The selectivity of protein kinase inhibitors: a further update - PubMed (original) (raw)

The selectivity of protein kinase inhibitors: a further update

Jenny Bain et al. Biochem J. 2007.

Abstract

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.

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Figures

Figure 1

Figure 1. Effect of MKK1/MKK5 inhibitors on the activation of ERK1/ERK2 and ERK5

HeLa cells were serum-starved for 16 h and incubated for a further 1 h in the presence (+) or absence (−) of PD184352, PD 0325901-Cl [(R) steroisomer], PD 0325901 [(S) steroisomer] or PD 0325901 at the concentrations indicated. The cells were then stimulated for 10 min with 100 ng/ml EGF, lysed, and 30 μg of the extract protein was denatured in SDS, subjected to SDS/PAGE, transferred to nitrocellulose membranes and immunoblotted with an antibody recognizing phosphorylated ERK1 and ERK2 in (A) and immunoblotted with an antibody that recognizes all forms of ERK5 equally well in (B).

Figure 2

Figure 2. Effect of PI 103 on the activities of PI3K superfamily members in HEK-293 cells

(A) HEK-293 (human embryonic kidney-293) cells were incubated for 1 h with (+) or without (−) 0.5 μM PI 103, then stimulated for 30 min with IGF-1 (50 ng/ml) and lysed. Cell extracts (60 μg of protein) were denatured in SDS, subjected to SDS/PAGE and, after transfer to PVDF membranes, immunoblotted with a phosphospecific antibody that recognizes PKB phosphorylated at Ser473 [pPKB (S473)]. (B) HEK-293 cells were incubated for 1 h with (+) or without (−) 0.5 μM PI 103, then treated for 2 h with the DNA-alkylating agent MMS (2 mM) and lysed. Cell extracts (60 μg of protein) were denatured in SDS, subjected to SDS/PAGE and, after transfer to PVDF membranes, immunoblotted with phosphospecific antibodies that recognize CHK1 phosphorylated at Ser345 [pChk1 (S345)]and CHK2 phosphorylated at Thr68 [pChk2 (T68)].

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