Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Discovery and development of antiandrogens wikipedia , lookup
Metalloprotease inhibitor wikipedia , lookup
Discovery and development of integrase inhibitors wikipedia , lookup
DNA-encoded chemical library wikipedia , lookup
Discovery and development of ACE inhibitors wikipedia , lookup
Theralizumab wikipedia , lookup
Neuropsychopharmacology wikipedia , lookup
Neuropharmacology wikipedia , lookup
Drug design wikipedia , lookup
Chemoproteomic applications in kinase drug discovery di Kinase 2014: past, present and beyond y Douglas Thomson Overview – Introduction and Overview of the Cellzome Proteomic Platform – Application of the Platform to the Discovery of a PI3K in vivo Probe 2 Cellzome’s technologies use cell or tissue lysate drug g resin Proteome expression effects: Measure proteome dynamics upon drug treatment Target deconvolution: Identify targets of active compounds from disease relevant screens Target class profiles: Test compounds against entire target classes (e.g. KinobeadsTM) Pathway effects: Follow changes in signaling upon drug treatment 3 Target Class Profiling – Binding g affinityy measurement of compounds p to native kinases directly in cellular or tissue lysate – No recombinant protein used Lysates can be prepared that allow acess to activated kinases – Access to difficult targets e.g. lipid kinases Itk ZAP 70 Lck Directly from tissue – e.g. mouse brain gives acess to other proteins T-cell NFAT GATA-3 STAT6 – One experiment can simutaneously screen against whole captured proteome – Kinases -KinobeadsTM – Epigenetic targets -EpisphereTM – Rapid assay development and screening 4 Compound target profiling Inhibito or concentration 1. Add inhibitor to cells or cell lysate over a range of concentrations 2. Incubate - inhibitor binds to targets in cells or lysate 3. Add affinity matrix to lysates 4. Elute beads, digest, label with TMT6 (Tandem Mass Tag) 0 0 µM 0.0 (DMSO, Control) TMT 131 0.039 µM TMT 130 5. Mix, run LC-MS/MS and quantify in MS/MS spectrum Protein does bindsnot to drug bind to drug 0.156 µM TMT 129 0.625 µM TMT 128 2 5 µM 2.5 M TMT 127 10 µM TMT 126 5 Cellzome Chemoproteomic Platform (screening) Matrix Capture compounds on beads compound library Total protein extracts are prepared from cell or tissue samples Pulldown step: Reduction of target binding to beads in the presence of excess "free" compound IC50 values for target Eluted material is spotted on microarray membrane detection of target proteins on dot-microarrays by antibody Beads are separated and Beadbound proteins are eluted by SDS elution buffer Screen > 100 000 compounds 6 Kinobeads™ Combination of broad spectrum ATP mimetics Approx. 50 kinase inhibitor probes Bantscheff et al. (2007) Nat Biotechnol 25:1035-44 • • • • > 350 human protein kinases >15 lipid kinases (PI3K, PI4K, PIP5K) Many other purine-binding proteins Exact number depends on lysate 7 Selectivity profiles of clinical kinase inhibitors Glivec p(IC50) p Dasatinib Bosutinib Bantscheff et al. (2007) Nat Biotechnol 25:1035-44 8 Kinase protein complexes are preserved Glivec, Dasatinib, Bosutinib – Complex partners are quantified in proteomic experiment – These indirect binders to matrix have similar apparent pIC50 to direct binders Kruse et al., Leukemia 2011, 25: 89-100 9 Application of Platform to the Discovery of a PI3K in vivo Probe PI3K inhibition has anti-inflammatory effects COPD Thomas, M. et al, Eur. J. Immunol., 2005 Allergic Rhinitis Pinho et al, J. Leukoc. Biol. 2005 Asthma Pinho et al, J. Leukoc. Biol. 2005 Rheumatoid Arthritis Camps et al al, Nat Nat. Med Med, 2005 Sepsis Martin et al, Am J Respir Crit C Care M Med. d 2010 Systemic Lupus Erythrematosus (SLE) Barber et al., Nat. Med. 2005 ANCA-induced Glomerulonephritis Inflammatory Bowel Disease (IBD): Schreiber et al al, kidney intern. 