Targeting Phosphorylation Signalling Networks Stefan Knapp - PowerPoint PPT Presentation

Targeting Phosphorylation Signalling Networks Stefan Knapp Structural Genomics Consortium Phosphorylation Dependent Signalling Group Oxford University, Nuffield Department of Medicine Oxford, United Kingdom International Conference on Structural Genomics (ISGO) GO), Toronto, May 10-14, 2011 SGC Toronto SGC Oxford SGC Stockholm

Complexity of Cellular Signalling Limited understanding of cellular signalling  Large number of mutations in tumours  Best entry point of pharmacological intervention is not known  Detection of an oncogenic kinase mutation does not guarantee sensitivity to inhibition Cascades vs Networks

(Un)-explored Kinome – what we know about the Network Kinases:> 500 000 papers in PubMed Coveringmainly~10% Kinome > 10 000 US patents Patents followpublicdata

Consequences Limited understanding of cellular signalling  Large number of mutations in tumours  Best entry point of pharmacological intervention is not known  Detection of an oncogenic kinase mutation does not guarantee sensitivity to inhibition Clinical POC is main motivation for target selection > 50% of clinical inhibitors target kinases for which inhibitors have been already approved Unbiased Target Selection using tool molecules “probes”

Overcoming Selectivity Problems ? SBDD strategies  Out of the “Box” inhibitors (Allosteric Inhibitors, Reg. Domains)  Targeting inactive conformations (DFG out)  Targeting unique kinases  Targeting unique binding modes  Targeting unique active site features Requirements:  Complex with inhibitor scaffolds with target and cross reacting kinases  Sufficiently large (representative) screening panel

Targeting the Active Site Hinge Upper lobe ATPsite DFG Hinge P P P ATP Substrate site P lower lobe Kinase Inhibitors ATP mimetic: type I ATP competitive binding inactive conformation: type II Substrate competitive: type III

Inhibitors Types (Type I/II)

Targeting Intermediate Conformations

Kinase Family Wide Structural Analysis  61 Structure in PDB fromAcademia  42 Structures in PDB fromIndustry 56 Human Kinase Structures by SGC since 2004

Early Parallel Screening Kinase Target I n R1 h i R2 b i t o r Subset of Kinases that can be targeted with tested scaffold ID cross reacting kinases early Cell based … Screens Chemical array of well characterized inhibitor selectivity ~300 kinases screened

Parallel Screening of KTLs Pyrimido-diazepines (~60 compounds made and screened against AMBIT panel (~350 kinases) CollaborationwithGray Lab, Dana Faber, Harvard

Mechanisms of Cross-Reactivity K00135 Originally identified as PIM1 inhibitor (hit from a purchased library / Biofocus) Main cross reactivity : ACVR1, BMPR1, CLK1, BIKE, GAK, Haspin KIT,FLT, DYRK1  Selectivity for closely related isoforms (e.g. Pim1 vs Pim2; Clk1 vs Clk3)  Cross reactivity with diverse kinases Screening data ~300 kinases and 180 in D Tm assay

Unique Active Site Features Binding relies on 2 hydrophobic anchors Gatekeeper xDFG Gatekeeper :L, F or V xDFG : V, I or L Scaffold binds to two hydrophobic anchorpoints  xDFG is rarely a large  No classical hinge H-bond interaction hydrophobic residue  ATP sites that contain xDFG anchorare very diverse  Some bulky ligands will not fit into all sites

Cross-Reactivity – Non ATP mimetic CLK/ DYRK Haspin PIM DRAK DAPK3 D Tm>6 o C D Tm>6 o C D Tm>6 o C D Tm>6 o C PIM1/2/3, DRAK1, ACVR1 ACVR1, PIM1/3 PIM1/3, CLK1, DAPK3, PIM1/3, DYRK2, TGFBR2, LOC340156A BMPR1, CLK1, KIT, FLT D Tm>4 o C D Tm>4 o C CLK1, DRAK1 D Tm>4 o C Haspin, DRAK1, DYRK1/2 CLK1, DAPK3, DYRK1/2 BIKE, GAK, Haspin, PIM2 BMPR2 DRAK1, DAPK3, DYRK1/2

