The study hypothesizes that since the ADI proteins partake in bacterial ATP production, hydroquinine disrupts metabolic energy generation consequently leading to reduced cell division and growth. Further testing will focus on the exact mechanism of its activity.
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These interactions followed by RT-qPCR showed that a half MIC (1.25 mg/mL) of hydroquinine can result in substantial decrease in the mRNA expression of ADI proteins in the MDR PA-S4, S5 and reference strain. Therefore, proving hydroquinine stops bacterial growth by the downregulation of said genes.

Molecular docking was performed with the two solved structures and the AI generated ones. Table 4 shows an estimation of the binding energies of hydroquinine and the proteins. The lowest negative energy value, between AOA and hydroquinine, suggest the greatest favourable interaction.

Five models were generated for each of the two proteins (OTC, AOA) and evaluated by gene sequence alignment with existing homologous sequences and pLDDT. The models with a pLDDT score of >90 indicate high confidence prediction and thus were selected for docking. Model 1 for OTC, Model 3 for AOA.

To establish the confidence of the interaction between hydroquinine and ADI proteins for proving its inhibition mechanism, 3D structures of all four (ADI, OTC, CK, AOA) proteins were created. AlphaFold was used to make models for the two proteins that did not have solved x-ray crystal structures.

Moreover, partial synergistic effect of hydroquinine combined with ceftazidime (a cephalosporin antibiotic) demonstrated a two-fold decrease in its MIC value for the resistant strains. Otherwise, the MDR strains had a high MIC of ≥32 mg/mL and hence significant resistance to ceftazidime.

Hydroquinine could impede the growth and kill all six clinical isolates, including the MDR ones, and the reference strain at an MIC value of 2.50 mg/mL and MBC value of 5.00 mg/mL as shown in the following table. These values are lower and consistent compared to the previously profiled antibiotics.

Phenotypic antibiotic susceptibility test of P. aeruginosa for six different strains isolated from various sources and one reference strain (PA ATCC 27853) revealed two multidrug resistant strains, named PA-S4 and PA-S5, due to their resistance to ≥1 antibiotic in ≥3 classes of antimicrobials.

Hydroquinine is an organic cinchona alkoid compound, that has shown brilliant antimicrobial potential. This study is the first to exhibit the Arginine Deiminase pathway proteins to be the target molecules of hydroquinine for its inhibitory action against clinically isolated P. aeruginosa strains.