Ian Collins

CHK2 kinase: cancer susceptibility and cancer therapy – two sides of the same coin?

In the past decade, CHK2 has emerged as an important multifunctional player in the DNA-damage response signalling pathway. Parallel studies of the human CHEK2 gene have also highlighted its role as a candidate multiorgan tumour susceptibility gene rather than a highly penetrant predisposition gene for Li–Fraumeni syndrome. As discussed here, our current understanding of CHK2 function in tumour cells, in both a biological and genetic context, suggests that targeted modulation of the active kinase or exploitation of its loss in tumours could prove to be effective anti-cancer strategies.

Probing the Probes: Fitness Factors For Small Molecule Tools

Chemical probes for interrogating biological processes are of considerable current interest. Cell permeable small molecule tools have a major role in facilitating the functional annotation of the human genome, understanding both physiological and pathological processes, and validating new molecular targets. To be valuable, chemical tools must satisfy necessary criteria and recent publications have suggested objective guidelines for what makes a useful chemical probe. Although recognizing that such guidelines may be valuable, we caution against overly restrictive rules that may stifle innovation in favor of a “fit-for-purpose” approach. Reviewing the literature and providing examples from the cancer field, we recommend a series of “fitness factors” to be considered when assessing chemical probes. We hope this will encourage innovative chemical biology research while minimizing the generation of poor quality and misleading biological data, thus increasing understanding of the particular biological area, to the benefit of basic research and drug discovery.

Anticancer therapy with checkpoint inhibitors: what, where and when?

Research into inhibitors of the protein kinases controlling the cellular response to DNA damage has reached an exciting stage, particularly for the checkpoint kinases CHK1 and CHK2. Selective inhibitors are now being tested in clinical trials in cancer patients. In this review, we highlight recent data from cellular and in vivo preclinical models that provide insight into the clinical contexts for checkpoint kinase inhibition (e.g. the timing of treatment and what type of inhibitor would be most appropriate). Although it has been shown that CHK1 inhibition potentiates the efficacy of various DNA-damaging therapies, the context for selective CHK2 inhibition is not yet as well defined. Distinct effects of selective CHK1 or CHK2 inhibition are observed when combined with DNA-damaging agents. It has also been shown that both CHK1 and CHK2 inhibitors potentiate the effects of other molecular targeted therapeutics [e.g. poly(ADP-ribose) polymerase inhibitors]. We also consider the single-agent activity of checkpoint kinase inhibitors for tumours with defined genetic backgrounds.

Identification of Novel Small Molecule Inhibitors of Hypoxia-Inducible Factor-1 That Differentially Block Hypoxia-Inducible Factor-1 Activity and Hypoxia-Inducible Factor-1α Induction in Response to Hypoxic Stress and Growth Factors

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that is activated in response to hypoxia and growth factors. HIF-1 plays a central role in tumor progression, invasion, and metastasis. Overexpression of the HIF-1alpha subunit has been observed in many human cancers and is associated with a poor prognostic outcome with conventional treatments. Targeting HIF-1 using novel small molecule inhibitors is, therefore, an attractive strategy for therapeutic development. We have generated U2OS human osteosarcoma cells stably expressing a luciferase reporter construct under the control of a hypoxia response element (U2OS-HRE-luc). The U2OS-HRE-luc cells were robustly and reproducibly sensitive to hypoxic stress in a HIF-1-dependent manner. We developed an automated U2OS-HRE-luc cell-based assay that was used in a high-throughput screen to identify compounds that inhibited HIF-1 activity induced by treatment with the hypoxia mimetic, deferoxamine mesylate. We performed a pilot screen of the National Cancer Institute Diversity Set of 2,000 compounds. We identified eight hit compounds, six of these were also identified by Rapisarda et al. in an independent hypoxia screen. However, there were two novel hit compounds, NSC-134754 and NSC-643735, that did not significantly inhibit constitutive luciferase activity in U2OS cells (U2OS-luc). We showed that both NSC-134754 and NSC-643735 significantly inhibited HIF-1 activity and HIF-1alpha protein induced by deferoxamine mesylate. Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1alpha protein induced by hypoxia and significantly inhibited Glut-1 expression. Finally, we showed that both NCS-134754 and NCS-643735 inhibited HIF-1alpha protein induced by insulin-like growth factor-1. Our cell-based assay approach has successfully identified novel compounds that differentially target hypoxia and/or growth factor-mediated induction of HIF-1alpha.

