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

Nature Chemical Biology volume 4, pages 700–707 (2008)Cite this article

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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Figure 1: NOS inhibitors structures, inhibition and crystallographic data.
Figure 2: Quinazoline and aminopyridine binding in iNOSox and eNOSox.
Figure 3: Selective aminopyridine compound 9 binding to eNOS versus iNOS.
Figure 4: Isozyme-specific induced fit upon inhibitor binding.
Figure 5: Bicyclic thioenooxazepine inhibitor binding in iNOSox.

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Acknowledgements

We thank K. Panda (Cleveland Clinic) and S. Ghosh (Cleveland Clinic) for preparation of the mouse iNOSox and bovine eNOSox proteins used in this study. Part of this work is based on research conducted at CHESS, SSRL, ESRF and MAX-LAB (Lund University). This work was supported in part by US National Institutes of Health grants (E.D.Getzoff and D.J.S.), and by the Skaggs Institute for Chemical Biology (E.D.Garcin).

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Author notes

  1. Elsa D Garcin, Matt D Kroeger, Brian R Crane, Peter J Hamley & Anders Åberg

    Present address: Present addresses: University of Maryland Baltimore County, Department of Chemistry and Biochemistry, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA (E.D.Garcin); Takeda San Diego, 10410 Science Center Drive, San Diego, California 92121, USA (M.D.K.); Cornell University, Chemistry and Chemical Biology Department, Ithaca, New York 14853, USA (B.R.C.); Sanofi Aventis, Industriepark Höchst, G838, 65926 Frankfurt am Main, Germany (P.J.H.); and Sidec AB, Torshamnsgatan 30A, SE-164 40 Kista, Sweden (A.Å.).,

  2. Alan C Tinker: Deceased.

Authors and Affiliations

  1. Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB4, La Jolla, 92037, California, USA

    Elsa D Garcin, Andrew S Arvai, Robin J Rosenfeld, Matt D Kroeger, Brian R Crane, John A Tainer & Elizabeth D Getzoff

  2. AstraZeneca Structural Chemistry Laboratory, AstraZeneca R&D Mölndal, Pepparedsleden 1, Mölndal, S-431 83, Sweden

    Gunilla Andersson & Anders Åberg

  3. AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, Leicestershire, UK

    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 & Alan V Wallace

  4. Department of Pathobiology, The Cleveland Clinic, 9500 Euclid Avenue, Cleveland, 44106, Ohio, USA

    Dennis J Stuehr

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  1. Elsa D Garcin
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Contributions

E.D.Garcin, crystallization, X-ray data collection and refinement, mutant design, binding assays, interpretation of structural and biochemical results, and preparation of figures and manuscript. A.S.A., crystallization, X-ray data collection and refinement. R.J.R., crystallization, X-ray data collection and refinement, and interpretation of structural results. M.D.K., mutagenesis, expression and purification of double mutant protein, binding assay and crystallization. B.R.C., crystallization, X-ray data collection and refinement. G.Andersson and A.Å., cloning, expression and purification of human iNOSox protein used in structural studies. G.Andrews and D.J.N., biochemical assays on wild-type and mutant proteins, experimental design and interpretation of biochemical results. N.P.G., experimental design and interpretation of chemical data. P.J.H., A.C.T., A.M., D.R.C. and S.C., design and synthesis of novel selective inhibitors and interpretation of chemical data. P.R.M., cloning, expression and purification of wild-type and mutant full-length enzymes and oxygenase modules used in biochemical assay. S.A.St-G., interpretation of chemical data. D.J.S., preparation of mouse iNOSox and bovine eNOSox used for structural studies. A.V.W., experimental design, interpretation of biochemical and structural data, and mutant design. J.A.T., direction and interpretation of structural experiments. E.D.Getzoff, direction and interpretation of structural and biochemical results, and preparation of manuscript.

Corresponding authors

Correspondence to Elsa D Garcin or Elizabeth D Getzoff.

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Competing interests

G. Andersson, G. Andrews, P.J.H., P.R.M., D.J.N., S.A.St-G., A.C.T., N.P.G., A.M., D.R.C., S.C., A.Å. and A.V.W. were employed by AstraZeneca when involved in this study.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1, Supplementary Methods and Supplementary Discussion (PDF 12630 kb)

Supplementary Movie 1

The Quicktime movie illustrates the cascade of conformational changes that occur in human iNOSox upon aminopyridine compound 9 binding (Quicktime, 582 kB). (MOV 587 kb)

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Garcin, E., Arvai, A., Rosenfeld, R. et al. Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase. Nat Chem Biol 4, 700–707 (2008). https://doi.org/10.1038/nchembio.115

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