The nuclear enzyme poly(ADP-ribose) polymerase (PARP) facilitates the repair of DNA strand breaks and is implicated in the resistance of cancer cells to certain DNA-damaging agents. Inhibitors of PARP have clinical potential as resistance-modifying agents capable of potentiating radiotherapy and the cytotoxicity of some forms of cancer chemotherapy. The preclinical development of 2-aryl-1H-benzimidazole-4-carboxamides as resistance-modifying agents in cancer chemotherapy is described. 1H-Benzimidazole-4-carboxamides, particularly 2-aryl derivatives, are identified as a class of potent PARP inhibitors. Derivatives of 2-phenyl-1H-benzimidazole-4-carboxamide (23, Ki = 15 nM), in which the phenyl ring contains substituents, have been synthesized. Many of these derivatives exhibit Ki values for PARP inhibition < 10 nM, with 2-(4-hydroxymethylphenyl)-1H-benzimidazole-4-carboxamide (78, Ki = 1.6 nM) being one of the most potent. Insight into structure−activity relationships (SAR) for 2-aryl-1H-benzimidazole-4-carboxamides has been enhanced by studying the complex formed between 2-(3-methoxyphenyl)-1H-benzimidazole-4-carboxamide (44, Ki = 6 nM) and the catalytic domain of chicken PARP. Important hydrogen-bonding and hydrophobic interactions with the protein have been identified for this inhibitor. 2-(4-Hydroxyphenyl)-1H-benzimidazole-4-carboxamide (45, Ki = 6 nM) potentiates the cytotoxicity of both temozolomide and topotecan against A2780 cells in vitro (by 2.8- and 2.9-fold, respectively).
Automated synthesis of oligodeoxyribonucleotides using in situ prepared methylchlorophosphite intermediates from amidine protected purine nucleosides is described. Phosphoramidite method using 1-methylimidazole trifluoromethanesulfonate as mediator is described.
Abstract The ribonuclease H (RNase H) is an essential activity associated with HIV reverse transcriptase (RT). RNase H cleaves the RNA in hybrid RNA-DNA intermediates during minus-strand DNA synthesis initiated from the tRNALys, 3 primer. In the course of this ordered degradation of the genomic RNA template, the enzyme also generates a specific RNA fragment in the polypurine tract (ppt) region, which is then used as a primer for initiation of the plus-strand DNA synthesis. RNase H is also involved in the specific removal of both tRNA and ppt primers so that synthesis of the double-stranded DNA of the provirus can be completed. These well-defined functions of RNase H during reverse transcription can not be substituted by cellular enzymes. Since inhibition of any of these functions would block virus replication, RT-associated RNase H represents a valid target for antiretroviral therapy (1).
Poly(ADP-ribose) polymerase-1 (PARP) inhibitors (PARPi) exploit tumour-specific defects in homologous recombination DNA repair and continuous dosing is most efficacious. Early clinical trial data with rucaparib suggested that it caused sustained PARP inhibition. Here we investigate the mechanism of this durable inhibition and potential exploitation. Uptake and retention of rucaparib and persistence of PARP inhibition were determined by radiochemical and immunological assays in human cancer cell lines. The pharmacokinetics and pharmacodynamics of rucaparib were determined in tumour-bearing mice and the efficacy of different schedules of rucaparib was determined in mice bearing homologous recombination DNA repair-defective tumours. Rucaparib accumulation is carrier mediated (Km=8.4±1.2 μ M, Vmax=469±22 pmol per 106 cells per 10 min), reaching steady-state levels >10 times higher than the extracellular concentration within 30 min. Rucaparib is retained in cells and inhibits PARP ⩾50% for ⩾72 h days after a 30-min pulse of 400 nM. In Capan-1 tumour-bearing mice rucaparib accumulated and was retained in the tumours, and PARP was inhibited for 7 days following a single dose of 10 mg kg−1 i.p or 150 mg kg−1 p.o. by 70% and 90%, respectively. Weekly dosing of 150 mg kg−1 p.o once a week was as effective as 10 mg kg−1 i.p daily for five days every week for 6 weeks in delaying Capan-1 tumour growth. Rucaparib accumulates and is retained in tumour cells and inhibits PARP for long periods such that weekly schedules have equivalent anticancer activity to daily dosing in a pre-clinical model, suggesting that clinical evaluation of alternative schedules of rucaparib should be considered.
Novel tricyclic benzimidazole carboxamide poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been synthesized. Several compounds were found to be powerful chemopotentiators of temozolomide and topotecan in both A549 and LoVo cell lines. In vitro inhibition of PARP-1 was confirmed by direct measurement of NAD+ depletion and ADP-ribose polymer formation caused by chemically induced DNA damage.
<div>Abstract<p>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 (<i>K</i><sub>i</sub>, 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; <i>in vitro</i> 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 <i>in vitro</i>, 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 <i>in vivo</i> than AG14361 were also more potent <i>in vitro</i>, validating <i>in vitro</i> assays as a prescreen. These studies have identified a compound, AG14447, as a PARP inhibitor with outstanding <i>in vivo</i> 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]</p></div>
A gene for cow colostrum trypsin inhibitor (CTI) was constructed from synthetic oligonucleotides using a novel method of solid-phase gene assembly. In the first step an anchor oligonucleotide was covalently bound to the CNBr-activated Sephacryl S-500 support. Next, triads or tetrads of separately annealed oligonucleotides were stepwise hybridized to the immobilized complementary sequence, with washing after each step. In the last step a linearized vector molecule was ligated to the assembled gene. The whole construct was released from the solid support with a restriction enzyme, circularized, and used for transformation, with a high yield of recombinant clones being obtained. The method represents a generally applicable approach to rapid and efficient assembly of extended DNA duplexes.
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