Specificity and mechanism of action of some commonly used protein kinase inhibitors

The specificities of 28 commercially available compounds reported to be rel atively selective inhibitors of particular serine/threonine-specific protei n kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many prote in kinases, sometimes much more potently than their presumed targets, and c onclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA107 7 and Y 27632, were more selective inhibitors, but still inhibited two or m ore protein kinases with similar potency. LY 294002 was found to inhibit ca sein kinase-2 with similar potency to phosphoinositide (phosphatidylinosito l) 3-kinase. The compounds with the most impressive selectivity profiles we re KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and S B 202190. U0126 and PD 184352, like PD 98059, were found to block the mitog en-activated protein kinase (MAPK) cascade in cell-based assays by preventi ng the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activit y directly. Apart from rapamycin and PD 184352, even the most selective inh ibitors affected at least one additional protein kinase. Our results demons trate that the specificities of protein kinase inhibitors cannot be assesse d simply by studying their effect on kinases that are closely related in pr imary structure. We propose guidelines for the use of protein kinase inhibi tors in cell-based assays.