Selective Targeting of Protein Interactions Mediated by Epigenetic - PowerPoint PPT Presentation

Selective Targeting of Protein Interactions Mediated by Epigenetic Effector Domains Stefan Knapp Structural Genomics Consortium Oxford University, Nuffield Department of Clinical Medicine Oxford, United Kingdom Seminar at the Universität Duisburg-Essen Essen, Germany September 5, 2013

Exploring New Target Areas Modification Write Read Erase HDAC Acetyl HAT Bromo HDAC HDAC Methyl KMT Tudor , MBT, Chromo KDM Probe Criteria Divide each protein family into subfamilies, and generate at least one chemical probe for each subfamily with: • Kd <100nM • >30-fold selectivity versus other subfamilies • Demonstration of “on - target” effect in cells at ≤1uM

Chemical Probe Consortium  Too large an undertaking and too high risk for individual Pharma companies  Large global academic community, but lack of quality chemical tools in the public domain  => Open Access Chemical Probes Consortium combines expertise in academia and Pharma, and pools resources to effectively evaluate the field A Large Private Public Partnership GlaxoSmithKline NIH Chemical Genomics Pfizer Centre, Bethesda Eli Lilly & Many Academic Novartis Collaborators Abbvie Takeda Boehringer J&J Toronto SGC UNC Center for Integrative Oxford : Chemical Biology and Drug SGC Discovery Chemistry Department

Acetyl-Lysine Readers (Bromodomains)  61 domains in Human  ~120 residue Kac interaction module  Clinical POC targeting Kac regulation (HDACs)  Linked to many diseases  Druggable pocket BRD2: 2DVQ Cell, March 30, 2012

Identifying High Affinity Substrates Peptide library screen using SPR Peptide array screens using dot blots Histone peptide Targets  Affinities can be determined by SPR but not using dot blots  Many BRDs show no interaction with Histone marks  36 BRDs screened against all possible histone Kac sites and combinations of marks.  485 new target sequences identified  BRDs recognize multiple marks Cell, March 30, 2012

B ROMODOMAIN B INDING A SSAYS

Benzo(thieno)diazepines as selective BET inhibitors GSK WO 2009/54844 (phenotypic screen)

Benzo(thieno)diazepines as selective BET inhibitors GSK 525762 RVX-208 OTX015 Oncoethix Resverlogix Nature Drug Discovery, Sep 2013

JQ1: Impact (Basic Biology) Basic understanding of transcription IFN-induced recruitment of P-TEFb is under control of pausing complex  NELF/DSIF.  BRD4 is associated with small set of exceptionally large super- enhancers associated with genes that feature key oncogenic drivers. BET mediates RNA polymerase II (Pol II) S2 phosphorylation.  RNA polymerase II stalling promotes nucleosome occlusion and pTEFb  recruitment to drive immortalization by Epstein-Barr virus.  BRD4 is required for DNA damage response : Loss of BRD4 results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation. Cell. 2013 Apr 11;153(2):320-34. Mol Cell Biol. 2013 Jun;33(12):2497-507. J Biol Chem. 2012 Dec 14;287(51):43137-55 PLoS Pathog. 2011 Oct;7(10):e1002334. Nature. 2013 Jun 13;498(7453):246-50.

JQ1 probe Impact (Cancer) Cancer Biology: BRD4/NUT driver tumours are sensitive to JQ1 resulting in  terminal differentiation of cancer cells. Brd4 as a promising therapeutic strategy in AML   BET bromodomain proteins regulate c-Myc expression in myeloma . Down regulation of c-Myc and IL7R in B-cell acute lymphoblastic  NMC leukaemia (B-ALL) BET inhibition suppresses FOSL1 expression in lung cancer   Suppression of key oncognic drivers (c-Myc, p21(CIP1/WAF1), hTERT, Bcl-2, and Bcl-xL) in glioblastoma BET inhibition increases sensitivity of standard therapy  (Rituximab resistance in lymphoma and dexamethasone in ALL) Glioma Nature. 2010 Dec 23;468(7327):1067-73. Nature. 2011 Aug 3;478(7370):524-8. Cell. 2011 Sep 16;146(6):904-17. Nat Med. 2011 Nov 7;17(11):1325. Nature. 2012 Aug 9;488(7410):148-50. Blood. 2012 Oct 4;120(14):2843-52. PNAS 2012; 109(47):19408-13. Clin Cancer Res. 2013 Apr 1;19(7):1748- 59. EMBO Mol Med. 2013 Aug;5(8):1180-95.

