Gribbon, P.

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Kiel 28th June 2012contact@screeningport.com www.screeningport.comAcademic drug discovery in EuropeDrug Discovery Living with FailureAll figs from Munos 2009 Nature Drug DiscoveryOne solution to constant NME output:Harness the ‘global brain’ to access the best science and ideaswherever they may be. Such open architecture for R&D has keyadvantages: it heightens competition, reduces costs and increasesagility by making it easier to initiate and terminate projects. Moreimportantly, it makes it easier to manage ‘disruptive innovation’ bylocating it outside the corporate walls (Munos 2009),The innovation gap updatedTop level figures• 4300 companies involved in R+D • 261 organizations = 1,222 NMEs. (6% of all companies)• 21 companies = ~600 NMEs.• 70% Pharma mergers reduce NME output• Small company (not top 15) success rate < 0.1 NME paIndustry response to the innovation gapData and IP pooling New collaboration models        Talking the talk> 70 Screening CentresFrearson and Collie 2009Clinical and Translational Science Centres USAPlanned 60 centres> $500 millionof NIH fundsInitiatives in Europehttp://www.ncrr.nih.gov/http://imi.europa.euhttp://www.eatris.eu/http://www.eu-openscreen.eu/Academic Initiatives in Drug DiscoveryLead Finding in Big PharmaEuropean Academic Screening Centres – 2011 figuresEuropean, approx 27% of the total  (64% USA) Typically cover all main target classesFor all centres, average of 13 targets p/a Most centres screen < 1 million wells per yearAll figures courtesy of John Comley - HTStec’s ‘Academic Outreach and Screening Trends 2011’ Report Regional and centralised drug discovery facilities•Assay development, compound logistics and Screening facilities on par with what might be found in small and medium Biotechnology organisation•Assay biology and targets originate from Institutional or regional networks (eg Max Planck), Scottish Universities etc) but also free to bring in external targets from other institutions•Large libraries > 200k  and facilities to profile (ADME, tox, Med-chem, Computational)•Diverse, fragment and focussed sets with emphasis on small molecules•Integrated robotics and workstation based infrastructures•Emphasis on drug discovery (eg tropical diseases) as well as Chemical Biology•Staff led typically by experienced ex-Pharma scientistsPan European Initiatives on horizonImplementation  Phase Oct 2011 Implementation 2013 http://imi.europa.euhttp://www.eatris.eu/http://www.eu-openscreen.eu/European Lead factoryScale will be less than MLP, but still order of magnitude > than previous efforts (ChemBioNet etc)Implementation  2013/2014ESP Centralized Screening HubCompound ManagementHTS SystemTarget +Biol. IPProject Basic Funding byFunding BMBF and Shareholders Validated Hit:•xC50•Cytotox•Cyp P450•Apoptosis•Biol. IP + Chem. IPChemistryEvotec Library• 250.000 cpds• Proven trackrecordESP Library• proof of concept /known drug library• Joint academicChemBioNetViSoR• Virtual system formolecular dockingESP infrastructureInfrastructure ServicesProject development:• Funding support• Grant applications• Build up consortiaProject prosecution:• Assay  Development• Screening (prim., sec., HC, fragmentbased)• Hit ValidationIn Operation August 2008Microsoft Office PowerPoint PresentationChemical Libraries at ESP• Access to Evotec Library• Σ = 250,000 compounds (cpds)• Enamine Library hosted  • Σ = 200,000 compounds (cpds) (70% diverse, gene and target family including PPI’s)• ESP Library • Σ = 35,000 compounds (NP’s, lead-like synthetic, 10% blinded)• Access to Hypha Discovery• Σ = 10,000 compounds 1Evotec small molecule libraryR small molecule13Access to Evotec Library•Σ = 250,000 compounds (cpds)•20k Additional fragment based library•All cpds QC-checked (LC/MS)•Optimized