Abstract:
The progressive development of resistance in Neisseria gonorrhoeae to almost all available
antibiotics has made it crucial to develop novel approaches to tackling multi-drug resistance (MDR).
One of the primary causes of antibiotic resistance is the over-expression of the MtrCDE efflux pump
protein, making this protein a vital target for fighting against antimicrobial resistance (AMR) in N.
gonorrhoeae. This study was aimed at evaluating the potential MtrCDE efflux pump inhibitors (EPIs)
and their stability in treating gonorrhoea infection. This is significant because finding novel EPIs
would allow for the longer maintenance of antibiotics at therapeutic levels, thereby prolonging the
susceptibility of currently available antibiotics. A virtual screening of the selected Helichrysum populifolium
compounds (4,5-dicaffeoylquinic acid, apigeninin-7-glucoside, and carvacrol) was conducted to
evaluate their potential EPI activity. An integrated computational framework consisting of molecular
docking (MD), molecular mechanics generalized born, and surface area solvation (MMGBSA) analysis,
molecular dynamics simulations (MDS), and absorption, distribution, metabolism, and excretion
(ADME) properties calculations were conducted. Of the tested compounds, 4,5-dicaffeoylquinic acid
revealed the highest molecular docking binding energies (−8.8 kcal/mol), equivalent MMGBSA
binding free energy (−54.82 kcal/mol), indicative of consistent binding affinity with the MtrD protein,
reduced deviations and flexibility (root mean square deviation (RMSD) of 5.65 Å) and, given by root
mean square fluctuation (RMSF) of 1.877 Å. Carvacrol revealed a docking score of −6.0 kcal/mol
and a MMGBSA computed BFE of −16.69 kcal/mol, demonstrating the lowest binding affinity to
the MtrD efflux pump compared to the remaining test compounds. However, the average RMSD
(4.45 Å) and RMSF (1.638 Å) of carvacrol-bound MtrD protein showed no significant difference from
the unbound MtrD protein, except for the reference compounds, implying consistent MtrD conformation
throughout simulations and indicates a desirable feature during drug design. Additionally,
carvacrol obeyed the Lipinski rule of five which confirmed the compound’s drug-likeness properties
making it the most promising EPI candidate based on its combined attributes of a reasonable binding
affinity, sustained stability during MDS, its obedience to the Lipinski rule of five and compliance
with drug-likeness criteria. An in vitro validation of the potential EPI activities of H. populifolium
compounds confirmed that 4,5-dicaffeoylquinic acid reduced the expulsion of the bis-benzimide dye
by MtrCDE pump, while carvacrol showed low accumulation compared to other compounds. While
4,5-dicaffeoylquinic acid demonstrated the highest binding affinity in computational analysis and
an EPI activity in vitro, it showed lower stability compared to the other compounds, as indicated in
MDS. This leaves carvacrol, as a better EPI candidate for the management of gonorrhoea infection.
