Question

. Gleevec (Imatinib) inhibits protein kinase BCR-ABL, which is constitutively active in patients with Chronic Myelogenous Leukemia (CML). The structure of Gleevec is shown below:

Imatinib (Gleevec, Novartis), CH.NO: MW = 494

a) Based on our discussion in lecture and the structure above, explain how the “lead” compound structure for Gleevec was designed and how it led to the development a compound that could bind ABL with higher affinity than ATP.

b) Using a broad kinase inhibition assay, you discover that Gleevec inhibits another tyrosine kinase called KIT. In order to examine the specificity of Gleevec on kinases in cancer cells, you decide to use the “bump-hole” approach with mutated alleles of BCRABL and a well-studied bumped kinase inhibitor called 4-amino-1-tert-butyl-3-(1- naphthyl)pyrazolo[3,4-D]pyrimidine (NaPP1).

(i) Based on your lecture notes, describe how you would decide which residue to mutate in BCR-ABL to make a “hole” in the ATP binding site. Which amino acid would you mutate this residue to in order to create a “hole” in mutant BCR-ABL? Do you expect this mutation will affect the kinase activity of BCR-ABL?

(ii) Hypothesize how a bump-hole approach would give you insight about the effect of inhibition of only BCR-ABL, and not KIT, by NaPP1 in cells.

c) After deciding on a solution to part B, you decide to compare total protein phosphorylation in cells expressing wildtype OR mutant BCR-ABL (the mutant has a “hole” in the ATP binding site). To do so, you analyze proteins from lysed cells by SDSPAGE and perform a western blot using an antibody that detects phosphorylation marks on Tyr residues (this antibody is called aPY). You detect expression of BCR-ABL in these cell lines using an antibody called aABL). The resulting blots are shown below.

i) What is the motivation behind monitoring phosphorylation of Tyr residues in cellular proteins by this experiment?

ii) Based on the results in the western blot above, does Gleevec likely inhibit both wildtype and mutant BCR-ABL in cells? Briefly explain your answer.

ii) Based on the results in the western blot above, does NaPP1 inhibit both the wild-type and mutant BCR-ABL in cells? Explain your answer and hypothesize why or why not.

d) In order to tease apart whether Gleevec is inhibiting BCR-ABL in conjunction with the kinase KIT, you express the mutant BCR-ABL from part B in primary splenic CML-type cancer cells and treat them with no drug (DMSO), Gleevec, NaPP1 or ACK45 (an antibody that specifically inhibits KIT) and measure the number of cancer cells that survive, as shown below:

Based on all of the information above and the data shown in part D, does Gleevec potentially inhibit other kinases besides BCR-ABL in cells? Please briefly explain your answer.

Answer 1

ANSWER:

a) The "lead" structure for Gleevec for designed by the fusion of BCR-ABL with tryosin kinase. An enzyme chronic myeloid leukemia cells is produced with contain philadelphia chromosomes. Its receptors are used as growt factors ad stem cell factors.

It could lead to develop the compound which could bind to ABL with higher affinity because the inhibitor present are active against the mutant of ABl kinase.