Zdenek Hostomsky

The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site.

During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.

Therapeutic Potential of Poly(ADP-ribose) Polymerase Inhibitor AG014699 in Human Cancers With Mutated or Methylated BRCA1 or BRCA2

BACKGROUND Mutations in BRCA1 and BRCA2 (BRCA1/2), components of the homologous recombination DNA repair (HRR) pathway, are associated with hereditary breast and ovarian cancers. Poly(ADP-ribose) polymerase (PARP) inhibitors are selectively cytotoxic to animal cells with defective HRR, but results in human cancer cells have been contradictory. We undertook, to our knowledge, the first comprehensive in vitro and in vivo investigations of the antitumor activity of the PARP inhibitor AG014699 in human cancer cells carrying mutated or epigenetically silenced BRCA1/2. METHODS We used nine human cell lines, four with nonmutated BRCA1/2 (MCF7, MDA-MB-231, and HCC1937-BRCA1 [breast cancer] and OSEC-2 [ovarian surface epithelial]), two with mutated BRCA1 (MDA-MB-436 and HCC1937 [breast cancer]), one with mutated BRCA2 (CAPAN-1 [pancreatic cancer]), one that was heterozygous for BRCA2 (OSEC-1 [ovarian surface epithelial]), and one with epigenetically silenced BRCA1 (UACC3199 [breast cancer]), and two Chinese hamster ovary cell lines, parental AA8 and XRCC3 mutated IRS 1SF. We assessed cytotoxicity, DNA damage, and HRR function. Antitumor activity of AG014699 was determined by growth of xenograft tumors (five mice per treatment group). Long-term safety of AG014699 was assessed. RESULTS AG014699 (≤10 μM) was cytotoxic to cells with mutated BRCA1/2 or XRCC3 and to UACC3199 cells with epigenetically silenced BRCA1 but not to cells without BRCA1/2 or XRCC3 mutations or that were heterozygous for BRCA2 mutation. AG014699 induced DNA double-strand breaks in all nine cell lines studied. HRR was observed only in cells with functional BRCA1/2 proteins. Growth of xenograft tumors with BRCA1/2 mutations or with epigenetically silenced BRCA1 was reduced by AG014699 treatment, and combination treatment with AG014699 plus carboplatin was more effective than either drug alone. AG014699 was not toxic in mice with nonmutated or heterozygous BRCA2. CONCLUSION Human cancer cells or xenograft tumors with mutated or epigenetically silenced BRCA1/2 were sensitive to AG014699 monotherapy, indicating a potential role for PARP inhibitors in sporadic human cancers.

Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial

Poly(ADP-ribose) polymerase (PARP)-1 (EC 2.4.2.30) is a nuclear enzyme that promotes the base excision repair of DNA breaks. Inhibition of PARP-1 enhances the efficacy of DNA alkylating agents, topoisomerase I poisons, and ionizing radiation. Our aim was to identify a PARP inhibitor for clinical trial from a panel of 42 potent PARP inhibitors (Ki, 1.4–15.1 nmol/L) based on the quinazolinone, benzimidazole, tricyclic benzimidazole, tricyclic indole, and tricyclic indole-1-one core structures. We evaluated chemosensitization of temozolomide and topotecan using LoVo and SW620 human colorectal cells; in vitro radiosensitization was measured using LoVo cells, and the enhancement of antitumor activity of temozolomide was evaluated in mice bearing SW620 xenografts. Excellent chemopotentiation and radiopotentiation were observed in vitro, with 17 of the compounds causing a greater temozolomide and topotecan sensitization than the benchmark inhibitor AG14361 and 10 compounds were more potent radiosensitizers than AG14361. In tumor-bearing mice, none of the compounds were toxic when given alone, and the antitumor activity of the PARP inhibitor-temozolomide combinations was unrelated to toxicity. Compounds that were more potent chemosensitizers in vivo than AG14361 were also more potent in vitro, validating in vitro assays as a prescreen. These studies have identified a compound, AG14447, as a PARP inhibitor with outstanding in vivo chemosensitization potency at tolerable doses, which is at least 10 times more potent than the initial lead, AG14361. The phosphate salt of AG14447 (AG014699), which has improved aqueous solubility, has been selected for clinical trial. [Mol Cancer Ther 2007;6(3):945–56]

Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors

Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors selectively target homologous recombination (HR)–defective cells and show good clinical activity in hereditary breast and ovarian cancer associated with BRCA1 or BRCA2 mutations. A high proportion (up to 50%) of sporadic epithelial ovarian cancers (EOC) could be deficient in HR due to genetic or epigenetic inactivation of BRCA1/BRCA2 or other HR genes. Therefore, there is a potential for extending the use of PARP inhibitors to these patients if HR status can be identified. We developed a functional assay of HR status in primary cultures of EOCs based on Rad51 focus formation that correlates well with sensitivity to the potent PARP inhibitor AG014699. Experimental Design: Primary cultures were derived from ascitic fluid from patients with EOCs. HR status was investigated by γH2AX and Rad51 focus formation by immunofluorescence. Cytotoxicity to PARP inhibitors was tested by sulforhodamine B and survival assay. Results: Twenty-five cultures were evaluated for HR status and cytotoxicity to PARP inhibitor. Following exposure to AG014699, there was an increase in Rad51 foci (HR competent) in 9 of 24 (36%) but no increase (HR deficient) in 16 of 24 (64%) cultures. Cytotoxicity was observed in 15 of 16 (93%) HR-deficient samples but not in 9 of 9 HR-competent samples following 24-hour exposure to 10 μmol/L AG014699. Conclusion: HR status can be determined in primary cancer samples by Rad51 focus formation, and this correlates with in vitro response to PARP inhibition. Use of this assay as a biomarker now needs testing in the setting of a clinical trial. Clin Cancer Res; 16(8); 2344–51. ©2010 AACR.

2.3 Å crystal structure of the catalytic domain of DNA polymerase β

The crystal structure of the catalytic domain of rat DNA polymerase beta (pol beta) has been determined at 2.3 A resolution and refined to an R factor of 0.22. The mixed alpha/beta protein has three subdomains arranged in an overall U shape reminiscent of other polymerase structures. The folding topology of pol beta, however, is unique. Two divalent metals bind near three aspartic acid residues implicated in the catalytic activity. In the presence of Mn2+ and dTTP, interpretable electron density is seen for two metals and the triphosphate, but not the deoxythymidine moiety. The principal interaction of the triphosphate moiety is with the bound divalent metals.

Novel Poly(ADP-ribose) Polymerase-1 Inhibitor, AG14361, Restores Sensitivity to Temozolomide in Mismatch Repair-Deficient Cells

Purpose: Mismatch repair (MMR) deficiency confers resistance to temozolomide, a clinically active DNA-methylating agent. The purpose of the current study was to investigate the reversal mechanism of temozolomide resistance by the potent novel poly(ADP-ribose) polymerase (PARP)-1 inhibitor, AG14361, in MMR-proficient and -deficient cells. Experimental Design: The effects of AG14361, in comparison with the methylguanine DNA methyltransferase inhibitor, benzylguanine, on temozolomide-induced growth inhibition were investigated in matched pairs of MMR-proficient (HCT-Ch3, A2780, and CP70-ch3) and -deficient (HCT116, CP70, and CP70-ch2) cells. Results: AG14361 enhanced temozolomide activity in all MMR-proficient cells (1.5–3.3-fold) but was more effective in MMR-deficient cells (3.7–5.2-fold potentiation), overcoming temozolomide resistance. In contrast, benzylguanine only increased the efficacy of temozolomide in MMR-proficient cells but was ineffective in MMR-deficient cells. The differential effect of AG14361 in MMR-deficient cells was not attributable to differences in PARP-1 activity or differences in its inhibition by AG14361, nor was it attributable to differences in DNA strand breaks induced by temozolomide plus AG14361. MMR-deficient cells are resistant to cisplatin, but AG14361 did not sensitize any cells to cisplatin. PARP-1 inhibitors potentiate topotecan-induced growth inhibition, but AG14361 did not potentiate topotecan in MMR-deficient cells more than in MMR-proficient cells. Conclusions: MMR defects are relatively common in sporadic tumors and cancer syndromes. PARP-1 inhibition represents a novel way of selectively targeting such tumors. The underlying mechanism is probably a shift of the cytotoxic locus of temozolomide to N7-methylguanine and N3-methyladenine, which are repaired by the base excision repair pathway in which PARP-1 actively participates.

Structures of DNA and RNA polymerases and their interactions with nucleic acid substrates

DNA and RNA polymerases are enzymes that are primarily responsible for copying genetic material in all living systems. The four polymerases whose structures have been determined by X-ray crystallographic methods have significant similarities at the polymerase active site that are indicative of common requirements for polynucleotide synthesis. Structural studies of complexes of the Klenow fragment of Escherichia coli DNA polymerase I, HIV type 1 reverse transcriptase, and rat DNA polymerase beta with DNA are leading to generalized models for catalysis.

Inhibition of Poly (ADP-Ribose) Polymerase-1 Enhances Temozolomide and Topotecan Activity against Childhood Neuroblastoma

