Presentation on theme: "Jason Witherington EpiNova DPU"— Presentation transcript:
1 Jason Witherington EpiNova DPU “Bromodomains: A new class of epigenetic targets ripe for small molecule drug discovery”ELRIG – Manchester 2012Jason WitheringtonEpiNova DPU
2 Outline Brief introduction to Epigenetics Luck strikes!.....discovery of small molecule bromodomain inhibitorsExploiting serendipity through SBDD/FBDDBrief overview of preclinical iBET biology
3 Epigenetics - Chromatin DNA is packaged around histones and other proteins to form chromatinChromatin is highly dynamic material which undergoes remodelling to allow suppression or activation of genesA number of Epigenetic mechanisms control chromatin remodelling including post-translational modifications (PTMs) on histone tailsDysregulation of histone PTMs implicated in human disease
4 Epigenetics : Histone post-translational modifications PTM of histone tails>70 sites are known mostly located in the unstructured N- terminal tails.> 8 types of modification have been reported.AA modified include : K, R, S, T, Y, H, EMost of these are reversible and dynamic.PTM rarely occur in isolation => complex pattern of modification = histone code.Reader domains rarely occur in isolationPTMs can have a direct impact on physical properties of individual nucleosomes e.g. neutralisation of chargePTM are recognised by specialised reader domains.
5 Apo-A1 phenotypic assay Apo-A1 target for dyslipidemiaUpregulator reporter HTS identified several lead series including a BZD series.Medicinal chemistry successfully optimised molecules to candidate selection without knowledge of molecular target.Extensive profiling of compounds did not identify target for these molecules Chemoproteomics5’-UTRApoA13’-UTR-1.4kbHuman ApoA1 promoterFirefly luciferaseBenzodiazepine
6 How were new medicines discovered ? Between :More first-in-class drugs were discovered by phenotypic screeningMore follower drugs were discovered by target-based screeningNat. Rev Drug Discovery 10, 507 (2011)
7 Chemoproteomics – Overview of approach Biologically relevant systemHepG2 & THP1J Med Chem (2011) 54, 3827Derivatised CompoundsActive BZDInactive BZDMatrixWash and ElutionStringencyCompound / SDSPMMLC/MS/MSPROTEINIDENTITYAISeparate on 1-D Gelactive compound specific bands & low backgrounds
8 Chemoproteomics BET (BromoDomain & Extra Terminal) proteins identified +Series X inactive+ Series X active+ BZD inactive2004050150kDa302010075BZD active matrixBZD Inactive matrixMatrix alone+ BZD activeCompound KeyRED = ActiveBLACK = InactivekDa200150All bands identified asBET family proteinsBrd2, Brd3, Brd41007550403020Competition experiments suggest that actives from BZD and other series specifically interact with BET proteins
9 Brd4 knockdown induces Apo-A1 upregulation Apo-A1 activators are ligands for the BET proteinsIs this interaction responsible for Apo-A1 upregulation?Increase in ApoA1 mRNA on addition of BZDIncrease in ApoA1 mRNA on BRD4 knockdownIncrease in Apo-A1 mRNAActive BZD – 1mMBRD4 siRNA: 500nMDCT24hr48hr96hr0hr72hr-1.6-1.4-1.2-1-0.8-0.6-0.4-0.20.21We were therefore pretty convinced that the Apo-A1 activators were ligands for the Bet proteins.Which leads naturally to the question is this the interaction that is responsible for Apo-A1 upregulation?The histogram below shows in blue that application of the BZD cmpd to HepG2 cells results in an increase in Apo-A1 mRNA. In red you can see that this response can be induced using siRNA knockdown of Brd4.Clearly linking this BET protein with Apo-A1.
10 BET binding correlates with Apo-A1 cellular activity BZDsApo-A1 pec170BRD4 FP p IC50Theoretical difficulties in tackling epigenetic PPI were not realisedMany diverse and potent compound obtained using cellular activity to guide SAR.
