Optimization of a non-canonical anti-infective: interrogation of the target binding pocket for a small molecule inhibitor of E. coli polysaccharide capsule expression
The polysaccharide capsule in E. coli is a major pathogenic factor. Loss of encapsulation results in complete attenuation in an otherwise lethal systemic murine infection model. We have previously identified a small molecule inhibitor of capsule biogenesis (designated DU011) and identified its target as MprA, a transcriptional repressor of multi-drug efflux pumps. Unlike other proposed MprA ligands such as salicylate and 2,4-dinitrophenol (DNP), DU011 does not alter E. coli antibiotic resistance and has significantly enhanced inhibition of capsule expression. We hypothesized that DU011 interacts uniquely in the MprA binding pocket relative to other ligands to produce a different phenotype and sought to define critical residues in the binding pocket to optimize inhibitor design.
To identify mutations within MprA that confer resistance to DU011, a plasmid carrying mprA was randomly mutated in DNA repair deficient XL-Red cells. In a non-mutator strain of E.coli, plasmids were screened for DU011 and DNP resistance by using a capsule dependent phage assay. Individual clones were selected and the plasmids sequenced.
Mutant MprA clones were identified that conferred shared and specific resistance to DU011 and DNP. Mutations mapped to the predicted binding pocket as modeled in silico. Biophysical studies demonstrated altered and abrogated binding of DU011 and DNP in purified mutant MprA proteins.
DNP and DU011 use different residues for binding to MprA, which may explain their differential effects on multi-drug efflux pump and capsule regulation. Molecules that target the amino acid residues that are part of the DU011 binding pocket would be good candidates as therapeutics that would inhibit a major pathogenic factor in E .coli while maintaining the repression of multi-drug efflux pumps and thereby eliminating a major mechanism of resistance to such molecules.
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