Computational Chemistry Mechanistic Enzymology

Biomarker Development

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Medicinal Chemistry & Synthesis

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Benefits of Contracting CDT

Problem Solvers– Experienced drug discovery team– Broad Technology Base

Mechanistic enzymology – industry leading Biochemical and cell biology assay development Biomarker assay development Computational and Medicinal Chemistry

Partners– Communication– Versatile and flexible– Project leadership/consultation – partner in strategy and execution

Research Partnership from Target ID through IND and beyond

Computational & Medicinal Chemistry

Experience where it counts:– Drug Design– Synthetic Route Analysis– Compound Synthesis– Structure-Based Drug Design – Molecular Modeling– Virtual Screening

Confluence can enable at all stages:– Understanding the Competitor Landscape– Discovering Areas with Freedom To Operate– ID Leads from Virtual Screen– Parachute Opportunity Exploitation– Designing Initial Chemical Matter– Identifying Synthetic Routes– Providing Active Pharmaceutical Ingredients– Writing Patent Protocols– Optimizing Current Lead Matter– Singleton and Library Production

Lys 53

Asp 168

Glu 71

Met 109

Ala 51

Thr 106

Asp 112 Ser 154

Leu 167

Lys 53

Asp 168

Glu 71

Met 109

Ala 51

Thr 106

Asp 112 Ser 154

Leu 167

Assay Establishment and ValidationWe can deliver a validated assay where no off the shelf option exists

Assay Development Expertise– Flexible screening formats– Biochemical and Cellular assays – Direct HTS translation

Appropriately Equipped– 96 and 384 well enabled– Multiple liquid handling stations– Versatile plate readers

Data Analysis and Secondary Assays– Hit validation and IC50 values– Mechanistic characterization of hits– Follow-up orthogonal assays

Problem Solving– New targets – need entire testing funnel– Complex signaling systems– Challenging targets– Exclusion of artifacts

Medium Throughput Specialty Screens

Biochemistry/Enzymology

Mechanism of Action Studies

Enzyme Activity

Steady State Kinetics

Inhibitory Mechanism

CompetitiveNoncompetitiveUncompetitive

Irreversible (kinact & Ki)Slow tight binding (kon & koff)

Aggregate (non drugable)

Thermodynamics

Isothermal Titration Calorimetry

Independent of Catalysis

Kd and stoichiometry DH, DS, DG

Direct Binding

RadioligandBiacore

TR-FRETFluorescence (FP)

AbsorbanceLuminescence

Filtration

Independent of Catalysis

Binding kinetics (kon & koff)

Key to Increased Efficiency of Lead Discovery & Development is to Link SAR to Mechanism of Action!

Cell-Based Assay Design & Development

Cell-based assays are invaluable for drug discovery:– Identification of biologically important signal transduction

pathways and key point(s) for therapeutic intervention– Understanding compound mechanism of action– Quantitation of efficacy and selectivity– Understanding safety issues linked to pathways

Cell-based assays have inherent problems in:– Simulating ex vivo the microenvironment/disease

state/species and/or tissue– Choosing disease relevant cell types and/or stimuli– Genetic drift, inter-donor variability, cross-species, etc.– Understanding effects of enzyme isoforms, scaffolding,

substrate selectivity, etc.

Confluence scientists have experience with these issues:– We can identify & design validated assays for translatable

target modulation & efficacy biomarkers– Offer broad assay technologies including DELFIA/FRET,

AlphaScreen, Westerns, FACS etc.– Biomarkers selected for assay development based on

pertinent scientific literature and our collective experience

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Coupling Assay Technology with Biological Relevance

Biomarker Development

CDT is Experienced With:– Target, Mechanism and Disease Biomarkers– Biomarker selection– Biomarker assay design, development,

validation and interpretation– Cell surface, intracellular and secreted analytes

CDT Can Deliver:– Assays developed across species and in

various biological matrices– Complete statistical evaluation of data– Seamless assay transfer to the clinical CRO

Reliable interpretation of preclinical disease and pharmacology models and human clinical studies requires suitable biomarker analysis to optimally relate PK/PD, PK/TD and outcomes.

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We can help you generate an interpretable and decision-enabling cellular, animal or human study though effective use of translatable biomarkers.

Biology Facilities and Instrumentation

Facilities

• General Biology Lab• Cell Culture Lab• WUSTL Screening

Core• Radiation Laboratory

Analytical

• Biacore 2000• Spectramax Plus• Spectramax M2• Analyst HT 96/384• Caliper LabChip• Packard Top Count• Tecan GENios• Microcal ITC• BMG Polar Star Omega• Waters & Agilent HPLC• FACScan Cytometer• MesoScale Discovery• Luminex

Other

• SDS PAGE/Western• Cell culture hoods• CO2 Incubators

• Various Microscopes• Centrifuges of All

Types• Various Balances• Market Forge

Autoclave• Thermocyclers• Shaking Incubators• Static Incubators• Biological Safety

Hoods

Automation

• Biomek NX:96/384• Precision 2000• Multidrop Combi

Chemistry Facilities & Instrumentation

Facilities & Infrastructure

• Synthetic Chemistry Lab• Analytical/Purification

Lab• Washington University facilities agreement• Waste and storage

facilities for chemicals

Synthesis Purification & Analytical• Air Sensitive Reactions• Customized Quantities (mg-g)• Library

Design/Production• Intermediate

Preparation• Route Identification• Route Optimization

• HPLC• Normal Phase

Purification• Reverse Phase Purification• LC/MS• NMR

Management: Joseph Monahan, PhD, President Michael Kuo, MD, Board Member Al Beardsley, PhD, Business Development Walter Smith, MS, Vice President Research

Scientist Founders: Gabriel Mbalaviele, PhD Shaun Selness, PhD

Business Professionals: Jennifer Bohnert – Controller Lisa Gabel - Bookkeeper Polsinelli Shughart - General Counsel Harness, Dickey & Pierce - IP Attorney Stone Carlie - Tax Advisor

Confluence Team

Staff Scientists: Barry Burnette, PhD Jeff Hirsch, BS Balekudru Devadas, PhD Susan Hockerman, MS John Springer, MS Jerry Cubbage, PhD Jim Blinn, MS Heidi Hope, PhD Arthur Wittwer, PhD William Hood, MS Sheri Bonar, BA Matt Saabye, BS Jon Jacobsen, PhD Taryn Syrtees, BS Kristin Sauer, BS

Joseph Monahan, PhDPresidentConfluence Discovery TechnologiesCORTEX Bioscience District4320 Forest Park Avenue, Suite 303St. Louis MO 63108josephmonahan@confluencelifesciences.comwww.confluencediscovery.com314.932-4032 x-305

Walter SmithVice President ResearchConfluence Discovery TechnologiesCORTEX Bioscience District4320 Forest Park Avenue, Suite 303St. Louis MO 63108waltersmith@confluencelifesciences.comwww.confluencediscovery.com314.932-4032 x-305