Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

AbstractCysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy

Photochemical reactions of thiols with organic nitrates \u2014 Oxygen atom transfer via a thionitrate

Nitroglycerin is an organic nitrate that has been used in the clinical treatment of angina for 130 years, yet important details of its mechanism of action remain unanswered. The biological activity of nitrates suggests that they are bioactivated to NO via a three-electron reduction. The involvement of free or bound protein thiols in this reduction has often been proposed. To examine the involvement of thiyl radicals in such a process, the photochemical generation of benzenethiyl radical from thiol and disulfide precursors was studied in the presence of isopropyl nitrate. Analysis of reaction products and kinetics led to the conclusion that photolysis of the nitrate to NO2 dominated the observed photochemistry. Formation of sulfonothioate and NO as products, and trapping of NO2 by 4-chlorophenol, indicated a mechanism involving oxygen atom transfer from N to S via a thionitrate intermediate. The results of the study did not indicate a rapid reaction between thiyl radical and organic nitrate. Despite weak nitrate absorption of light >300 nm and a relatively high BDE for homolysis to give NO2, the photochemistry under thiyl-generating conditions was driven by nitrate photolysis to NO2. A novel nitrate, containing a phenyl disulfanyl group linked to nitrate groups, did not undergo photolysis to NO2 or generate sulfonothioate, but did yield NO. These observations suggest that reaction between thiyl radicals and nitrates leading to NO release is a viable pathway, but it is subservient to other competing reactions, such as photolysis, in the case of IPN, and reaction with thiolate, in the case of the novel nitrate.NRC publication: Ye

Is S-Nitrosocysteine a True Surrogate for Nitric Oxide?

S-Nitrosothiol (RSNO) formation is one manner by which nitric oxide (NO) exerts its biological effects. There are several proposed mechanisms of formation of RSNO in vivo: auto-oxidation of NO, transnitrosation, oxidative nitrosylation, and from dinitrosyliron complexes (DNIC). Both free NO, generated by NO donors, and S-nitrosocysteine (CysNO) are widely used to study NO biology and signaling, including protein S-nitrosation. It is assumed that the cellular effects of both compounds are analogous and indicative of in vivo NO biology. A quantitative comparison was made of formation of DNIC and RSNO, the major NO-derived cellular products. In RAW 264.7 cells, both NO and CysNO were metabolized, leading to rapid intracellular RSNO and DNIC formation. DNIC were the dominant products formed from physiologic NO concentrations, however, and RSNO were the major product from CysNO treatment. Chelatable iron was necessary for DNIC assembly from either NO or CysNO, but not for RSNO formation. These profound differences in RSNO and DNIC formation from NO and CysNO question the use of CysNO as a surrogate for physiologic NO. Researchers designing experiments intended to elucidate the biological signaling mechanisms of NO should be aware of these differences and should consider the biological relevance of the use of exogenous CysNO. Antioxid. Redox Signal. 00, 000–000

Estrogen Receptor α Enhances the Rate of Oxidative DNA Damage by Targeting an Equine Estrogen Catechol Metabolite to the Nucleus*

Exposure to estrogens increases the risk of breast and endometrial cancer. It is proposed that the estrogen receptor (ER) may contribute to estrogen carcinogenesis by transduction of the hormonal signal and as a “Trojan horse” concentrating genotoxic estrogen metabolites in the nucleus to complex with DNA, enhancing DNA damage. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in estrogen replacement formulations, autoxidizes to a redox-cycling quinone that has been shown to cause DNA damage. 4-OHEN was found to be an estrogen of nanomolar potency in cell culture using a luciferase reporter assay and, using a chromatin immunoprecipitation assay, was found to activate ERα binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells by comparing ERα(+) versus ERα(-) cells and 4-OHEN versus menadione, a reactive oxygen species (ROS)-generating, but non-estrogenic, quinone. 4-OHEN selectively induced DNA damage in ERα(+) cells, whereas menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ERα(+) cells, whereas ER status had no effect on the rate of menadione-induced damage. Imaging of ROS induced by 4-OHEN showed accumulation selective for the nucleus of ERα(+) cells within 5 min, whereas in ERα(-) or menadione-treated cells, no selectivity was observed. These data support ERα acting as a Trojan horse concentrating 4-OHEN in the nucleus to accelerate the rate of ROS generation and thereby amplify DNA damage. The Trojan horse mechanism may be of general importance beyond estrogen genotoxins