David Peters Baran Group Meeting Kinase Inhibitors: An Introduction 2/2/19 INTRODUCTION SIGNAL TRANSDUCTION KINASES ARE IMPORTANT IN HUMAN BIOLOGY/DISEASE: The process describing how a signal (chemical/physical) is transmitted through a - Kinases are a superfamily of proteins (5th largest in humans) cell ultimately resulting in a cellular response - 518 genes and 106 pseudogenes - Diverse in size, subunit structure, and cellular location RECEPTOR TRANSDUCERS EFFECTORS - ~260 residues make up their conserved catalytic core - dysregulation of kinases occurs in many diseases (cancer, inflamatory, degenerative, and autoimmune diseases) - signals can originate inside or outside the cell - 244 of the 518 map to disease loci - signals generally passed through a series of steps (signal transduction pathway) often consisting of multiple enzymes and messengers protein O - pathways open possibility for signal aplification (>1x106) kinase ATP + PROTEIN OH ADP + PROTEIN O P O - Extracellular signals transduced by RTKs, GPCR’s, guanlyl cyclases, or ligand-gated Ion channels O - Phosphorylation is the most prominent covalent modification/signal in KINASES INHIBITORS ARE IMPORTANT IN DISEASE THERAPY/RESEARCH: cellular regulation - currently 51 FDA approved small molecule kinase inhibitors - “converter enzymes” (protein kinases and phosphoprotein phosphorlyases) - 4 FDA approved antibody kinase inhibitors are regulated; conserve ATP/maintain desired target protein state - thousands of known inhibitors spanning the kinome - pathway ends by affecting biomolecule function or gene expression - used to treat a broad range of disease types; primarily cancer - inhibitors used as tools for biological discovery A Brief History - 1984: Staurosporine first kinase inhibitor discovered (non-selective) - 1991: first 3D structure of kinase solved (PKA) - 1999: First FDA approved antibody kinase inhibitor (Trastuzumab, Herceptin™; Genentech) - 2001: First FDA approved small molecule kinase inhibitor (Imatinib, Gleevec™; Novartis) Protein Kinases: GROUP MEETING INCLUDES: H - generally highly specific often recognizing consensus sequences in their - Introduction to signal transduction O N target (contains target residue) - Introduction to therapeutically relevant kinase targets - can phosphorylate one or many residues on a protein - Introduction to kinase structure - residues phosphorylated bear a free hydroxyl/phenol (serine, threonine, - Case studies in targets and their inhibitors tyrosine) - Introduction to drug resistance to kinase inhibitors - tyrosine kinases only exist in multi-cellular organisms − important for - Introduction to drug discovery in kinase inhibitors N N intercellular communication - Introduction to perspectives on the future of kinase O - usually locked in their “inactive conformations” − blocked by intrasteric Me control/pseudosubstrates inhibitors as therapeutics 2+ - Summary of all FDA approved kinase inhibitors OMe - turned on by interaction with messengers (cyclic nucleotides, Ca , etc.) or phosphorylation/dephosphorylation HN GROUP MEETING DOES NOT INCLUDE: Me - tyrosine kinases broken into two broad categories (RTK’s and NRTK’s) - an exhaustive discussion of any of the above topics Staurosporine - 90 tyrosine kinases - 58 RTK’s and 32 NRTK’s David Peters Baran Group Meeting Kinase Inhibitors: An Introduction 2/2/19 KINASE-MEDIATED SIGNAL TRANSDUCTION PATHWAY: THERAPEUTIC TARGETS 1. Receptor binding - results in conformational change/dimerization of RTK 2. Phosphorlyation on RTK - (trans)autophosphorylation via now active kinase Below is a list of clinically validated kinase targets: 3. Recognition of phosphoprotein - SH-2 domain binds phospho-RTK BCR-Abl - fusion kinase in some cancers; NRTK; activates numerous pathways 4. Transphosphorylation - newly bound protein is phosphorylated cKit - RTK; activates numerous MAP-Kinase pathways 5. Cascade continues - successive recognitions and phosphorylations EGFR - epidermal growth factor receptor; also ErB1; RTK 6. Effector reached - cascade ends by modulating final target and its function VEGFR - vascular endothelial growth factor receptor; RTK PDGFR - platelet derived growth factor receptor; RTK ErbB - (also called HER) other RTK’s important in regulating growth ALK - anaplastic lymphoma kinase; NRTK B-raf - Ser/Thr kinase; MAP/ERK pathway JAK - Janus kinase; NRTK; part of JAK-STAT pathway BTK - Bruton’s tyrosine kinase; important in B-cell development MET - cMET or HGFR; RTK; activates numerous pathways CDK - cyclin-dependent kinase; Ser/Thr kinase; help regulate cell cycle Rho - also ROCK; Ser/Thr kinase; associated with degenerative diseases TRK - tropomyosin receptor kinase; RTK; activates MAP and PI3 pathways PROTEIN KINASE CLASSES: Syk - spleen tyrosine kinase; NRTK 