A novel peptide mimetic, brilacidin, for combating multidrug-resistant Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is classified by the Centers for Disease Control and Prevention as an urgent public health threat due to rising infections and rapid resistance development. N. gonorrhoeae has developed resistance to nearly all FDA-approved drugs, with ceftriaxone being the only remaining effective treatment for gonococcal infections. Alarmingly, ceftriaxone-resistant N. gonorrhoeae strains were isolated worldwide, raising the potential of untreatable gonorrhea in the near future. Hence, the critical need to develop new anti-N. gonorrhoeae therapeutics cannot be overemphasized. In this study, we identified the peptide mimetic brilacidin as an effective anti-gonococcal agent. Brilacidin completed phase 2 clinical trials for treating skin infections, oral mucositis, and COVID-19. Herein, brilacidin displayed potent activity against a panel of 22 drug-resistant strains of N. gonorrhoeae, inhibiting 50% of the strains tested (MIC50) at the concentration of 4 µg/mL. The peptide exhibited rapid bactericidal activity, reducing N. gonorrhoeae high inoculum within two hours. Moreover, brilacidin was superior to the drug of choice, ceftriaxone, in eliminating the intracellular N. gonorrhoeae harbored within endocervical cells. Additionally, brilacidin showed high tolerability in mammalian cells and lacked hemolytic activity in human erythrocytes. Altogether, the results demonstrate that brilacidin is a promising anti-gonococcal agent that warrants further in-depth investigation.

Fig 1. Time-kill curve illustrating the bactericidal effect of brilacidin and azithromycin (both at 4 × MIC) against N. gonorrhoeae WHO-X over a 24-hour period.

Each point is the mean of Log₁₀ CFU/mL, and the error bars in each point are for the standard deviation of the mean.

Fig 2. The high safety profile of brilacidin.

(A) ME-180 cell viability of after incubation with different concentrations of brilacidin for 24 h. Results are shown as a percentage of cell viability relative to negative control (DMSO). (B) Hemolytic activity of brilacidin against human RBCs. The results are shown as percentage of RBCs hemolysis for each concentration of brilacidin relative to 0.1% Triton X-100 (positive control with complete hemolysis of RBCs). Error bars represent the standard deviation of the mean. **** (P < 0.0001), ns stands for not significant.

Fig 3. Intracellular clearance activity of ceftriaxone, azithromycin, and brilacidin (at 4 × MIC) against N. gonorrhoeae FA1090 in infected ME-180 cells.

DMSO served as a negative control. Asterisks (*) denote statistically significant differences between test agents and DMSO (untreated) (P < 0.05). Pound signs (#) indicate statistically significant differences (P < 0.05) between brilacidin and azithromycin in comparison to ceftriaxone.

Fig 4. Effect of brilacidin on N. gonorrhoeae cytoplasmic membrane integrity and ATP leakage.

A) Propidium iodide fluorescence after treating N. gonorrhoeae with either azithromycin or brilacidin to predict the permeabilization of the cytoplasmic membrane. B) Percentage of ATP leakage from N. gonorrhoeae treated with brilacidin or azithromycin relative to Triton X-100 (positive control with complete ATP leakage). Asterisks denote statistically significant differences between test agents and DMSO (untreated), * (P < 0.05), ** (P < 0.01), and **** (P < 0.0001) as determined by one-way ANOVA.