Purpose: High-risk neuroblastoma is characterized by poor survival rates, and the development of improved therapeutic approaches is a priority. Temozolomide and topotecan show promising clinical activity against neuroblastoma. Poly(ADP-ribose) polymerase-1 (PARP-1) promotes DNA repair and cell survival following genotoxic insult; we postulated that its inhibition may enhance the efficacy of these DNA-damaging drugs in pediatric cancers. Experimental Design: We evaluated the chemosensitizing properties of the PARP inhibitor AG014699 (Pfizer, Inc.) in combination with temozolomide and topotecan, against human neuroblastoma cells and xenografts, alongside associated pharmacologic and toxicologic indices. Results: Addition of PARP-inhibitory concentrations of AG014699 significantly potentiated growth inhibition by both topotecan (1.5- to 2.3-fold) and temozolomide (3- to 10-fold) in vitro, with equivalent effects confirmed in clonogenic assays. In two independent in vivo models (NB1691 and SHSY5Y xenografts), temozolomide caused a xenograft growth delay, which was enhanced by co-administration of AG014699, and resulted in complete and sustained tumor regression in the majority (6 of 10; 60%) of cases. Evidence of enhanced growth delay by topotecan/AG014699 co-administration was observed in NB1691 xenografts. AG014699 metabolites distributed rapidly into the plasma (Cmax, 1.2-1.9 nmol/L at 30 min) and accumulated in xenograft tissues (Cmax, 1-2 μmol/L at 120 min), associated with a sustained suppression of PARP-1 enzyme activity. Doses of AG014699 required for potentiation were not toxic per se. Conclusions: These data show enhancement of temozolomide and topotecan efficacy by PARP inhibition in neuroblastoma. Coupled with the acceptable pharmacokinetic, pharmacodynamic, and toxicity profiles of AG014699, our findings provide strong rationale for investigation of PARP inhibitors in pediatric early clinical studies.

Central nervous system penetration and enhancement of temozolomide activity in childhood medulloblastoma models by poly(ADP-ribose) polymerase inhibitor AG-014699.

Background:Temozolomide shows activity against medulloblastoma, the most common malignant paediatric brain tumour. Poly(ADP-ribose) polymerase (PARP) inhibitors enhance temozolomide activity in extracranial adult and paediatric human malignancies.Methods:We assessed the effect of AG-014699, a clinically active PARP inhibitor, on temozolomide-induced growth inhibition in human medulloblastoma models. Pharmacokinetic, pharmacodynamic and toxicity assays were performed in tumour-bearing mice.Results:Sensitivity to temozolomide in vitro was consistent with methylguanine methyltransferase (MGMT) and DNA mismatch repair (MMR) status; MGMT+ MMR+ D384Med cells (temozolomide GI50=220 μM), representative of most primary medulloblastomas, were sensitised fourfold by AG-014699; MGMT− MMR+ D425Med cells were hypersensitive (GI50=9 μM) and not sensitised by AG-014699, whereas MGMT+ MMR− temozolomide-resistant D283Med cells (GI50=807 μM) were sensitised 20-fold. In xenograft models, co-administration of AG-014699 produced an increase in temozolomide-induced tumour growth delay in D384Med xenografts. Consistent with the in vitro data, temozolomide caused complete tumour regressions of D425Med xenografts, whereas D283Med xenografts were relatively resistant. AG-014699 was not toxic, accumulated and reduced PARP activity ⩾75% in xenograft and brain tissues.Conclusion:We show for the first time central nervous system penetration and inhibition of brain PARP activity by AG-014699. Taken together with our in vitro chemosensitisation and toxicity data, these findings support further evaluation of the clinical potential of AG-014699–temozolomide combinations in intra-cranial malignancies.

NF-κB mediates radio-sensitization by the PARP-1 inhibitor, AG-014699

The stress-inducible transcription factor, nuclear factor (NF)-κB induces genes involved in proliferation and apoptosis. Aberrant NF-κB activity is common in cancer and contributes to therapeutic-resistance. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated during DNA strand break repair and is a known transcriptional co-regulator. Here, we investigated the role of PARP-1 function during NF-κB activation using p65 small interfering RNA (siRNA), PARP siRNA or the potent PARP-1 inhibitor, AG-014699. Survival and apoptosis assays showed that NF-κB p65−/− cells were more sensitive to ionizing radiation (IR) than p65+/+ cells. Co-incubation with p65 siRNA, PARP siRNA or AG-014699 radio-sensitized p65+/+, but not p65−/− cells, demonstrating that PARP-1 mediates its effects on survival via NF-κB. Single-strand break (SSB) repair kinetics, and the effect SSB repair inhibition by AG-014699 were similar in p65+/+ and p65−/− cells. As preventing SSB repair did not radio-sensitize p65−/− cells, we conclude that radio-sensitization by AG-014699 is due to downstream inhibition of NF-κB activation, and independent of SSB repair inhibition. PARP-1 catalytic activity was essential for IR-induced p65 DNA binding and NF-κB-dependent gene transcription, whereas for tumor necrosis factor (TNF)-α-treated cells, PARP-1 protein alone was sufficient. We hypothesize that this stimulus-dependent differential is mediated via stimulation of the poly(ADP-ribose) polymer, which was induced following IR, not TNF-α. Targeting DNA damage-activated NF-κB using AG-014699 may therefore overcome toxicity observed with classical NF-κB inhibitors without compromising other vital inflammatory functions. These data highlight the potential of PARP-1 inhibitors to overcome NF-κB-mediated therapeutic resistance and widens the spectrum of cancers in which these agents may be utilized.