MIT OpenCourseWare http://ocw.mit.edu 5.36 Biochemistry Laboratory Spring 2009 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms.__________________________________________________________________________ ________________________________________________________________________________ 5.36 Lecture Summary #1 February 3, 2009 Reading assignment for Thursday: Weisberg, E. et al. Second Generation Inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukamia. Nature Rev. Cancer 7, 345-356 (2007). Posted on : Next Laboratory Session: #1 Topics: I. Background chemistry for Modules 4 and 5 (kinases!)A. Review of kinases and phosphatases B. Abelson kinase (Abl) and Bcr-Abl C. Kinase inhibitors as cancer drug targets II. Overview of Modules 4 and 5 I. BACKGROUND CHEMISTRY FOR MODULES 4 AND 5 (KINASES!) A) REVIEW OF KINASES AND PHOSPHATASES A kinase is an enzyme that catalyzes the transfer of a _______________ group from ATP onto a _____________ (target molecule). substrateOsubstrateOHATP+NNNNNH2OOPOO-OPOP-OOO-OO-OH OHA phosphatase is an enzyme that catalyzes the ______________ of a phosphate group from a target molecule: the reverse reaction. substrateOHsubstrateOPO-O-OphosphataseSerine/threonine-kinases phosphorylate target proteins on a Ser or Thr residue. Tyrosine-kinases phosphorylate target proteins on a Tyr residue. H3NOOHOserine (Ser, S) threonine (______ , T) tyrosine (Tyr, ___ ) MIT serverBiological Relevance of Protein Kinases. Kinases (and phosphatases) can regulatethe activity of a target protein in various ways. For example, phosphorylation of a target protein can: kinasephosphatasePPinteraction withadditional signaling molecules• Create new ____________________ for that protein. Signaling pathways are ___________ or inhibited. • Result in __________________ changes that affect binding Signaling pathways areactived or inhibited. kinasephosphatasePinteraction withadditional signaling moleculesPor modulate enzyme ____________. OR Depending on the enzyme, the phosphorylated form may be active OR inactive. kinasephosphataseP inactive form active formkinasephosphataseP inactive form active formPhosphorylation by kinases can “switch on” an enzyme substrate to produce significant quantities of active enzyme on timescale of _____________ to ___________. Compare this to protein expression, which can take ___________ to ________ to produce significant quantities of protein. Phosphorylation (and other post-translational modifications) thereby allows cells to respond to their environment on a much faster timescale than if they relied solely onexpression to modulate quantities of active protein. B) ABELSON KINASE (Abl) Abl kinasedomainSH3 SH2capC-terminaldomainsN-terminus ------------------------------------------------------C-terminusAbl is a 120 kDa (________ amino acid) protein tyrosine kinase (PTK). The Abl “kinase domain” is ______ kDa (amino acids 229-551). See Appendix B for the Abl kinase domain DNA and amino acid sequences. Sub domains bind signaling proteins, DNA, and actin. Abl is _____________________ by the binding of the “cap” domain and other N-terminal domains to the kinase active site. The Abl default setting is _______.Abl in healthy cells. The precise biological roles of Abl are still unknown. However Abl appears to be involved in • cell division. • stress response. • cell adhesion and migration. Abl in disease. Mutations and / or overexpression of kinases lead to a wide range of diseases. A reciprocal ________________ between the Abl-enconding chromosome 9 and the Bcr-encoding chromosome 22 results in a fused ____________ gene. ABLBCRBCR-ABLchromosome ____chromosome ______________________ chromosomeThe fused BCR-ABL gene product is the mutant protein Bcr-Abl. The Bcr-Abl protein lacks the residues responsible for Abl inhibition and is ________________________ (always ON). Bcr-Abl. Constitutively active. Abl. Auto-inhibited. Activity is ___________. Abl kinasedomainSH3 SH2capC-terminaldomainsAbl kinasedomainSH3 SH2C-terminaldomainsBCRAberrant kinase activity of Bcr-Abl is the underlying cause chronic myloid leukemia (______), a cancer of the bone marrow. • CML white blood cells do not function correctly and take up room, resultingin ___________ normal white blood cells and red blood cells. • Incidence: ~5000 new cases each year in US. This represents ________% of allcases of adult leukemia in Western populations • The only well-described risk factor is exposure to ionizing radiation. C) ABL INHIBITORS AS CANCER DRUG TARGETS Chemists at Novartis used rational drug design combined with high throughputscreening technologies to find drug targets that inhibit Abl activity. These efforts culminated in the development of the drug _____________, which wasapproved by the FDA in _______ for CML treatment.NNHNNNHONNGleevec• Gleevec binds to the active site of the Abl kinase domain and stabilizes the _____________ conformation of the protein. • The Gleevec-Abl interaction is highly ___________. Gleevecinhibits only ____ other kinases at physiological levels,neither of which result in problematic side effects. • Other names for Gleevec are ____________ and ___________. • Approximately ____% of CML patients diagnosed in thechronic stage experience remission. Gleevec is the first drug that selectively inhibits a __________________. This is incredibly exciting from the standpoint of drug discovery! Resistance to Gleevec: As of 2006, _____% of patients diagnosed in the __________ stage showed Gleevecresistance. More than half of patients in advanced stages of CML show Gleevecresistance. In patients with Gleevec resistant CML, mutations are found in the Bcr-Abl gene, usually at just __ ______________ in the kinase domain. BcrAblACTIVE kinaseuncontrolled cell proliferation (cancer)uncontrolled cell proliferation (cancer)XBcr AblACTIVE kinaseBcrAbl____________ kinaseGleevecGleevecuncontrolled cell proliferation (cancer)BcrAblACTIVE kinaseBcrAbl____________ kinaseGleevecGleevec*mutant*Over 30 different ___________ mutations in the Abl kinase domain of Bcr-Abl have been identified in Gleevec-resistant CML patients to date. (229)SP NYDKWEMERT HF V V V E RH K 260 270 GA280 A 300 DITMKHKLGG GQYGEVYEGV WKKYSLTVAV KTLKEDTMEV EEFLKEAAVM KEIKHPNLVQ N A 310 L I L320 330 340 T 350 T G V LLGVCTREPP FYIITEFMTY