Anxiogenic properties of an inverse agonist selective for α3 subunit‐containing GABAA receptors

1 α3IA (6‐(4‐pyridyl)‐5‐(4‐methoxyphenyl)‐3‐carbomethoxy‐1‐methyl‐1H‐pyridin‐2‐one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype. 2 α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15‐1788 in vivo binding assay. 3 At doses in rats that produce relatively low levels of occupancy (12%) in the cerebellum (i.e. α1‐containing receptors), α3IA (30 mg kg−1 i.p.), like the nonselective partial inverse agonist N‐methyl‐β‐carboline‐3‐carboxamide (FG 7142), not only caused behavioural disruption in an operant, chain‐pulling assay but was also anxiogenic in the elevated plus maze, an anxiogenic‐like effect that could be blocked with the benzodiazepine antagonist Ro 15‐1788 (flumazenil). 4 Neurochemically, α3IA (30 mg kg−1 i.p.) as well as FG 7142 (15 mg kg−1 i.p.) increased the concentration of the dopamine metabolite 3,4‐dihydroxyphenylacetic acid in rat medial prefrontal cortex by 74 and 68%, respectively, relative to vehicle‐treated animals, a response that mimicked that seen following immobilisation stress. 5 Taken together, these data demonstrate that an inverse agonist selective for GABAA receptors containing an α3 subunit is anxiogenic, and suggest that since α3‐containing GABAA receptors play a role in anxiety, then agonists selective for this subtype should be anxiolytic.

Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response

Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N‐terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C‐terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1α bound to ADP, revealing the ‘phosphoryl‐transfer’ competent dimeric face‐to‐face complex, which precedes and is distinct from the back‐to‐back RNase ‘active’ conformation described for yeast Ire1. We show that the Xbp1‐specific ribonuclease activity depends on autophosphorylation, and that ATP‐competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro, also inhibit Xbp1 splicing in vivo. Furthermore, we demonstrate that activated Ire1α is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1α as a target for development of ATP‐competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies.

Measuring and interpreting the selectivity of protein kinase inhibitors

Protein kinase inhibitors are a well-established class of clinically useful drugs, particularly for the treatment of cancer. Achieving inhibitor selectivity for particular protein kinases often remains a significant challenge in the development of new small molecules as drugs or as tools for chemical biology research. This review summarises the methodologies available for measuring kinase inhibitor selectivity, both in vitro and in cells. The interpretation of kinase inhibitor selectivity data is discussed, particularly with reference to the structural biology of the protein targets. Measurement and prediction of kinase inhibitor selectivity will be important for the development of new multi-targeted kinase inhibitors.

CCT241533 Is a Potent and Selective Inhibitor of CHK2 that Potentiates the Cytotoxicity of PARP Inhibitors

CHK2 is a checkpoint kinase involved in the ATM-mediated response to double-strand DNA breaks. Its potential as a drug target is still unclear, but inhibitors of CHK2 may increase the efficacy of genotoxic cancer therapies in a p53 mutant background by eliminating one of the checkpoints or DNA repair pathways contributing to cellular resistance. We report here the identification and characterization of a novel CHK2 kinase inhibitor, CCT241533. X-ray crystallography confirmed that CCT241533 bound to CHK2 in the ATP pocket. This compound inhibits CHK2 with an IC(50) of 3 nmol/L and shows minimal cross-reactivity against a panel of kinases at 1 μmol/L. CCT241533 blocked CHK2 activity in human tumor cell lines in response to DNA damage, as shown by inhibition of CHK2 autophosphorylation at S516, band shift mobility changes, and HDMX degradation. CCT241533 did not potentiate the cytotoxicity of a selection of genotoxic agents in several cell lines. However, this compound significantly potentiates the cytotoxicity of two structurally distinct PARP inhibitors. Clear induction of the pS516 CHK2 signal was seen with a PARP inhibitor alone, and this activation was abolished by CCT241533, implying that the potentiation of PARP inhibitor cell killing by CCT241533 was due to inhibition of CHK2. Consequently, our findings imply that CHK2 inhibitors may exert therapeutic activity in combination with PARP inhibitors.

Creating an antibacterial with in vivo efficacy: synthesis and characterization of potent inhibitors of the bacterial cell division protein FtsZ with improved pharmaceutical properties.

3-Methoxybenzamide (1) is a weak inhibitor of the essential bacterial cell division protein FtsZ. Alkyl derivatives of 1 are potent antistaphylococcal compounds with suboptimal drug-like properties. Exploration of the structure−activity relationships of analogues of these inhibitors led to the identification of potent antistaphylococcal compounds with improved pharmaceutical properties.

Identification of 4-(4-Aminopiperidin-1-Yl)-7H-Pyrrolo[2,3-D]Pyrimidines as Selective Inhibitors of Protein Kinase B Through Fragment Elaboration.

Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.