MIT OpenCourseWare http://ocw.mit.edu5.36 Biochemistry LaboratorySpring 2009For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms.Massachusetts Institute of Technology Department of Chemistry Gleevec / Abl kinase domain PDB: 1IEP Imatinib (Gleevec, STI571)HNOClSNNHNNNNOHDasatinib (Sprycel, BMS-354825)NNHNNNHONN5.36 Laboratory Manual Biochemistry Modules 4 and 5 Spring 2009Laboratory Manual for URIECA Modules 4 and 5 Course 5.36 U Table of Contents Introduction and background.………..……………………………................................2 Overview and outline of sessions..…………………………………………..……….….4 Procedures Session 1………………………………………………………………......8 Session 2………………………………………………………………....10 Session 3………………………………………………………………....13 Session 4………………………………………………………………....18 Session 5………………………………………………………………....21 Session 6………………………………………………………………....24 Session 7………………………………………………………………....26 Session 8………………………………………………………………....27 Session 9………………………………………………………………....28 Session 10………………………..……………………………………....30 Session 11…..…………………………………………………………....33 Session 12..……………………………………………………………....35 Session 13..……………………………………………………………....37 Session 14..……………………………………………………………....37 Session 15..……………………………………………………………....41 List of abbreviations…………...……………..……………………………………..….48 Appendix A: Common biochemistry laboratory procedures.……….....……………..…49 Appendix B: Protein and Nucelotide sequences……………...………..………...…..….52 Appendix C: Primer design for site-directed mutagenesis.……..………..…...……..….53 Appendix D: Abl inhibitors.……..……………………………………….……...…..….56 References……………………………………………………..……………………..…57An Introduction and Background for URIECA Modules 4 and 5 Abl and Bcr-Abl Proteins Abelson (c-Abl or Abl) is a protein tyrosine kinase that is involved in a number of highly-regulated cellular processes, including cell division, differentiation and adhesion. In healthy cells, c-Abl is auto-inhibited by domains at its amino (N)-terminus. This means that the kinase activity of c-Abl is tightly regulated, and the default activity setting is “off”. A chromosomal abnormality implicated in chronic myleloid leukemia (CML) causes the reciprocal translocation of genetic material from two different chromosomes, 9 and 22, which results in the formation of a mutant gene that contains part of the BCR (break cluster region) gene from chromosome 22 and part of the ABL gene from chromosome 9. This mutant gene is called BCR-ABL, and the protein it encodes, denoted Bcr-Abl, contains the kinase domain of c-Abl, but lacks the residues responsible for auto-inhibition. Bcr-Abl is therefore a constitutively active kinase, which means that the enzyme activity is permanently “on”. This aberrant kinase activity is responsible for uncontrolled cell proliferation, which leads to cancer. Abl kinasedomainSH3 SH2capC-terminaldomainsAbl kinasedomainSH3 SH2C-terminaldomainsBCRAbl kinasedomain c-Abl kinase (or Abl)The activity of c-Abl is auto-inhibited by itsN-terminal domains. Activity is controlled.N-terminus ------------------------------------------------------C-terminus Bcr-AblThe Bcr-Abl protein is constitutively active.The uncontrolled kinase activity of Bcr-Abl is implicated in chronic myeloid leukemia (CML). Abl kinase domainThe kinase domain of Abl is sufficient for kinaseactivity, but lacks auto-inhibition. We will use it as a mimic of Bcr-Abl for our investigations. Like Bcr-Abl,itis constitutively active.BCR AblWe will abbreviate this below as A Small Molecule Drug for CML Treatment Bcr-Abl activity is the underlying cause for CML, and the identification of the Bcr-Abl oncoprotein led to high throughput screening and the “rational design” of potential small molecule inhibitors. These efforts culminated in the development of the drug Gleevec by chemists at the pharmaceutical company Novartis (a branch of which is a few doors down from us on Mass Ave). Gleevec (also known as Glivec, imatinib, and STI-571) showed excellent efficacy against CML and was approved by the FDA in 2001. Gleevec inhibits Bcr-Abl tyrosine kinase activity by competitively binding in the ATP binding pocket of the kinase domain and stabilizing the inactive conformation of the protein. This development was particularly thrilling to the scientific community because Gleevec is the first example of a small molecule tyrosine kinase inhibitor to treat human disease. Also exciting is the striking specificity of Gleevec for Abl. Gleevec only inhibits two other proteins at physiological levels, neither of which result in problematic side effects. 2Bcr Abluncontrolled cell proliferation (cancer)ACTIVE kinaseBcrAblACTIVE kinaseBcr AblINACTIVE kinaseGleevecGleevecunc However….A PERCENTAGE OF CML PATIENTS DO NOT RESPOND TO GLEEVEC TREATMENT, AND OTHER PATIENTS THAT INITIALLY RESPOND TO TREATMENT EVENTUALLY DEVELOP GLEEVEC RESISTANCE. ontrolledliferationX cell pro (cancer) The majority of these Gleevec-resistant cases can be linked to a single amino acid mutation in the Abl kinase domain of the Bcr-Abl protein. Over 30 different point mutations have been identified in Gleevec-resistant CML patients (see appendix B3). Bcr AblACTIVE kinaseBcr AblINACTIVE kinaseGleevecGleevecuncontrolledliferationcer)X cell pro (can The Abl kinase domain is often used as a model for the full-length Bcr-Abl protein. The Abl