts.

0,7% 3,5% proteins per year to our clients.

In addition to purification of proteins Uses of protein All main expression hosts are by SOPs or according to literature supported by Evotec with shut­tle precedents, Evotec has extended vectors available to rapidly move methods commonly employed in expression to higher organ­ isms as structural genomic facilities so Assays Crystallo required. Gene synthesis is performed that the team can perform high33% 47% by a series of secure third party rela- throughput (“HTP”) expression screening in E. coli, insect and tionships with confirmed timelines. mammalian hosts to determine The experienced team of scientists optimal conditions for production will determine optimal host for ahead of parallel purification or large Other NMR 6% 14% expression and yield and develop scale production runs. Large scale suitable protocols for purification expressions can be carried out in using a suite of tech­niques, including parallel, either in shake flasks or in semi-automated expression analysis WAVE cellbag bioreactors. and purification.

E. coli up to 295 L/week + 450 L fermentation vessel

Tags: no tag, His (N- or C- terminal), GST, MBP, Trx Strains: BL21 (DE3)/pLysS, ArcticExpress, BL21-AI Secretion signal peptide (if needed): pelB

Pichia pastoris up to 40L/week

Tags: no tag or C- terminal His-tag Strain: X-33 Secretion signal: α-Factor

Tags: no tag, His (N- or C- terminal), GST, MBP Insect cells Strains: Sf21, Sf9, T. ni (Baculovirus) up to 160 L/week + Secretion signals (if needed): gp67, honeybee 100 L wave system melittin, wild type Mammalian cells up to 80 L/week + 100 L wave system

Tags: no tag, His (N- or C- terminal), GST, MBP Strains: CHO, HEK293, CAP-T, EXPi293 Secretion signals (if needed): wild type, lgk

SECTION 9

Option 2: 8/week

Option 1: 16/week

IMAC HisTrap

IMAC HisTrap

(ÄKTAxpress)

(ÄKTAxpress)

Insufficient yield

QC expression optimisation

NO

SEC

Insufficient yield

SDS-PAGE, Western blot

Yield and Quality OK?

YES

Dispatch

With a range of ÄKTA purification modules (from Purifiers to Pilots) and HTP methods suitable for costeffective parallelisation, Evotec has the capacity to deliver significant quantities of proteins in parallel using a variety of purification schemes (see figure above).

All proteins pass stringent in-process quality controls, including Mass Spectroscopy, Dynamic Light Scattering, Differential Scanning Fluorimetry, Activity Testing and NMR, before final release to the end user. In summary, by tailoring the purification schemes to the problem to be solved and using appropriate input from scientists on the projects, Evotec delivers the target protein optimised for the end use.

3000 [PH]-ZM241335 binding [cpm]

Demonstrated delivery of crystal grade membrane proteins (here for example the Adenosine A2a) with comprehensive QC package including Radio Ligand Binding assays available to the production teams

2500 2000 1500 1000 500 0 0

2

4

6

8

10

[PH]-ZM241385 Concentration [n/M]

LABELLING CHEMISTRY

Evotec offers labelling of biological relevant molecules and reagents for use in ELISA, FACS, FCS, plate readers and other fluorescence based detection systems. Our service helps clients to improve their research results in all areas of biological and pharmacological assays, as well as clinical diagnostics and analytics.

Our expertise covers the labelling of the following: Small organic molecules e.g. hormones, neurotransmitters Peptides and chemokines (as enzyme substrates, GPCR ligands, etc.)

Proteins including antibodies Site-specific mutagenesis of proteins (for site-specific labelling) Glycoproteins

Carbohydrates Lipids (for membrane studies) Beads and surfaces Evotecâ&#x20AC;&#x2122;s labelling chemistry team has acquired a comprehensive expertise having successfully designed and labelled components for more than 150 different assays representing a broad range of assay classes. For labelling we use the optimally suited dyes and tags for your application, such as: Proprietary dyes (EVOblueâ&#x201E;˘ family) Commercially available fluorescent dyes and the in-licensed MR121 dye Non-fluorescent markers and probes like affinity tags, biotin, reporter enzymes, etc.

SECTION 10

IN VITRO

PHARMACOLOGY, MICROBIOLOGY & TRANSLATIONAL SCIENCE In addition to its extensive capabilities in mammalian biological systems, Evotec has in depth in vitro microbiology expertise spanning a broad range of pathogens from bacteria to fungi and viruses.

