Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase - PubMed (original) (raw)

doi: 10.1038/nchembio.115. Epub 2008 Oct 12.

Andrew S Arvai, Robin J Rosenfeld, Matt D Kroeger, Brian R Crane, Gunilla Andersson, Glen Andrews, Peter J Hamley, Philip R Mallinder, David J Nicholls, Stephen A St-Gallay, Alan C Tinker, Nigel P Gensmantel, Antonio Mete, David R Cheshire, Stephen Connolly, Dennis J Stuehr, Anders Aberg, Alan V Wallace, John A Tainer, Elizabeth D Getzoff

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Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

Elsa D Garcin et al. Nat Chem Biol. 2008 Nov.

Abstract

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation.

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Figures

Figure 1

Figure 1. NOS inhibitors structures, inhibition and crystallographic data

For all inhibitors, including quinazolines (left column: compounds 1–5), aminopyridines (middle column: compounds 6–12) and bicyclic thienooxazepines (right column: compounds 14–16), the chemical structure is shown in black (core with red cis-amidine nitrogens) and magenta (tail), together with IC50 values in the three human NOS isozymes. The resolution (d in Å), crystallographic R and Rfree values are indicated for each structure of murine iNOSox (unlabeled), human iNOSox (hiNOS), bovine eNOSox (beNOS) and human eNOSox (heNOS) complexes.

Figure 2

Figure 2

Quinazoline and aminopyridine binding in iNOSox and eNOSox. (a) Potent but non-selective aminopyridine compound 6 (ref. 28) bound to murine iNOSox. (b) Highly-selective quinazoline compound 3 (ref. 26) bound to murine iNOSox. (c) Selective aminopyridine compound 12 (ref. 28) bound to murine iNOSox. (d) Aminopyridine 9 (ref. 28) bound to human iNOSox. For all structures, critical hydrogen bonds (dots) and iNOS residues are shown: active-site residues (peach), first-shell residues (yellow, residues interacting directly with the inhibitor), second-shell residues (orange, residues interacting with first-shell residues) and third-shell residues (green, residues interacting with second-shell residues). The Fo-Fc electron density map contoured at 3 σ (blue mesh) is shown around each inhibitor (pink). (e) Key iNOS residues involved in inhibitor binding are colored according to a–d.

Figure 3

Figure 3

Selective aminopyridine compound 9 binding to eNOS versus iNOS. (a) Solvent-accessible surfaces for the iNOS (left) and eNOS (right) active sites colored according to Fig. 2. The core of compound 9 binds closer and more parallel to the heme in eNOS. In iNOS, side-chain rotations of Gln, Arg, and Arg388 open the Gln specificity pocket for binding of the bulky inhibitor tail. (b) Stereoview of the superimposition of bovine eNOS:compound 9 (yellow) and human iNOS:compound 9 (blue) x-ray structures, highlighting the cascade of conformational changes of first-shell and second-shell residues upon inhibitor binding to iNOS.

Figure 4

Figure 4

Isozyme-specific induced-fit upon inhibitor binding. Schematic of the cascade of conformational changes associated with inhibitor binding in the three human NOS isozymes. The van der Waals surfaces for the isozyme-specific triads are shown in orange (second shell) and green (third shell). In human iNOS (hiNOS), inhibitor binding first induces the Gln-closed to Gln-open conformation and Arg rotation, which in turn leads to rotation of second-shell Asn towards third-shell Phe286 and Val305. In human eNOS (heNOS), bulkier third-shell residues (Ile269 and Leu288) prevent the Asn rotation (overlap of van der Waals surfaces). In human nNOS (hnNOS), partial rotation of Asn towards third-shell residues Phe506 and bulky Leu525 may be possible.

Figure 5

Figure 5

Bicyclic thioenooxazepine inhibitor binding in iNOSox. Moderately selective compound 16 binds to murine iNOSox similarly to bulky quinazoline and aminopyridine inhibitors and induces the Gln-open conformation. Residues are colored according to Fig. 2. The Fo-Fc electron density map contoured at 3 σ (blue mesh) is shown around the inhibitor (pink).

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