Mechanisms of Cross-Reactivity Binding to Active and Inactive Conformations Imidazo-pyridazines change orientation when bindingto active kinases Selectivitycan be increased by avoidingbindingto ATPmimetic mode Kinome wide analysis rationalizes most cross reactivity and suggests strategies for selective inhibitor development ? f DFG Non ATP mimetic ATP mimetic binding mode

Targeting Splicing: CLK1 Selective for CLK1 (150 kinases screened by DSF Collaboration : F. Bracher, University Munich & 80 kinases screened by enzymatic assays)

CLK1 Regulation of TF Splicing K01874 inhibits S/R phosphorylation in endothelia cells and splicing of TF. 1.5 EC 50 180 nM IC 50 20 nM 1.0 responce 0.5 0.0 -10 -8 -6 -4 -2 log(conc) A. Eisenreich and U. Rauch Charite Berlin

SRPK2 Inhibitors and Regulation of VEGF Splicing  SRPK1/2 suppressed HCV replication  SPRK1 regulates vascular endothelial growth factor (VEGF) splicing frompro-angiogenic to anti- angiogenic isoforms K02466 highly selective forSRPKs Collaboration : D . Bates, Bristol ,UK

Targeting Unique Kinases (Haspin)  Low sequence homology with ePKs (less than 20%)  Lack motifs that are invariant in ePKs  Specific substrate : H3T3  Depletion of Haspin leads to: • Premature chromatid separation • Activation of spindle checkpoint • Block in mitosis  Activator of Aurora B

Targeting Unique Kinases (Haspin) IC 50 : 5nM in vitro Iodotubercidin  137 kinases screened  Only cross reactivity that has been observed is to DYRK2 and CLK kinases

New Approaches: Octasporines Collaboration : E. Meggers (Marburg)

New Approaches: Octasporines Collaboration : E. Meggers (Marburg)

New Approaches: Octasporines  Octasporines are highly potent & target selective Collaboration : E. Meggers (Marburg)

A CKNOWLEDGEMENTS SGC SGCcont. Harvard Oxford Chemistry Aled Edwards Susanne Muller-Knapp James Bradner Chris Schofield Chas Bountra Frank von Delft Jun Qi NathanRose Cheryl Arrowsmith Joao Muniz Andrew Kung Akane Kawamura Johan Weigelt MartinPhilpott Christopher A. French Oliver King Oleg Fedorov William B. Smith Lars Hillringhaus Udo Oppermann Frank Niesen ElizabethM. Morse Esther Woon StanNg Tony Tumber Tyler T. Hickman Alice Grabbe Jing Yang Michael McKeown Oxford Biochemistry Michelle Daniel Yuchung Wang RobKlose AtulGadhave Amanda L. Christie Shirley Li NathanWest Panagis Fillipakopoulos Michael J. Cameron NCGC GSK SarahPicaud BrianSchwartz Anton Simeonov TimWillson TracyKeates DaveMaloney Bill Zuecher Ildiko Felletar Ajit Jadhav DavidDrewry AmyQuinn BrianMarsden Minghua Wang Charite, Berlin BaselUniversity Hospital University ofMunich CNRS Roscoff URauch Juerg Schwaller Franz Bracher Laurent Meijer A. Eisenreich Kilian Huber FUNDING PARTNERS Canadian Institutes for Health Research, Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Knut and Alice Wallenberg Foundation, Merck & Co., Inc., Novartis Research Foundation, Ontario Innovation Trust, Ontario Ministry for Research and Innovation, Swedish Agency for Innovation www.thesgc.org Systems, Swedish Foundation for Strategic Research, and Wellcome Trust.