Andreas Scheel Executive Vice President In vitro Biology

OFFERING The cornerstone of Evotec’s in vitro pharmacology function is disease and target biology expertise coupled with state-of-the-art technology platforms. A large team of scientists with extensive industrial experience supports the in vitro pharmacological characterisation of compounds as part of hit expansion, lead finding and lead optimisation projects. Our team routinely generates project-relevant high-quality data in short turnaround times. In addition, we support in vivo studies with PD readouts and engage in early translational biology research.

Typical activities include: Development of biochemical and functional assays Secondary and tertiary characterisation of screening hits Compound profiling as part of hit-to-lead and lead optimisation programmes: — Design and implementation of target-relevant assays (screening cascades) — Potency and selectivity testing — Mode of action studies (e.g. competitive versus allosteric mechanisms, reversibility, usedependent mechanisms for ion channel modulators) — Translational cellular assays to bridge the gap between in vitro and in vivo studies: testing of compound potency and mechanism using disease-relevant primary cells from rodents, primates or human — Translational biomarkers: building assays in the discovery phase that allow measuring target engagement in patients and thus de-risking of clinical projects In more than 15 years of compound profiling at Evotec, >400 assays have been developed and executed.

Such activities are part of Evotec’s integrated lead finding and optimisation projects but are also frequently used to support medicinal chemistry projects that are executed in the labs of Evotec’s partners.

AVAILABLE READ-OUT AND ASSAY TECHNOLOGIES

Evotec has access to a wealth of assay technologies that can be utilised to assess compound activity. Appropriate technologies and expert teams are selected to answer key project questions and to ensure project advancement.

Beyond standard and established read-out technologies for biochemical and cellular assays, Evotec has built key expertise in a number of technological areas that have shown to be drivers for the success of our partner’s projects.

These include: The use of stem cells to derive neurons and primary neuronal cultures to build disease-relevant cellular models and to identify and characterise new compounds with disease-modifying properties Studying compounds modulating beta cell proliferation

Insulin

BrdU

An extensive knowledge of highcontent screening and a state-ofthe-art hardware platform to run complex and disease-relevant imaging assays, e.g. using primary neuronal cultures, kidney cells, muscle cells as well as rodent and human beta cells

A world-leading ion channel discovery platform including fluorescence-based assays, auto-

mated and manual patch clamp methods An excellent suite of bio­physical methods including SPR and NMR that are utilised as part of our structure-based drug design projects but also LC-MS-based methods that are utilised to assess difficult-to-assay enzyme targets In the oncology area, the setup of integrated assays based on tumour

cells, fibroblasts, macrophages, endothelial cells, or adipocytes in 2D, co-culture or 3D culture to recapitulate tumour and microenvironment conditions The use of extensive read-outs, from signalling to metabolic pathway engagement, to develop new ways to address pathologies and overcome resistance to treatments

Neuroprotective compound

Read-out technologies CELL-BASED ASSAY TECHNOLOGIES

Fluorescence read-outs —H TRF, standard dyes, ligand binding Second messengers (Ca2+, cAMP, IP3) Membrane potential (GPCRs, ion channels and transporters) Manual and automated patch clamp Reporter gene assays ELISA (standard and Mesocale) Imaging (HCS) — OPERA®, Operetta, Zeiss and ArrayScan Radioactive binding and uptake Flow cytometry and cell sorting Migration and invasion assays Whole cell blood assays Metabolic analysis (seahorse, metabolomics, mitochondrial OXPHOS function) Primary cell culture Stem cells Established cell lines — Co-culture — 3D culture

BIOCHEMICAL ASSAY TECHNOLOGIES

FCS+plus Fluorescence polarisation Fluorescence intensity HTRF/Delfia AlphaScreen ELISA Mesoscale electrochemiluminescence Singulex single protein molecule counting Luminescence LC/MS BIOPHYSICAL READ-OUT TECHNOLOGIES

Surface Plasmon Resonance (SPR) Mass Spectrometry (LC-MS) Nuclear Magnetic Resonance (NMR) Radiometric Thermal Shift

SECTION 10

OVERVIEW TARGET CLASSES

Our in-depth experience in the biology and pharmacology of diseaserelevant target classes is what our partners come to us for. This expertise is a key driver to the successful and rapid execution of lead finding and optimisation processes. Evotecâ&#x20AC;&#x2122;s in vitro pharmacology team has gained expertise across a wide area of disease targets. Beyond a large number of projects that have successfully been run in the classical target areas such as GPCRs, ion channel and kinases, we have also worked on a large diversity of other target areas such as transporters, proteinprotein interactions and multiple enzyme families, including novel target classes such as epigenetic regulators and immune and metabolic pathways modulators.

