Andrew Jobson

Repair of Topoisomerase I-Mediated DNA Damage

Publisher Summary This chapter discusses the use of inhibitors of tyrosyl-DNA phosphodiesterase (Tdp1) and Chk1/2 in combination with Topoisomerase I (TopI) inhibitors. TopI is an abundant and essential enzyme. It is the selective target of camptothecins, which are effective anticancer agents. TopI–DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions, including mismatches, abasic sites, and carcinogenic adducts. Tdp1 is one of the repair enzymes for Top1–DNA covalent complexes. It forms a multiprotein complex that includes poly (Adenosine diphosphate (ADP)–ribose) polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. This chapter reviews the developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast, and fission yeast.

Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide]

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Identification of a Bis-guanylhydrazone [4,4'-Diacetyldiphenylurea-bis(guanylhydrazone); NSC 109555] as a novel chemotype for inhibition of Chk2 kinase.

Chk2 is a protein kinase involved in the ATM-dependent checkpoint pathway (http://discover.nci.nih.gov/mim). This pathway is activated by genomic instability and DNA damage and results in either cell cycle arrest, to allow DNA repair to occur, or cell death (apoptosis). Chk2 is activated by ATM-mediated phosphorylation and autophosphorylation and in turn phosphorylates its downstream targets (Cdc25A, Cdc25C, BRCA1, p53, Hdmx, E2F1, PP2A, and PML). Inhibition of Chk2 has been proposed to sensitize p53-deficient cells as well as protect normal tissue after exposure to DNA-damaging agents. We have developed a drug-screening program for specific Chk2 inhibitors using a fluorescence polarization assay, immobilized metal ion affinity-based fluorescence polarization (IMAP). This assay detects the degree of phosphorylation of a fluorescently linked substrate by Chk2. From a screen of over 100,000 compounds from the NCI Developmental Therapeutics Program, we identified a bis-guanylhydrazone [4,4′-diacetyldiphenylureabis(guanylhydrazone); NSC 109555] as a lead compound. In vitro data show the specific inhibition of Chk2 kinase activity by NSC 109555 using in vitro kinase assays and kinase-profiling experiments. NSC 109555 was shown to be a competitive inhibitor of Chk2 with respect to ATP, which was supported by docking of NSC 109555 into the ATP binding pocket of the Chk2 catalytic domain. The potency of NSC 109555 was comparable with that of other known Chk2 inhibitors, such as debromohymenialdisine and 2-arylbenzimidazole. These data define a novel chemotype for the development of potent and selective inhibitors of Chk2. This class of drugs may ultimately be useful in combination with current DNA-damaging agents used in the clinic.

Inhibition of human tyrosyl-DNA phosphodiesterase by aminoglycoside antibiotics and ribosome inhibitors

DNA topoisomerase I (Top1) is the target of camptothecin, and novel Top1 inhibitors are in development as anticancer agents. Top1 inhibitors damage DNA by trapping covalent complexes between the Top1 catalytic tyrosine and the 3′-end of the broken DNA. Tyrosyl-DNA phosphodiesterase (Tdp1) can repair Top1-DNA covalent complexes by hydrolyzing the tyrosyl-DNA bond. Inhibiting Tdp1 has the potential to enhance the anticancer activity of Top1 inhibitors (http://discover.nci.nih.gov/pommier/pommier.htm) and to act as antiproliferative agents. In the present study, we report that neomycin inhibits Tdp1 more effectively than the related aminoglycosides paromomycin and lividomycin A. Inhibition of Tdp1 by neomycin is observed both with single- and double-stranded substrates but is slightly stronger with duplex DNA, which is different from aclarubicin, which only inhibits Tdp1 with the double-stranded substrate. Inhibition by neomycin can be overcome with excess Tdp1 and is greatest at low pH. To our knowledge, aminoglycoside antibiotics and the ribosome inhibitors thiostrepton, clindamycin-2-phosphate, and puromycin are the first reported pharmacological Tdp1 inhibitors.

Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor

Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM‐Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA‐damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC50 = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP‐competitive inhibitor, as the electron density clearly reveals that it occupies the ATP‐binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.

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

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.

X-Ray Structures of Checkpoint Kinase 2 in Complex with Inhibitors that Target its Gatekeeper-Dependent Hydrophobic Pocket.

The serine/threonine checkpoint kinase 2 (Chk2) is an attractive molecular target for the development of small molecule inhibitors to treat cancer. Here, we report the rational design of Chk2 inhibitors that target the gatekeeper‐dependent hydrophobic pocket located behind the adenine‐binding region of the ATP‐binding site. These compounds exhibit IC50 values in the low nanomolar range and are highly selective for Chk2 over Chk1. X‐ray crystallography was used to determine the structures of the inhibitors in complex with the catalytic kinase domain of Chk2 to verify their modes of binding.

Cellular Inhibition of Chk2 Kinase and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor Pv1019.

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Abstract A205: Chk2 status (activation, proteomic, gene, and exon expression) and insights into the activity of its inhibitor PV1019 across the 60 cell lines from the NCI Screen

Chk2 is a multi‐substrate kinase involved in the DNA damage response and genomic stability. Part of the ATM‐Chk2‐p53 axis, it is also activated in various malignant tumors, possibly in a “gene addiction” context. Chk2 inhibitors are in development as anticancer agents based on their selectivity for enhancing the activity of DNA targeting agents in p53‐deficient tumors. Here we determined the activation of Chk2, its protein and gene expression levels, and the activity of PV1019 (a new and specific Chk2 inhibitor; see abstract by Jobson et al.) across the 60 lines of the NCI DTP screen. Chk2 expression data were obtained from five microarray platforms (Affymetrix HG‐U95, U133, U133 Plus 2.0, HuEx 1.0, and Agilent WHG). Levels of total and activated Chk2 (i.e., phosphorylated on threonine 68) were analyzed by Western blotting. PV1019 was tested in duplicate, and GI50 was interpolated from five serial dilutions of the drug. Our analyses demonstrate that a number of cell lines have high endogenous Chk2 activation (ovarian OVCAR‐3 and OVCAR‐4, renal RFX‐393, lung NCI‐H322M and EKVX, melanoma UACC‐62 and ‐257, leukemia RPMI‐8226 and breast BT‐549) whereas the wild‐type p53 tumors all showed low Chk2 activation. Expression of total Chk2 protein and mRNA were generally positively correlated to each other and to the activity of PV1019. Exon array analyses across the NCI‐60 suggest exon 3 splicing in approximately half of the cell lines and transcript interruption between exons 8 and 10 in the ovarian SKOV‐3 cells, which could be related to the low level of Chk2 expression in this cell line. Together, these data demonstrate a complex phenotype, which underlies the susceptibility of tumor cell lines to Chk2 inhibitors. Such phenotype may relate to potential patient selection for the optimal use of Chk2 inhibitors as single agents or modulators of DNA targeting therapies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A205.

Abstract A120: Cellular inhibition of Chk2 kinase and potentiation of camptothecins and radiation by the novel Chk2 inhibitor PV1019

Chk2 is a checkpoint kinase involved in the ATM pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported NSC 109555 as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039), which is a selective sub‐micromolar inhibitor of Chk2 in vitro. The co‐crystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against IR‐induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 as well as Chk2 siRNA can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A120.