1. Ser/Thr Protein Kinases a. Cyclic nucleotide dependent Flt3 - also CD135; RTK; one of most frequently mutated genes in AML b. Ca2+-calmodulin dependent (CaM) PI3K - phosphoinositol 3 kinase; lipid kinase; PI3 involved in diverse signaling c. Protein kinase C (PKC) d. mitogen activated protein kinases (MAP) FKBP/mTor - mammalian target of rapamycin; Ser/Thr kinase; FKBP e. G-protein-coupled/Rhodopsin/BARK association necessary for activity 2. Ser/Thr/Tyr Protein Kinases a. MAP kinase kinase (MAPK) 3. Tyr Protein Kinases - TARGETS MUST BE LINKED TO PHENOTYPE a. Cytosolic tyrosine kinases - most targets are oncogenes discovered by clinical/biochemical investigation b. Receptor tyrosine kinases (RTKs) - some above targets are markers for certain cancer (or disease) types - not all kinase targets are oncogene products Kinome - the complete set of kinases encoded in a genome - all kinase targets are involved in cell cycle/cell growth regulation Protein Kinase - enzyme that transfers a phosphate to a target protein in an ATP- - many are in or are upstream of MAP-kinase pathways (stimulate cell division) dependent manner - certain disease states demonstrate activated kinase forms (abnormally Phosphoprotein Phosphatase - enzyme that catalyzes the hydrolysis of phosphates active or constitutively) from phosphoproteins Intrasteric control - regulation of enzyme activity in which an internal sequence of - some cancer cells rely heavily on the above targets/pathways protein resembling substrate (pseudosubstrate) binds the active site, - makes them particularly sensitive to inhibition - therapeutic index blocking activity - some inhibitors target a single kinase (selective/mono-specific) Receptor Tyrosine Kinase (RTK) - membrane-bound tyrosine kinase who’s activity is - some inhibitors target multiple kinases (poly-specific) dictated by interaction with an extracellular stimulus Non-receptor Tyrosine Kinase (NRTK) - completely cytosolic tyrosine kinase Mitogen - a chemical substance that encourages cell division (mitosis) Oncogene - a gene that has the potential to cause cancer via mutation/overexpression SH-2 domain - conserved domain among intracellular signal transducing proteins; Proto-oncogene - a gene in its normal state that could become an oncogene recognizes phosphotyrosine Phenotype - physiological/observable characteristics of an organism David Peters Baran Group Meeting Kinase Inhibitors: An Introduction 2/2/19 KINASE STRUCTURE [overlay of active/inactive kinase] [depiction of C- and R- spines] - activation usually result of phosphorylation - the “spines” are linear arrangements or binding a “signal” of hydrophobic residues - activation changes DFG orientation (below) - formation necessary for function - functions validated by mutation - ACTIVE = AS open; !C in; DFG-Asp in - some tolerance R-spine gatekeeper residue IMPORTANT STRUCTURAL MOTIFS: N-Lobe - N-terminal small lobe of mostly β-sheet ACTIVE INACTIVE C-Lobe - C-terminal large lobe of mostly !-helix Hinge - short portion connecting the N- and C-lobes; important for inhibitor binding INHIBITOR BINDING MOTIFS: K/E/D/D - signature motif defining functional kinases (stabilizing/active-site residues) - figure at left depicts pockets/regions Activation Segment - regulatory loop that changes conformation upon activation generally utilized by inhibitors - most inhibitors make 1-3 hydrogen DFG - three residue motif at beginning of activation segment; large role in catalysis bonds w/ hinge residues C-Spine - 8 hydrophobic residues that facilitate ATP binding - specificity defined largely by alternative R-spine - 4 hydrophobic residues that position substrate appropriately pockets Shell residues - 3 residues surrounding/stablizing the R-spine - gatekeeper residue size determines access to hydrophobic pocket II SH2 (Gatekeeper) - shell residue controlling access to an important binding pocket - binding active vs. inactive state !C-helix - dynamic motif whose conformation dictates binding of some inhibitors provides access to different pockets David Peters Baran Group Meeting Kinase Inhibitors: An Introduction 2/2/19 INHIBITOR TYPES BCR-Abl - BCR-Abl is a fusion gene (hybrid of two previously separate genes) - two original genes normally exist on two different chromosomes - expression of fusion results in a constitutively active Abl kinase - first discovery of a genetic Philadelphia Chromosome abnormality directly associated with a cancer - decreases apoptotic potential and activates MAP kinase pathways at Ras - associated with various leukemias (CML and ALL) TYPE I - Binds in ATP binding pocket; active form; DFG-Asp IN; - first clinically validated kinase Type I 1/2 - Binds in ATP binding pocket; inactive form; DFG-Asp IN target; target of imantinib TYPE II - Binds