cpd storage for long term stability•Proven enhanced hit-rate from focussed sets•Cpd design guided by Lipinski’s Rule-of-Five and knowledge-based filters to enhance drug likeness•Privileged scaffolds and drug-like functionality complimented with extensive use of proprietary building blocks•More than 40 different structural cpd classes•Cpd preparation via validated, synthetic routes ensuring rapid access to analoguesESP Library  - Blinded• 2000 compounds Natural products and synthetic molecules from a German research Institute• 300 compounds  - anti infectives from a German research Institute• Future – an additional 1000 marine derived Natural products from a German research InstitutesThese compounds have a less straightforward IP position but are available for screening in all projects Hypha Discovery LibraryCase Study 1 North American University Indication Neurodegenerative diseaseTarget Protease•Assay development• Using full length protein substrate • TRF readout with antibody detection of cleavage site• enzyme titration, kinetics of substrate turnover, standard compound profiling• DMSO tolerance, day-to-day and plate-to-plate variability•HTS campaign• Primary screen 23k compounds• Hit Confirmation in Primary 11pt dose response• Hit characterization 2 additional orthogonal assay formats• Secondary assay 1 – Luminescence • Secondary assay 2 - FluorescenceProtein substrate [µM] log10 [inhibitor] MSubstrate Km5µl assay volumeTR- FRET signal TR-FRET signalZ’TR-FRET signalZ’Screening and Profiling 23k cpdsPharmacology DMSO toleranceScreen StatsPrimary screenSecondary screensLumi Fluor1.1% Hit rate @ 30% cut - offCase Study 2 German Research Institute Indication MalariaTarget Synthesis of an essential co-factorAssay development• Hetro-dimer complex• Coupled detection of synthaseproduct• Enzyme titration and kinetics, (no standard compounds)• DMSO tolerance, day-to-day and plate-to-plate variabilityHTS campaign• Primary screen 250k compounds• Hit Confirmation 2500 compounds• Hit Profiling 512 compounds• Secondary assay parasite proliferation assay in human rbc’s (Safety Level 3) 0200000400000600000800000100000012000001400000-4.5 -3.5 -2.5 -1.5 -0.5 0.5RFUlog(concentration) [µM] Protein 1 + protein 2 Protein 1Protein 2Assay Development (3 months)0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.500500100015002000250030003500Slope uncorrectedSlope correctedConc. of Pdx1 [µM]v [1/s]0 20 40 6002004006008001000Glutamine Concentration [mM]v [1/s]02000004000006000008000001000000120000014000001600000180000020000000.125 0.25 0.5 0.75 1 1.50 2 4 0 (High Ctrl)0 (Low Ctrl)RFUDMSO conc. [%]A BReagent production Enzyme titrations Heterodimer functional testingDMSO ToleranceMarker Mini-screen384 well Plates screened945 (plus DMSO sacrificial plates)Plate QC failures104Median Z’ 0.72    Screened cpds 251,000Hit Rate 1.4% (3607)ConfirmationPick size2500Primary Screening Statistics•Compound Triage by expert Medicinal Chemist  • Structural classification and selection based on potency and attractiveness as starting points• Prioritized hits from known drug library to facilitate re-purposingBECConfirmation and Counter AssayCounter Assay 1• Detection system only• 1uM Glutamate •>50% Compounds inhibit detection system• Crucial readoutPutative HitsInhibit ors of detectionConfirmation assay• Primary in triplicate•80% Hit recovery•Significant  # “super” inhibitors – artefacts?Counter Assay  2• Run reaction as primary • Add compound   then read•Quenchers false +ve•Fluorescent false -ve• Resorufin produces robust signal!Fluorescent cpds QuenchingcpdsDose Response + Hit profiling01020304050600.10.40.7 11.31.61.92.22.52.83.13.43.7 44.34.64.9FrequencyBinned Hill slope 020406080100120140FrequencyBinned pIC50 0246810121416180.10.50.91.31.72.12.52.93.33.74.14.54.9FrequencyBinned