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.
Download ReportTranscript 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.
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 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 2 Confidential 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) 3 Confidential Examples of GNF Technology Platforms High-Throughput Compound Screening High-Content Imaging (2.5 M compounds, 600K academic collaboration) (annual throughput of >10 M wells of confocal Opera images) Protein and Antibody Engineering Automated Protein Production and Purification Automated Cell/Compound Profiling Automated Functional Genomics (multiple cell lines/assays; fewer compounds) (cDNA, siRNA) Protein X-ray Structure Determination Core technology platforms provides much versatility and flexibility, allowing us to focus on interesting science. 4 Confidential Small Molecule Drug Discovery Infrastructure Target Validation D0 HTS D1 Exploratory Chemistry D2 Full Lead Optimization D3 Novartis Infrastructure Candidate Selection Point Clinic 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 5 Confidential Exploratory/Target ID Tools • Linker Chemistry & SAR • Affinity methods/ Target pull-down • Pathway profiling • Functional genomics collection • Solexa Sequencing 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 Lead Gen Translational Lead Opt Preclinical Novartis Novartis MK 4815 miniportfolio 2 Projects (Merck) GSK GSK GNF156 miniportfolio 1 Project Novartis Broad/Genzyme Aminoindole miniportfolio Broad/Genzyme AN3661 Anacor Many others … Many others … Phase I Tafenoquine GSK Development Phase IIa OZ 439 (Monash/UNMC/ STI) NITD609 Novartis Phase IIb/III Registration AZCQ Eurartesim™ Coartem®-D Pfizer sigma-tau Novartis Pyramax® Shin Poong/University of Iowa Phase IV ASAQ Winthrop sanofi aventis/DNDi IV artesunate Guilin 6 Confidential Malaria • Medical Need Infective mosquito bite – 500 million cases annually worldwide • Nearly 1 million deaths/yr, >75% under age of 5 Hepatic phase: Normal hepatic phase leading to primary infection Erythrocytic phase – Resistance to current therapies widespread with exception of artemisinin derivatives Primary infection • Increased parasite clearance times for Artemisininbased therapies seen in Thai-Cambodian border • Current artemisinin-based combination therapies (ACTs) contraindicated in 1st trimester – No current therapies address need for bloodstage, liver-stage, and transmission blocking activity to most marketed Drug resistance Ring -> Troph Protracted hepatic phase leading to relapse infection drugs widespread… Relapse infection 7 Confidential 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 8 Confidential Fully automated GNF Screening System Weigh station Dispensers Centrifuge FLIPR Viewlux Pintool Assay Plate Incubator 9 Confidential Compound Plate Storage (486 slots each) Fully automated GNF Screening System Weigh station Assay Protocol Plate Centrifuge Dispensers media Pintool in 10nl of compounds FLIPR Plate parasite/blood mixture Move plates offline for incubation for 3 days Dispense lysis buffer with SyBR Green Viewlux Pintool Assay Plate Incubator Read plates offline with Analyst GT 10 Confidential Compound Plate Storage (486 slots each) 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/inde x.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. 11 Confidential 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. • ~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) 12 Confidential 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 13 Confidential Scientific Approaches • Affinity chromatography/proteomics analysis • Lab-evolved resistant strains upon compound treatment • Tiling array analysis and whole-genome sequencing to help MoA determination 14 Confidential From an HTS hit to Phase I candidate • 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 NITD609 activity on clinical isolates of P. vivax (top) and P. falciparum (bottom) chloroquine artesunate NITD609 • 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 awarded MMV Project of the Year 2009 Rottmann et al Science, 2010 15 Confidential 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 16 Confidential 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 17 Confidential Acknowledgements – Malaria Project Team Chemistry • Arnab Chatterjee • Advait Nagle • Tao Wu • Tomoyo Sakata • Robert Moreau • Jason Roland • Pranab Mishra • David Tully • Valentina Molteni 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 Informatics • Jeff Janes • John Che • Yingyao Zhou 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 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 External collaborators Clinical team • Montip Gettayacamin (AFRIMS - Bangkok) • Jens Praestgaard • Robert Sauerwein (NCMLS - The Netherlands) • Ruobing Li • Ian Bathhurst (MMV) Assay Development HTS • Achim Brinker • Jason Matzen • Paul Anderson 18 Confidential GNF Management • Jennifer Taylor • Richard Glynne • Martin Seidel • Peter Schultz External support and advice • Marcel Tanner, Swiss Tropical Institute, • Nick White, Oxford University 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) 19 Confidential 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. 20 Confidential 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 21 Confidential Vesicle Detection Infection of mammalian cells Myotube Detection Acknowledgement Assay Development – HTS: Annie Mak’s group: Jason Chyba Vicki Zhou Sandra Gao Richard Brusch Amy Foraker Ingo Engels & his group 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 John Joslin & his group Ed Ainscow Jennifer Harris Thank you for your attention 22 Confidential 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) 23 Confidential 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 24 Confidential