Antimicrobial Prodrug Activation by the Staphylococcal Glyoxalase GloB

Abstract

With the rising prevalence of multidrug resistance, there is an urgent need to develop novel antibiotics. Many putative antibiotics demonstrate promising in vitro potency but fail in vivo due to poor drug-like qualities (e.g., serum half-life, oral absorption, solubility, and toxicity). These drug-like properties can be modified through the addition of chemical protecting groups, creating “prodrugs” that are activated prior to target inhibition. Lipophilic prodrugging techniques, including the attachment of a pivaloyloxymethyl group, have garnered attention for their ability to increase cellular permeability by masking charged residues and the relative ease of the chemical prodrugging process. Unfortunately, pivaloyloxymethyl prodrugs are rapidly activated by human sera, rendering any membrane permeability qualities absent during clinical treatment. Identification of the bacterial prodrug activation pathway(s) will allow for the development of host-stable and microbe-targeted prodrug therapies. Here, we use two zoonotic staphylococcal species, Staphylococcus schleiferi and S. pseudintermedius, to establish the mechanism of carboxy ester prodrug activation. Using a forward genetic screen, we identify a conserved locus in both species encoding the enzyme hydroxyacylglutathione hydrolase (GloB), whose loss-of-function confers resistance to carboxy ester prodrugs. We enzymatically characterize GloB and demonstrate that it is a functional glyoxalase II enzyme, which has the capacity to activate carboxy ester prodrugs. As GloB homologues are both widespread and diverse in sequence, our findings suggest that GloB may be a useful mechanism for developing species- or genus-level prodrug targeting strategies.

Structure-guided microbial targeting of antistaphylococcal prodrugs

“…We incubated each enzyme with POM-HEX and characterized the products via 31 P- 1 H-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR). We have previously shown that GloB removes only one POM moiety, resulting in an accumulation of mono-POM-HEX (Figure 1b) 27 . Similarly, FrmB is capable of removing only one POM-moiety (Supplementary Figure 4).…”

“…In the zoonotic staphylococcal species S. schleiferi and S. pseudintermedius , loss of the enzyme GloB, a hydroxyacylglutathione hydrolase, or glyoxalase II enzyme, confers resistance to carboxy ester prodrugs because carboxy ester prodrugs are not activated 27 . However, purified GloB alone is insufficient to activate carboxy ester prodrugs in vitro , suggesting that at least one additional cellular enzyme is required.…”

“…One of these strains had a transposon disrupting the gene that encodes the glyoxalase II enzyme, GloB. The GloB ortholog in S. scheiferi is a prodrug-activating enzyme, mutation of which confers resistance to POM-HEX 27 . The second POM-HEX-resistant strain harbored a transposon insertion in the locus encoding the predicted carboxylesterase annotated as FrmB.…”

“…FrmB and GloB are carboxylesterases with diverging substrate specificity GloB is predicted to be a type II glyoxalase and a member of the large metallo-β-lactamase protein superfamily (INTERPRO IPR001279). Glyoxalase II enzymes, including the closely related GloB ortholog from S. scheiferi, catalyze the second step in the glyoxalase pathway that is responsible for the cellular conversion of methylglyoxal (a toxic glycolytic byproduct) to lactic acid 27,[31][32][33] . Conversely, FrmB orthologs hydrolyze p-nitrophenyl esters of short-chain fatty acids (C2-C6) and are thought to mediate detoxification of cellular formaldehyde 34,35 .…”

“…We recently described the staphylococcal enzyme, GloB, which facilitates activation of carboxy ester prodrugs in S. schleiferi and S. pseudintermedius 27 . Notably, GloB is insufficient to fully activate prodrugs in vitro , suggesting that at least one additional enzyme is necessary for complete phosphonate release.…”