Presentation on theme: "Building a strong anti-malarial drug pipeline based on phenotypic"— Presentation transcript:
1Building a strong anti-malarial drug pipeline based on phenotypic whole organism screeningAnnie Mak, PhDpresenting on behalf of Malaria project team at GNFSeptember 22, 2011
2Outline An introduction to GNF Anti-malarial drug discovery Our small molecule drug discovery infrastructureAnti-malarial drug discoveryAD-HTS in the 2010s – which direction are we heading
3Genomics Institute of the Novartis Research Foundation GNFGenomics Institute of the Novartis Research FoundationMission: 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 pipelineFunded by the Novartis Research FoundationLocated in La Jolla, CaliforniaMoved into permanent 260,000 square foot research campus in 1Q 2002Additional 24,000 square foot manufacturing facility for our automation system(www.GNFSystems.com)
4Examples 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 EngineeringAutomated Protein Production and PurificationAutomated Cell/Compound Profiling(multiple cell lines/assays; fewer compounds)Automated Functional Genomics(cDNA, siRNA)Protein X-ray Structure DeterminationCore technology platforms provides much versatility and flexibility,allowing us to focus on interesting science.
5Small Molecule Drug Discovery Infrastructure NovartisInfrastructureTargetValidationExploratoryChemistryFull LeadOptimizationHTSClinicD0D1D2D3Candidate SelectionPointGNFMedicinal & Analytical ChemistryExperienced medicinal chemistry staffHigh-throughput analytical and purification technologySophisticated compound managementPharmacology/ADMETIn vitro and in vivo ADMET capabilitiesState-of-the-art bioanalytical technologyIn vivo efficacy models in metabolic disease, immunology and oncologyExploratory/Target ID ToolsLinker Chemistry & SARAffinity methods/ Target pull-downPathway profilingFunctional genomics collectionSolexa Sequencing
6Discovery 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, 2011ResearchTranslationalDevelopmentLead GenLead OptPreclinicalPhase IPhase IIaPhase IIb/IIIRegistrationPhase IVNovartisminiportfolioNovartis2 ProjectsMK 4815(Merck)TafenoquineGSKOZ 439(Monash/UNMC/STI)AZCQPfizerEurartesim™sigma-tauCoartem®-DNovartisGSKminiportfolioGSK1 ProjectGNF156NovartisNITD609NovartisPyramax®Shin Poong/University of IowaASAQ Winthropsanofi aventis/DNDiBroad/GenzymeminiportfolioAminoindoleBroad/GenzymeAN3661AnacorIV artesunateGuilinMany others …Many others …
7Drug resistance to most marketed drugs widespread… MalariaMedical Need500 million cases annually worldwideNearly 1 million deaths/yr, >75% under age of 5Resistance to current therapies widespread with exception of artemisinin derivativesIncreased parasite clearance times for Artemisinin-based therapies seen in Thai-Cambodian borderCurrent artemisinin-based combination therapies (ACTs) contraindicated in 1st trimesterNo current therapies address need for blood-stage, liver-stage, and transmission blocking activityInfective mosquito biteHepatic phase:Normal hepatic phase leading to primary infectionErythrocytic phasePrimary infectionProtracted hepatic phase leading to relapse infectionRelapse infectionRing -> TrophDrug resistance to most marketed drugs widespread…
8Scientific Approaches Cell-based screen approachMajority of anti-infectives discovered through cell-based screening by facilitating parallel interrogation of druggable targets and also addresses compound permeability issuesHTS on >2M compounds: P. falciparum infected human red blood cells (RBCs)Liver–stage infection assay (P. vivax infection)Target-based screen approachCollaboration with academic institutesPlasmodium kinases: such as CDPK1, CDPK5, GSK3, CK2αAdditional biochemical targets screening at GNF on cell-active compoundsTarget identification methodsLab-evolved resistant strains upon compound treatmentTiling array analysis and whole-genome sequencing to help MoA determinationAffinity chromatography/proteomics analysis
10Fully automated GNF Screening System DispensersAssay ProtocolPlate mediaPintool in 10nl of compoundsPlate parasite/blood mixtureMove plates offline for incubation for 3 daysDispense lysis buffer with SyBR GreenRead plates offline with Analyst GTWeigh stationCentrifugeFLIPRViewluxPintoolAssay Plate IncubatorCompound Plate Storage (486 slots each)
11Malaria 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 communityhttps://www.ebi.ac.uk/chembldb/index.php/compound1,256 < 200 nM EC50 vs 3d7>200 scaffolds represented in the reconfirmed compound setTremendous amount of chemical diversity from screenHTS reported in: Plouffe and co-workers PNAS 2008, 9059.