Identifying cpds that protect podocytes in chronic kidney disease

Neurite Total Length

setting up relevant in vitro models and translational biomarker assays for pre-clinical and clinical use are Evotecâ&#x20AC;&#x2122;s core expertise includes a key factor in our success when areas such as CNS, neurodegenera- working with our partners. tion, pain, inflammation, metabolic disease, oncology and immunology. Setting up complex in vitro assays Our scientific expertise and under- utilising rodent or human primary standing of disease mechanisms cells to confirm the potency and combined with our track record in mechanism of lead compounds is a prerequisite to build confidence in the translatability of any mechanism. We build these assays Co-culture system of stem cell derived motoneurons and astrocytes together with microglia to identify early in the drug discovery process cpds for the treatment ALS to gather disease-relevant data before assessing compound efficacy 100K in vivo. Such assays are also utilised to identify read-outs for in vivo 80K target engagement. We routinely 60K utilise various neuronal and stem cell cultures, pancreas and beta cells, 40K kidney cells and various immune cells, tumour cells, endothelial cells, 20K fibroblasts, adipocytes and muscle cells to assess bespoke read-outs and 2 3 4 5 6 7 mechanisms, including cell health Days After Plating D3 After Plating GFAP/GFP/DNA and survival, metabolism, apoptosis, DISEASE EXPERTISE, TRANSLATIONAL ASSAYS AND BIOMARKERS

reduce the risk of a failed trial due to inadequate clinical read-outs.

Robust and objective biomarker read-outs that are applicable to clinical samples are therefore needed A key challenge for bringing a new to stratify patient populations, therapy to the market is to provide quantify disease progression and to proof of efficacy in the clinic and to demonstrate target engagement and differentiation, de- / regeneration; neurite outgrowth, retraction and synaptic density; cellular signalling and secretion.

dosing response. Together with our partners, we utilise state-of-the-art single molecule counting Singulex technology to routinely develop, validate and apply ultra-sensitive protein biomarker quantification read-outs for clinical samples such as human blood, plasma and CSF.

Extensive expertise with translational cellular assays using primary cells and tissues to investigate compound potency and mechanism

Rodent glomeruli

Synaptic connectivity

Human kidney cell fibrosis

Replicating pig beta cells

ISOLATION, CULTIVATION AND MANIPULATION OF PRIMARY CELLS

Neurobiology: neurons (CNS, DRGs, moto­neurons), astrocytes, microglia including co-cultures Blood: PBMC, TH1/TH2 populations, B cells Pancreas: islets, beta cells Kidney: podocytes, glomeruli

ASSAY READ-OUTS

Cell density, degeneration, regeneration, survival Cytokine secretion Transcrptional activity Protein phosphorylation

ASSAY TECHNOLOGIES

Imaging MSD LC-MS Standard methods

SECTION 10

Streptococcus pyogenes cultured on Columbia Blood Agar

MICROBIOLOGY Evotec’s specialist group in the infectious disease therapeutic area boasts state-of-the-art microbiology facilities including a unique and highly characterised strain bank, EVOStrAInTM. Our team provides bespoke anti-infective drug discovery and development services to a growing number of global partners and has an established track record in collaborating to discover and develop new therapies and vaccines to treat and prevent serious and life-threatening infections resulting from multi-drug resistant pathogens including Gram positive and Gram negative bacteria, including the ESKAPE organisms, fungi, and viruses.

a fully integrated anti-infective drug discovery capability. Core strenghts of Evotec’s microbiology group fall into the four areas of EVOStrAInTM, Microbiology, Pharmacology and ADME/PK/PD as follows: EVOStrAInTM is a highly valuable collection of clinical isolates that can be used to establish the activity profile of lead compounds and candidates. A key feature is that the isolates are highly characterised and, in many cases, mechanisms of resistance defined. EVOStrAInTM contains an extensive range of geographically diverse human bacterial and fungal pathogens that cover isolates susceptible and resistant to current antimicrobial drugs.

The group’s offering is fully integrated with the wider discovery platform at Evotec enabling either a standalone microbiology service or

We employ industry-standard methods such as CLSI, EUCAST and BSAC to test compounds for antimicrobial activity against

strains and clinical isolates from EVOStrAInTM, or strains provided by our collaborators. This includes the ability to conduct whole-cell screening for antimicrobial activity for hit identification in an HTS format, MIC, MBC/MFC, timekill and PAE studies using single or combinations of agents, hollow fibre PK/PD or bioreactor human cell systems for detailed profiling for characterisation of novel antiinfective agents, and compound/ drug combination studies for assessment of synergistic, antagonistic and additive effects.

Bespoke methods for susceptibility profiling can be developed for testing novel agents where standardised methods may not be appropriate. In parallel, mechanism of action determination studies and resistance frequency assays can be performed.

