Current Enzyme Inhibition - Volume 3, Issue 2, 2007

The past decade has witnessed an explosive growth of literature related to the role of nitric oxide (NO) in renal physiology and pathophysiology. Alterations of renal NO generation have been incriminated in various renal disorders. Excessive production of NO by NOS II in infiltrating immune cells and mesangial cells was described in acute glomerulonephritides and inhibition of NOS II resulted in protection from such renal injury [1]. Sustained high output generation of NOS II mediated NO, often accompanied by consequent suppression of NOS III derived NO is characteristic of endothelial dysfunction in ischemic acute renal injury [2]. Selective NOS II inhibition with L-N6-(1-iminoethyl) lysine or NOS II knockout rats demonstrated protection in acute ischemic renal failure models [3]. Diabetic nephropathy is characterized initially by increased renal NO production, primarily derived from NOS III, which is believed to account for glomerular hyperfiltration and microalbuminuria. On the other hand advanced diabetic nephropathy as well as most forms of chronic renal injury are associated with incremental renal NO deficiency, a consequence of NOS inhibition (mainly NOS II and NOS III) by hyperglycemia [4], and several other mediators [5]. In animal models of diabetic nephropathy, inhibition of NOS II and III has been shown to accelerate renal structural damage and functional impairment. Dietary supplementation of L-arginine, a precursor of NO, in diabetic rats resulted in reduction in proteinuria and renal injury [6]. Metabolic acidosis, a common consequence of renal failure may contribute to decreased intrarenal NO synthesis, since reduced extracellular pH impairs oxidation of nicotinamide dinucleotide phosphate (NADPH), an important post-translational mechanism in NOS II activation [7]. Urea in general leads to inhibition several enzyme systems in the body including NOS enzymes which could account for macrophage dysfunction in uremia [8]. In summary, inhibition of NOS has variable effects of renal structure and function, being renoprotective in acute glomerular and ischemic renal injury while accelerating structural damage, proteinuria, and renal failure in most forms of chronic renal failure [9], particularly in diabetes. These observations are not only crucial in understanding the pathophysiology but also provide potentially novel therapeutic targets in various renal disorders.

Nitric oxide (.NO, EDRF), a potent vasodilator is formed from arginine by NO synthases. Under physiological conditions endothelial NO synthase (NOS3) yields low steady-state .NO levels dilating the smooth muscle of the vasculature via activation of the soluble guanylyl cyclase. Superoxide (O2.-), a toxic reactive oxygen species that easily reacts with metal-sulfur-clusters and causes oxidative damage via Fenton chemistry, is formed by NADPH oxidases, xanthine oxidase, uncoupled NOS3 and mitochondria. Under physiological conditions excessive activation of these sources is suppressed. Accordingly, steady-state levels of O2.- are low. In aging or diseases such as inflammation, hypertension and atherosclerosis O2.- formation increases dramatically and at the same time inducible NO synthase (NOS2) generates high concentrations of .NO. Both radicals react in a diffusion-controlled fashion to yield the toxic and highly reactive nitrogen species peroxynitrite (ONOO-) which has been shown to oxidize all kinds of biomolecules like proteins, DNA, lipids as well as low molecular weight antioxidants. The most prominent protein modifications are the nitration and dimerization of tyrosine residues, the oxidation of cysteine thiol-groups as well as the oxidation of methionine sulfur-groups. Moreover, disruption of metal-sulfur-clusters has been demonstrated to result in oxidative inactivation of enzymatic catalysis. These oxidative modifications by ONOO- may modulate or inhibit enzymatic activity. However, since ONOO- has been shown to contribute to pathophysiological conditions in various cardiovascular, neurodegenerative and inflammatory diseases, the majority of ONOO-- mediated oxidations obviously must have functional consequences that result in interference with cellular redox-signaling, cell damage or in death of the entire organism.