JQ1 probe Impact (Inflammation) Inflammation and Viral Infection: BET inhibition reactivate HIV from latency via a Tat-independent  mechanism  JQ1 impairs mouse macrophage inflammatory response Enhanced migration, proliferation, and IL-6 release observed in  LFs from Idiopathic pulmonary fibrosis patients are attenuated by JQ.  BRD4 is essential for human papillomavirus type 16 DNA and polyomavirus DNA replication BET inhibition potently suppresses cardiomyocyte hypertrophy  in vitro and pathologic cardiac remodeling in vivo Phenylephrine (PE) induced cellular hypertrophy Cell Cycle. 2013 Feb 1;12(3):452-62. Am J Pathol. 2013 Aug;183(2):470-9. J Immunol. 2013 Apr 1;190(7):3670-8. Cell. 2013 Aug 1;154(3):569-82 J Virol. 2013 Apr;87(7):3871-84. Cell. 2013 Aug 1;154(3):569-82. Haldar lab/Cleveland

Consequences of BET inhibition in vivo

Consequences of BET inhibition: Spermatogenesis (BRDT) Ap Spermatogonium Type B Primary/Secondary Ad Spermatogonium Spermatocyte Spermatogonium Mature spermatid BTB Spermatid Spermatocyte Spermatogonia Sertoli Cell Basal lamina Lumen

Consequences of BRDT Inhibition  Genetic studies of BRDT in mice have demonstrated that deletion of the BRDT(1) is sufficient to confer sterility.  JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting serum hormone levels.  GWAS linked SNPs in BRDT to male infertility.  JQ1 crosses BTB and accumulates in testis (AUC testis /AUC plasma = 259%) with a rapid (T max = 0.25 hr) and pronounced exposure (C max = 34 μg /mL)  50 mg/kg of JQ1/daily showed 75% reduction of testis volume after 3 weeks and 54% reduction after 6 weeks Cell August, 2012

Consequences of BRDT Inhibition

Recovery After BRDT Inhibition

Hopes & Promises

Clinical BET Inhibitors (I)

RVX-208 is a Clinical BET Inhibitor Specific for the Second BRDs  Currently in phase IIb for treatment of atherosclerosis and trial in AD listed (Resverlogix).  Increases endogenous ApoA-1 production and HDL levels and thereby augment reverse cholesterol transport.  Identified as BET inhibitor (based on GSK publication) and advertised as such on web- page (but no data have been disclosed).  Compound structure disclosed by Resverlogix but currently not available.  Site specific inhibitors will help to unravel function of the individual bromodomains in BETs and will further or knowledge of the design for isoform specific targeting.

RVX-208 is Selective for the Second Bromodomain of BETs D Tm data ITC data First BRDs  Good selectivity for second bromodomain  Strongest binding to BRD4(2) (130 nM)  Selectivity in and BRD2 and BRD3 is 21 fold (ITC)  Weaker inhibition for BRDT

RVX-208 is Selective for the Second Bromodomain of BETs BRD2(1) L383 L381 BRD2(2) Q35 K374 RVX-208 K91 P430 V435 DMSO W370 D94/H433  Residue differences (H433/D) provide rational for tighter binding to BRD2(2)  Good shape complementarity with Kac site but “shelf” region not occupied by compound (DMSO binding) 2FoFc BRD2(2)  Binding mode conserved in BRD4(1) (not shown) (Reso: 1.6 Å) Reso: 1.7 Å

Variant of RVX-208 (E25190) has different binding mode resulting in loss of selectivity for the second BRD E25190 Overlay E25190 ITC: E25190  E25190 is selective for BETs in D Tm panel (46 BRDs) but selectivity for second BRDs is lost  ITC showed that Kds are between 150 and 350 nM (BRD2 and BRD4)  Co-Crystal Structures revealed a second binding mode that is sterically excluded for RVX-208  In E25190 the dimethyl phenol acts as a acetyl lysine mimetic

Transcription of small Subset of BET Target Genes is Affected by Inhibiting BD(2)

Clinical BET Inhibitors (unintentional targeting)

Clinical JAK and PLK Inhibitors with Strong BET Activity

Example of a BioMAP System “ BioMAP ” Profile SAg: TCR Stimulation Model Human Primary Cell Type Stimulation Assay Readouts Cocktail of two factors: Superantigens 24-hour stimulation MCP-1, CD38, CD40, Relevance to human E-selectin, CD69, IL-8, disease: MIG, T cell HuVEC + PBMC Auto-immunity (RA, IBD, Proliferation, PBMC co-culture COPD etc.) cytotoxicity, SRB Inflammation Asthma/Allergy Oncology Safety 26 Alison O'Mahony& Team /DiscoverX

Clinical JAK/BET and PLK/BET Inhibitors have Dual Phenotypes in Human Primary Cells Venular Cornorary artery endothelia cells smooth muscle

BRD inhibitors beyond BET