EvostrAInTM: A collection of highly characterised clinical isolates available to establish a detailed activity profile of lead compounds, both in vitro and in vivo models of infection BACTERIA: Gram positive pathogens

BACTERIA: Gram negative pathogens

FUNGI

Staphylococcus aureus including MRSA, VISA and VRSA strains

E. coli including Extended Beta lactamase producing strains

Aspergillus spp. (including strains resistant to azoles, polyenes and echinocandins with known mechanisms of resistance)

硫-Haemolytic streptococci groups A, B, C and G

Klebsiella pneumoniae Carba足penemase producing strains (KPCs & MDR)

Candida spp. (including strains resistant to azoles, polyenes and echinocandins with known mechanisms of resistance)

Streptococcus pneumoniae (in足cluding penicillin, macrolide, fluoroquinolone, cephalosporin and MDRSP resistant strains)

Acinetobacter baumannii including MDRAB

Mucorales

Vancomycin Resistant Enterococci (VRE)

Pseudomonas spp. including multi-resistant strains

Cryptococcus

Bacillus species

Haemophilus influenzae

Dermatophytes including Malassezia spp and Trichophyton spp

Listeria species

Bacteroides spp.

Fusarium

Corynebacterium and Propioni足bacterium species

Neisseria gonorrhoeae and N. meningitidis

Protozoa Acanthamoeba spp

Clostridium difficile (multiple ribotypes including 012, 027 and 078)

Intestinal pathogens: Vibrio spp, Campylobacter spp, Salmonella spp, Shigella spp, Yersinia spp.

Other Clostridia (including C. perfringens)

Legionella spp.

Mycobacterium

SECTION 11

ION CHANNELS OFFERING Evotec’s world-class ion channel platform combines industry-driven expertise with state-of-the-art instrumentation.

In routine use are multiple setups for manual patch clamp studies and all of the most popular automated patch clamp instruments. The automated patch clamps or FLIPRs can be used as entry points for HTS, and the manual and automated patch clamps enable on-going support of medicinal chemistry

programmes progressing from HTS through hit expansion, hit-to-lead, lead optimisation but are also utilised for safety pharmacology.

We now offer brain and spinal cord slice electrophysiology to measure the effects of test substances or manipulation of CNS targets on synaptic function.

Stephen Hess Research Leader Ion Channels

The electrophysiology platforms in place at Evotec

SyncroPatch384PE

IonWorks® Quattro™

Qpatch HTX®

PatchLiner®

Manual Patch-Clamp

PLATFORM SynchroPatch384PE

1

384-well

4,000-10,000

High-throughput screening, hit-to-lead

IonWorks Quattro™

2

384-well

4,000-10,000

High-throughput screening, hit-to-lead

Q-patch HTX®

1

48-well

150-450

Hit-to-lead, lead optimisation and early safety assessment

PatchLiner©

3

16-well

75-150

Hit-to-lead, lead optimisation and early safety assessment

Port-A-Patch®

2

1-well

25-50

Manual Electro physiology (DynaflowTM optional)

4

1-well

1-25

Slice Electrophysiology

1

1-well

Varies with assay

®

Hit-to-Lead, Lead optimisation Lead optimisation, safety pharmacology Detailed MOA studies on brain & spinal cord synaptic function

OVERVIEW OF ION CHANNEL SCREENING AT EVOTEC

Evotec has a strong background in running hit identification campaigns against ion channel targets. On average more than 235,000 compounds per target

1

Voltage gated ion channels Potassium ion channels: Kv family Calcium channels: Cav family Sodium channels: Nav family

2

Additional potassium ion channels KCa (e.g. SK, BK, IK) Inward rectifiers (e.g. Kir2.1) Tandem pore domain (e.g. TREK, TASK)

3

Ligand-gated ion channels P2X family (e.g. P2X3, P2X7) Glutamate receptors (e.g. NMDA, AMPA) TRP family (e.g. TRPV1, V3, V4) GABA-A

4

Others CRAC Chloride channel (e.g. CFTR)

are screened in singlicate at a fixed compound concentration.

Campaigns resulted in “primary” hit rates in the range of 1 % and confirmation rates are generally good (> 50 %), underlining robust assay performance. Secondary assays

using electrophysiology to confirm compound activity is part of the routine HTS follow-up and are key to the successful identification of relevant starting points for medicinal chemistry projects.

