Molecular Interactions of Cephalosporins with the Deep Binding Pocket of the RND Transporter AcrB.

The drug/proton antiporter AcrB, part of the major efflux pump AcrABZ-TolC in Escherichia coli, is characterized by its impressive ability to transport chemically diverse compounds, conferring a multidrug resistance phenotype. However, the molecular features differentiating between good and poor substrates of the pump have yet to be identified. In this work, we combined molecular docking with molecular dynamics simulations to study the interactions between AcrB and two representative cephalosporins, cefepime and ceftazidime (a good and poor substrate of AcrB, respectively). Our analysis revealed different binding preferences of the two compounds toward the subsites of the large deep binding pocket of AcrB. Cefepime, although less hydrophobic than ceftazidime, showed a higher affinity than ceftazidime for the so-called hydrophobic trap, a region known for binding inhibitors and substrates. This supports the hypothesis that surface complementarity between the molecule and AcrB, more than the intrinsic hydrophobicity of the antibiotic, is a feature required for the interaction within this region. Oppositely, the preference of ceftazidime for binding outside the hydrophobic trap might not be optimal for triggering allosteric conformational changes needed to the transporter to accomplish its function. Altogether, our findings could provide valuable information for the design of new antibiotics less susceptible to the efflux mechanism.

Identification and characterization of carbapenem binding sites within the RND-transporter AcrB.

Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-OprM, the major efflux pump transporter in Pseudomonas aeruginosa. However, not much is known regarding recognition and transport of these compounds by AcrAB-TolC, which is the MexAB-OprM homologue in Escherichia coli and by definition the paradigm model for structural studies on efflux pumps. Prompted by this motivation, we unveiled the molecular details of the interaction of imipenem and meropenem with the transporter AcrB by combining computer simulations with biophysical experiments. Regarding the interaction with the two main substrate binding regions of AcrB, the so-called access and deep binding pockets, molecular dynamics simulations revealed imipenem to be more mobile than meropenem in the former, while comparable mobilities were observed in the latter. This result is in line with isothermal titration calorimetry, differential scanning experiments, and binding free energy calculations, indicating a higher affinity for meropenem than imipenem at the deep binding pocket, while both sharing similar affinities at the access pocket. Our findings rationalize how different physico-chemical properties of compounds reflect on their interactions with AcrB. As such, they constitute precious information to be exploited for the rational design of antibiotics able to evade efflux pumps.

Molecular Determinants of the Promiscuity of MexB and MexY Multidrug Transporters of Pseudomonas aeruginosa.

Secondary multidrug transporters of the resistance-nodulation-cell division (RND) superfamily contribute crucially to antibiotic resistance in Gram-negative bacteria. Compared to the most studied transporter AcrB of Escherichia coli, little is known about the molecular determinants of distinct polyspecificities of the most important RND transporters MexB and MexY of Pseudomonas aeruginosa. In an effort to add knowledge on this topic, we performed an exhaustive atomic-level comparison of the main putative recognition sites (access and deep binding pockets) in these two Mex transporters. We identified an underlying link between some structural, chemical and dynamical features of the binding pockets and the physicochemical nature of the corresponding substrates recognized by either one or both pumps. In particular, mosaic-like lipophilic and electrostatic surfaces of the binding pockets provide for both proteins several multifunctional sites for diffuse binding of diverse substrates. Specific lipophilicity signatures of the weakly conserved deep pocket suggest a key role of this site as a selectivity filter as in Acr transporters. Finally, the different dynamics of the bottom-loop in MexB and MexY support its possible role in binding of large substrates. Our work represents the first comparative study of the major RND transporters in P. aeruginosa and also the first structure-based study of MexY, for which no experimental structure is available yet.

Molecular Rationale behind the Differential Substrate Specificity of Bacterial RND Multi-Drug Transporters.