2009 Gonzalez Garcia, Gonzalez-Garcia Gastroenterology, 2010 PI3K -/- mice published 2000 (Hirsch et al Science) 11 Selectivity is key: off-target effects mediated by PI3K family members PI3K PI3K Heart: contractile dysfunction Lu, Circulation, 2009 PI3K Insulin resistance Knight, Cell, 2006 PI3K PI3K Eosinophilic infiltration in different organs, particularly gastritis Ji, Blood, 2007 Reproductive organs: Testes reduced in size, degeneration of germ cells Ciraolo, Mol Cell Biol, 2010 PI3K Increased IgE production Zhang, J Allergy Clin Immunol 2008 12 Establising a Chemoproteomic Assay Cell lysate 3 Sources of the proteins were established Human blood immobilize probe PI3K mTOR Tissue lysate design probe PI3K identify cell source PI3K DNAPK format binding assay Multiplex assay setup allowing simultaneous analysis of 6 closely related kinases PI3K screen lead opt Antibody validated for each h target t t enabling bli dot-microarray readout Know PI3K inhibitors PIK-93 and LY294002 were linked to beads Hit ID: library screening 16,146 compounds 13 From multiplexed screening to selective probes: PI3K inhibitors Library screening DNAPK PI3K PI3K Compoun nds mTor Bergamini et al, NatChemBiol, June 2012 14 Identification of a selective PI3K inhibitor CZC24832 MW 364 LogD 1.36 Solubility 65 M LE 0.4 PPB (h/m) 34%/24% Cl in Liver microsomes (h/m/r) 0/1/0.4 l/min/mg hERG No inhibition at 100 M CYP1A, 2C19, 2C9, 2D6 and 3A4 No inhibition at 25 mM PK in rat: 0.2 mg/kg iv and 10 mg/kg po Cl: 14 ml/min/kg F: 37% Bergamini et al, NatChemBiol, June 2012 15 KinobeadsTM Profiling of CZC24832 PI3K F HN N O N N S O NH2 N Bergamini et al, NatChemBiol, June 2012 Protein pKdapp PI3K 7.6 PI3K 6.1 PI3K 51 5.1 PI3K <5 PIP4K2C 5.8 CK1 5.5 16 Affinity Purification Imoblised analogue was used as matrix CZC24832 was used as competitor p at 280 nM Either HL-60 or PBMC cells were used as lysate source No additional proteins were captured and competed Bergamini et al, NatChemBiol, June 2012 17 KinobeadsTM Profoling of CZC24832 in Different Species Potency drop for PI3K PI3K, of 2-5 2 5 fold in rodents Selectivity maintained Bergamini et al, NatChemBiol, June 2012 18 CZC24832 shows efficacy in mouse RA model Collagen induced arthritis model 50 5,0 4,5 Disease control Mean±SE Clinical Arthritis Sco ore (Scored 0-5) 4,0 35 3,5 CZC24832 3 mg/kg * 3,0 * 2,5 CZC24832 * 20 2,0 * * * * * * * * * * * CZC24832 10 mg/kg Dex 0.01mg/kg * 1,5 1,0 05 0,5 0,0 0 1 2 3 4 5 6 7 8 Arthritis Day 9 10 11 12 13 14 – Dose-dependent Dose dependent efficacy mouse CIA model – Reduction in severity of arthritis is comparable to PI3K-/- mice 19 Summary – An in vivo probe (CZC24832) for PI3K was discovered using our Chemoproteomic Platform – CZC24832 is potent for PI3K in both proteomic competition binding assays and cell based assays – CZC24832 has excellent selectivity for PIK3 within the PIK3 family and the rest of kinome – CZC24832 can be used for the further pharmacological investigation and validation of PI3K – However CZC24832 has limitations that stop p it´s further p progression g as a drug g candidate – Low solubility – Non-linear PK at higher doses – Moderate efficacy y in CIA models 20 Thank you! All Cellzomers past and presentt In particular Gitte Neubauer Giovanna Bergamini Andrew Cansfield Kathryn Bell Thilo Werner Marcus Bantscheff Gerard Drewes Our collaborators in particular: Emilio Hirsch Alison O‘Mahoni 21