12Selection 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); andhas pharmacokinetic properties compatible with once-daily oral dosing.~5K reconfirmed blood-stage hits from HTS identified for further evaluation were determined byLess than 1.25 µM EC50 with activity in 15 strain resistance panelLess than 5-fold potency shift between strainsHigh selectivity in 6-cell line toxicity panel (SI > 20-fold)Novel chemotype for malariaEase of synthesis (less than 7 steps)Good solubility, metabolic stability and limited CYP450 inhibitionMultiple actives within a scaffold (if library diversity allows)
13Scientific Approaches Cell-based screen approachMajority of anti-infectives discovered through cell-based screening by facilitating parallel interrogation of druggable targets and also addresses compound permeability issuesHTS on >2M compounds: P. falciparum infected human RBCsLiver–stage infection assay (P. vivax infection)Target-based screen approachCollaboration with academic institutesPlasmodium kinases: such as CDPK1, CDPK5, GSK3, CK2αAdditional biochemical targets screening at GNF on cell-active compoundsTarget identification methodsLab-evolved resistant strains upon compound treatmentTiling array analysis and whole-genome sequencing to help MoA determinationAffinity chromatography/proteomics analysis
14Scientific Approaches Affinity chromatography/proteomics analysisLab-evolved resistant strains upon compound treatmentTiling array analysis and whole-genome sequencing to help MoA determination
15From 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 NITD609Potent (IC50< 1 nM) blood stage compoundPotent transmission-blocking activityLow clearance, moderate-to-long half-life: predicted human efficacious dose < 100 mgCurrently in healthy volunteer trialsClinical efficacy studies in mid-2011First in class compound with efficacy superior to standard anti-malarial drugs in mouse efficacy modelGenome wide scanning of drug resistant mutant reveals that NITD609 targets PfATP4 and inhibits protein synthesis, a distinct MoA different from Arteminisin.chloroquineartesunateNITD609chloroquineartesunateNITD609(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 studiesawarded MMV Project of the Year 2009Rottmann et al Science, 2010
16On-going activity in the pipeline Hit series among others: ImidazolopiperazinesNot identified in >100 HTS screens performed at GNFNovel chemotype with a mechanism of action distinct from that of NITD609Simple chemical synthesisFirst-in-class compound that has broad activity across parasite life cycleFavorable 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
17MedChem lessons learnt so far SAR can be challenging, but getting hits from HTS with good SAR is criticalBetter to have SAR than absolute potencies off the deckPotencies can be optimized relatively quickly compared to other parametersMolecular 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
19AD-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)
20Continual upgrades to support various screening modalities New readers to improve sensitivity and speedCompound transfer via Acoustic Droplet Ejectionmore 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.
21High content imaging in HTS Interesting Phenotype (Blackbox!)Rigorous Assay DevelopmentRobust and accurate automation for HTSImager to capture sufficient resolution and statisticsImage analysis algorithm in placeIT infrastructure to handle dataNov 2010 – Feb 2011: Completed full deck HCI-HTSNuclear translocation assay detected by IF stainingAutomation handled 200 plates/batch, 5 week campaign3.9 million wells imaged by Opera (confocal microscope)Read time: 80 daysData generated: 9 TBData analysis script developed in-house Infrastructure and expertise in place to handle HCI in a HTS scaleNew assays under development:Cellular differentiation – specific marker expression or morphological changesInfection – host vs pathogen“New tricks to old questions” – DNA repair assayed by chromosome breakpoint detection; cell cycle analysis by multiplexing specific markersVesicleDetectionMyotubeInfectionof mammalian cells
22Thank you for your attention AcknowledgementAssay Development – HTS:Annie Mak’s group:Jason ChybaVicki ZhouSandra GaoRichard BruschAmy ForakerIngo Engels & his groupJohn Joslin & his groupEd AinscowJennifer HarrisAutomation/Engineering:Dan SipesJason MatzenPaul AndersonMike GarciaTim SmithDrew GundersonDan Rines… many othersInformatics:Jeff JanesJulia TurnerGhislain BonamyThank you for your attention
23Various modalities are supported LuminescenceFluorescenceAbsorbanceTR-FRETHigh content imagersCompound/Library transfer via:Acoustic Droplet Ejection, PintoolFLIPR, Lumilux(kinetic fluorescence/luminescence)
24Innovative 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 systemOn-the-fly data analysis + system alerts to monitor progressExtensive informatics support for data analysis (kinetic reads, imaging assays)Large hitpick capacityTypical full deck HTS = 4M wells in ~2-3 weeks Hitpick of 20KOverall, lower opportunity cost means more opportunistic approach to HTSGantt ChartTrace file from on-the-fly data analysisTimePlate