building a strong anti-malarial drug pipeline based on phenotypic whole organism screening annie...
TRANSCRIPT
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