Hill slope01020304050607080Frequency8.500.10.20.30.40.50.60.70.80.910 50 100 150 200Ratio IC50 (Primary / Counter)CompoundPrimary Assay  - 497 compounds (of 512) with curve fitsCounter  Assay - 200 compounds (of 512) with curve fits (IC-50 Primary) / (IC-50 Counter)    Marine Fungi Project – Drug discovery24.09.2013 Marine Fungi - WP7 MGA May 201223The aim is to identify specific marine fungi derived compounds which are suitablestarting points for drug discovery. Parties in Drug Discovert Worrking groups: ESP,GEOMAR, UIO (Oslo), DTI (Denmark), Hypha discovery (UK)Cell line panel• NCI cell line panel24.09.2013 Marine Fungi - WP7 MGA May 201224sourced all 60 cell linesPreliminary panel consisting of M14, 786-0, MCF-7 and HL-60 cell culture protocols set upfor 20 cell linesEfficacy assessment assays at 3 sites• Profile compounds effects on growth and viability of NCI panel24.09.2013 Marine Fungi - WP7 MGA May 201225ESP UIO HDCell Titer Glo Luminescence Viability Assay (Promega Corp., US)Neutral Red Assaytoluylene redstains lysosomes in living cellsMethods24.09.2013 Marine Fungi - WP7 MGA May 201226 NCI cancer cell line screenConcentrations at 50% cell growth (GI50), 0% cell growth (TGI) and -50% cell growth (LC50) were collected for each cell line per compound.Aim: Screening of interesting compounds in the whole panel to obtain GI50, TGI and LC50 in each cell line Optimisation of protocols• 3D cell culture (BioLevitator)24.09.2013 Marine Fungi - WP7 MGA May 201227SF-593 A549-ATCCM14 786-0Bioinformatics tools for Cpd analysis24.09.2013 Marine Fungi - WP7 MGA May 201228dataGI50TGILC50COMPAREonline toolNCIMeangraphOutputCorrelationof test compounds to known compoundsMoAstudiesTarget directedstudiesNCI Mean Graph TGIs of compound 954 in 8cell lines displayed relative to the mean (MGMID) of -4.48 log molar . Unit: log molarAssay Validation24.09.2013 Marine Fungi - WP7 MGA May 201229 NCI cancer cell linesCell growth inhibition and Cytotoxicity assay validated for 17 cell lines Validation includes:- DMSO tolerance- Titration/Linearity experiments- Signal stability experiments- Cell growth evaluation- Standard compound dose-response experiments(cisplatin, paclitaxel, staurosporine, 6-mercaptopurine) Selected fractions and pure compounds screened in preliminary panel and all other validated cell linesPurified compound – Example results24.09.2013 Marine Fungi - WP7 MGA May 201230About 300 compounds screened in the preliminary panelHits identified and profiled in 17 cell lines (ESP&UIO)• Compound ADose-response curvecompound 525n in SF-53924.09.2013 Marine Fungi - WP7 MGA May 201231Profiling results (DRC)pure compoundsmean graphs in all screened cell lines for COMPARE algorithmMean graph of compound B in 8 cell linesPurified compound studiesMarine Fungi Project 24.09.2013 Marine Fungi - WP7 MGA May 201232Project on track to deliver in-vitro Proof of Concept (2013)1H 20122013 2011 / 20122011ESP Enabling Technologies groupPartner with Bioassay Discovery Tools Company (USA)Validation of new reagent portfolio for epigenetic targets H D ACS and thei r i nhibiti ons in c ancer cells  (Witt et al; Canc er L etters  277 (2009); 8-21)Chuang et al ; Trends  in Neuros c .; Vol 32, No 11, 591-601H D AC inhibitiors  i n CNS di sorders (Kaz ants ev, Thompson; Nature Rev. D rug D is c.  