Structural basis for the inhibition of bacterial multidrug exporters
Ryosuke Nakashima1, Keisuke Sakurai, Seiji Yamasaki
1Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Abstract:
The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.
Insights
Pyridopyrimidine derivatives block multidrug efflux pumps AcrB and MexB by binding to a hydrophobic trap, hindering their function. This discovery aids in developing new antibiotics against Gram-negative pathogens.
Area of Science:
- Structural Biology
- Microbiology
- Drug Discovery
Background:
- Multidrug efflux transporters like AcrB are key to multidrug resistance in Gram-negative bacteria.
- Existing efflux pump inhibitors lack clinical utility, necessitating novel therapeutic strategies.
- MexB and MexY are critical multidrug exporters in Pseudomonas aeruginosa, a significant pathogen.
Purpose of the Study:
- To determine the structural basis of inhibition for AcrB and MexB by pyridopyrimidine derivatives.
- To elucidate the mechanism by which pyridopyrimidines hinder the functional rotation of these efflux pumps.
- To identify structural features that enable specific inhibitor binding and guide the development of new drugs.
Main Methods:
- Determination of crystal structures of AcrB and MexB in complex with a pyridopyrimidine derivative.
- Analysis of inhibitor-binding sites, focusing on the distal pocket and associated hydrophobic features.
- Comparison of binding interactions in AcrB, MexB, and the related transporter MexY.
Main Results:
- Pyridopyrimidine derivatives bind to a specific hydrophobic trap in the distal pocket of AcrB and MexB.
- This binding sterically hinders the functional rotation essential for drug efflux.
- Phenylalanine residues (Phe178) in AcrB and MexB facilitate tight inhibitor binding via π-π interactions, while tryptophan in MexY (Trp177) prevents it.
Conclusions:
- The identified hydrophobic trap is a critical target for pyridopyrimidine-based inhibitors.
- Understanding these structural interactions is crucial for designing potent and specific inhibitors of AcrB and MexB.
- This work lays the foundation for developing universal inhibitors targeting MexB and MexY in Pseudomonas aeruginosa.
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