Crystal Structures of Constitutive Nitric Oxide Synthases in Complex with De Novo Designed Inhibitors

Abstract

New nitric oxide synthase (NOS) inhibitors were designed de novo with knowledge gathered from the studies on the nNOS-selective dipeptide inhibitors. Each of the new inhibitors consists of three fragments: an aminopyridine ring, a pyrrolidine, and a tail of various length and polarity. The in vitro inhibitory assays indicate good potency and isoform selectivity for some of the compounds. Crystal structures of these inhibitors bound to either wild type or mutant nNOS and eNOS have confirmed design expectations. The aminopyridine ring mimics the guanidinium group of L-arginine and functions as an anchor to place the compound in the NOS active site where it hydrogen bonds to a conserved Glu. The rigidity of the pyrrolidine ring places the pyrrolidine ring nitrogen between the same conserved Glu and the selective residue nNOS Asp597/eNOS Asn368 which results in similar interactions observed with the α-amino group of dipeptide inhibitors bound to nNOS. These structures provide additional information to help in the design of inhibitors with greater potency, physico-chemical properties, and isoform selectivity.

Minimal Pharmacophoric Elements and Fragment Hopping, an Approach Directed at Molecular Diversity and Isozyme Selectivity. Design of Selective Neuronal Nitric Oxide Synthase Inhibitors

“…Crystallographic analysis of new NOS inhibitors generated from the proposed fragment hopping method is described in detail elsewhere. 72 To briefly summarize, the structure of nNOS complexed with 4 (PDB accession 3B3N) was solved to 1.98 Å resolution with R/R free = 0.23/0.27, and the complex with 6 (PDB accession 3B3M) was solved to 1.95 Å with R/R free = 0.20/0.23). Only one of the two cis enantiomers, the (3′S,4′S)-isomer, was bound to the active site.…”

Case Study 3: Fragment Hopping to Design Highly Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors

“…These selectivities are comparable to those attained with nitroarginine-containing dipeptide derivatives 1-3 [28]. The crystallographic analysis shows that only the designed enantiomer was bound to the active site (PDB id, 3B3N) [77]. Figure 18.3a shows a superimposition of the binding conformations and the predicted bioactive conformation of 4 with nNOS, and Figure 18.3b shows a superimposition of the binding conformation of 2 (PDB id, 1P6I [71]) and 4 with nNOS.…”

Nitric oxide synthase and structure-based inhibitor design

“…The aminopyridine end of the inhibitor should interact with the active site Glu while the pyrrolidine N atom should interact with Asp597 in nNOS but much more weakly with Asn368 in eNOS. The crystal structures showed that compounds like 8 bound to rat nNOS very close to what was predicted (PDB 3B3M and 3B3N) [41]. …”