Microbial efflux pump inhibition: tactics and strategies

Abstract

Traditional antimicrobials are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. To overcome these deficiencies, a range of novel approaches to control microbial infections are under investigation as potential alternative treatments. Multidrug efflux is a key target of these efforts. Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents as well as antimicrobials. Efflux occurs due to the activity of membrane transporter proteins widely known as Multidrug Efflux Systems (MES). They are implicated in a variety of physiological roles other than efflux and identifying natural substrates and inhibitors is an active and expanding research discipline. One plausible alternative is the combination of conventional antimicrobial agents/antibiotics with small molecules that block MES known as multidrug efflux pump inhibitors (EPIs). An array of approaches in academic and industrial research settings, varying from high-throughput screening (HTS) ventures to bioassay guided purification and determination, have yielded a number of promising EPIs in a series of pathogenic systems. This synergistic discovery platform has been exploited in translational directions beyond the potentiation of conventional antimicrobial treatments. This venture attempts to highlight different tactical elements of this platform, identifying the need for highly informative and comprehensive EPI-discovery strategies. Advances in assay development genomics, proteomics as well as the accumulation of bioactivity and structural information regarding MES facilitates the basis for a new discovery era. This platform is expanding drastically. A combination of chemogenomics and chemoinformatics approaches will integrate data mining with virtual and physical HTS ventures and populate the chemical-biological interface with a plethora of novel chemotypes. This comprehensive step will expedite the preclinical development of lead EPIs.

Fig. 1

Representative members of the five characterized families of MES [3]. The ABC family including ATP-driven multidrug pumps such as P-glycoprotein and LmrA from Lactococcus lactis. The MFS consists of secondary transporters driven by chemiosmotic energy and includes proton/drug antiporters such as QacA from S. aureus. Both the resistance/nodulation/cell division (RND) and the small multidrug resistance (SMR) families include proton-driven drug efflux pumps such as E. coli AcrB and EmrE, respectively. AcrB functions as a multisubunit complex in association with the outer membrane channel TolC and the membrane fusion protein AcrA. The multidrug and toxic compounds efflux (MATE) family consists of sodium ion-driven drug efflux pumps such as NorM from Vibrio parahaemolyticus.

Fig. 2

(a,b) Lateral and axial view of the overlap of the crystal structures of Multidrug Resistance Protein D (red) Lactose Permease (green) and the Glycerol-3-Phosphate Transporter (blue) from MFS family; (c,d) Lateral and axial view of the crystal structure of EmrE multidrug transporter from SMR family (Calpha atoms only);(e,f) Lateral and axial view of the crystal structure of the MATE transporter NorM from Vibrio cholerae. Crystal structures of (g) MexA, (h) MexB, (i) OprM, and (j) a model of the assembly of AcrAB-TolC from RND family; (k,l) Perpendicular views of the crystal structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus.

Fig. 3

Structures of representative microbial EPIs

Fig. 4

Duplex format flow cytometric assay for identification of ABCB1, ABCC1 and ABCG2 EPIs

Fig. 5

R6G (A) and Nile red (B) content of S. cerevisiae strain AD/pABC3 (vector only) and strains expressing C. albicans Cdr1p (AD/CDR1), Cdr2p (AD/CDR2), or Mdr1p(AD/MDR1) MES genes. Yeast cells were incubated with 15 μM R6G (A) or 7 μM Nile red (B). Enniatin (50 μM) was added to strains preloaded with substrate and incubated for 20 min. Each bar represents the median ± standard deviation (n = 3).