RESUMEN
The emerging COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised a global catastrophe. To date, there is no specific antiviral drug available to combat this virus, except the vaccine. In this study, the main protease (Mpro) required for SARS-CoV-2 viral replication was expressed and purified. Thirty-six compounds were tested as inhibitors of SARS-CoV-2 Mpro by fluorescence resonance energy transfer (FRET) technique. The half-maximal inhibitory concentration (IC50) values of Ebselen and Ebsulfur analogs were obtained to be in the range of 0.074-0.91 µM. Notably, the molecules containing furane substituent displayed higher inhibition against Mpro, followed by Ebselen 1i (IC50 = 0.074 µM) and Ebsulfur 2k (IC50 = 0.11 µM). The action mechanism of 1i and 2k were characterized by enzyme kinetics, pre-incubation and jump dilution assays, as well as fluorescent labeling experiments, which suggested that both compounds covalently and irreversibly bind to Mpro, while molecular docking suggested that 2k formed an SS bond with the Cys145 at the enzymatic active site. This study provides two very potent scaffolds Ebsulfur and Ebselen for the development of covalent inhibitors of Mpro to combat COVID-19.
Asunto(s)
Antivirales/metabolismo , Azoles/metabolismo , Compuestos de Organoselenio/metabolismo , SARS-CoV-2/metabolismo , Compuestos de Azufre/metabolismo , Proteínas de la Matriz Viral/metabolismo , Antivirales/química , Antivirales/uso terapéutico , Azoles/química , Azoles/uso terapéutico , Sitios de Unión , COVID-19/patología , COVID-19/virología , Dominio Catalítico , Transferencia Resonante de Energía de Fluorescencia , Humanos , Concentración 50 Inhibidora , Isoindoles , Cinética , Simulación del Acoplamiento Molecular , Compuestos de Organoselenio/química , Compuestos de Organoselenio/uso terapéutico , SARS-CoV-2/aislamiento & purificación , Relación Estructura-Actividad , Compuestos de Azufre/química , Compuestos de Azufre/uso terapéutico , Proteínas de la Matriz Viral/antagonistas & inhibidores , Proteínas de la Matriz Viral/genética , Tratamiento Farmacológico de COVID-19RESUMEN
The bacteria, harboring metallo-ß-lactamases (MßLs), become resistant on most ß-lactam antibiotics, specifically New Delhi metallo-ß-lactamase-1 (NDM-1), which hydrolyzes almost all ß-lactam antibiotics leading to bacterial multiple-drug resistance. It is highly desirable to develop effective NDM-1 inhibitors in reviving the efficacy of existing antibiotics. Here, we report a potent covalently reversible scaffold, 3-Bromopyruvate (3BP) to target the NDM-1 in vitro and in vivo. Enzymatic kinetic studies revealed that 3BP is capable of inhibiting the B1 and B2 MßLs and exhibited the best inhibition on NDM-1 with an IC50 of 2.57 µM, also, it was found to be a dose- and time-dependent inhibitor. The study of inhibition mechanism suggested that 3BP reversibly inactivate NDM-1, and may form a dynamic reversible covalent bond with cysteine at active site of the enzyme. Besides, 3BP effectively restored the activity of five ß-lactam antibiotics on three clinical strains expressing NDM-1, resulting in 2-8-fold reduction in MIC. Moreover, the toxicity evaluation of 3BP against L929 mouse fibroblastic cells indicated that 3BP had low cytotoxicity, implying it may be used as lead molecule for future drug candidate.
Asunto(s)
Antibacterianos/farmacología , Piruvatos/farmacología , beta-Lactamasas/metabolismo , Animales , Bacterias/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Línea Celular , Cisteína/metabolismo , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Fibroblastos/microbiología , Cinética , RatonesRESUMEN
The superbug infection caused by New Delhi metallo-ß-lactamase (NDM-1) has grown into an emerging threat, labelling and inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. Here, we report a potent covalent scaffold, ebsulfur, for targeting the protein in vitro and in vivo. Enzymatic kinetic study indicated that eighteen ebsulfurs gained except 1a-b and 1f inhibited NDM-1, exhibiting an IC50 value ranging of 0.16-9⯵M, and 1g was found to be the best, dose- and time-dependent inhibitor with an IC50 of 0.16⯵M. Also, these ebsulfurs effectively restored the antibacterial activity of cefazolin against E. coli expressing NDM-1, and the best effect was observed to be from 1g, 1i and 1n, resulting in an 256-fold reduction in MIC of the antibiotic at a dose of 16⯵g/mL. The equilibrium dialysis study implied that the ebsulfur disrupted the coordination of one Zn(II) ion at active site of NDM-1. Labelling of NDM-1 using a constructed fluorescent ebsulfur Ebs-R suggested that the inhibitor covalently bound to the target through SDS-PAGE analysis in vitro. Also, labelling NDM-1 in living E. coli cells with Ebs-R by confocal microscopic imaging showed the real-time distribution change process of intracellular recombinant protein NDM-1. Moreover, the cytotoxicity of these ebsulfurs against L929 mouse fibroblastic cells was tested, and their capability to restore antibacterial activity of antibiotic against clinical strains E. coli EC08 producing NDM-1 was determined. The ebsulfur scaffold proposed here is valuable for development of the covalent irreversible inhibitors of NDM-1, and also for labelling the target in vitro and in vivo.
Asunto(s)
Antibacterianos/farmacología , Azoles/farmacología , Escherichia coli/efectos de los fármacos , Compuestos de Azufre/farmacología , beta-Lactamasas/metabolismo , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Azoles/síntesis química , Azoles/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Escherichia coli/citología , Escherichia coli/enzimología , Fibroblastos/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Microscopía Confocal , Estructura Molecular , Relación Estructura-Actividad , Compuestos de Azufre/síntesis química , Compuestos de Azufre/químicaRESUMEN
We report the discovery of ebselen-based dual covalent inhibitors of metallo-ß-lactamases. Fluorescence and MALDI-TOF analysis suggested that the scaffold could bind to NDM-1 by forming a S-Se bond with Cys221 and an amide bond with Lys224 of NDM-1, thereby exhibiting selective inhibition and labeling against B1 and B2 subclass enzymes in vitro and in vivo.