RESUMEN
FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/antagonistas & inhibidores , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Metilación , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Transgénicos , Terapia Molecular Dirigida , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Relación Estructura-Actividad , Células U937 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sortase A (SrtA) anchors surface proteins to the cell wall and aids biofilm formation during infection, which functions as a key virulence factor of important Gram-positive pathogens, such as Staphylococcus aureus. At present researchers need a way in which to validate whether or not SrtA is a druggable target alternative to the conventional antibiotic targets in the mechanism. In this study, we performed a high-throughput screening and identified a new class of potential inhibitors of S. aureus SrtA, which are derived from natural products and contain the quinone skeleton. Compound 283 functions as an irreversible inhibitor that covalently alkylates the active site Cys184 of SrtA. NMR analysis confirms the direct interaction of the small-molecule inhibitor towards SrtA protein. The anchoring of protein A (SpA) to the cell wall and the biofilm formation are significantly attenuated when the S. aureus Newman strain is cultured in the presence of inhibitor. Our study indicates that compound 283 could be a potential hit for the development of new anti-virulence agents against S. aureus infections by covalently targeting SrtA.
Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Benzoquinonas/química , Inhibidores de Cisteína Proteinasa/química , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Transferencia Resonante de Energía de Fluorescencia , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Péptidos/análisis , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimologíaRESUMEN
ATP-dependent Clp protease (ClpP), a highly conserved serine protease in vast bacteria, could be converted into a noncontrollable enzyme capable of degrading mature proteins in the presence of acyldepsipeptides (ADEPs). Here, we design such a gain-of-function mutant of Staphylococcus aureus ClpP (SaClpP) capable of triggering the same level of dysfunctional activity that occurs upon ADEPs treatment. The SaClpPY63A mutant degrades FtsZ in vivo and inhibits staphylococcal growth. The crystal structure of SaClpPY63A indicates that Asn42 would be an important domino to fall for further activation of ClpP. Indeed, the SaClpPN42AY63A mutant demonstrates promoted self-activated proteolysis, which is a result of an enlarged entrance pore as observed in cryo-electron microscopy images. In addition, the expression of the engineered clpP allele phenocopies treatment with ADEPs; inhibition of cell division occurs as does showing sterilizing with rifampicin antibiotics. Collectively, we show that the gain-of-function SaClpPN42AY63A mutant becomes a fairly nonspecific protease and kills persisters by degrading over 500 proteins, thus providing new insights into the structure of the ClpP protease.
Asunto(s)
Endopeptidasa Clp/metabolismo , Mutación , Alelos , Cristalografía por Rayos X , Endopeptidasa Clp/química , Endopeptidasa Clp/genética , Microscopía Electrónica , Conformación Proteica , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
The diterpenoid compound 5 was identified as an antibacterial lead in our screening of small synthetic natural product-like (NPL) library. A series of novel diterpene derivatives were synthesized and investigated for their activity against Staphylococcus aureus Newman strain and multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). Among the compounds tested, 42 and 43 showed highest activity with a MIC of 1 µg/mL against strain Newman, 45 and 52 showed the most potent activity with MIC values of 0.71-3.12 µg/mL against five multidrug-resistant S. aureus. All high-antimicrobial active compounds showed no obvious toxicity to human fibroblast (HAF) cells at the MIC concentration.