386 Current Medicinal Chemistry, 2012, 19, 386-405 0929-8673/12 $58.00+.00 © 2012 Bentham Science Publishers Nitric Oxide: State of the Art in Drug Design R.A.M. Serafim, M.C. Primi, G.H.G. Trossini and E.I. Ferreira* LAPEN: Laboratory of Design and Synthesis of Chemotherapeutic Potentially Active against Neglected Diseases, Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo – FCF/USP, Brazil Abstract: Since the great discovery of Furchgott, Ignarro and Murad in the late 90´s, nitric oxide (NO) is considered one of the most versatile endogenous molecules, which is involved in important signaling biochemistry pathways of the human body. Thus, it is directly related to pathological processes and its over- or low-production is able to cause damage in systems that are involved. By using certain functional groups present in molecules that already have potential therapeutic value, hybrid compounds, by means of inclusion of NO- donors (e.g., ester nitrates, furoxans, benzofuroxans, NONOates, S-nitrosothiols, metal complexes), can be generated that have a NO release benefit along with maintaining the activity of the native drug. This approach has proved to be useful in many spheres of Medicinal Chemistry, such as cardiovascular, inflammatory, bacterial, fungal, viral, parasitic, ocular diseases and cancer. Potent and selective nitric oxide synthase inhibitors are being designed, mainly through enzyme structure based process, however, due to high homology between the isoforms, these studies have proved to be very difficult. The objective of the research is to achieve a balance between the release of therapeutic amounts of NO, especially in specific site of action, and maintaining the native drug activity. The search for new and effective NO-donor hybrid drugs, as well as selective nitric oxide synthase inhibitors, is an important focus in modern drug design in order to manipulate biochemical pathways involving NO that influence many dysfunctions of the human organism. Keywords: Drug design, nitric oxide, NO-releasing agents, nitric oxide synthase inhibitors, immune system, hybrid compounds, mutual prodrugs, medicinal chemistry, therapeutic potential, biochemical pathways. 1. INTRODUCTION Nitric Oxide (NO) is an uncharged diatomic molecule containing 11 electrons in its valence shell, one of which is unpaired [1,2]. The molecule is produced by bacteria, plants and animals and notably, the majority of the chemical interactions involving NO in biological systems entail the stabilization of the unpaired electron. NO solubility is higher in nonpolar media and therefore tends to dissolve in cell membrane and lipid phases [1,3]. In its pure state, and under ambient temperature and pressure conditions, NO remains in gaseous form. However, within organisms it acts in the form of a dissolved non-electrolyte, except at the lung, where NO can also be found in its gaseous state [1]. The importance of research in NO has been recognized by the scientific community at several junctures. In 1992, Science magazine elected NO as the molecule of the year [4], and in 1998 the Nobel Prize in Physiology and Medicine was awarded to the researchers R. Furchgott, L. Ignarro and F. Murad, whose work led to the discovery of NO as a biological mediator produced by mammalian cells [5,6]. Further, in 1999, Salvador Moncada was the most cited scientist in the biomedical field for his group of work on mammalian NO [5]. The Nitric Oxide Society was founded in 1996, publishers of the official journal on NO, “Nitric Oxide: Biology and Chemistry” [7]. An online journal about NO is also available called “The Open Nitric Oxide Journal”, which seeks to fast-track the publication of quality articles and provides ease of access to researchers worldwide [8]. 1.1. Historical Aspects NO was originally regarded as an environmental pollutant but in the late 1970s Robert Furchgott, upon analyzing vascular relaxation by acetylcholine in blood vessels, noted the obligatory role of endothelial cells to enable vasodilation [9,10,11]. Thus, Furchgott identified the interference of a factor needed in vasodilation which he termed endothelium-derived relaxing factor (EDRF) [9,10]. Furchgott later suggested that EDRF could in fact be NO, and in 1986 found several similar properties shared by both EDRF and NO. In 1987, the research groups of Ignarro and Moncada demonstrated that EDRF and NO were indeed the same molecule [9,12,13]. *Address correspondence to this author at the Av. Prof. Lineu Prestes, 580 05508-900 Sao Paulo – SP, Brazil; Tel: 55 11 3091-3793; Fax: 55 11 3815-4418; E-mail: elizabeth.igne@gmail.com 1.2. NO Generation and Nitric Oxide Synthases NO is generated from L-arginine by a family of enzymes denominated nitric oxide synthases (NOSs) [14,15,16]. These enzymes were originally identified and described in 1989 and the first crystallographic structures of their domains were determined in 1998 and published in 1999 [5]. The NOSs present as three distinct isoforms (Fig. 1), two of which are defined as constitutive and the other induced [14,16]. The constitutive isoforms include: neuronal (nNOS), present in nerve cells, skeletal muscle and heart muscle; and endothelial (eNOS), present in the cells coating blood vessels [14,3]. The inducible isoform (iNOS) occurs in many types of cells as an immune system response to inflammatory processes [14,3]. Fig. (1). Isoforms of NOSs. The isoforms of NOS are dimeric, where each monomer has two domains, namely the reductase and oxygenase domains. The structure of the reductase domain comprises FAD, FMN and NADPH whereas the oxygenase domain comprises the heme group and BH 4 . These domains are linked by calmodulin, a calcium sensing portion, which can act as the enzyme activator [14]. The need for calcium in order to activate the enzyme is a factor differentiating the two types of NOS: the constitutive synthases (eNOS and nNOS) activated during high intracellular calcium, and the induced synthase (iNOS) which is calcium independent [17]. 1.3. Biological Properties NO can regulate a wide array of biological processes, in many cases through activation of guanylyl cyclase and increase of cyclic guanosine monophosphate (cGMP) synthesis from cyclic guanosine triphosphate (cGTP) [18,19,6]. However, there are many independent effects from cGMP, such as NO interactions with nitric oxide synthases (NOSs) constitutive inducible (iNOS) neuronal (nNOS) endothelial (eNOS)