High-Throughput Screening, Alexander Böcker, Sabine Schaertl and Stephen D. Hess, Ion Channel Drug Discovery, 2014, 16-41 DOI:10.1039/9781849735087-00016

EXPERTISE IN IDENTIFYING CHANNEL OPENERS AND BLOCKERS

TRACK RECORD OF SUCCESS WITH A RANGE OF ION CHANNEL CLASSES

DEMONSTRATED CAPABILITY IN ION CHANNEL DRUG DISCOVERY ACROSS VARIOUS PLATFORMS FROM 1 TO 384 WELL FORMAT

NDG neurons 6h after preparation

Evotec has electrophysiology and ion channel pharmacology expertise using transiently- & stably-expressing cell lines or cells from primary tissues (e.g. DRGs, NDGs). In addition, we have capabilities and experience in the generation of transient and stable formats for ion channel targets.

NaV current

Electrophysiological recordings from primary cells provide additional mechanistic insight beyond that possible using recombinant cells. In this case we used neurons isolated from dorsal root and nodose ganglia.

On the right is an example taken from our efforts using mouse nodose ganglion neurons:

TTX 300nM

NaV channel X: 1.00 ms

Y: 2.00 nA

Voltage-gated sodium channels (NaV) are expressed in neurons The selective NaV blocker, TTX (300nM) inhibited NaV signal in big diameter cells (arrows)

SECTION 12

IN VIVO PHARMACOLOGY

Fraser McIntosh Executive Vice President In vivo Pharmacology

OFFERING Evotec has developed considerable in vivo pharmacology expertise in our key therapeutic areas of CNS, metabolic disease and complications, oncology and infectious disease. Our in vivo pharmacology team (> 70 staff), consisting of highly experienced scientists with extensive biopharmaceutical and drug hunting expertise, supports in vivo studies from early target validation through to candidate selection.

Furthermore, we have the flexibility to

support defined stand-alone studies in addition to in vivo work conducted as part of larger integrated drug discovery programmes. All experimental procedures involving animals are carried out to the highest standards of animal welfare in state-of-the-art facilities and approved according to European Union and national regulations.

tions are typically co-localised with our in vitro pharmacology units and this complementarity leads to disease biology platforms with a stronger disease focus and breadth of expertise.

Evotec will work with you to identify the most appropriate in vivo pharmacology solution for your drug discovery programme; whether that’s an off-the-shelf standard protocol or developing bespoke assays / readouts for your specific requirements.

We utilise a plethora of acute, mechanistic, pharmacodynamic models relevant to the target biology to assess target engagement and establish PK / PD relationships. Our efficacy models are used to assess biological effects on pathophysiology’s relevant to the disease of interest and are validated with appropriate standards of care and supported by rigorous statistical methods. At Evotec we are continually striving to develop in vivo models and endpoints with greater translational relevance to the clinic and all our models can be supported by relevant pharmacokinetic measurements to determine exposure to effect relationship. Importantly, Evotec’s in vivo pharmacology func-

Evotec offers a range of acute and chronic pain models in rodents with different read-outs depending on the model of interest. Importantly, our scientists are fully blinded to reduce the potential for operator bias.

CENTRAL NERVOUS SYSTEM (CNS) ACUTE / CHRONIC PAIN

Pain Models Spinal Nerve Ligation model (SNL); Visceral pain: colorectal / vaginal distension-induced visceromotor reflex; CFA-induced Inflammatory pain: Formalin induced nocifensive behavior, mustard-oil induced neurogenic inflammation Key Read-outs Thermal sensitivity: Hargreaves Test, hot plate, tail flick Mechanical sensitivity: Von Frey test, pressure application measurement device, dynamic weight bearing Laboras automated recording for formalin paw and locomotor activity Visceral motor reflex-EMG activity

ONCOLOGY Evotec offers a range of rodent models, spanning xenograft, syngeneic and orthotopic models coupled with the possibility of a broad range of efficacy and biomarker read-outs.

In addition, we have access to a range of relevant human tumour tissues via the Institut Universitaire du Cancer Toulouse Biobank which can be utilised for biomarker studies or ex vivo evaluations including compound treatment.

Microdialysis reveals differential kinetic profiles of endocannabinoid release in the hypothalamus upon treatment with a CB1 agonist.

Red arrow indicates dosing of drug. % 250 200 150 100 50 0 -90

-60

-30

0

30

60

90

120

150

180

210

240

270

time (min) rimonabant (10mg/kg) (CB1 antagonist)

NEURODEGENERATIVE DISEASE

Evotec offers phenotypic screening in a variety of models of neurodegeneration, primarily in transgenic animals for Chorea Huntington and for Alzheimer’s disease.

