Technical study: battle at the gates – combating mutant kinases

Molecular therapeutics that target proteins kinases represent a major advance in the treatment of disease, particularly cancer. These drugs have proven successful in treating some cancer patients, particularly those with pathologies derived by oncogenic forms of tyrosine kinases. One of the first successful drugs developed against protein kinases is Gleevec (imatinib), which inhibits the Abl and Kit tyrosine kinases. The initial success of Gleevec was tempered by patients who had responded initially to the drug but subsequently developed clinical resistance. The molecular basis of this resistance was soon identified – patients were shown to possess previously undetected mutant forms of Abl and Kit. Shortly thereafter during clinical studies of the EGF receptor (EGFR) inhibitors Iressa (gefitinib) and Tarceva (erlotinib), a similar phenomenon was observed and mutant forms of EGFR resistant to the inhibitors were identified.

It was soon realized that several of these drug resistant protein kinases achieved their resistance through similar mechanisms. One mutation was identified in Abl, Kit and EGFR: a mutation that changes a conserved threonine in the recess of the ATP-binding pocket to another amino acid. Remarkably, this mutation does not affect the catalytic activity of the enzyme but interferes with the ability of the inhibitor to bind effectively, leaving the kinase active but resistant to these new drugs. This aptly named ‘gatekeeper’ residue appears to play a role in influencing the sensitivity of inhibitors in numerous protein kinases (Figure 1).

However, not all protein kinase inhibitors developed are influenced by the amino acid at the gatekeeper position. This important realization implies testing various chemotypes for sensitivity to different gatekeeper sequences may be prudent to minimize the risks this type of selected clinical resistance may evolve.

The availability of three dimensional crystal structures of the protein kinases in complex with their respective inhibitors provides a glimpse of nature’s defense mechanism (Figure 2). For example, the amino acid change identified in an imatinib-resistant form of Abl appears to sterically block the inhibitor from binding productively. Generally, the resulting amino acid substitutions at the gatekeeper appear to either sterically block inhibitor binding and/or interfere with a hydrogen bond interaction between protein and inhibitor.

Once the mechanism of resistance was identified, the quest for rapid discovery of a second generation of inhibitors that were not susceptible to gatekeeper mutations was underway. To facilitate this search, Upstate has made available several disease-relevant mutant forms of protein kinases suitable for high throughput screening and enzymology studies to understand the mechanism of action for new inhibitors.

Upstate is committed to offering disease-relevant kinases and has the largest commercially available selection of inactive and mutant kinases, including a growing collection of gatekeeper mutants. As new drug resistant mutants are discovered, this important target class will undoubtedly aid in the development of successful second-generation drugs.