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1.
Biochemistry ; 57(16): 2383-2393, 2018 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-29601187

RESUMEN

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 ß-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 ß-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.


Asunto(s)
Esterasas/antagonistas & inhibidores , Lactonas/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Esterasas/química , Humanos , Hidrólisis , Cinética , Lactonas/química , Lipasa/química , Lípidos/antagonistas & inhibidores , Lípidos/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/patogenicidad , Orlistat , Tuberculosis/enzimología , Tuberculosis/microbiología
2.
Sci Rep ; 11(1): 22240, 2021 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-34782676

RESUMEN

Ergothioneine (EGT) is a low molecular weight histidine betaine essential in all domains of life but only synthesized by selected few organisms. Synthesis of EGT by Mycobacterium tuberculosis (M. tb) is critical for maintaining bioenergetic homeostasis and protecting the bacterium from alkylating agents, oxidative stress, and anti-tubercular drugs. EgtD, an S-adenosylmethionine-dependent methyltransferase (AdoMet), catalyzes the trimethylation of L-Histidine to initiate EGT biosynthesis and this reaction has been shown to be essential for EGT production in mycobacteria and for long-term infection of murine macrophages by M. tb. In this work, library screening and structure-guided strategies identified multiple classes of M. tb EgtD inhibitors that bind in various regions of the enzyme active site. X-ray crystal structures of EgtD-inhibitor complexes confirm that L-Histidine analogs bind solely to the L-Histidine binding site while drug-like inhibitors, such as TGX-221, and S-Glycyl-H-1152 span both the L-Histidine and AdoMet binding sites. These enzyme-inhibitor complexes provide detailed structural information of compound scaffolds useful for developing more potent inhibitors that could shorten Tuberculosis treatment regimens by weakening important bacterial defenses.


Asunto(s)
Antituberculosos/química , Betaína/análogos & derivados , Sitios de Unión , Vías Biosintéticas/efectos de los fármacos , Ergotioneína/química , Histidina/análogos & derivados , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/farmacología , Betaína/química , Betaína/metabolismo , Relación Dosis-Respuesta a Droga , Ergotioneína/biosíntesis , Histidina/química , Histidina/metabolismo , Histidina/farmacología , Conformación Molecular , Estructura Molecular , Mycobacterium tuberculosis/metabolismo , Relación Estructura-Actividad
3.
ACS Infect Dis ; 7(10): 2876-2888, 2021 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-34478259

RESUMEN

Tetrahydrolipstatin (THL, 1a) has been shown to inhibit both mammalian and bacterial α/ß hydrolases. In the case of bacterial systems, THL is a known inhibitor of several Mycobacterium tuberculosis hydrolases involved in mycomembrane biosynthesis. Herein we report a highly efficient eight-step asymmetric synthesis of THL using a route that allows modification of the THL α-chain substituent to afford compounds 1a through 1e. The key transformation in the synthesis was use of a (TPP)CrCl/Co2(CO)8-catalyzed regioselective and stereospecific carbonylation on an advanced epoxide intermediate to yield a trans-ß-lactone. These compounds are modest inhibitors of Ag85A and Ag85C, two α/ß hydrolases of M. tuberculosis involved in the biosynthesis of the mycomembrane. Among these compounds, 10d showed the highest inhibitory effect on Ag85A (34 ± 22 µM) and Ag85C (66 ± 8 µM), and its X-ray structure was solved in complex with Ag85C to 2.5 Å resolution. In contrast, compound 1e exhibited the best-in-class MICs of 50 µM (25 µg/mL) and 16 µM (8.4 µg/mL) against M. smegmatis and M. tuberculosis H37Ra, respectively, using a microtiter assay plate. Combination of 1e with 13 well-established antibiotics synergistically enhanced the potency of few of these antibiotics in M. smegmatis and M. tuberculosis H37Ra. Compound 1e applied at concentrations 4-fold lower than its MIC enhanced the MIC of the synergistic antibiotic by 2-256-fold. In addition to observing synergy with first-line drugs, rifamycin and isoniazid, the MIC of vancomycin against M. tuberculosis H37Ra was 65 µg/mL; however, the MIC was lowered to 0.25 µg/mL in the presence of 2.1 µg/mL 1e demonstrating the potential of targeting mycobacterial hydrolases involved in mycomembrane and peptidoglycan biosynthesis.


Asunto(s)
Mycobacterium tuberculosis , Animales , Antituberculosos/farmacología , Isoniazida , Pruebas de Sensibilidad Microbiana , Orlistat
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