Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of N. gonorrhoeae necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of N. gonorrhoeae anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC50 value of C7-3 and C7-3m2 against anaerobically grown N. gonorrhoeae was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development.
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Funding for this work was provided to A.E.S. by General Research Funds (OSU), by the Medical Research Foundation of Oregon, and partly by grant R01-AI117235 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. R.H.M. was supported by an ASM undergraduate research fellowship. The research reported in this publication was partially supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers P20GM103486 and P30GM110787 and by the College of Medicine, University of Kentucky, to K.V.K.
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00186-17.
Sikora, Aleksandra E.; Mills, Robert H.; Weber, Jacob V.; Hamza, Adel; Passow, Bryan W.; Romaine, Andrew; Williamson, Zachary A.; Reed, Robert W.; Zielke, Ryszard A.; and Korotkov, Konstantin V., "Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA" (2017). Molecular and Cellular Biochemistry Faculty Publications. 115.