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Building a strong anti-malarial drug pipeline based on phenotypic

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Presentation on theme: "Building a strong anti-malarial drug pipeline based on phenotypic"— Presentation transcript:

1 Building a strong anti-malarial drug pipeline based on phenotypic
whole organism screening Annie Mak, PhD presenting on behalf of Malaria project team at GNF September 22, 2011

2 Outline An introduction to GNF Anti-malarial drug discovery
Our small molecule drug discovery infrastructure Anti-malarial drug discovery AD-HTS in the 2010s – which direction are we heading

3 Genomics Institute of the Novartis Research Foundation
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)

4 Examples of GNF Technology Platforms
High-Throughput Compound Screening (2.5 M compounds, 600K academic collaboration) High-Content Imaging (annual throughput of >10 M wells of confocal Opera images) Protein and Antibody Engineering Automated Protein Production and Purification Automated Cell/Compound Profiling (multiple cell lines/assays; fewer compounds) Automated Functional Genomics (cDNA, siRNA) Protein X-ray Structure Determination Core technology platforms provides much versatility and flexibility, allowing us to focus on interesting science.

5 Small Molecule Drug Discovery Infrastructure
Novartis Infrastructure Target Validation Exploratory Chemistry Full Lead Optimization HTS Clinic D0 D1 D2 D3 Candidate Selection Point GNF 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 Exploratory/Target ID Tools Linker Chemistry & SAR Affinity methods/ Target pull-down Pathway profiling Functional genomics collection Solexa Sequencing

6 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 Research Translational Development Lead Gen Lead Opt Preclinical Phase I Phase IIa Phase IIb/III Registration Phase IV Novartis miniportfolio Novartis 2 Projects MK 4815 (Merck) Tafenoquine GSK OZ 439 (Monash/UNMC/ STI) AZCQ Pfizer Eurartesim™ sigma-tau Coartem®-D Novartis GSK miniportfolio GSK 1 Project GNF156 Novartis NITD609 Novartis Pyramax® Shin Poong/University of Iowa ASAQ Winthrop sanofi aventis/DNDi Broad/Genzyme miniportfolio Aminoindole Broad/Genzyme AN3661 Anacor IV artesunate Guilin Many others … Many others …

7 Drug resistance to most marketed drugs widespread…
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 activity 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 Drug resistance to most marketed drugs widespread…

8 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

9 Compound Plate Storage (486 slots each)
Fully automated GNF Screening System Dispensers Weigh station Centrifuge FLIPR Viewlux Pintool Assay Plate Incubator Compound Plate Storage (486 slots each)

10 Fully automated GNF Screening System
Dispensers 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 Weigh station Centrifuge FLIPR Viewlux Pintool Assay Plate Incubator Compound Plate Storage (486 slots each)

11 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.

12 Selection criteria for cell-based hit-to-lead optimization
A new antimalarial should ideally meet the following criteria: kills parasite blood stages; is active against drug-resistant parasites; is safe (i.e., no cytotoxicity, genotoxicity, and/or cardiotoxicity); and has pharmacokinetic properties compatible with once-daily oral dosing. ~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)

13 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

14 Scientific Approaches
Affinity chromatography/proteomics analysis Lab-evolved resistant strains upon compound treatment Tiling array analysis and whole-genome sequencing to help MoA determination

15 From an HTS hit to Phase I candidate
NITD609 activity on clinical isolates of P. vivax (top) and P. falciparum (bottom) 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. chloroquine artesunate NITD609 chloroquine artesunate NITD609 (B) Ex vivo sensitivity of P. vivax and (C) P. falciparum (9 and 10 clinical isolates, respectively) to NITD609 compared with the reference drugs chloroquine (CQ) and artesunate (AS). The antimalarial sensitivity of these two species was measured after exposing ring (unshaded boxes) and trophozoite stages (shaded boxes) to drug for 20 hours. Data are shown as box plots. Maximum-minimum IC50 values are indicated, respectively, by the top and bottom horizontal thin bars, with the solid internal line indicating the median. Boxed areas indicate 75% confidence intervals. Inhibition of parasite growth was determined after 42 hours. Only chloroquine-treated P. vivax displayed a significant stage-specific sensitivity (P < 0.001). ns, not significant. : NOAEL are respectively 0.5 mg/kg and 3 mg/kg in 2-week toxicology dog and rats toxicology studies awarded MMV Project of the Year 2009 Rottmann et al Science, 2010

16 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

17 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

18 Acknowledgements – Malaria Project Team
Chemistry Arnab Chatterjee Advait Nagle Tao Wu Tomoyo Sakata Robert Moreau Jason Roland Pranab Mishra David Tully Valentina Molteni 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 Swiss Tropical and Public Health Institute Matthias Rottmann Christoph Fischli Sonja Maerki 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 NITD Bryan Yeung Zou Bin Anne Goh Suresh B. Lakshminarayana Veronique Dartois Thomas Keller Thierry Diagana 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 External collaborators Montip Gettayacamin (AFRIMS - Bangkok) Robert Sauerwein (NCMLS - The Netherlands) Ian Bathhurst (MMV) Clinical team Jens Praestgaard Ruobing Li External support and advice Marcel Tanner, Swiss Tropical Institute, Nick White, Oxford University Assay Development - HTS Achim Brinker Jason Matzen Paul Anderson GNF Management Jennifer Taylor Richard Glynne Martin Seidel Peter Schultz Informatics Jeff Janes John Che Yingyao Zhou

19 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, (July 2011)

20 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.

21 High content imaging in HTS
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 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 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 Myotube Infection of mammalian cells

22 Thank you for your attention
Acknowledgement Assay Development – HTS: Annie Mak’s group: Jason Chyba Vicki Zhou Sandra Gao Richard Brusch Amy Foraker Ingo Engels & his group John Joslin & his group Ed Ainscow Jennifer Harris Automation/Engineering: Dan Sipes Jason Matzen Paul Anderson Mike Garcia Tim Smith Drew Gunderson Dan Rines … many others Informatics: Jeff Janes Julia Turner Ghislain Bonamy Thank you for your attention

23 Various modalities are supported
Luminescence Fluorescence Absorbance TR-FRET High content imagers Compound/Library transfer via: Acoustic Droplet Ejection, Pintool FLIPR, Lumilux (kinetic fluorescence/luminescence)

24 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 Gantt Chart Trace file from on-the-fly data analysis Time Plate


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