Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug resistant strains of P. aeruginosa and E. coli.
Masuda N, Sakagawa E, Ohya S, Gotoh N, Tsujimoto H, et al. (2000) Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother 44: 3322–3327. doi: 10.1128/aac.44.12.3322-3327.2000
Lomovskaya O, Warren MS, Lee A, Galazzo J, Fronko R, et al. (2001) Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob Agents Chemother 45: 105–116. doi: 10.1128/aac.45.1.105-116.2001
Hirakata Y, Kondo A, Hoshino K, Yano H, Arai K, et al. (2009) Efflux pump inhibitors reduce the invasiveness of Pseudomonas aeruginosa. Int J Antimicrob Agents 34: 343–346. doi: 10.1016/j.ijantimicag.2009.06.007
Sastry GM, Adzhigirey M, Day T, Annabhimoju R, Sherman W (2013) Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des 27: 221–234. doi: 10.1007/s10822-013-9644-8
Du J, Sun H, Xi L, Li J, Yang Y, et al. (2011) Molecular modeling study of checkpoint kinase 1 inhibitors by multiple docking strategies and prime/MM-GBSA calculation. J Comput Chem 32: 2800–2809. doi: 10.1002/jcc.21859
Coldham NG, Webber M, Woodward MJ, Piddock LJ (2010) A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli. J Antimicrob Chemother 65: 1655–1663. doi: 10.1093/jac/dkq169
Yu EW, Aires JR, McDermott G, Nikaido H (2005) A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study. J Bacteriol 187: 6804–6815. doi: 10.1128/jb.187.19.6804-6815.2005
Aparna V, Mohanalakshmi N, Dineshkumar K, Hopper W (2014) Identification of inhibitors for RND efflux pump of Pseudomonas aeruginosa using structure-based pharmacophore modeling approach. International Journal of Pharmacy and Pharmaceutical Sciences 6: 84–89.
Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, et al. (2006) Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49: 6177–6196. doi: 10.1021/jm051256o
Kumar A, Khan IA, Koul S, Koul JL, Taneja SC, et al. (2008) Novel structural analogues of piperine as inhibitors of the NorA efflux pump of Staphylococcus aureus. J Antimicrob Chemother 61: 1270–1276. doi: 10.1093/jac/dkn088
Zhang Z, Liu ZQ, Zheng PY, Tang FA, Yang PC (2010) Influence of efflux pump inhibitors on the multidrug resistance of Helicobacter pylori. World J Gastroenterol 16: 1279–1284. doi: 10.3748/wjg.v16.i10.1279