Pharmacological modulation of nitric oxide synthesis by mechanism-based inactivators and related inhibitors

Abstract

Nitric oxide synthase (NOS) (EC 1.14.13.39) is a homodimeric cytochrome P450 monooxygenase analog that generates nitric oxide (NO) from the amino acid l-arginine. Enzymatically produced NO acts as an intracellular messenger in neuronal networks, blood pressure regulatory mechanisms, and immune responses. Isoform-selective pharmacological modulation of NO synthesis has emerged as a new therapeutic strategy for the treatment of diverse clinical conditions associated with NO overproduction. Mechanism-based inactivators (MBIs) represent a class of NOS mechanistic inhibitors that require catalytic turnover to produce irreversible inactivation of the ability of NOS to generate NO. Diverse isoform-selective NOS MBIs have been characterized with respect to their kinetic parameters and chemical mechanisms of inactivation. In studies with isolated and purified NOS isoforms, MBIs produce irreversible inactivation of NOS enzymatic activities. The inactivation process is associated with covalent modification of the NOS active site and proceeds either through heme destruction, its structural alteration, or covalent modification of the NOS protein chain. The behavior of NOS MBIs in intact cells is different from their behavior observed with the isolated NOS isoforms. In cytokine-induced RAW 264.7 macrophages, treatment with MBIs produces a complete loss of cellular NOS synthetic competence and inducible NOS activity. However, following drug removal, cells can recover at least partially in the absence of protein synthesis. In GH₃ cells containing the neuronal NOS isoform, calcium transients are too low and abbreviated to allow significant NOS inactivation; hence, the cellular effects of MBIs on the neuronal isoform are almost completely and immediately reversible.

Introduction

Since the original discovery of nitric oxide (NO) synthetic activity in mammalian cell cultures and tissues more than a decade ago, our understanding of the enzymology of NO production and the role of NO in human physiology, as well as in the pathology of numerous diseases, has advanced dramatically. Biochemical studies, which started with the purification of three NO synthase (NOS) isoforms, led to their detailed characterization, successful cloning, and crystallization of the inducible NOS (iNOS) oxygenase dimeric core. The ubiquitous role of NO in human physiology and pathology, which is clearly established now, hardly could have been imagined little more than a decade ago. While a vast variety of physiologically important biological actions accomplished by NO is well appreciated, its ability to mediate tissue damage suggested the potential clinical utility of the pharmacological modulation of NO enzymatic production by NOS isoforms. A search for potent, nontoxic, cell-permeable isoform-selective NOS inhibitors opened the field of NOS mechanism-based inactivators (MBIs), agents that depend on NOS catalytic turnover to produce their enzyme inactivation.

The current review will focus on NOS MBIs as a class of highly efficient and potentially valuable therapeutic tools, and will discuss in detail the kinetics and mechanisms of NOS inactivation by the known MBIs in vitro and in intact cells. The recent advances in our understanding of NOS, its biochemistry, chemical mechanism of catalysis, active site structure, and regulation of NOS enzymatic activities will be discussed as a prelude to our consideration of NOS mechanism-based inactivation. The physiology and pathology of NOS-derived NO will be considered only briefly with relation to the potential clinical utility of isoform-selective NOS inhibitors for the treatment of diverse human pathological conditions.