Key Read-outs Locomotor activity (rotarod) Emotion (Elevated Zero Maze, fear conditioning) Cognition (Novel Object Recognition, spatial memory) Irwin test Pre-Pulse Inhibition Gait analysis ADENO-ASSOCIATED VIRUS (AAV) TARGET VALIDATION PLATFORM

Neonatal and adult (stereotaxic) injections into specific brain regions in rodents using AAV-vectors designed for knock down or overexpression of specific proteins to support target identification.

WIN55,212-2 (2.5mg/kg) (CB1 antagonist)

Key Read-outs Behavioral assays, histology and immuno-histochemistry (IHC), morphometric analysis High-content IHC analysis Biomarkers (e.g. cytokines using Mesoscale) EX VIVO RADIOLIGAND BINDING AUTORADIOGRAPHY (ARG) PLATFORM

Beta Imager allows fast screening, Phosphoimager for high resolution images. Multiple applications: KD and Bmax determinations in specific tissue regions Receptor / protein localisation Functional responses: GTPγS radioligand binding as a marker of G protein-coupled receptor (GPCR) receptor activation Ex vivo receptor occupancy to determine target engagement

Oncology models Subcutaneous human xenograft models. Syngeneic models suitable for the evaluation of immuno-oncology therapies Orthotopic models including: Bladder, brain, breast, colon, kidney, liver, lung ovary, skin.

Suitable for metastasis studies.

Angiogenesis models (in mice and rats), with Laser Doppler

Adjacent sections (20 µm), male Sprague Dawley rat, 8 weeks old 1 nM 3H-naloxone

SECTION 12

METABOLIC AND DIABETIC COMPLICATION

Metabolic diseases like Diabetes mellitus, obesity or the metabolic syndrome belong to the major chronic diseases in the world. Long term Diabetes mellitus results in a number of severe diabetic complications impairing kidney, eyes, heart and peripheral nerve function.

Animal models of metabolic diseases

include genetic models (ie. ZDF rats, Ins2 Akita mice), diet-induced models using different diets, chemically-induced models with Streptozotocin (STZ) or Alloxan as well as a humanised beta-cell model where human islets are transplanted into STZ-treated diabetic NOD-SCID mice.

UUO results in the upregulation of the fibrosis marker α-Smooth Muscle Actin UUO as a model for interstitial fibrosis

15000 Fold expression rel. to DCT (REF36)

Key Read-outs Efficacy evaluation by tumour volume and / or Bioluminescence (IVIS Spectrum) imaging for 3D tomography and metastasis.

Full Hematological analysis including FACS analysis Bioanalysis for associated pharmacokinetic studies (PK / PD) Tissue sampling for: - Genomics studies including: RT-PCR; Taqman; TLDA - Protein analysis (total and phosphorylated protein profiling) - ELISA, Western Blotting, dot-blotting, histology, immunohistochemistry - Quantitative image analysis (Definiens technology) Mitochondrial metabolism: - Cellular ATP generation: Glycolytic vs mitochondrial pathways - Oxygen Consumption Rate (OCR) and Extracellular acidification rate (ECAR) - Oxphography: a way to analyse the isolated mitochondrial function - Mitochondrial membrane potential, respiration rat, phosphorylation rate - Metabolite analysis for quantification of lactate, glucose, glutamine, glutamate Microdialysis - Detection of metabolities and/ or drug levels by ex vivo microdialysis in tumour, brain, adipose tissue or biological fluids

α-SMA TaqMan analysis (whole kidney)

α-SMA IHC

10000

5000

0

Sham

Vehicle Compound X Compound Y

Chronic kidney disease (CKD) is a

progressive loss in renal function over a period of months or years resulting from e.g. Diabetes mellitus or genetic predisposition. In order to identify new therapeutic options clinically relevant animal models for acute and chronic kidney disease have been developed which include surgery models (e.g. Acute and Chronic Ischemia Reperfusion Injury models (IRI, CRI), Unilateral Ureter Obstruction model (UUO), genetic models of Diabetes mellitus (ie. BTBRob / ob mice on different diets, Ins2-Akita mice) and models of chemically induced Diabetes mellitus with STZ and combinations thereof.

Sham control

Obstructed kidney

Relevant animal models of eye disease for proliferative and diabetic

retinopathy have been developed and validated. These include models of chemically induced diabetes (ie.

STZ) as well as a model of Oxygen Induced Retinopathy (OIR).

All metabolic and diabetic complication animal models are run under standardised conditions and profiled with a variety of supporting read-outs: Standard Read-outs

Standard clinical and metabolic read-outs including functional glucose/insulin tolerance test; ex vivo read-outs including RT-PCR, biochemical assays, hormonal status and histology.

