Structure-Based Design and Synthesis ofN^ω-Nitro-l-Arginine-Containing Peptidomimetics as Selective Inhibitors of Neuronal Nitric Oxide Synthase. Displacement of the Heme Structural Water

Abstract

The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOSselective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N′-nitroguanidine (1) and 4-N-(N ω -nitro-L-argininyl)-trans-4-amino-L-proline amide (2), led to the discovery of a conserved structural water molecule that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). Based on this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of 2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues 3-5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized. X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety (Figures 4 and 5). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1). Rationalizations for the small increase in potency are consistent with other changes in the crystal structures.

Design of Selective Neuronal Nitric Oxide Synthase Inhibitors for the Prevention and Treatment of Neurodegenerative Diseases

“…This should enhance the potencies of both of these inhibitors. Consequently, compounds 27 and 28 were synthesized and tested for inhibition of the three isozymes xxx. Surprisingly, there is little, if any, difference between the potency and selectivities for 20 (n = 1) compared with 27 (R = OH; when R = NH 2 , it was least potent) and for 12 compared with 28 .…”

A cellular model for screening neuronal nitric oxide synthase inhibitors

“…Hence, inhibition of NOS to decrease NO biosynthesis has been an attractive approach for the design of potential new drugs for diseases caused by NO overproduction [7], [8], [9], and [10]. Many NOS inhibitors have been developed and tested based on an in vitro assay using recombinant enzymes [8], [10], [11], [12], [13], and [14]. An in vitro assay is important for initial inhibitor screening and for enzyme mechanism studies.…”

Open challenges in structure-based virtual screening: Receptor modeling, target flexibility consideration and active site water molecules description

“…Water displacement might, in fact, have unpredictable consequences on ligand binding affinity, as well as on ligand physicochemical properties, pharmacokinetics, specificity and safety [255]. In many cases, as previously reported, replacing tightly bound bridging waters only resulted in a slight improvement in the binding energy [244,[256][257][258], or even in a loss of binding affinity [259]. Overall, promoting enthalpic with respect to pre-supposed entropic gain could represent the winning strategy.…”