Role of zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase .

Nitric oxide synthases (NOSs) catalyze the oxidation of L-arginine to nitric oxide (NO) and L-citrulline (1). Mammals contain three NOS isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) (2). NO produced from these different NOS isoforms is involved in a wide range of physiologic functions in the nervous, immune, and cardiovascular systems (3). Unregulated NO production can lead to pathologic conditions such as stroke (4), inflammation (5), and hypertension (6). Therefore, the control of NOS activity by isoform selective NOS inhibitors has great potential for therapeutic treatments of NO-related diseases (7). The three NOS isoforms share a similar domain architecture with an N-terminal catalytic domain containing the heme active site and a tetrahydrobiopterin (H4B) nearby as a redox active cofactor and a C-terminal reductase domain consisting of FMN, FAD, and NADPH binding sites (8). The active sites for the various NOS isoforms, however, are nearly identical, which has presented a serious challenge in the development of isoform selective inhibitors. Nevertheless, we found (9) that a single amino acid difference between nNOS and eNOS, Asp597 in nNOS vs. Asn368 in eNOS, is responsible for why a series of dipeptide inhibitors (10-12) bind much more tightly to nNOS than eNOS. In this earlier work we observed that a series of dipeptide inhibitors bind quite differently in eNOS and nNOS. In nNOS the inhibitors adopt a “curled” conformation which enables the didpeptide α-amino group to be in position to interact with Asp597. However, in eNOS the dipeptide inhibitors adopt an extended conformation since Asp597 is replaced by Asn368 in eNOS and thus there is no additional electrostatic incentive in eNOS for the inhibitors to “curl”. The N368D mutant in eNOS and D697N mutant in nNOS confirmed that the Asn/Asp difference is the primary structural basis for why the dipepetide inhibitors bind much better to nNOS (9).This led to the design of a series of chiral pyrrolidine inhibitors (Fig. 1) that exhibit Ki values in the low nanomolar range with some exhibiting up to 4,000-fold selectivity for nNOS over eNOS (13-16). The most potent of these inhibitors have dramatic in vivo effects and can protect newborn rabbit kits from experimentally induced ischemic brain damage (14). Figure 1 Two different binding orientations of cis-pyrrolidine compounds. (A) The aminopyridine of (3′S, 4′S)-2 interacts (dashed lines) with Glu592 while (B) the aminopyridine of (3′R, 4′R)-2 is flipped and interacts with heme ... We had anticipated that these pyrrolidine inhibitors would bind so that the aminopyridine group would mimic the substrate L-Arg guanidinium and be situated over the heme and hydrogen bond/ion pair with the active site Glu592. Indeed, this is the case for the (3′S, 4′R) and (3′R, 4′S)-trans inhibitors and the cis-(3′S, 4′S) inhibitors (e.g., (3′S, 4′S)-2, Fig. 1A) whose structures were determined in a previous study (17). However, the crystal structures (17) showed that cis-(3′R, 4′R) compounds (e.g., (3′R, 4′R)-2) bind with the inhibitor flipped 180° so that the “tail” fluorophenyl end is situated over the heme, leaving the aminopyridine in position to hydrogen bond/ion pair with heme propionate D (Fig. 1B). Binding in the flipped orientation requires the movement of Tyr706. Because the aminopyridine portion of the inhibitor can bind in two flipped orientations, we reasoned that a symmetric, “double headed” inhibitor that has an aminopyridine at each end should bind especially well, displacing Tyr706 for direct interactions between one aminopyridine and the heme propionate and the other aminopyridine with Glu592. A series of double headed inhibitors now have been designed, synthesized, and Ki values measured (18). Here we report the crystal structures of these inhibitors bound to both nNOS and eNOS.