Oct 2008;  Vol  7;  854-868)c las s i soformplay s rol e insubstratespan inhibitors clas s I  i nhibitors c lass  I I inhibitorsfunc tion in c ancer cells func tion in CN S k nock  out phenoty pes  (i n mice)c ancerCN S inflam-mation TSA SAH A L AQ824Panobi -nostatBeli -nostatPCI-24781MS-275MG CD-0103Depsi -peptideApic idinVPA Trapoxi nS B-429201Bi s-Py ri-di ni-um dieneS H I-1:2 R306465SB-379-278APCI-34051Cpd2APH A deriv ativ esTuba-ci nMercapto-acet-amideN CT-10a/ 14aS urami nN ic otinamideTS AN a-Buty-rateVPAbenz -amidesM344 EX-527I1 Y Y Yp53, MyoD,  E2F -1, Stat3, androgencerv ic al  c ancer cells  :  H DAC1 knoc k down res ults  in i nhibiti on of proliferati on and i nducti on of autophagy ; os teos arcoma and breas t c anc er cell s : k noc kdown c aus es  cell  cy cl e arres t,  growth i nhibiti on, apoptosi s;  c olon c ancer cells  :  knock down suppres ses  growth; pros tate c ancer:  ov erexpress ion increases prol iferation and dedifferentiati on; neurobl as toma cell s: knock down sensi tiz es  for chemotherapy ; CLL  c ells  : knock down sensi tiz es  for TRAIL apoptos isRett Sy ndromeac c umulating ev idenc e supports  the notion that his tone hy poac etyl ation and transc ri ptional dy sfunction are i nv ol ved in a large number of neurodegenartiv e c onditi ons  in v iv o and in v itro; targeti ng  his tone ac etylati on may prov ide benefi t for the treatment of depress ion,  sc hi zophrenia, drug addic tion and anx iety  dis orders ; Rubinstei n-Taybi s yndrome: ubiqui tous  roles  of CBP and p300 in al l tiss ues , and the rec ent data suggestingan improvement in long-term memory  and s ynapti c pl as tic ity by  the H DAC i nhibitor tric ho-s tati n A (TS A) through its effect on CBP and CREB; Friedreic h’ s atax ia : ex pans ion of a  tri pl et repeat reg ion wi thi n an intron in the frataxi n gene (F XN ),  whi ch enc odes a hi ghly  c ons erved mitoc hondri al protein, l eads to trans cript-i onal  s ilenc ing i n the neuro-degenerativ e di seas eFriedreic h’s atax ia  (s ee Benz amide H DAC inhibitors);  F ragil e X syndrome:  benz ami de-bas ed H DAC i nhibitors  rev ersed frataxi n-medi ated s ilenc ing,  S IRT1 inhi bi tion revers es  hetero-chromati n-medi ated s ilenc ing of the F MR1 loc us  by increasi ng  ac etyl ati on and dec reas ing methyl ation of his tones.  Therefore, pharmaco-l og ic al  i nhibiti on of S IRT1 c oul d provide an a lternativ e s trategy to reac tiv ate F MR1 ex pres si on and to rev erse the los s of a requi red neuronal  protein; a ls o involv ed i n Stroke,  H unti ngton' s di seas e, ALS , SMA, Parkins ons di seas es,  Al zhei mers  dis eas eembryonic  lethal day  9.5, p21 and p27 upregulation, reduc ed ov erall  H DAC ac tiv ity2 Y Y YBcl -6, S tat3, g lucoc ortic oi d, rec eptor, YY-1cerv ic al  c ancer cells : H DAC2 k noc kdown res ul ts  in di fferentiation,  apoptosi s and p53 independent p21 ex press ion;  breas t c anc er cell s: increased p53 acti vi ty , inhi bi tion of prol iferation, induction of senes cence, i nduc ti on of apoptos is ; c olon c anc er c el ls:  k nock down c auses  growth arrest; neurobl as toma cells  :  k nock down induc es  apoptos is ; genetic H DAC2 mutation reduc es  intestinal  tumor dev el opment i n APC mic e i n vi vo; CL L c ells  : knock down sensi tiz es  for TRAIL-apoptos isRett Sy ndrome, S pi nal Mus cular Atrophy  (S MA)viable unti l perinatal peri od, fatal  muli tple,  c ardiac  defec ts , exc ess iv e hy perplasi a of heart mus cle, arry thmi a3 Y Y YG ATA-1, RelA, S tat3, MEF2D, YY-1,  S HPAPL  c ell s:  H DAC3 as soc iated with PML -RARa fusi on protei n,  knock down induc es  differentiation genes ; AML :AML -1-ETO binds HDAC3 (and HDACs  1, 2),  di srupts  c el l c yc leembryonic  lethal before day 9. 5, defective c ell c y cl e, DN A repai r and apoptosi s in embry onic  fibroblas ts.  