Resistance-Nodulation-cell Division (RND) transporters AcrB and AcrD of Escherichia coli expel a wide range of substrates out of the cell in conjunction with AcrA and TolC, contributing to the onset of bacterial multidrug resistance. Despite sharing an overall sequence identity of ~66% (similarity ~80%), these RND transporters feature distinct substrate specificity patterns whose underlying basis remains elusive. We performed exhaustive comparative analyses of the putative substrate binding pockets considering crystal structures, homology models and conformations extracted from multi-copy µs-long molecular dynamics simulations of both AcrB and AcrD. The impact of physicochemical and topographical properties (volume, shape, lipophilicity, electrostatic potential, hydration and distribution of multi-functional sites) within the pockets on their substrate specificities was quantitatively assessed. Differences in the lipophilic and electrostatic potentials among the pockets were identified. In particular, the deep pocket of AcrB showed the largest lipophilicity convincingly pointing out its possible role as a lipophilicity-based selectivity filter. Furthermore, we identified dynamic features (not inferable from sequence analysis or static structures) such as different flexibilities of specific protein loops that could potentially influence the substrate recognition and transport profile. Our findings can be valuable for drawing structure (dynamics)-activity relationship to be employed in drug design.

Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections are becoming increasingly difficult to treat due to intrinsic antibiotic resistance and the propensity of this pathogen to accumulate diverse resistance mechanisms. Hyperexpression of efflux pumps of the Resistance-Nodulation-Cell Division (RND)-type multidrug efflux pumps (e.g., MexAB-OprM), chromosomally encoded by mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY (-oprA) is often detected in clinical isolates and contributes to worrying multi-drug resistance phenotypes. Not all antibiotics are affected to the same extent by the aforementioned RND efflux pumps. The impact of efflux on antibiotic activity varies not only between different classes of antibiotics but also between members of the same family of antibiotics. Subtle differences in physicochemical features of compound-pump and compound-solvent interactions largely determine how compounds are affected by efflux activity. The combination of different high-resolution techniques helps to gain insight into the functioning of these molecular machineries. This review discusses substrate recognition patterns based on experimental evidence and computer simulations with a focus on MexB, the pump subunit of the main RND transporter in P. aeruginosa.

Recognition of imipenem and meropenem by the RND-transporter MexB studied by computer simulations.

Basic understanding of the means by which multidrug efflux systems can efficiently recognize and transport drugs constitutes a fundamental step toward development of compounds able to tackle the continuous outbreak of new bacterial strains resistant to traditional antibiotics. We applied a series of computational techniques, from molecular docking to molecular dynamics simulations and free energy estimate methods, to determine the differences in the binding properties of imipenem and meropenem, two potent antibiotics of the carbapenem family, to MexB, the RND transporter of the major efflux system of Pseudomonas aeruginosa. We identified and characterized two affinity sites in the periplasmic domain of the transporter, sharing strong similarities with the distal and proximal binding pockets identified in AcrB, the homologue of MexB in Escherichia coli. According to our results, meropenem has a higher affinity to the distal binding pocket than imipenem while both compounds are weakly bound to the proximal pocket. This different behavior is mainly due to the hydration properties of the nonpharmacophore part of the two compounds, being that of imipenem less bulky and hydrophobic. Our data provide for the first time a rationale at molecular level for the experimental evidence indicating meropenem as a compound strongly affected by MexB contrary to imipenem, which is apparently poorly transported by the same pump.

Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli.

Multi-drug-resistant bacteria can cause serious infections that are extremely difficult to treat. Bacterial efflux pumps are known to contribute to multi-drug resistance and, thus, constitute a promising target for novel antibacterial agents. Resazurin is widely used to monitor bacterial growth because resazurin is reduced to the fluorescent resorufin by live cells. We have shown by flow cytometric analysis and by accumulation studies with wild type and efflux deficient strains that resazurin is a substrate of efflux pumps in Escherichia coli and Pseudomonas aeruginosa. Our investigations showed that the conversion rate of resazurin to resorufin is affected by efflux pumps. This finding was used to design an assay useful to detect efflux pump activity and to find potential efflux-pump inhibitors in a microtiter plate format. Mefloquine was detected as efflux-pump inhibitor when a panel of selected chemical compounds was tested for assay validation purposes.

Crystallographic analysis of bacterial signal peptidase in ternary complex with arylomycin A2 and a beta-sultam inhibitor.

