Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates

Abstract

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Microbial esterases and ester prodrugs: An unlikely marriage for combating antibiotic resistance

“…(d) Desthiobiotin–fluorophosphonate (Tallman et al, ). (e and f) Complementary fluorogenic ester substrates used to measure dynamic esterase activity under disease‐related growth conditions (Bassett et al, ; Tallman et al, ; Tallman & Beatty, ; Tallman, Levine, & Beatty, ). Cleavage of ester moieties (labeled red) by a bacterial esterase transitions the fluorophore from a stable nonfluorescent form into a highly fluorescent state.…”

“…As a complementary approach to proteome‐wide ABPP, fluorogenic ester probes have showcased the intragenus and intrastrain variation of esterase activity and pinpointed the unique hydrolase activity that was upregulated during dormant growth conditions (Figure e,f) (Bassett et al, ; Tallman et al, , ; Tallman & Beatty, ). Complementary turn‐on fluorogenic ester scaffolds have each highlighted the broad reactivity of mycobacterial esterases with the ability to hydrolyze short‐chain, long‐chain, branched, and polar esters (Bassett et al, ; Johnson, Hoops, Savas, Kartje, & Lavis, ; Lukowski et al, ; Tallman et al, ; Tallman et al, ; Tallman & Beatty, ). Importantly for prodrug ester design, dormant mycobacterial esterase activity showed a skewed substrate distribution, with its esterases preferring longer, more hydrophobic esters (Figure f) (Bassett et al, ).…”

“…Complementary turn‐on fluorogenic ester scaffolds have each highlighted the broad reactivity of mycobacterial esterases with the ability to hydrolyze short‐chain, long‐chain, branched, and polar esters (Bassett et al, ; Johnson, Hoops, Savas, Kartje, & Lavis, ; Lukowski et al, ; Tallman et al, ; Tallman et al, ; Tallman & Beatty, ). Importantly for prodrug ester design, dormant mycobacterial esterase activity showed a skewed substrate distribution, with its esterases preferring longer, more hydrophobic esters (Figure f) (Bassett et al, ). These differentially activated esters represent promising starting points for introducing selectivity into ester prodrugs.…”

“…Cleavage of ester moieties (labeled red) by a bacterial esterase transitions the fluorophore from a stable nonfluorescent form into a highly fluorescent state. Changing the ester moieties and screening the resulting fluorogenic libraries have identified ester moieties with specificity to pathogenic Mycobacteria species (e) (Tallman et al, 2016a(Tallman et al, , 2016b and to dormant growth conditions (f ) (Bassett et al, 2018). (e) DDAO (7-hydroxy-9H-1,3-dichloro-9,9-dimethylacridin-2-one)-acyloxymethyl ether probe (Tallman et al, 2016a(Tallman et al, , 2016b.…”

“…(g) Two antimalarial prodrugs selectively activated by PfPARE (Istvan et al, 2017). The ester prodrug moiety that increases cell permeability and is selectively removed by PfPARE within P. falciparum is shown in red more hydrophobic esters ( Figure 2f ) (Bassett et al, 2018). These differentially activated esters represent promising starting points for introducing selectivity into ester prodrugs.…”