Pseudomonas Cytochrome c551 at 2.0 angstrom Resolution: Enlargement of the Cytochrome c Family

The structure of respiratory cytochrome c551 of Pseudomonas aeruginosa, with 82 amino acids, has been solved by x-ray analysis and refined to a crystallographic R factor of 16.2%. It has the same basic folding pattern and hydrophobic heme environment as cytochromes c, c2, and c550, except for a large deletion at the bottom of the heme crevice. This same "cytochrome fold" appears to be present in photosynthetic cytochromes c of green and purple sulfur bacteria, and algal cytochromes f, suggesting a common evolutionary origin for electron transport chains in photosynthesis and respiration

Structure of the Human Cytomegalovirus Protease Catalytic Domain Reveals a Novel Serine Protease Fold and Catalytic Triad

AbstractProteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 Å crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other

Resistance-modifying agents. 9. Synthesis and biological properties of benzimidazole inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase

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)

Anticancer chemosensitization and radiosensitization by the novel poly(ADP-ribose) polymerase-1 inhibitor AG14361

Background: Poly(ADP-ribose) polymerase-1 (PARP-1) facilitates the repair of DNA strand breaks. Inhibiting PARP-1 increases the cytotoxicity of DNA-damaging chemotherapy and radiation therapy in vitro. Because classical PARP-1 inhibitors have limited clinical utility, we investigated whether AG14361, a novel potent PARP-1 inhibitor (inhibition constant <5 nM), enhances the effects of chemotherapy and radiation therapy in human cancer cell cultures and xenografts. Methods: The effect of AG14361 on the antitumor activity of the DNA alkylating agent temozolomide, topoisomerase I poisons topotecan or irinotecan, or x-irradiation or gamma-radiation was investigated in human cancer cell lines A549, LoVo, and SW620 by proliferation and survival assays and in xenografts in mice by tumor volume determination. The specificity of AG14361 for PARP-1 was investigated by microarray analysis and by antiproliferation and acute toxicity assays in PARP-1(-/-) and PARP-1(+/+) cells and mice. After intraperitoneal administration, the concentration of AG14361 was determined in mouse plasma and tissues, and its effect on PARP-1 activity was determined in tumor homogenates. All statistical tests were two-sided. Results: AG14361 at 0.4 muM did not affect cancer cell gene expression or growth, but it did increase the antiproliferative activity of temozolomide (e.g., in LoVo cells by 5.5-fold, 95% confidence interval [CI] = 4.9-fold to 5.9-fold; P = .004) and topotecan (e.g., in LoVo cells by 1.6-fold, 95% CI = 1.3-fold to 1.9-fold; P = .002) and inhibited recovery from potentially lethal gamma-radiation damage in LoVo cells by 73% (95% CI = 48% to 98%). In vivo, nontoxic doses of AG14361 increased the delay of LoVo xenograft growth induced by irinotecan, x-irradiation, or temozolomide by two- to threefold. The combination of AG14361 and temozolomide caused complete regression of SW620 xenograft tumors. AG14361 was retained in xenografts in which PARP-1 activity was inhibited by more than 75% for at least 4 hours. Conclusion: AG14361 is, to our knowledge, the first high-potency PARP-1 inhibitor with the specificity and in vivo activity to enhance chemotherapy and radiation therapy of human cancer