Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA

AniA is expressed in a diverse panel of N. gonorrhoeae isolates. N. gonorrhoeae wild-type strain FA1090, the isogenic ΔaniA/P_lac::aniA strain, and the ΔaniA/P_lac::aniA D137A H280A strain, as well as 36 additional strains of N. gonorrhoeae, as indicated above the immunoblots, were grown concurrently on solid medium for 20 h in 5% CO₂ at 37°C. The ΔaniA/P_lac::aniA and ΔaniA/P_lac::aniA D137A H280A strains were grown on solid medium supplemented with IPTG. Samples containing the whole-cell lysates were matched by equivalent OD₆₀₀ units, resolved in a 4 to 20% Tris-glycine gel, and transferred onto nitrocellulose. The immunoblotting was performed using polyclonal rabbit antiserum against sAniA.

Targeting AniA using a phage display approach. (A) The recombinant version of AniA, sAniA, was purified to homogeneity and used in an affinity capture method as the bait during three panning experiments with two phage display libraries expressing either randomized linear 12-mer peptides (Ph.D.-12) or randomized 7-mer peptides flanked by a pair of cysteine residues (Ph.D.-C7C). As a first step in every round of the panning experiment, both peptide libraries were precleared against Ni-NTA magnetic chitin resin to remove nonspecifically binding peptides. The supernatants from this step were then added to 200 μg of sAniA bound to the resin. After incubation with the protein, unbound phages were washed away using TBST, with an increasing stringency of the washing being used in consecutive rounds. The elutions were performed with glycine-HCl (pH 2.2). (B) Deduced peptide sequences of the 7-mer and 12-mer peptides obtained by sequencing of the DNA of 24 randomly selected phages from each group eluted after the third round of biopanning. These studies revealed 26 unique peptides, with 1 of them, C7-3, being identified multiple times. Synthesized peptides are shown in red. (C) Evaluation of the identified peptides through phage ELISA. Phage clones were purified and tested separately to measure their affinity to sAniA in the phage ELISA. Purified sAniA (2 μM) was coated overnight at 4°C on 96-well flat-bottom plates. After the coating step, unbound sAniA was removed and the wells were thoroughly washed with the storage buffer. The wells were blocked and incubated with 10¹⁰ PFU per well from each amplification. After washing, the anti-M13 monoclonal antisera coupled to horseradish peroxidase were added. Unbound antisera were removed by excessive washing, and enzymatic activity was exerted by the addition of Turbo TMB-ELISA substrate. The absorbance at 450 nm was measured. Readings were compared to those of wells which underwent an identical treatment but lacked sAniA (control) and to the signal from a wild-type phage, M13KE (10¹⁰ PFU), that did not display peptides (wild type). The means and SEMs from seven independent experiments are shown. Clones 3* and 5 (in red) were identified multiple times. *, P < 0.05.

Normal mode analysis (NMA) of the conformational transition of the homotrimer AniA structure. The motion of the combined four lowest normal modes of vibrations described the opening and closing states of cupredoxin domain II (Cu²⁺ catalytic active site) of the AniA structure. Thus, NMA revealed the tendency of the protein to move in a certain direction, even though this movement was not fully explored during the MD simulations. The view on the top of the catalytic active site of the AniA structure shows that the flexibility of the protein is well oriented and can dramatically change the accessible area of the catalytic metal ion Cu²⁺ and its potential interaction with a ligand (NO₂ or peptide). The open state of the homotrimer AniA structure was used to identify several binding pockets. These conformations essentially depended on the sequence of the residue composing the peptide. Afterward, the initial conformation of the peptide (which is in a stable conformation in the solvent) was slightly perturbed during the docking procedure due to the interactions with the receptor.

Stick view of the interaction of the peptide 12-5 Tyr side chain in the catalytic active site of AniA homotrimer. The Tyr side chain of the peptide fits in the cavity and established strong van der Waals contact with the hydrophobic residues of the cavity, while the hydroxyl group of the Tyr formed an H bond with the Asp137 residue.