11 Bromodomains bind to acetylated lysine residues
12 Challenges with targeting epigenetic readers Reader domains often bind PTM weakly => no hot spots?Multi-valency of protein-protein interactions => Tethered ligandsMegaDalton protein-protein/DNA complexes => will inhibiting a single interaction be enough for biological efficacy?If protein-protein inhibition is poorly tractable => how tractable are targeting epigenetic readers?PREVIOUS PHARMACEUTICAL FOCUS ON EPIENZYMES NOT EPIREADERS
13 BRDs control gene transcription ET domainbromodomain2bromodomain1Transcriptional co-regulators involved in histone binding complexesBrd4 binds to cdk9/cyclinT (pTef-B) to positively regulate RNA pol II mediated transcription at multiple promotersPol IIpTef-BPtranscriptionBRDAcAcAcAcetylated lysines on Histones within euchromatinApoA1 compounds bind to BET BUT where specifically do the compounds interact?
15 Biophysical data demonstrates specific binding 1Brd2(1-473)INACTIVE XINACTIVE YACTIVE XINACTIVE X0.8ACTIVE XACTIVE Y0.6normalised CDACTIVE YINACTIVE Y0.4Tool compounds stabilise all Brd2 bromodomain constructs0.2405060Temp (oC)KDBRD2_1KD °CBRD2_2BZD tool binds both N and C-terminal domains but kinetics and affinity at 25°C are different for each
16 Isothermal Titration Calorimetry demonstrates specific binding to both BRDs 1:1 46nM(16°C)Brd1Brd32 1:1 52nM(26°C) 2:1 30nM(26°C)C-terminalbromodomainN-terminalN-terminalbromodomainC-terminalbromodomain
17 I-BET762 is a highly selective inhibitor of BET bromodomains Tm profiling5-7oC1-3oC<1oCI-BET762Not all protein-protein are difficult. This protein surface is filled with crevices and pockets.A yellow histone peptide binds along one of these with the Ac-K buried deep into a recognition pocket.Overlaying this with the white BZD compound shows that points into the Ac-K whilst this runs along the peptide grove and this pendant benzyl group binds in a hydrophobic pocket that is not occupied by the peptideOverlaying this with the purple ATAQ compound shows a similar heterocyle in the Ac-K pocket and the compound now occupying space to the left of the natural peptide ligandOverlay it with the green isoxazole compound shows that this compound doesn’t really use the peptide groove at all and the isoxazole is the moiety occupying the Ac-K pocket
18 iBET Broader Selectivity Profiling Inactive against a wide range of proteins
19 Where do the compounds bind? N-terminal bromodomain of Brd2 is typical helical structureTheir role is to recognise acetylated marks on histones and other proteinsCompounds shown to displace the tetraAcH4 peptide Antagonise protein-protein interactionFRET assay for displacement of tetraacetylated H4
20 First Small Molecule X-ray co-crystal confirms binding in the acetylated lysine pocket H4 peptideRecognition of carbonyl of AcK preserved (N156,Y113)F-(VP)-Y-(CAS)-N AcK binding siteCommon to 44 out of 58 bromodomainsH2O structure in pocket preserved.NH interactions of AcK not preserved
21 Interactions of BZD outside the AcK pocket BrdT – Nature (2009)
22 Bromodomains can deliver both probes and drug like molecules iBET 762clogP, PSA, MWt~2, ~80, ~400BRD2/3/4 pIC506.8/6.7/6.7hERG EC50Ion Works (Dof)100uMPatch Express61uMRat (Mouse) PK*Clb (mL/min/Kg);Vss (L/kg); t½ (h), %Fpo63 (24), 1.8 (1.7), 0.5 (0.8), 27 (22)Dog PK*5, 1.8, 5.9, 44Unbound fraction in blood (R/D/Mou/H)0.18 /0.24/0.21/0.19CYP inhibition IC50s (uM)> 33P450 TDI<2-fold*3mg/kg p.o.; 1mg/kg i.v.