Advanced Read-outs

Hyperinsulinemic euglycemic clamp, food intake profiles, pairfeeding, body composition analysis by Nuclear Magnetic Resonance (NMR) spectroscopy, morphometric analysis with customised algorithms e.g. with pancreas and adipose tissue, blood pressure analysis, assessment of Glomerular Filtration Rate (GFR).

Pseudomonas aeruginosa, Escherichiacoli, Acinetobacter baumanii and others), anaerobes (including Clostridium difficile) or fungal species (including Candida sp., Aspergillus sp., Mucorales, Malassezia sp.).

In particular, Evotec specialises in PK/PD profiling and modelling of antimicrobial agents in multiple disease models in order to understand the key drivers for efficacy and translation of data into clinical trial design.

Biomarker approaches Plasma and tissue analysis (e.g. ELISA, quantitative RT-PCR), high-content tissue imaging for bright as well Key Read-outs as immune histochemistry, single and multiplexing on the Mesoscale Efficacy evaluation by: platform, e.g.cytokines, mass spec- Burden at site of infection and troscopy (ie. LC-MS/MS). in multiple biological matrices including quantitative culture, QPCR and biomarkers INFECTIOUS DISEASE Evotec specialises in assessing the Microbiome analysis efficacy of lead and candidate and sequencing compounds in highly relevant and Bioluminescence validated models of infection. Our (IVIS Spectrum) imaging. extensive range of established models Host response endpoints are well-suited to the development of including ELISA, Western multiple classes of agent including Blotting, dot-blotting, histology, small molecules, natural products, immunohistochemistry, cytokine peptides, antibodies, other biologics profiling and cytometry and vaccines. We provide a highly bespoke service, customising studies to meet the exact requirements based on programme needs and parametersâ&#x20AC;&#x2030;/ endpoints of interest.

Models of Infection For efficacy assessment against bacterial and fungal infections, our models address different sites of infection (localised and systemic) and pathogens by Gram-positive (including Staphylococcus aureus, Streptococcus pneumoniae, and others), Gram-negative (including

Bioanalysis for associated pharmacokinetic studies (PK/PD) and pathogen-associated biomarkers HISTOLOGY, IMMUNOHISTOCHEMISTRY AND MORPHOMETRIC ANALYSIS PLATFORM

The combination of in vivo pharmacology with histology techniques offers a powerful tool to further analyse disease relevant biomarkers.

Whereas histology and IHC helps to identify disease and target relevant changes, automated morphometric analysis can yield unbiased quantitative information amenable to statistical analysis, which adds significant value to the customers drug discovery effort. Furthermore, histopathology on critical organ systems will allow early risk assessment.

Immunohistochemistry and morphometric analysis of male ZDF rat pancreas: Islet of Langerhans fragmentation in male ZDF rats

SECTION 13

EVOMAB

ANTIBODY GENERATION AND SELECTION

as GPCRs and ion channels. Evotec offers high-throughput antibody cloning and expression which can be combined with all standard immunisation and selection technologies.

The offering includes reformatting antibody pools from phage display selections or animal immunisations into suitable screening formats.

Evotec’s own “AbExpress” technology combines specific B-cell enrichment with high-throughput cloning and expression of antibodies in mammalian cells. We provide optimal cellbased HTS/HCS screening formats support­ing fast development cycles.

Evotec offers a turnkey solution for antibody development up to the production of mg quantities necessary for testing in animal models.

Arnd Steuernagel Senior Vice President Biologics

OFFERING Antibody identification including B-cell cloning and high-throughput expression platform is embedded in Evotec’s pre-clinical development expertise.

As a partner in antibody drug development, Evotec utilises more than 15 years of cell-based functional screening expertise on various targets such

CAPABILITIES AND PLATFORM

Evotec has established its antibody screening platform EVOmAb to incorporate functional screening early in the antibody discovery and selection process. Attractive targets such as transmembrane proteins need to be altered in their activity in order to achieve a meaningful pharmacological effect. The most critical challenge in functional screening is the availability of sufficient antibody

in assay compatible media or buffers.

AbExpress combines high-throughput cloning with high parallel expression and purification strategies. Isolating antibodies directly from single B cell lineages overrides hybridoma technologies due to efficiency in isolating rare functional clones. The limiting factor in high parallel expression of antibodies is the cost of cloning and handling thousands of individual sequences while preserving cognate pairs of heavy and light chain during the primary screening process. In our pool cloning and expression strategy each heavy chain is coexpressed with a limited, pool of light chains including its cognate partner during screening. This primary expression step yields several hundred microgram of antibody. Following the primary screen, interesting heavy chains are paired with their cognate light chains using sequence information obtained from primary plates.