Lipoxygenases (LOXs), cytochromes P450 (CYPs) and cyclooxygenases (COXs) catalyze peroxidation of unsaturated fatty acids. In humans they convert arachidonic acid into a variety of eicosanoids, which play a role in all inflammatory responses, cardiovascular and kidney diseases, Alzheimer's, cancer and other ailments. Blocking one pathway can prompt the body to switch to the available alternatives. In contrast to CYP and COX, LOX has a non-heme iron co-factor. Several LOXs are produced or stress-induced in the human body. They share the same mechanism, but differ in sequence causing catalysis on the same substrate to be regio- and stereospecific. The action of 15-LOXs could be pro- or anti-inflammatory, and pro- or anticarcinogenic. Depending on the dose, LOXs inhibitors can induce or inhibit other oxygenases. Inhibition of these enzymes presents a great challenge in solving the problem of how to control their action and treat diseases, without causing severe side effects and maintaining/restoring a delicate equilibrium between them. Research on CYPs and COXs is more advanced, while studies of LOXs are lagging behind. This article presents a brief review about LOX structures and inhibition, their involvement in human diseases, and their interplay with other oxidoreductases.

Parasitic nematodes are major causes of human, animal, and plant diseases worldwide. Although a number of therapeutics are available as treatments, reported resistance to certain anthelmintics, severe side-effects, or limited efficacy resulting from differences in the life cycles of target organisms underscore the need for the continued development of nematicidal compounds. Identifying biochemical targets that differ between the parasite and host species is essential for finding effective new molecules. The free-living nematode Caenorhabditis elegans serves as a useful model system for studying nematode biology and for analyzing the biochemistry of enzymes in potential target pathways. Providing a major component of cellular membranes, the core metabolic pathways of phosphatidylcholine synthesis in eukaryotes are well conserved; however, recent studies suggest that nematodes (and Plasmodia) use a different metabolic route to this phospholipid than mammals. In addition, phosphatidylcholine is a precursor in the production of glycoconjugates secreted by parasitic nematodes to avoid host immune responses. RNA-mediated interference experiments in C. elegans suggest that the enzymes of phosphatidylcholine biosynthesis are essential for nematode normal growth and development. Therefore, small molecule inhibitors of these enzymes may be valuable as medical, veterinary, and agricultural nematicides. This review examines the current state of knowledge of phosphatidylcholine biosynthesis in the model organism C. elegans.

Alterations in protein kinase C (PKC) isozymes and mitochondrial functions such as oxidative phosphorylation (OXPHOS) and apoptosis have each been implicated in human diseases. However, relatively little is currently understood regarding the physiologic roles of individual PKC isozymes or their substrates for phosphorylation in mitochondria. Recent advances in mitochondrial localization of PKC isozymes and methods of PKC isozyme-selective inhibition have made possible a more focused pursuit of these relationships. Studies of PKC isozyme involvement in areas such as mitochondrial ROS production, antioxidant defense, energetics, and apoptosis are now possible and hold the potential to provide promising pharmaceuticals for therapies against cardiovascular, diabetic, cancer and other diseases. The purpose of this review is to present the current state of knowledge illustrating a link between PKC isozymes, mitochondria and human disease. Novel strategies for PKC isozyme-selective inhibition will also be discussed with an emphasis on the rational targeting of mitochondrial PKC isozymes in research and clinical settings.

Recent years have seen the development of the latest generation of inhibitors of the topoisomerase enzymes. New discoveries and improvements to methodologies underpinning drug design and discovery have caused a renewed interest in the ubiquitously expressed DNA topoisomerases. Virtually every aspect of nucleic acid physiology is influenced by the topological state of DNA, which is managed predominantly by these enzymes. For this reason, they have been the targets of clinically important anti-cancer drugs for a number of years. Although the emergence of targeted therapies may have diminished the interest in topoisomerases, clinically established drugs such as irinotecan and etoposide will remain the mainstay treatment of cancer both as single agents and more recently, in combinatorial programs. By today's standards, these first generation inhibitors are crude, and their applications limited by non-ideal drug characteristics. However, the new inhibitors, such as diflomotecan, gimatecan, ICRF-193 and XR5000 offer enhanced activity through increased metabolic stability and improvements in pharmacokinetic and pharmacodynamic profiles. These developments have resulted in improved drug delivery and in reductions in adverse effects. This review will revisit this class of anti-cancer agent, and will summarise and update the exciting developments in the field.