Characterization of Tetrahydrolipstatin and Stereoderivatives on the Inhibition of Essential Mycobacterium tuberculosis Lipid Esterases

Abstract

Tetrahydrolipstatin (THL) is a covalent inhibitor of many serine esterases. In mycobacteria, THL has been found to covalently react with 261 lipid esterases upon treatment of Mycobacterium bovis cell lysate. However, the covalent adduct is considered unstable in some cases because of the hydrolysis of the enzyme-linked THL adduct resulting in catalytic turnover. In this study, a library of THL stereoderivatives was tested against three essential Mycobacterium tuberculosis lipid esterases of interest for drug development to assess how the stereochemistry of THL affects respective enzyme inhibition and allows for cross enzyme inhibition. The mycolyltransferase Antigen 85C (Ag85C) was found to be stereospecific with regard to THL; covalent inhibition occurs within minutes and was previously shown to be irreversible. Conversely, the Rv3802 phospholipase A/thioesterase was more accepting of a variety of THL configurations and uses these compounds as alternative substrates. The reaction of the THL stereoderivatives with the thioesterase domain of polyketide synthase 13 (Pks13-TE) also leads to hydrolytic turnover and is nonstereospecific but occurs on a slower, multihour time scale. Our findings suggest the stereochemistry of the β-lactone ring of THL is important for cross enzyme reactivity, while the two stereocenters of the peptidyl arm can affect enzyme specificity and the catalytic hydrolysis of the β-lactone ring. The observed kinetic data for all three target enzymes are supported by recently published X-ray crystal structures of Ag85C, Rv3802, and Pks13-TE. Insights from this study provide a molecular basis for the kinetic modulation of three essential M. tuberculosis lipid esterases by THL and can be applied to increase potency and enzyme residence times and enhance the specificity of the THL scaffold.

Methyl arachidonyl fluorophosphonate inhibits Mycobacterium tuberculosis thioesterase TesA and globally affects vancomycin susceptibility

“…As expected, we observed that THL is a potent inhibitor of TesA TE activity (Fig. 3C), further confirming that THL might be an interesting anti-TB drug candidate [17,37]. Indeed, THL was shown to reduce mycobacteria cell wall lipid biosynthesis, targeting various serine enzymes involved among others in sulpholipids, mycolic acids, or PDIM biosynthesis [25,[37][38][39].…”

Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria

“…Chemical inhibitors can interfere with lipid metabolism by impairing the activity of mycobacterial lipolytic enzymes 43–45 . Pharmacological lipid inhibitors, such as serine-hydrolase inhibitors, have been widely used to decipher the molecular aspects of TAG metabolism and their relationship with the different mycobacterial metabolic stages 19,28,46,47 .…”

A FRET-Based Fluorogenic Trehalose Dimycolate Analogue for Probing Mycomembrane-Remodeling Enzymes of Mycobacteria

“…21,22 Mtb Ag85C and its catalytic mutant Ag85C S124A carrying His 6 tags were expressed and purified from E. coli as previously reported by Ronning. 42 Purity of recombinant proteins was assessed by SDS-PAGE. Commercially available hydrolases from yeast, P. cepacia , B. subtilis esterase, and C. rugosa were obtained from Sigma.…”