Endogenous Enzymes Enable Antimicrobial Activity

Abstract

In light of the continued threat of antimicrobial-resistant bacteria, new strategies to expand the repertoire of antimicrobial compounds are necessary. Prodrugs are an underexploited strategy in this effort. Here, we report on the enhanced antimicrobial activity of a prodrug toward bacteria having an enzyme capable of its activation. A screen led us to the sulfurol ester of the antibiotic trans-3-(4-chlorobenzoyl)acrylic acid. An endogenous esterase makes Mycolycibacterium smegmatis sensitive to this prodrug. Candidate esterases were identified, and their heterologous production made Escherichia coli sensitive to the ester prodrug. Taken together, these data suggest a new approach to the development of antimicrobial compounds that takes advantage of endogenous enzymatic activities to target specific bacteria.

Figure 1.

Microbial esterase activity assays. (A) A small panel of esters of 2-thiopheneacetic acid was assessed for cleavability in E. coli DH10B or M. smegmatis mc²155 lysate. Only the phenolic ester was cleaved in E. coli lysate whereas all four esters initially tested were cleaved in M. smegmatis lysate. Consequently, a larger panel of esters was screened in M. smegmatis lysate, and it was found that all esters were subject to cleavage. (B) Synthetic route to esters of trans-3-(4-chlorobenzoyl)acrylic acid. Note that acid 1 and esters 2–4 are labeled with the corresponding color for viability curves in panel C. (C) Viability curves for bacteria incubated with acid 1 and esters 2–4 (100 μM), which have high antimicrobial activity. Viability was assessed through the addition to the medium of resazurin dye, which fluoresces upon reduction in the presence of live, metabolizing cells. Medium: E. coli and B. subtilis, CAMHB; M. smegmatis, 7H9. Lines are the average of triplicate experiments; ribbons depict one standard deviation.

Figure 2.

LC–MS extracted ion chromatograms (EICs) of sulfurol and sulfurol ester 3 after incubation with a cell lysate. Red EIC has m/z 144 (sulfurol), and black EIC has m/z 336 (sulfurol ester 3) from the same LC–MS run. The small peak in the black trace is likely the cis isomer of sulfurol ester 3. Standard traces are from a solution containing 200 μM of both sulfurol and sulfurol ester 3. Inset: Sulfurol ester 3 % hydrolyzed was determined from calibration curves as described in the Supporting Information. Note: In addition to hydrolysis, the 7H9 medium led to isomerization.

Figure 3.

Identification of pnbA and effect of its expression in E. coli DH10B. (A) Sequence similarity network of annotated carboxylesterases from B. subtilis 168, E. coli DH10B, and M. smegmatis mc²155 (Table S1). The black arrow indicates the pnbA carboxylesterase from B. subtilis 168; the gray arrow indicates carboxylesterase A0QYI2 from M. smegmatis. (B) LC–MS extracted ion chromatograms (EICs) of dichloromethane extract from cultures of E. coli exposed to 200 μM of sulfurol ester 3. The “Authentic substances” traces are from a solution containing 200 μM of both sulfurol and sulfurol ester 3. The red EIC has m/z 144 (sulfurol), and the black EIC has m/z 336 (sulfurol ester 3) from the same LC–MS sample. Inset: The sulfurol ester 3 % hydrolyzed was determined from calibration curves as described in the Supporting Information. (C) Viability curves for E. coli expressing the identified carboxylesterase incubated with acid 1 and ester 3 (100 μM). (D) Viability curves for E. coli expressing the identified carboxylesterase incubated with esters 2 and 3 (100 μM). In panels C and D, the ordinate was adjusted such that the bacteria-only growth curves from each gene expression condition have the same inflection point, allowing for a comparison between the antimicrobial compound dose curves. Lines are the average of triplicate experiments; ribbons depict one standard deviation.