To date, eight inhibitors of protein kinases are approved by the FDA for treating cancer (). Although each was developed with a drug target in mind, off-target effects of these drugs have assumed significant importance in their clinical use. lists the number of kinases that is affected by each drug in biochemical assays of binding [16
] or activity (Selectscreen®, Invitrogen). Conventionally, drug targets have been identified through the isolation of stable ligand-enzyme interactions or screening in activity-based assays across large panels of recombinant kinases [16
]. Both methods have exposed variable promiscuity among ostensibly selective protein kinase inhibitors. Among the FDA approved agents, sunitinib (kidney cancer) has the greatest number of protein kinase targets, whereas lapatinib (Her2+ breast cancer) appears to be highly selective for inhibition of EGFR1/Her2 (). While useful, such in vitro
assays often deviate significantly from in vivo
reality, due to the lability of the drug-enzyme complex and/or omission of physiologically relevant regulators and substrates. Thus, validating drug selectivity in living cells and organisms remains a challenging but essential step in drug development. Fortunately, chemical genetics provides several strategies for achieving this objective.
FDA approved inhibitors of protein kinases
For kinase inhibitors, the selectivity, or lack thereof, has affected clinical development, as illustrated by imatinib (Gleevac) and sorafenib. Imatinib was developed as a selective inhibitor of Abl kinase for CML, and has provided a highly effective (but not curative) alternative to bone marrow transplant [17
]. However, imatinib was subsequently found to be a high affinity inhibitor of c-Kit and PDGFRα/β [18
]. This made it an attractive drug for treating GI stromal tumors (GIST) which harbor activating mutations in c-Kit, and myeloproliferative disorders mediated by PDGFRβ. Thus, other targets were useful in expanding use of the drug to other indications. This allowed rational use of a drug with limited selectivity.
Sorafenib is another case study in the importance of off-target functions. Sorafenib (BAY 43-9006) was developed as a potent inhibitor of Raf kinase, selected as a drugable target mediating oncogenic signaling from KRAS [19
]. Combinatorial libraries were screened with Raf kinase assay. Active compounds were secondarily analyzed by detecting a Raf-mediated phosphorylation and ability to block KRAS mutant cancer cells from proliferation and growth in soft agar. Next, hits were counterscreened for blocking downstream elements of the KRAS-Raf pathway. Finally preclinical data supported use of sorafenib for KRAS+ tumors and this was moved into clinical development. At the same time, effect of this drug on other protein kinases was analyzed in biochemical assays, defining other targets[20
]. Ultimately, the focus of clinical development included kidney and liver cancer, in which no effective chemotherapies were available. Sorafenib was approved in the U.S. for unselected patients with kidney and liver cancer, although KRAS mutations are rare in these diseases (~1% of kidney cancers and 4% of liver cancers according to COSMIC database [21
]). At present, the mechanism of sorafenib’s action against these malignancies remains obscure. Its efficacy has been variously attributed to effects on VEGFR2/3, PDGFRβ, Flt-3, and c-Kit, although in vitro
assays have defined up to 32 protein kinase targets.
These case studies demonstrate that development of ‘targeted drugs’ can proceed rationally or empirically. Drugs with moderate selectivity, such as imatinib can be rationally used for a number of targets, but when selectivity is poor, clinical development is empiric. Identification and validation of drug targets allows a rational approach to drug development. Below we discuss how such measures can be established through chemical genetics.