Molecular Medicine: Gleevec and Chronic Myelogenous Leukemia

Transcription

1 Molecular Medicine: Gleevec and Chronic Myelogenous Leukemia Dec 14 & 19, 2006 Prof. Erin Shea Prof. Dan Kahne Cancer, Kinases and Gleevec: 1. What is CML? a. Blood cell maturation b. Philadelphia Chromosome c. Bcr and Abl 2. Protein kinases a. Structure b. Mechanism of catalysis c. egulation 3. Kinases important in CML a. Src Family b. egulation of Src family kinases c. Abl and its regulation d. Bcr-Abl and misregulation e. Gleevec structure, selectivity, and mechanism of action f. Le Chatelier s Principle g. Gleevec resistance Lecture eadings Alberts p McMurry p

2 Kinase-Mediated Phosphate Transfer - H + P P P H H H Bring substrates together H 2 H H H - - P P P Mg 2+ H 3 Aspartic Acid esidue from Kinase H H H rient substrates H 2 Lysine esidue from Kinase eutralize charge in TS Increase nucleophilicity of - H P P H H P - - H 2 Proximity Effect: A Kinase Brings the Two Substrates Together ATP protein substrate 2

3 The Mg-ATP binding pocket Glu 91 Glycinerich loop (P-loop) Lys 72 Mg 2+ Mg 2+ ATP Ser 53 Mg 2+ The Catalytic Center ATP Mg 2+ Asp 184 Mg 2+ Asn 171 Tyrosine substrate Asp 166 Lys 168 3

4 A Minimalist View of Kinase-Mediated Phosphate Transfer - H + P P P H H H H 2 H H H - - P P P Mg 2+ H 3 Aspartic Acid esidue from Kinase H Asp 166 H H H 2 Lys 72 Lysine esidue from Kinase - H P P H H P - - H 2 Kinases are egulated by Phosphorylation of an Activation Loop Inactivated form Active site not accessible Activated form Active site accessible P Unphosphorylated; inactive Phosphorylated; active 4

5 The Activation Loop Modulates Active Site Conformation Inactivated form Active site not accessible Mg-ATP not oriented Activated form Active site accessible Mg-ATP oriented Salt Bridge Unphosphorylated Threonine (Thr 197) Phosphorylated Threonine Src Family of Protein Kinases 5

6 The Architecture of Src: The Closed Conformation The Latch, the Clamp, and the Switch : The egulatory Apparatus of Src Src closed Active 6

7 Abl Uses a Different Latch Mechanism from Src Src Abl ormal egulation of Abl Abl closed Active 7

8 Bcr-Abl Lacks a Latch Missing in fusion! The Missing Latch Has Disastrous Consequences kinase Destabilized closed conformation (missing latch!) Active 8

9 Gleevec (STI-571; imatinib) CH 3 H 3 C H H Gleevec binds to and stabilizes this conformation Gleevec Binds the Form of Bcr-Abl kinase closed Active 9

10 Gleevec Binds to the Closed, Conformation of Abl Steric clash! Gleevec + inactive conformation Gleevec + active conformation o clash w/ closed activation loop Clashes w/ open activation loop!!! Le Chatelier s Principle and Gleevec Gleevec binds to and stabilizes this conformation kinase closed Active 10

11 Gleevec is ot a Cure for All CML Patients 90% of patients in early stages of CML respond to treatment with Gleevec 96% of responding patients still exhibit Bcr- Abl ma expression - stem cells? 16% of patients relapse within 42 months of treatment Main cause of relapse are Bcr-Abl mutations Modeling CML Treatment and Gleevec esistance S = stem cell P = progenitor D = differentiated TD = terminally differentiated S P D TD 11

12 Gleevec esistant Mutations Analyze 32 patients who have relapsed - find Abl kinase domain mutations in 29/32! esistant mutations must preserve kinase activity Mutations found in 13 different residues in the Abl kinase domain Contact with Gleevec P-loop (glycine-rich loop) Activation loop How do mutations in P-loop and activation loop cause resistance? P-loop * * Gleevec esistant Mutants * activation loop * contact with Gleevec 12

13 Dasatinib Dasatinib Gleevec (imatinib) Structure of Abl-Dasatinib Compared to Active Kinase 13

14 Abl Bound to Imatinib (Gleevec) and Dasatinib Summary Protein kinases contain a domain that folds into a conserved structure Protein kinases catalyze phosphorylation through proximity, orientation, and electronic effects Multiple mechanisms regulate the Src family of kinases Bcr-Abl is missing a critical mode of control, causing it to be constitutively active Gleevec binds to the inactive, closed form of Abl, shifting the equilibrium towards this form Gleevec resistant mutants destabilize the closed conformation, destabilizing Gleevec binding Dasatanib binds the active conformation of Abl, explaining its efficacy against Gleevec-resistant mutants Combination therapy may be the best treatment for CML 14