Building a strong anti-malarial drug pipeline based on phenotypic whole organism screeningAnnie Mak, PhD

presenting on behalf of Malaria project team at GNF

September 22, 2011

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Outline

• An introduction to GNF–Our small molecule drug discovery infrastructure

• Anti-malarial drug discovery• AD-HTS in the 2010s – which direction are we heading

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GNF

Genomics Institute of the Novartis Research Foundation

Mission: To apply innovative technologies to the discovery of new biological processes and the underlying mechanisms of disease, and to develop new or improved human therapeutics

which contribute to the NIBR preclinical pipeline

• Funded by the Novartis Research Foundation• Located in La Jolla, California• Moved into permanent 260,000 square foot research campus in 1Q 2002 • Additional 24,000 square foot manufacturing facility for our automation

system(www.GNFSystems.com)

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Examples of GNF Technology Platforms

High-Throughput Compound Screening(2.5 M compounds, 600K academic collaboration)

Automated Functional Genomics(cDNA, siRNA)

Automated Protein Production and Purification

Automated Cell/Compound Profiling(multiple cell lines/assays; fewer compounds)

Protein and Antibody Engineering

Protein X-ray Structure Determination

High-Content Imaging(annual throughput of >10 M wells of

confocal Opera images)

Core technology platforms provides much versatility and flexibility,allowing us to focus on interesting science.

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Small Molecule Drug Discovery Infrastructure

ClinicCandidate Selection

PointD3D2D1D0

TargetValidation HTS

ExploratoryChemistry

Full LeadOptimization

Medicinal & Analytical Chemistry• Experienced medicinal chemistry

staff• High-throughput analytical and

purification technology• Sophisticated compound

management

Pharmacology/ADMET• In vitro and in vivo ADMET

capabilities• State-of-the-art bioanalytical

technology• In vivo efficacy models in

metabolic disease, immunology and oncology

Novartis Infrastructure

GNFExploratory/Target ID Tools• Linker Chemistry & SAR• Affinity methods/ Target pull-down• Pathway profiling• Functional genomics collection • Solexa Sequencing

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Discovery of Antimalarials

• GNF joined the malaria initiative led by Novartis Institute for Tropical Diseases (NITD) to fight against Malaria.

• The NGBS consortium partner includes:– Biomedical Primates Research Center (BPRC), Rijswijk (NL)– Swiss Tropical Institute (STI), Basel (CH)

MMV Portfolio, 1st Quarter, 2011

RegistrationPreclinicalResearch Translational Development

Lead Opt Phase IIaPhase ILead Gen Phase IVPhase IIb/III

Novartisminiportfolio

Novartis2 Projects

MK 4815(Merck)

IV artesunateGuilin

Eurartesim™ sigma-tau

Coartem®-DNovartis

GSKminiportfolio

Broad/Genzymeminiportfolio

GSK1 Project

AminoindoleBroad/Genzyme

NITD609Novartis

TafenoquineGSK

OZ 439(Monash/UNMC/

STI)

AZCQPfizer

Pyramax®Shin Poong/University

of Iowa

ASAQ Winthropsanofi aventis/DNDi

GNF156Novartis

Many others … Many others …

AN3661Anacor

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Malaria

• Medical Need– 500 million cases annually worldwide

• Nearly 1 million deaths/yr, >75% under age of 5– Resistance to current therapies widespread

with exception of artemisinin derivatives• Increased parasite clearance times for Artemisinin-

based therapies seen in Thai-Cambodian border• Current artemisinin-based combination therapies

(ACTs) contraindicated in 1st trimester– No current therapies address need for blood-

stage, liver-stage, and transmission blocking activityDrug resistance to most marketed drugs widespread…

Infective mosquito bite

Hepatic phase:Normal hepatic phase leading to primary infection

Erythrocytic phase

Primary infection

Protracted hepatic phase leading to relapse infection

Relapse infection

Ring -> Troph

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Scientific Approaches

Cell-based screen approach• Majority of anti-infectives discovered through cell-based screening by facilitating

parallel interrogation of druggable targets and also addresses compound permeability issues

• HTS on >2M compounds: P. falciparum infected human red blood cells (RBCs)• Liver–stage infection assay (P. vivax infection)

Target-based screen approach • Collaboration with academic institutes• Plasmodium kinases: such as CDPK1, CDPK5, GSK3, CK2α• Additional biochemical targets screening at GNF on cell-active compounds

Target identification methods• Lab-evolved resistant strains upon compound treatment• Tiling array analysis and whole-genome sequencing to help MoA determination• Affinity chromatography/proteomics analysis

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Weigh station

FLIPR

Viewlux

Centrifuge

Fully automated GNF Screening System

Dispensers

PintoolCompound

Plate Storage (486 slots

each)

Assay Plate Incubator

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Weigh station

FLIPR

Viewlux

Centrifuge

Fully automated GNF Screening System

Dispensers

PintoolCompound

Plate Storage (486 slots

each)

