Assessing and utilizing esterase specificity in antimicrobial prodrug development

Abstract

As a class of enzymes, esterases have been investigated for decades and have found use in industrial processes, synthetic organic chemistry, and elsewhere. Esters are functional groups composed of an alcohol moiety and a carboxylic acid moiety. Although much work has explored the influence of the carboxyl moiety of an ester on its susceptibility to esterases, little work has explored the influence of the alcohol moiety. Here, we describe an in vitro methodology to explore the influence of changing the alcohol moiety of an ester on its enzymatic hydrolysis, including strategies for analyzing such data. We then describe leveraging data from these assays to develop targeted antimicrobial prodrugs that activate in certain species due to the discriminatory activity of species-specific esterases. We envisage the potential of genomics and machine learning to further these efforts. Finally, we anticipate the potential future uses of these ideas, including developing targeted anti-cancer compounds.

Keywords: Antimicrobial compounds; Data visualization; Esterases; Genomics; Prodrugs; Targeted therapeutics.

Figure 1.

The ester prodrug concept. Carboxylate-containing drugs are poorly permeable due to Coulombic repulsion. Masking the negative charge makes the drug more readily permeable in a nonspecific fashion. The active drug is revealed only in cells that contain an enzyme capable of hydrolyzing the ester bond to reveal the active moiety. In a targeted prodrug strategy, only cells that contain such an esterase would incur the effects of the drug.

Figure 2.

Scheme for the development of targeted ester prodrugs. (A) The in vitro 2TA–terbium luminescence assay is used to identify alcohol moieties that modulate esterase activity in desired cell lysates. (B) Candidate carboxylate compounds are unearthed through literature mining, machine learning, or other approaches. (C) Carboxylate compounds are condensed with the alcohol compounds of interest and their activity against target and off-target cell lines assessed using a viability assay. (D) Genomic and bioinformatic analyses are used to determine which esterase(s) present in the target strain(s) are responsible for the observed cleavage. These data further suggest which strains are susceptible to the compounds and which strains are not. Additional viability assays can then be employed to confirm conclusions.

Figure 3.

Scheme for the synthesis of 2TA esters for the in vitro 2TA–terbium luminescence assay. (A) 2-Thiopheneacetic acid (1) is readily converted into esters through condensation with alcohols either via boiling in the alcohol with sulfuric acid (Fischer esterification) or through the use of DCC and DMAP. (B) Compounds that influence the luminescent response of ester cleavage.

Figure 4.

Graphs shows raw individual replicates, averaged replicate, and concentration results for the hydrolysis of the 2TA–sulfurylester by pig liver esterase (PLE). The raw data as well as the R code for creating the graphs is available at https://github.com/hetrickk/Plots_for_MIE.

Figure 5.

Graph of the fitted progress curve for the hydrolysis of the 2TA–sulfurylester by pig liver esterase (PLE). Raw data and the script for making this plot and fit are available at https://github.com/hetrickk/Plots_for_MIE. Note that the progress curve data is saved as comma-separated values (csv).

Figure 6.

Carboxylate-containing compounds of interest for the ester prodrug strategy.

Figure 7.

Fit of a growth curve of E. coli in CAMHB.

Figure 8.

Example plot of viability assays using gghighlight to generate a visual comparison of the viability profiles of select esters of trans-3-(4-chlorobenzoyl)acrylic acid (inset).