The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

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The specificity of 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase-2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr-or Tyr-specific. In the presence of 10 W WM TBB (and 100 W WM ATP) only CK2 was drastically inhibited ( s 85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3L L and cyclin-dependent kinase 2/cyclin A) underwent moderate inhibition, with IC 50 values one^two orders of magnitude higher than CK2 (IC 50 = 0.9 W WM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell-based assays. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

1 Contraction of guinea-pig ileum to muscarinic agonists is mediated by M3 receptors, even though they account for only 30% of the total muscarinic receptor population. The aim of this study was to characterize the biochemical and functional effects of stimulation of the predominant M2 muscarinic receptor (70%) and to investigate the hypothesis that M2 receptors specifically oppose P-adrenoceptormediated effects in the ileum.2 In guinea-pig ileal longitudinal smooth muscle slices, isoprenaline, a non-selective P-adrenoceptor agonist, and BRL 37344 (sodium-4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylaminolpropyl]-phenoxyacetate sesquihydrate), a P3-adrenoceptor selective agonist, increased cyclic AMP accumulation with -log EC";, values of 6.6 ± 0.1 and 5.8 ± 0.1 respectively. Maximal stimulation by BRL 37344 (10 gM) was 26.4 ± 5.2% of that observed with isoprenaline (10 iM). Isoprenaline (10 gM)-stimulated cyclic AMP accumulation was significantly, but not completely, inhibited by propranolol (5 PM), with a propranololresistant component of 28.2 ± 6.8% of the maximal stimulation to isoprenaline. In contrast, basal and BRL 37344 responses were resistant to this antagonist. These data provide evidence that both Pl-and B3-adrenoceptors activate adenylyl cyclase in guinea-pig ileum.3 Isoprenaline (10 gM)-stimulated cyclic AMP accumulation was inhibited (67.4 ± 0.9%) by the muscarinic agonist (+)-cis-dioxolane (-log EC50 = 7.3 ± 0.1). The rank order of antagonist affinities against the (+)-cis-dioxolane response was (-log KB values in parentheses): atropine (9.0 ± 0.2) > methoctramine (7.1 ± 0.1) >p-fluoro-hexa-hydrosilaphenidol (p-F-HHSiD; 6.5 ± 0.2) ) pirenzepine (6.3 ± 0.2). (+)-cis-dioxolane also significantly inhibited BRL 37344 (10 IM; 56.5 +2.4%) stimulated cyclic AMP accumulation. These data suggest that M2 receptors mediate inhibition of cyclic AMP accumulation in response to both Pl-and P3-adrenoceptor stimulation in guinea-pig ileum. appear to be restricted to P-adrenoceptor-stimulated cyclic AMP accumulation. 5 The potential for involvement of activation of M2 receptors on responses to fradrenoceptor agonists was also studied functionally. Selective M3 receptor alkylation was achieved by pretreatment of tissues with 4-DAMP mustard (40 nM), in the presence of methoctramine (1 riM; to protect M2 receptors). After washing, tissues were pre-contracted with histamine (0.3 gM) and relaxed with isoprenaline (0.6 AM).Under these conditions, oxotremorine M caused concentration-dependent contractions (-log EC50 of 7.8 ± 0.1), that were surmountably antagonized by methoctramine (1 gM) with a -log KB estimate of 7.4 ± 0.1. Similar observations were seen versus relaxation produced by BRL 37344 (1 tM), where the -log KB value for methoctramine was 7.8 ± 0.2. These data suggest that M2 receptors mediate a functional inhibition of relaxant responses to isoprenaline and BRL 37344.6 These findings are consistent with Pl-and P3-adrenoceptors coupling to stimulation of adenylyl cyclase in guinea-pig ileum; a response that is inhib...