Assay Plate Incubator

Assay Protocol

Plate media

Pintool in 10nl of compounds

Plate parasite/blood mixture

Move plates offline for incubation for 3 days

Dispense lysis buffer with SyBR Green

Read plates offline with Analyst GT

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Malaria Cell-based Screening Summary

• P. falciparum infected human RBCs: Screen measured production of new DNA over 72 hours using SYBR green

• > 2M compounds screened at 1.25 mM (3d7)

• 4851 hits < 1.2 mM EC50 vs W2 and/or 3d7 – “Malaria Box” public resource for

malaria research community– https://www.ebi.ac.uk/chembldb/index

.php/compound• 1,256 < 200 nM EC50 vs 3d7• >200 scaffolds represented in the

reconfirmed compound set

Tremendous amount of chemical diversity from

screen

HTS reported in: Plouffe and co-workers PNAS 2008, 9059.

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• ~5K reconfirmed blood-stage hits from HTS identified for further evaluation were determined by

– Less than 1.25 µM EC50 with activity in 15 strain resistance panel

• Less than 5-fold potency shift between strains

– High selectivity in 6-cell line toxicity panel (SI > 20-fold)

– Novel chemotype for malaria

– Ease of synthesis (less than 7 steps)

– Good solubility, metabolic stability and limited CYP450 inhibition

– Multiple actives within a scaffold (if library diversity allows)

Selection criteria for cell-based hit-to-lead optimization

A new antimalarial should ideally meet the following criteria:(i) kills parasite blood stages; (ii) is active against drug-resistant parasites; (iii) is safe (i.e., no cytotoxicity, genotoxicity, and/or cardiotoxicity); and(iv) has pharmacokinetic properties compatible with once-daily oral dosing.

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Scientific Approaches

Cell-based screen approach• Majority of anti-infectives discovered through cell-based screening by facilitating

parallel interrogation of druggable targets and also addresses compound permeability issues

• HTS on >2M compounds: P. falciparum infected human RBCs• Liver–stage infection assay (P. vivax infection)

Target-based screen approach • Collaboration with academic institutes• Plasmodium kinases: such as CDPK1, CDPK5, GSK3, CK2α• Additional biochemical targets screening at GNF on cell-active compounds

Target identification methods• Lab-evolved resistant strains upon compound treatment• Tiling array analysis and whole-genome sequencing to help MoA determination• Affinity chromatography/proteomics analysis

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Scientific Approaches

• Affinity chromatography/proteomics analysis • Lab-evolved resistant strains upon compound treatment• Tiling array analysis and whole-genome sequencing to help

MoA determination

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chloroquineartesunate NITD609

• Collaboration with Novartis Natural Products Unit and NITD chemistry on the development of NITD609

• Potent (IC50< 1 nM) blood stage compound

• Potent transmission-blocking activity• Low clearance, moderate-to-long half-life: predicted human

efficacious dose < 100 mg• Currently in healthy volunteer trials• Clinical efficacy studies in mid-2011• First in class compound with efficacy superior to standard

anti-malarial drugs in mouse efficacy model

• Genome wide scanning of drug resistant mutant reveals that NITD609 targets PfATP4 and inhibits protein synthesis, a distinct MoA different from Arteminisin.

NITD609 activity on clinical isolates of P. vivax (top) and P. falciparum (bottom)

Rottmann et al Science, 2010

chloroquineartesunate NITD609

From an HTS hit to Phase I candidate

awarded MMV Project of the Year 2009

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On-going activity in the pipeline

Hit series among others: Imidazolopiperazines• Not identified in >100 HTS screens performed at GNF• Novel chemotype with a mechanism of action distinct from that of NITD609• Simple chemical synthesis• First-in-class compound that has broad activity across parasite life cycle• Favorable preclinical safety profile, TI > 30 (rat, dog)

Taken from MMV.org:• GNF 156, displays pharmacological properties compatible with a target product

profile for the development of an uncomplicated malaria treatment. • Good Laboratory Practice toxicology studies will commence in the first quarter of

2011 to more carefully assess the safety profile of this compound with the aim of starting a Phase I study in healthy human volunteers by the end of 2011/early 2012.

Imidazolopiperazines: Hit to Lead Optimization of New Antimalarial Agents J. Med. Chem., 2011, 54 (14), pp 5116–5130

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MedChem lessons learnt so far

• SAR can be challenging, but getting hits from HTS with good SAR is critical • Better to have SAR than absolute potencies off the deck• Potencies can be optimized relatively quickly compared to

other parameters• Molecular weights greatly effect the cellular optimization

Forces the chemistry to be very atom economical (high ligand efficiencies)

• Weekly cytotoxicity, protein binding shift potency, and cross-resistance data to other scaffold lab-evolved resistance strains really allows for better optimization of other parameters

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Biology • Kelli Kuhen • Carolyn Francek • Zhong Chen • Kerstin Henson• Rachel Borboa• James Gilligan• Tae-gyu Nam • Neekesh Dharia• David Plouffe• Case McNamara• Stefan Meister• Elizabeth Winzeler