23 Optimisation of dimethyl isoxazole HTS lead to in vivo probe I-BET 151 CLi microsomes (mL/min.Kg)CLb ml/min.kgVd L/kgT½ hF %Rat<0.53182.11.766Dog17383.01.216minipig151.665Human1.1BMCL, 2012, 2963BMCL, 2012, 2968
24 GSK525762 and GSK1210151 bind BET proteins using similar “hot spots” WPFZA ChannelAcK pocketI-BET 762I-BET 151
25 Bromodomain Family and Structural Coverage BRPF1BRD2_1BRD3_1BRD1CREBBPBRD3_2BRD4_1BPRF3EP300BRD2_2BRD9BRDT_1>50 bromodomainsIn isolation or combination with other domainsMultiple opportunities for clinical utilityBRDT_2BRD7BRD4_2BAZ1AKIAA1240ATAD2BRD8WDR9_2PHIP_2BRWD3_2TAF1_1BAZ1BPRKCBP1TAF1L_1TAF1_2CECR2TAF1L_2FALZBAZ2BGCN5L2BAZ2APCAFZMYND11TRIM33MLLTTTRIM28WDR9_1TIF1YTTRIM66SP110YASH1LBRWD3_1YPHIP_1SP140TSP100LOC93349PB1_1YPB1_3PB1_5PB1_2SMARCA4PB1_4SMARCA2Structure knownAtypical AcK Binding Residue
26 Across the family there is significant structural divergence outside of the AcK binding region ZA LoopBC Loop
27 Exploiting Structural Knowledge : Fragments – Generation of a Hit-ID platform for Bromodomains Knowledge of key ligand-protein interactions derived from the Bet programme lead-like compoundsGeneration of a pharmacophore modelSelection of a focussed screening setConfirmation of the binding mode using crystallography>20% inhib at 200uMCreation of a fragment toolchest that binds in the AcK recognition pocket of the bromodomain
28 Fragment based discovery 1400 Fragments screened>40 Fragments crystallisedKey Structural waters identifiedPharmacophore refined"Fragment-based discovery of bromodomain inhibitors part 1: Inhibitor Binding Modes and Implications for lead discoveryAuthor(s): Chung, Dean, Woolven and Bamborough
30 Application of Encoded Library Technology (ELT) Structural knowledgeConstruction and screening of librariesLIBRAEStargetsIdentification of FeaturesExploitation of Screening outputELT hits against target 2HitsScreening toolsProbeslibrariesenrichment
32 Nodal AND gene specific intervention? TNFaIL-6IFNbpI:CLPSI-BETTNFaXXIFNbIL6unaffectedblockedblocked
33 BET compound displaces BRD4 from IFNb and IL-6 promoters (ChIP) BRD4 / H3LPS drives recruitment of Brd4 to selective promotersCompounds prevent this recruitment and block transcriptional activationSoren Beinke
35 Use of Chemoproteomics for target class expansion Pharma industry mainly reliant on recombinant platformsLarge screening panels required for selectivity profiling (human/rat etc)Brds occur in isolation & combination with other domainsProtein complexes modify functionDifferent complexes may form under different activation states &/or different tissuesEpiNova-Cellzome alliance provides a complementary screening platform to address the above
36 Triple purification strategy BET interacting proteins: MS-proteomic analysisTriple purification strategyAcetylatedH4 tail (K4,K8,K12)AcNature (2011) 478, 529H4proteins binding directly or indirectly to histone marks= BET inhibitor(I-BET)Antibodyagainst BRD2/3/4proteins binding directly or indirectly to I-BETBET protein imuno-complexes
37 BET interacting proteins: MS-proteomic analysis
38 I-BET762 is effective in multiple models of Multiple Myeloma **log10 [I-BET762][I-BET762]
39 BET interacting proteins: MS-proteomic analysis
40 I-BET151 is a novel & selective inhibitor of BET proteins with improved PK properties
42 I-BET151 mediates disease control in MLL leukaemia models Transplanthuman MV411leukaemia cellsNOD-SCIDTransplantsyngeneic MLL-AF9leukaemia cellsC57BL/6
43 SummaryChemoproteomics has been employed to identify a chemical opportunities against a previously intractable target classChemoproteomics has been utilised to allow the efficient selectivity profiling across the “Bromonome” using endogenous cell lysatesChemoproteomics has demonstrated utility in defining clinical opportunities through complex identification
44 Effect of BET inhibition on LPS induced shock preventativeI-BET-1htherapeuticI-BET1.5hLPS0hNature, 468, p1119, 2010
45 Summary…..Use of chemoproteomics can be a powerful way to identify output of phenotypic screeningPreviously “undruggable” reader class of epigenetic proteins are ripe for drug discoveryThe iBET bromodomain family of proteins have profound preclinical biology (more this afternoon)