Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

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Abstract

Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.

Introduction

Checkpoint kinase 2 (Chk2) is a serine/threonine protein kinase which, when activated by DNA damage, leads to the downstream phosphorylation of various substrates that are involved in cell cycle arrest, DNA damage repair, and apoptosis (Hirao et al., 2000, Hirao et al., 2002, Matsuoka et al., 2000, Pommier et al., 2005, Stolz et al., 2010). Chk2 is activated primarily by ATM or DNA-PK (also ATR and hMPs1) via phosphorylation of Thr68 in the SQ/TQ cluster domain (Ahn et al., 2000), which initiates homodimerization of Chk2 monomers followed by trans-activating autophosphorylation of residues Thr383 and Thr387 (Ahn et al., 2002, Oliver et al., 2006, Oliver et al., 2007) and then cis-phosphorylation of Ser516 (Wu and Chen, 2003). Upon activation, Chk2 phosphorylates a number of downstream targets involved in regulation of the cell cycle (e.g. Cdc25a and Cdc25c) (Bartek and Lukas, 2003, Matsuoka et al., 1998), in DNA repair and chromosome stability (e.g. BRCA1 and FOXM1) (Stolz et al., 2010, Tan et al., 2007, Zhang et al., 2004) and/or proteins that have functional roles in apoptosis such as p53, PML, and E2F1 (Antoni et al., 2007, Pommier et al., 2006).

Chk2 has attracted much attention in recent years as a potential therapeutic target for anti-cancer drug design in part because studies have shown that Chk2 may play an important role in the proliferation of cancer cells (Antoni et al., 2007, Poehlmann and Roessner, 2010, Pommier et al., 2006, Stolz et al., 2010, Zhou et al., 2003). For instance, Chk2 is activated in precancerous lesions with genomic instability and in cancer cells grown in culture (Bartkova et al., 2005, Gorgoulis et al., 2005). Chk2 has also been implicated in playing important functional roles in tumor cell adaptation to changes that result from the cycling nature of hypoxia and reoxygenation found in solid tumors (Freiberg et al., 2006), the activation of BRCA1 (Stolz et al., 2010, Zhang et al., 2004), and in the release of survivin, a protein which is involved in tumor survival (Ghosh et al., 2006). Therefore, a selective inhibitor of Chk2 could potentially abrogate the proliferation of cancer cells with endogenously activated Chk2. Indeed, prior studies with the selective cell-permeable Chk2 inhibitor PV1019 demonstrated that this compound exerts anti-proliferative effects in tumor cell lines from the NCI-60 (Shoemaker, 2006) that exhibit high endogenous levels of activated Chk2 in contrast to those with low levels of Chk2 (Jobson et al., 2009).

A second and arguably more compelling reason to be interested in Chk2 inhibitors is that selective inhibition of Chk2 in p53-defective tumor cell lines may increase their sensitivity to DNA-damaging drugs and radiation by targeting the G2 checkpoint (Levesque and Eastman, 2007, Pommier et al., 2006, Zhou and Sausville, 2003, Zhou et al., 2003). In principle, such a dual-therapy approach could increase the therapeutic efficacy of radiation and current drugs used in chemotherapy. This rationale is supported by prior evidence demonstrating that down-regulation of Chk2 in p53-mutant tumor cells results in enhanced apoptotic activity in response to ionizing radiation (Yu et al., 2001). Furthermore, the inhibitor PV1019 was shown to potentiate the cytotoxicity of camptothecin and topotecan in three ovarian cancer cells lines that had high levels of endogeneous activated Chk2 (Jobson et al., 2009). The compound CCT241533 was also shown to be a selective inhibitor of Chk2 and to potentiate the cytotoxicity of PARP inhibitors (Anderson et al., 2011). Finally, selective Chk2 inhibition may also afford some protection of normal cells (Hirao et al., 2000). As a result of therapies involving ionizing radiation and chemotherapeutics, the p53-mediated apoptotic pathway often leads to the initiation of cell death in normal tissues as a consequence of exposure to these agents (Pommier et al., 2006). Therefore, selective inhibition of Chk2 may help alleviate undesirable side effects due to radiation and chemotherapy. Indeed, such effects were demonstrated in Chk2−/− transgenic mice that showed resistance to apoptosis after exposure to ionizing radiation (Takai et al., 2002), and the treatment of mouse thymocytes and human T-cells with selective Chk2 inhibitors also provided radioprotective effects (Arienti et al., 2005, Carlessi et al., 2007, Jobson et al., 2009).

Because only a small number of Chk2 inhibitors have been identified to date (Arienti et al., 2005, Caldwell et al., 2011, Carlessi et al., 2007, Curman et al., 2001, Hilton et al., 2010, Janetka and Ashwell, 2009, Jobson et al., 2007, Jobson et al., 2009, Sharma et al., 2007, Yu et al., 2002), the discovery of novel chemotypes remains of significant interest. We previously described a novel Chk2 inhibitor obtained from a high-throughput screen, NSC 109555 (Fig. 1A), and reported co-crystal structures of this inhibitor and one of its derivatives, PV1019 (Fig. 1B), with the catalytic domain of Chk2 (Jobson et al., 2009, Lountos et al., 2009). In the present study, we present crystal structures of the Chk2 catalytic domain in complex with five more inhibitors (Fig. 1C–G) derived from NSC 109555 that were designed on the basis of information obtained from previous co-crystal structures to improve their potency and selectivity for Chk2. The new structures reported here should lead to the development of even more potent and selective inhibitors by iterative structure-assisted optimization.

Section snippets

Protein expression and purification

The catalytic domain of human Chk2 (Ser210-Glu531) was expressed and purified as previously reported (Lountos et al., 2009). The purified fraction was concentrated to 35–45 mg/mL (estimated at 280 nm using a molar extinction coefficient of 32,890 M−1 cm−1) and aliquots were flash frozen in liquid nitrogen and stored at −80 °C. The final product was judged to be > 90% pure by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The molecular weight was confirmed by LC electrospray mass

Biochemical characterization of inhibitors

The compounds were tested for inhibition against Chk2 using the IMAP Screening Express Kit (Molecular Devices, Sunnyvale, CA). They were also screened against Chk1 and RSK2 kinases to test for specificity. The results are presented in Table 1. In the assay, a fluorescently labeled peptide is phosphorylated in a kinase reaction. The addition of the IMAP binding reagent stops the kinase reaction and binds specifically to the phosphorylated peptides through a high affinity interaction of trivalent

Acknowledgments

This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research and by the Developmental Therapeutics Program of the Division of Cancer Treatment and Diagnosis. We thank Dr. Dominic Scudiero, Michael Selby and Julie Laudeman for conduction of the kinase inhibition studies. Electrospray mass spectrometry experiments were conducted on the LC/ESMS instrument maintained by the Biophysics Resource in the

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