Conditional l iver knoc k  out results i n hepatocy te and i nduc ti on of metaboli c genes hy pertrophy8 Y Y Y ndneuroblas toma c ells  : H DAC8 k noc kdown i nduc es di fferentiation,  c ell c y cl e arres t and inhibits c lonogenic  growth; lung, c olon, cerv ic al  c ancer cells  :  knock down of H DAC8 reduces proli ferationRett Sy ndrome ndIIa4 Y YG CMa,  G ATA-1,  H P-1APL  c ell s : H DAC4 interacts  wi th PL ZF-RARa fus ion protein, repres ses  differentiation genes ; renal c arci noma cells : knock down inhi bi ts ex pres s ion and func tional ac tiv ity of H IF -1aviable, premature and ec topic  os si fic ation,  chondrocy te hypertrophy ; from the related c ardi acfield, i t is  noteworthy  that k noc kdown of HDAC4 reduc ed i nfarc t si ze foll owing  my ocardia l i sc hemia-induc ed reperfusi on i njury  5 Y Y S mad7, H P-1, G CMaerythroleukemia : HDAC5 s huttles  from nuc leus  to c y toplas m upon differenti ation, interacts wi th G ATA-1seems  to mediate antidepres sant activ ity  in animal s tudies  (N a-Buty rate)my ocardia l hypertrophy , abnormal cardiac s tres s respons e7 Y Y F LAG  1 and 2endothelia l cells : HDAC7 s ilencing  a lters  morphology , migration and tube-formi ng  c apaci tyembryonic  lethal , l ac k of endothelia l cell-cell  adhes ion9 Y nd ndviable at birth, spontaneous  my ocardia l hy pertrophyIIb6 Y Ya-Tubul in, H S P90, S H P,  S mad7Targeted i nhibiti on of H DAC6 l eads  to ac etylati on of H S P90 and disrupti on of i ts chaperone function, resulting i n depletion of pro-growth and pro-s urv ival cl ient proteins  incl uding the Bc r-Abl  onc oprotein i n K562 l eukemic  c el ls;  H DAC6 targeting  bloc k s EG F  induc ed nuc lear transl oc ation of ß-cateni n and c-myc  express ion in c olon c arc inoma c ells ; knock down of H DAC6 caus es donwregul ation of HI F-1a, VEG F R1/2; HDAC6 inv ov led in TG F b induc ed epi theli al -mes enc hy mal  transi tion of lung c arc inoma cell sviable, no si gnifi cant defec ts , i nc reas e in global  tubuli n ac etylati on. MEF s fai l to rec over from ox idativ e stres s10 Y H S P90 ? Knock down of H DAC10 downregulates VEG FR ndIV 11 nd nd ndIIISI RT1 Y Y i nhibited inhi bited i nhibited fragi le X sy ndromeSI RT2 Y i nhibited inhi bitedSI RT3 inhi bitedSI RT4 inhi bitedSI RT5 inhi bitedSI RT6 inhi bitedSI RT7 inhi bitedcl inic al tri al  phasesI, I I, III , approv ed CTCLII , I II I,  II I I II I, I I I, I Iin c linic al tri al s for treatment of S MAl eukemia, advanc ed solid tumors, ly mphomasnd:  no dataMEF:  mous e embry onic  fi broblastss trong inhibition (EC50 < 5fold EC50 relativ e to most sensitiv e HDAC isoformweak  i nhibiti on (EC50 > 5fol d EC50 rel ati ve to mos t s ens itiv e H DAC is oformno inhi bition (EC50 > 100fold EC50 relativ e to mos t s ens itiv e H DAC is oformno data publi shedEpigenetic targets  ESP Library  ESP Infrastructure  ESP Expertise  Novel assayLimited validationEnzyme (nM)Industrialised formatHTS compatibility Market Validation  Open Innovation in Practice:Neu2  - A Competence Consortium in MSNeu2 Project flowNeu2  - Competence CascadePositioning of Neu2 portfolioNeu2  Project portfolio April 2012Activities•Hit Finding – Phase  II trial •ScreeningPort involved in 3 running projects•2 new MS Biomarker related projects accepted for next round•1 new target for  comprehensive Hit findingScope and project finances•Acute focus on Multiple Sclerosis• Novel mechanisms favoured• Higher risk than typical portfolio• BMBF 25Mio first 3 years• all projects need counter-financing• No “double funding” allowed•Renewal process mid 2012, goal is to secure additional 3 – 5 years funding Summary• Pharma companies are increasingly moving out of early Discovery being replaced by Universities and Biotech• Academic drug discovery is a vibrant activity, but the impact in terms of addressing unmet patient needs has yet to be fully realised• Sources of compounds for Hi finding and Lead identification is increasingly varied and natural products still have a significant role to play• Academia should not try to replicate Industry activities, rather to complement and be prepared to take on greater risks

Academic drug discovery in Europe

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