Pharmacology/Analytical• Tove Tuntland• Perry Gordon• Jonathan Chang• Matthew Zimmerman• Liang Wang• Todd Groessl • Barbara Saechao • Bo Liu• Chun Li• David Jones• Wendy Richmond• Kevin Johnson• Tom Hollenbeck• Lucas Westling• Michael Kwok• Tiffany Chuan• John Isbell

Chemistry• Arnab Chatterjee• Advait Nagle• Tao Wu • Tomoyo Sakata• Robert Moreau• Jason Roland• Pranab Mishra• David Tully• Valentina Molteni

Swiss Tropical and Public Health Institute• Matthias Rottmann• Christoph Fischli• Sonja Maerki

NITD• Bryan Yeung• Zou Bin• Anne Goh• Suresh B. Lakshminarayana• Veronique Dartois• Thomas Keller• Thierry Diagana

Informatics• Jeff Janes • John Che• Yingyao Zhou

GNF Management• Jennifer Taylor• Richard Glynne• Martin Seidel• Peter Schultz

Acknowledgements – Malaria Project Team

External collaborators• Montip Gettayacamin (AFRIMS - Bangkok)• Robert Sauerwein (NCMLS - The

Netherlands)• Ian Bathhurst (MMV)Assay Development -

HTS• Achim Brinker• Jason Matzen• Paul Anderson

Core team• Margaret Weaver• Xingmei Han• Giancarlo Francese• Sreehari Babu• Rita Ramos• Karen Beltz• Bo Han• Wen Shieh• Markus Baenziger

Novartis Tropical Medicines• Heiner Grueninger• Anne-Claire Marrast• Paul Aliu• Gilbert Lefevre

Clinical team• Jens Praestgaard • Ruobing Li

External support and advice• Marcel Tanner,

Swiss Tropical Institute,

• Nick White, Oxford University

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AD-HTS in the 2010s

• New technology/assay format development• Closer to biology More complicated assays

“Contribution of phenotypic screening to the discovery of first-in-class small molecule drugs exceeded that of target-based approaches.”

“An additional challenge is to effectively incorporate new screening technologies in phenotypic screening approaches, which is important for addressing the traditional limitation of some of these assays: a considerably lower throughput than target-based assays.”

Nature Reviews Drug Discovery 10, 507-519 (July 2011)

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Continual upgrades to support various screening modalities

New readers to improve sensitivity and speed Compound transfer via

Acoustic Droplet Ejection more flexibility in

experimental design (eg. combinations; anaerobic organisms)

Improve washing capabilities to support non-homogeneous assays bead-based ELISA, FACS, high content imaging

*Note: this washer was not implemented after evaluation.

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High content imaging in HTS

Nov 2010 – Feb 2011: Completed full deck HCI-HTS• Nuclear translocation assay detected by IF staining• Automation handled 200 plates/batch, 5 week campaign• 3.9 million wells imaged by Opera (confocal microscope)• Read time: 80 days• Data generated: 9 TB• Data analysis script developed in-house Infrastructure and expertise in place to handle HCI in a HTS scale

Interesting Phenotype (Blackbox!)

Rigorous Assay Development

Robust and accurate

automation for HTS

Imager to capture

sufficient resolution and

statistics

Image analysis algorithm in

place

IT infrastructure to handle data

New assays under development:• Cellular differentiation – specific marker expression or

morphological changes• Infection – host vs pathogen• “New tricks to old questions” – DNA repair assayed by

chromosome breakpoint detection; cell cycle analysis by multiplexing specific markers

Vesicle Detection

MyotubeDetection

Infectionof mammalian cells

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Thank you for your attention

AcknowledgementAssay Development – HTS:Annie Mak’s group:Jason ChybaVicki ZhouSandra GaoRichard BruschAmy Foraker

Ingo Engels & his group

John Joslin & his group

Ed AinscowJennifer Harris

Automation/Engineering:Dan SipesJason MatzenPaul AndersonMike GarciaTim SmithDrew GundersonDan Rines … many others

Informatics:Jeff JanesJulia TurnerGhislain Bonamy

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Various modalities are supportedLuminescenceFluorescenceAbsorbanceTR-FRET

FLIPR, Lumilux(kinetic fluorescence/luminescence)

Compound/Library transfer via:Acoustic Droplet Ejection, Pintool

High content imagers

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Innovative Solutions that make Screening Work Specialized team of biologists in assay development and miniaturization (eg. from 24w

down to 1536w format) Dedicated and experienced system engineers to run the automation system On-the-fly data analysis + system alerts to monitor progress Extensive informatics support for data analysis (kinetic reads, imaging assays) Large hitpick capacity

Typical full deck HTS = 4M wells in ~2-3 weeks Hitpick of 20K Overall, lower opportunity cost means more opportunistic approach to HTS

Time

Plate

Trace file from on-the-fly data analysisGantt Chart