Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico High-Throughput Virtual Screening and In Vitro Validation
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.
References
[1]
Wise R, Hart T, Cars O, Streulens M, Helmuth R, et al. (1998) Antimicrobial resistance. Is a major threat to public health. BMJ 317: 609–610. doi: 10.1136/bmj.317.7159.609
[2]
Breidenstein EB, de la Fuente-Nunez C, Hancock RE (2011) Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol 19: 419–426. doi: 10.1016/j.tim.2011.04.005
[3]
Ball AR, Casadei G, Samosorn S, Bremner JB, Ausubel FM, et al. (2006) Conjugating berberine to a multidrug efflux pump inhibitor creates an effective antimicrobial. ACS Chem Biol 1: 594–600. doi: 10.1021/cb600238x
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
[6]
Van Bambeke F, Balzi E, Tulkens PM (2000) Antibiotic efflux pumps. Biochem Pharmacol 60: 457–470. doi: 10.1016/s0006-2952(00)00291-4
[7]
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
[8]
Askoura M, Mottawea W, Abujamel T, Taher I (2011) Efflux pump inhibitors (EPIs) as new antimicrobial agents against Pseudomonas aeruginosa. Libyan J Med 6..
[9]
Nelson ML (2002) Modulation of antibiotic efflux in bacteria. Current Medicinal Chemistry-Anti-Infective Agents 1: 35–54. doi: 10.2174/1568012023355054
[10]
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
[11]
Hemaiswarya S, Kruthiventi AK, Doble M (2008) Synergism between natural products and antibiotics against infectious diseases. Phytomedicine 15: 639–652. doi: 10.1016/j.phymed.2008.06.008
[12]
Poisson J, Le Hir A, Goutarel R, Janot MM (1954) Isolation of reserpine from roots of Rauwolfia vomitoria Afz. Comptes rendus hebdomadaires des seances de l'Academie des sciences 238: 1607–1609.
[13]
Stermitz FR, Tawara-Matsuda J, Lorenz P, Mueller P, Zenewicz L, et al. (2000) 5′-Methoxyhydnocarpin-D and pheophorbide A: Berberis species components that potentiate berberine growth inhibition of resistant Staphylococcus aureus. J Nat Prod 63: 1146–1149. doi: 10.1021/np990639k
[14]
Stermitz FR, Beeson TD, Mueller PJ, Hsiang J, Lewis K (2001) Staphylococcus aureus MDR efflux pump inhibitors from a Berberis and a Mahonia (sensu strictu) species. Biochem Syst Ecol 29: 793–798. doi: 10.1016/s0305-1978(01)00025-4
[15]
Stavri M, Piddock LJ, Gibbons S (2007) Bacterial efflux pump inhibitors from natural sources. J Antimicrob Chemother 59: 1247–1260. doi: 10.1093/jac/dkl460
[16]
Schr？dinger (2011) Version 9.2, LLC, New York, NY.
[17]
Sennhauser G, Bukowska MA, Briand C, Grutter MG (2009) Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa. J Mol Biol 389: 134–145. doi: 10.1016/j.jmb.2009.04.001
[18]
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
[19]
Jatana N, Sharma A, Latha N (2013) Pharmacophore modeling and virtual screening studies to design potential COMT inhibitors as new leads. J Mol Graph Model 39: 145–164. doi: 10.1016/j.jmgm.2012.10.010
[20]
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
[21]
CLSI (2012) Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing- Twenty-second informational supplement. M100-S22
[22]
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
[23]
Wang H, Joseph JA (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27: 612–616. doi: 10.1016/s0891-5849(99)00107-0
[24]
Bohnert JA, Schuster S, Seeger MA, Fahnrich E, Pos KM, et al. (2008) Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efflux pump AcrB. J Bacteriol 190: 8225–8229. doi: 10.1128/jb.00912-08
[25]
Chung SY, Sung MK, Kim NH, Jang JO, Go EJ, et al. (2005) Inhibition of P-glycoprotein by natural products in human breast cancer cells. Arch Pharm Res 28: 823–828. doi: 10.1007/bf02977349
[26]
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
[27]
Nakashima R, Sakurai K, Yamasaki S, Hayashi K, Nagata C, et al. (2013) Structural basis for the inhibition of bacterial multidrug exporters. Nature 500: 102–106. doi: 10.1038/nature12300
[28]
Peach ML, Nicklaus MC (2009) Combining docking with pharmacophore filtering for improved virtual screening. Journal of cheminformatics 1: 6. doi: 10.1186/1758-2946-1-6
[29]
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.
[30]
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
[31]
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
[32]
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
[33]
Melero CP, Medarde M, San Feliciano A (2000) A short review on cardiotonic steroids and their aminoguanidine analogues. Molecules 5: 51–81. doi: 10.3390/50100051
[34]
Lechner D, Gibbons S, Bucar F (2008) Plant phenolic compounds as ethidium bromide efflux inhibitors in Mycobacterium smegmatis. J Antimicrob Chemother 62: 345–348. doi: 10.1093/jac/dkn178
[35]
Limtrakul P, Khantamat O, Pintha K (2005) Inhibition of P-glycoprotein function and expression by kaempferol and quercetin. J Chemother 17: 86–95. doi: 10.1179/joc.2005.17.1.86
