Abstract
We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.
Highlights
MethodsMethionyl-tRNA synthetase inhibitors used in this study were previously described [12, 20, 21]
Brucella spp., small facultative intracellular coccobacilli, function as facultative intracellular bacteria causing the zoonotic disease, brucellosis
We have demonstrated that analogs of MetRS1 inhibitors for S. aureus and C. difficile can selectively target Trypanosoma brucei methionyl-tRNA synthetase (MetRS) (TbMetRS) with potent therapeutic activity in the mouse model of trypanosomiasis [12, 20]
Summary
Methionyl-tRNA synthetase inhibitors used in this study were previously described [12, 20, 21]. Protein expression and purification of Brucella MetRS. The complete coding region of methionyl-tRNA synthetase was PCR amplified from genomic DNA extracted from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156. Sequence similarities and conserved domain alignments of various Brucella spp. methionyl-tRNA synthetase open reading frames were performed using the online tools (BLAST and CDART) available at the National Center for Biotechnology Information (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Because the available Brucella species MetRS gene sequences seem to be highly conserved, we do not anticipate any structural differences or response to inhibitors between strains or species. Purified proteins were eluted in the same buffer supplemented with 250 mM imidazole
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