RESUMEN
The discovery of a bioactive inhibitor tool for human polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts), the initiating enzyme for mucin-type O-glycosylation, remains challenging. In the present study, we identified an array of quinic acid derivatives, including four new glycerates (1-4) from Tussilago farfara, a traditional Chinese medicinal plant, as active inhibitors of GalNAc-T2 using a combined screening approach with a cell-based T2-specific sensor and purified enzyme assay. These inhibitors dose-dependently inhibited human GalNAc-T2 but did not affect O-linked N-acetylglucosamine transferase (OGT), the other type of glycosyltransferase. Importantly, they are not cytotoxic and retain inhibitory activity in cells lacking elongated O-glycans, which are eliminated by the CRISPR/Cas9 gene editing tool. A structure-activity relationship study unveiled a novel quinic acid-caffeic acid conjugate pharmacophore that directs inhibition. Overall, these new natural product inhibitors could serve as a basis for developing an inhibitor tool for GalNAc-T2.
Asunto(s)
Inhibidores Enzimáticos/farmacología , N-Acetilgalactosaminiltransferasas/antagonistas & inhibidores , Ácido Quínico/farmacología , Tussilago/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Flores/química , Flores/metabolismo , Glicosilación , Células HEK293 , Humanos , Conformación Molecular , N-Acetilgalactosaminiltransferasas/aislamiento & purificación , N-Acetilgalactosaminiltransferasas/metabolismo , Ácido Quínico/química , Ácido Quínico/metabolismo , Relación Estructura-Actividad , Tussilago/metabolismo , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
H2S-producing enzymes in bacteria have been shown to be closely engaged in the process of microbial survival and antibiotic resistance. However, no inhibitors have been discovered for these enzymes, e.g., 3-mercaptopyruvate sulfurtransferase (MST). In the present study, we identified several classes of inhibitors for Escherichia coli MST (eMST) through high-throughput screening of â¼26,000 compounds. The thiazolidinedione-type inhibitors were found to selectively bind to Arg178 and Ser239 residues of eMST but hardly affected human MST. Moreover, the pioglitazone of this class concentration dependently accumulates the 3-mercaptopyruvate substrate and suppresses the H2S and reactive sulfane sulfur products in bacteria. Importantly, pioglitazone could potentiate the level of reactive oxygen species in cellulo and consequently enhance the antimicrobial effects of gentamicin and macrophages in culture. This study has identified the bioactive inhibitor of eMST, paving the way for the pharmacological targeting of eMST to synergistically control the survival of E. coli.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Sulfurtransferasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Escherichia coli/fisiología , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
AIM: Cell division cycle 25 (CDC25) phosphatases have recently been considered as potential targets for the development of new cancer therapeutic agents. We aimed to discover novel CDC25B inhibitors in the present study. METHODS: A molecular level high-throughput screening (HTS) assay was set up to screen a set of 48000 pure compounds. RESULTS: HTS, whose average Z' factor is 0.55, was finished and LGH00045, a mixed-type CDC25B inhibitor with a novel structure and relative selectivity for protein tyrosine phosphatases, was identified. Furthermore, LGH00045 impaired the proliferation of tumor cells and increased cyclin-dependent kinase 1 inhibitory tyrosine phosphorylation. In synchronized HeLa cells, LGH00045 delayed cell cycle progression at the G2-M transition. CONCLUSION: LGH00045, a novel CDC25B inhibitor identified through HTS, showed good inhibition on the proliferation of tumor cells and affected the cell cycle progression, which makes it a good hit for further structure modification.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Fosfatasas cdc25/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Regulación Enzimológica de la Expresión Génica , Humanos , Plásmidos/genéticaRESUMEN
Corosolic acid, a triterpenoid compound widely existing in many traditional Chinese medicinal herbs, has been proved to have antidiabetic effects on animal experiments and clinical trials. However, the underlying mechanisms remain unknown. Here, we investigate its cellular effects and related signaling pathway. We demonstrate that it enhances glucose uptake in L6 myotubes and facilitates glucose transporter isoform 4 translocation in CHO/hIR cells. These actions are mediated by insulin pathway activation and can be blocked by phosphatidylinositol 3-kinase (PI(3) Kinase) inhibitor wortmannin. Furthermore, Corosolic acid inhibits the enzymatic activities of several diabetes-related non-receptor protein tyrosine phosphatases (PTPs) in vitro, such as PTP1B, T-cell-PTP, src homology phosphatase-1 and src homology phosphatase-2.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Receptor de Insulina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Triterpenos/farmacología , Proteínas Quinasas Activadas por AMP , Androstadienos/farmacología , Animales , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Transportador de Glucosa de Tipo 4/efectos de los fármacos , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Insulina/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Tirosina Fosfatasas no Receptoras/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , WortmaninaRESUMEN
AIM: The aim of the present study was to discover novel protein tyrosine phosphatase 1B (PTP1B) inhibitors. We expressed and purified the human PTP1B catalytic domain and set up a molecular level high-throughput screening (HTS) assay to screen a set of 48,000 pure compounds. RESULTS: HTS was finished with an averaged Zo factor of 0.63, and LGH00081, a competitive inhibitor of PTP1B with novel structure and relatively good selectivity for receptor-type protein tyrosine phosphatases, was identified. CONCLUSION: We established a molecular level assay which is useful for the screening of PTP1B inhibitors with therapeutic potential. The novel competitive PTP1B inhibitor LGH00081 offers a good start for structure modification and cellular functional activity study.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Animales , Células CHO , Catálisis , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Humanos , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Tirosina/metabolismoRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) is a key element in the negative regulation of the insulin signaling pathway and may play an important role in diabetes and obesity. We identified ursolic acid, a natural pentacyclic triterpenoid that occurs widely in traditional Chinese medicinal herbs, as an inhibitor of PTP1B by screening an extract library of the traditional Chinese medicinal herbs used a diabetes clinic. By modifying urosolic acid, we designed and synthesized a derivative with a K(i) of 283 nM. As competitive inhibitors of PTP1B, ursolic acid and its derivative also inhibit T-cell protein tyrosine phosphatase and src homology phosphatase-2 but not leucocyte antigen-related phosphatase or protein tyrosine phosphatase alpha and epsilon, which are all possibly involved in the insulin pathway. The ursolic acid derivative enhanced insulin receptor phosphorylation in CHO/hIR cells and stimulate glucose uptake in L6 myotubes.