Characterization of the CHK1 Allosteric Inhibitor Binding Site

Abstract

Checkpoint kinase 1 (CHK1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G(2)-phase checkpoint. Inhibiting CHK1 is a therapeutic strategy involving abrogation of the G2/M mitotic checkpoint defense of tumor cells toward lethal damage induced by DNA-directed chemotherapeutic agents. To date, most CHK1 inhibition approaches have involved targeting the ATP site of this kinase. In this study, we provide crystallographic and kinetic characterization of two small molecule inhibitors that bind to an allosteric site in the proximity of the CHK1 substrate site. Analysis of kinetic and biophysical data has led to the conclusion that these small molecule allosteric site inhibitors of CHK1 are reversible and are neither ATP- nor peptide substrate-competitive. K(i) values of 1.89 and 0.15 microM, respectively, have been determined for these compounds using a mixed inhibitor kinetic analysis. Cocrystal structures of the inhibitors bound to CHK1 reveal an allosteric site, unique to CHK1, located in the C-terminal domain and consisting of a shallow groove linked to a small hydrophobic pocket. The pocket displays induced fit characteristics in the presence of the two inhibitors. These findings establish the potential for the development of highly selective CHK1 inhibitors.

Characterization of novel Checkpoint kinase 1 inhibitors by in vitro assays and in human cancer cells treated with topoisomerase inhibitors

“…The Km for ATP with GST-Chk1-FL was 3 μM, which is consistent with a report by Kawase (Carna Biosciences). The Km for ATP of the catalytic form of Chk1 is reported to be 38 μM, or 10 fold higher than that of the full-length version (Vanderpool et al, 2009). We are the first to report that Chk1 has maximal activity at pH 7.5 and 37°C, consistent with its role in human cells.…”

International Union of Basic and Clinical Pharmacology. XC. Multisite Pharmacology: Recommendations for the Nomenclature of Receptor Allosterism and Allosteric Ligands

“…It should be noted that lack of ATP-competition is not necessarily indicative of allosteric binding. Therefore it needs to be emphasized that only structural determination of the TK domain unambiguously reveals an allosteric KI as occupying a pocket outside of the ATP site (Vanderpool et al, 2009;Fabbro et al, 2012). Type 3 KIs, like the mitogenactivated protein kinase kinase 1/2 (Ohren et al, 2004) and ABL myristate pocket binders Fabbro et al, 2010), are truly non-ATP competitive.…”

“…Type 3 KIs, like the mitogenactivated protein kinase kinase 1/2 (Ohren et al, 2004) and ABL myristate pocket binders Fabbro et al, 2010), are truly non-ATP competitive. In contrast, the mixed-competition checkpoint kinase 1 inhibitors that, according to structure, also would classify as Type 3, reminds us that "competition" in binding and functional inhibition are not identical (Vanderpool et al, 2009); high negative cooperativity via an allosteric interaction is virtually indistinguishable from competition. The allosteric myr-pocket inhibitors assemble the inactive conformation of Abl or BcrAbl by promoting the docking of the SH3 and SH2 domains onto the Abl kinase domain, which seems to work also for the first generation TKI resistant T 315 I mutant Fabbro et al, 2010).…”

Factors influencing the inhibition of protein kinases

“…refs. [32][33][34], and some of those concern non-ATP competitive inhibitors. We therefore decided to determine β values for the two ATP competitive inhibitors, AEW541 and STAURO.…”

“…Vanderpool et al 34 have measured β > 1 for CHK1 inhibitors and they have proposed that the binding of the substrate (in their case ATP) affects the volume of the allosteric pocket where these molecules bind. These results together with our data show that measuring β brings valuable additional information on the protein kinase-inhibitor interaction, and we propose that the β parameter should be used by medicinal chemists to study the binding mode of compounds in more detail.…”