The EVOmAB workflow maximises the rapid capture of a diverse set of antibody clones and enables primary functional screening with recombinant antibodies.

ABEXPRESS: MULTIPLE AND FLEXIBLE ENTRY POINTS FOR FAST ANTIBODY DRUG DISCOVERY

Efficient recovery from B-cells

Massive parallel expression transfection

High-Throughput Screening

supernatant

HC

LC

Proprietary antibody discovery platform

Hundreds of recombinant antibodies at high microgram yield

Early screen for functional activity

GENERATE ANTIBODY POOLS by immunisation, phage display, yeast display.

Various platforms available, either in-house, through collaborations or from customer Typical output are thousands of clones (B-cells, Hybridomas, Yeast cells) expressing no or low amounts of soluble antibody

AbExpress

High parallel expression of antibodies

CELL LINES Ion Channels GPCRs ANTIBODIES 1,000 to >10,000 clones

Integrins Receptors Cytokines

Cell based screening platforms Established at Evotec in combination with target know-how and tools

CELL BASED ASSAYS Ion Channels – Automated electrophysiology GPCR – Ca2+ flux – cAMP/Arrestin HCS – Signalling cascades – Phenotypic changes – Translocation – Differentiation

EQUIPMENT

FLIPR384 FLIPR Tetra Tecan Safire II Perkin Elmer EnVision TopCount Opera™ SRU BIND IonWorks ® Quattro™ Q-patch HTX® PatchLiner ©

SECTION 14

TRACK RECORDS AND CLIENTS

Evotec, with its broad expertise and services, its repeat business and strategic alliances, is perfectly positioned to be your drug discovery services provider of choice. We are aware of the fact that it takes much more than just the highest quality services to develop new drugs, but nevertheless this is the most im­ portant starting point. Evotec delivers an industrialised, high-tech, and comprehensive infrastructure to our partners.

Evotec is one of the few drug discovery businesses that can execute a comprehensive outsourcing strategy.

In significant large-scale, multi-target deals, Evotec’s customers work with our scientists to progress discovery programmes from target through to lead optimisation using the Company’s integrated drug discovery platform.

Evotec has a broad client base ranging from venture capital firms to virtual biotechs to large pharmaceutical companies. We are a truly global company with clients in the United States, Europe and Japan. We have a loyal client base with many longterm agreements as well as regular repeat clients that keep coming back with project after project as Evotec delivers the drug discovery solutions and value they need to drive their discovery programmes forward.

Integrated, early stage collaboration in Alzheimer’s disease to identify and progress novel treatments, deal structure includes research funding, milestonesand royalties

Integrated chemistry, biology and DMPK team at Evotec, working with UCB scientists across various sites on multiple targets, deal structure includes research funding, milestones and royalties

Integrated five-year multi-target collaboration in the field of endometriosis, deal structure includes upfront payment plus milestones and royalties

Integrated chemistry and biology team at Evotec, including high-content assays and lead optimisation, recently extended to 2016 (started in 2006)

»Evotec’s knowledge in Alzheimer’s disease greatly enabled this collaboration«

»Evotec is the ideal partner to provide the resource bandwidth and drug-hunting expertise«

»Our new collaboration with Evotec will perfectly complement our activities in this field of high unmet medical need«

»We are impressed

by Evotec’s breadth of drug discovery expertise«

Imprint Editor: Evotec AG; Chief Editor: Michael Bayer Content: Mario Polywka,

Steven Hutchins Design: alessandridesign, Thomas Piribauer Programming: bgcc

Contact us:

Mario Polywka

Steven Hutchins

Chief Operating Officer mario.polywka@evotec.com

Executive Vice President, Business Development steven.hutchins@evotec.com

Pharmaceutical Accounts

North America

Christophe Muller

Ryan Brady

Vice President, Business Development Key accounts christophe.muller@evotec.com

Vice President, Business Development Venture Capital, Foundations & Not for Profits ryan.brady@evotec.com

Europe

Jeff Naroian Hermann-Josef Kaiser

Business Development Manager Cell and protein production hermann-josef.kaiser@evotec.com

Vice President, Business Development East Coast jeff.naroian@evotec.com

John Kamins Richard Law

Vice President, Business Development, Europe richard.law@evotec.com

Vice President Business Development Proteomics and biomarkers john.kamins@evotec.com

Itta MacNevin FrĂŠdĂŠric Somny

Vice President, Business Development, France frederic.somny@evotec.com

Japan Masahiko Otani

Vice President Business Development, Japan masahiko.otani@evotec.com

Director Business Development Venture Capital, Foundations & Not for Profits itta.macnevin@evotec.com