Bacterial type I signal peptidase (SPase I), an essential membrane-bound endopeptidase with a unique Ser/Lys dyad mechanism, is being investigated as a potential novel antibiotic target. We present here binding and inhibition assays along with crystallographic data that shows that the lipohexapeptide-based natural product arylomycin A2 and the morpholino-beta-sultam derivative (BAL0019193) inhibit SPase I by binding to non-overlapping subsites near the catalytic center. The 2.0 A resolution crystal structure of the soluble catalytic domain of Escherichia coli SPase I (SPase I Delta2-75) in ternary complex with arylomycin A2 and BAL0019193 reveals the position of BAL0019193 adjacent to arylomycin A2 within the SPase I binding site. BAL0019193 binds in a noncovalent manner in close proximity to SPase I residues Ser88, Ser90, Lys145, Asn277, Ala279, and Glu307, as well as atom O45 of arylomycin A2. The binding mode of arylomycin A2 in this 2.0 A resolution ternary complex is compared to that seen in the previous 2.5 A resolution arylomycin A2-SPase cocrystal structure. This work contributes to our understanding of SPase I inhibitor/substrate recognition and should prove helpful in the further development of novel antibiotics based on the inhibition of SPase I.

Activity of the novel macrolide BAL19403 against ribosomes from erythromycin-resistant Propionibacterium acnes.

BAL19403 is a macrolide antibiotic from a novel structural class with potent activity against propionibacteria in vitro. The antibacterial spectrum of BAL19403 covers clinical isolates with mutations in the 2057 to 2059 region of 23S rRNA that confer resistance to erythromycin and clindamycin. The basis of this improved activity was investigated by ribosome binding assays and by a coupled transcription and translation assay. The latter was specifically developed for the use of ribosomes from Propionibacterium acnes. BAL19403 inhibited protein expression by ribosomes from erythromycin-sensitive and erythromycin-resistant P. acnes with similar potencies if the resistance was due to G2057A or A2058G mutations. BAL19403 showed a >10-fold higher activity than erythromycin against ribosomes from a strain with the erm(X) gene. Erm(X) confers high levels of macrolide and lincosamide resistance by dimethylation of A2058. Assays with such ribosomes showed that BAL19403 was potent enough to inhibit half of the total activity with a 50% inhibitory concentration very close to the value measured with erythromycin-sensitive ribosomes. We concluded from our data that the P. acnes strain with the erm(X) gene had a mixed population of ribosomes, with macrolide-sensitive and macrolide-resistant species.

In vitro efficacy of new antifolates against trimethoprim-resistant Bacillus anthracis.

Bacillus anthracis is innately resistant to trimethoprim (TMP), a synthetic antifolate that selectively inhibits several bacterial dihydrofolate reductases (DHFRs) but not human DHFR. Previously, we were able to confirm that TMP resistance in B. anthracis (MIC > 2,048 microg/ml) is due to the lack of selectivity of TMP for the B. anthracis DHFR (E. W. Barrow, P. C. Bourne, and W. W. Barrow, Antimicrob. Agents Chemother. 48:4643-4649, 2004). In this investigation, 24 2,4-diaminopyrimidine derivatives, representing a class of compounds with dihydrophthalazine side chains, were screened for their in vitro effects on B. anthracis Sterne and their selectivities for the B. anthracis DHFR. MICs were obtained by a colorimetric (Alamar blue) broth microdilution assay. Purified human recombinant DHFR (rDHFR) and B. anthracis rDHFR were used in a validated enzyme assay to determine the 50% inhibitory concentrations (IC(50)s) and the selectivity ratios of the derivatives. The MICs ranged from 12.8 to 128 microg/ml for all but nine compounds, for which the MICs were > or =128 microg/ml. The IC(50) values for B. anthracis rDHFR ranged from 46 to 600 nM, whereas the IC(50) values for human rDHFR were >16,000 nM. This is the first report on the in vitro inhibitory actions of this class of antifolates against TMP-resistant B. anthracis isolates. The selective inhibition of B. anthracis rDHFR and the in vitro activity against B. anthracis demonstrate that members of this class of compounds have the potential to be developed into clinically important therapeutic choices for the treatment of infections caused by TMP-resistant bacteria, such as B. anthracis.

Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay.

Assays based on the bacterial SOS-response offer the possibility of automatization of genotoxicity testing for screening of large compound libraries. While existing assays use colorimetric detection or luminescence read-out, we describe here the use of a fluorescence-based system to achieve high sensitivity of detection required for assay miniaturization. Three commonly used fluorophores--fluorescein, DDAO and resorufin--are evaluated. Experimental evidence is given that S9 liver homogenate contains a heat-labile, reversible fluorophore-binding activity and therefore, significantly reduces fluorescence intensities. We have worked out simple solutions to overcome the S9 related interference in order to be able to establish a robust bacterial genotoxicity assay.