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Insights into the structural determinants for selective inhibition of nitric oxide synthase isoforms

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Abstract

Selective inhibition of the nitric oxide synthase isoforms (NOS) is a promising approach for the treatment of various disorders. However, given the high active site conservation among all NOS isoforms, the design of selective inhibitors is a challenging task. Analysis of the X-ray crystal structures of the NOS isoforms complexed with known inhibitors most often gives no clues about the structural determinants behind the selective inhibition since the inhibitors share the same binding conformation. Aimed at a better understanding of the structural factors responsible for selective inhibition of NOS isoforms we have performed MD simulations for iNOS, nNOS and eNOS complexed with Nω-NO2-L-Arg (1), and with the aminopyridine derivatives 2 and 3. The slightly better selectivity of 1 for nNOS may be assigned to the presence of extra charge–charge interactions due to its “extended” conformation. While the high affinity of 2 for iNOS can be explained by the formation of an iNOS-specific subpocket upon binding, the lack of affinity for eNOS is associated to a conformational change in Glu363. The strong van der Waals and electrostatic interactions between 3 and the active site of nNOS are most likely responsible for its higher affinity for this isoform. Owing to the elongated and narrow binding pocket of iNOS, the correct positioning of 3 over the heme group is difficult, which may account for its lower affinity toward this isoform. Brought together, our results might help to rationalize the design of selective NOS inhibitors.

Overall RMSD of the protein backbone over 8 ns simulation is shown for the complexes 3:eNOSmonomer and 3:eNOSdimer

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Acknowledgments

This work has been partially supported by the Fundação para a Ciência e Tecnologia (FCT), Portugal (PTDC/QUI-QUI/121752/2010). We would like to thank F.-Y. Dupradeau for advice on using the R.E.D. server and for helpful discussions.

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Authors and Affiliations

  1. Unidade de Ciências Químicas e Radiofarmacêuticas, IST/ITN, Instituto Superior Técnico, Universidade Técnica de Lisboa, Estrada Nacional 10, 2686-953, Sacavém, Portugal

    Bruno L. Oliveira, Isabel Santos & João D. G. Correia

  2. REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007, Porto, Portugal

    Irina S. Moreira, Pedro A. Fernandes & Maria J. Ramos

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  1. Bruno L. Oliveira
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  2. Irina S. Moreira
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  3. Pedro A. Fernandes
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  4. Maria J. Ramos
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  5. Isabel Santos
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Correspondence to Maria J. Ramos or João D. G. Correia.

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The following supplementary material is available: Fig. S1: Sequence alignment of NOS oxygenase domains. Fig. S2 Chemical structures of 1 – 3 and torsions free to rotate during the docking process. Fig. S3 Description of the R.E.D. server procedure used to derive RESP charges for 1. Fig. S4 Close-up view of the lowest energy binding conformation of 3 inside eNOS. Fig. S5 Snapshot after 0 ns, 1.5 ns, 5 ns and 8 ns of MD simulation of the unbound iNOS isoform. Fig. S6 Average structures of 3:eNOSdimer and 3:eNOSmonomer calculated using the last 4 ns of simulation (A) and superimposition of these structures (B). Fig. S7 Overall RMSD of the protein backbone over 8 ns simulation is shown for the complexes 1:iNOS, 1:nNOS and 1:eNOS (A) and for inhibitor 1 (B). Fig. S8 Structural micro-environment around the active site of the unbound isoforms. Fig. S9 Superimposition of the X-ray structures of the 1:nNOS (violet, PDB ID 1K2R) and 1:eNOS (cyan, PDB ID 8NSE) complexes. Table S1 Summary of distances involved in the main interactions between inhibitor 1 and the neighboring amino acids residues on NOS isoforms. Fig. S10: Ability of Gln to switch between “open” and “closed” conformations creating an iNOS-specific subpocket. Fig. S11 Overlay of the average structure of 3 from the simulation of 3:nNOS and 3:eNOS and the conformations observed in the corresponding X-ray structures. (DOC 3868 kb)

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Oliveira, B.L., Moreira, I.S., Fernandes, P.A. et al. Insights into the structural determinants for selective inhibition of nitric oxide synthase isoforms. J Mol Model 19, 1537–1551 (2013). https://doi.org/10.1007/s00894-012-1677-8

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