Unexpected binding modes of nitric oxide synthase inhibitors effective in the prevention of a cerebral palsy phenotype in an animal model

Abstract

Selective inhibition of the neuronal isoform of nitric oxide synthase NOS (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. However, given the high active site conservation among all three NOS isoforms, the design of selective inhibitors is an extremely challenging problem. Here we present the structural basis for why novel and potent nNOS inhibitors exhibit the highest level of selectivity over eNOS reported so far (approximately 3,800-fold). By using a combination of crystallography, computational methods, and site-directed mutagenesis, we found that inhibitor chirality and an unanticipated structural change of the target enzyme control both the orientation and selectivity of these novel nNOS inhibitors. A new hot spot generated as a result of enzyme elasticity provides important information for the future fragment-based design of selective NOS inhibitors.

Figure 1

The inhibitor binding pocket in NOS (A) and the active site structure of nNOS (C) and eNOS (D) showing the different binding modes of the dipeptide inhibitor 1 (B) that exhibits about 1500-fold selectivity for nNOS over eNOS (Table 1).

Figure 2

A series of 2Fo-Fc electron density maps contoured at 1.0σ for four various aminopyridine inhibitors bound to nNOS. Shown also their chemical formula and K_i values. Detailed refinement statistics are provided in Table S2. Briefly the resolution and R_work/R_free values for the 4 structures shown are A) 1.93Å 0.18/0.21; B) 1.98Å 0.18/0.21; C) 1.95Å 0.19/0.22; and D) 2.01Å 0.19/0.23.

Figure 3

(3′R, 4′R)-2 (yellow) modeled in the same orientation as (3′S, 4′S)-2 (cyan) with an emphasis placed on minimizing steric clashes. It is not possible to model (3′R, 4′R)-2 in the (3′S, 4′S)-2 orientation that still maintains optimal inhibitor-protein H-bonds and minimal steric clashes.