RESUMEN
A novel method for applying high-throughput docking to challenging metalloenzyme targets is described. The method utilizes information-based virtual transformation of library carboxylates to hydroxamic acids prior to docking, followed by compound acquisition, one-pot (two steps) chemical synthesis and in vitro screening. In two experiments targeting the botulinum neurotoxin serotype A metalloprotease light chain, hit rates of 32% and 18% were observed.
Asunto(s)
Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Inhibidores de Proteasas/química , Sitios de Unión , Toxinas Botulínicas Tipo A/metabolismo , Ácidos Carboxílicos/química , Dominio Catalítico , Química Farmacéutica , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Ácidos Hidroxámicos/química , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/metabolismo , Unión Proteica , Serotipificación , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small molecule furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochemical cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Molecular modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH(2)Cl. The compounds presented here are bona fide synthetic small molecule furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furin-dependent diseases.
Asunto(s)
Furina/antagonistas & inhibidores , Hexosaminas/química , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Animales , Línea Celular , Biología Computacional , Furina/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Estructura Molecular , Inhibidores de Serina Proteinasa/química , Relación Estructura-ActividadRESUMEN
The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Proteínas Quinasas/química , Animales , Inhibidores Enzimáticos/metabolismo , Humanos , Ligandos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Sirolimus/química , Sirolimus/metabolismo , Serina-Treonina Quinasas TORRESUMEN
To avoid detection and targeting by the immune system, the plague-causing bacterium Yersinia pestis uses a type III secretion system to deliver a set of inhibitory proteins into the cytoplasm of immune cells. One of these proteins is an exceptionally active tyrosine phosphatase termed YopH, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. Because Y. pestis strains lacking YopH are avirulent, we set out to develop small molecule inhibitors for YopH. We used a novel and cost-effective approach, in which leads from a chemical library screening were analyzed and computationally docked into the crystal structure of YopH. This resulted in the identification of a series of novel YopH inhibitors with nanomolar Ki values, as well as the structural basis for inhibition. Our inhibitors lack the polar phosphate-mimicking moiety of rationally designed tyrosine phosphatase inhibitors, and they readily entered live cells and rescued them from YopH-induced tyrosine dephosphorylation, signaling paralysis, and cell death. These inhibitors may become useful for treating the lethal infection by Y. pestis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Salicilatos/farmacología , Yersinia pestis/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Calorimetría , Diseño de Fármacos , Cinética , Plásmidos , Probabilidad , Proteínas Recombinantes/antagonistas & inhibidoresRESUMEN
Bid is a key member of the Bcl-2 family proteins involved in the control of the apoptotic cascade in cells, leading to cell death. Uncontrolled cell death is associated with several human pathologies, such as neurodegenerative diseases and ischemic injuries. Therefore, Bid represents a potential yet unexplored and challenging target for strategies aimed at the development of therapeutic agents. Here we show that a multidisciplinary NMR-based approach that we named SAR by ILOEs (structure activity relationships by interligand nuclear Overhauser effect) allowed us to rationally design a series of 4-phenylsulfanyl-phenylamine derivatives that are capable of occupying a deep hydrophobic crevice on the surface of Bid. These compounds represent the first antiapoptotic small molecules targeting a Bcl-2 protein as shown by their ability to inhibit tBid-induced SMAC release, caspase-3 activation, and cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Diseño de Fármacos , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Sitios de Unión , Bioensayo , Proteínas Portadoras/química , Línea Celular , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Preparaciones Farmacéuticas/síntesis química , Preparaciones Farmacéuticas/química , Estructura Terciaria de ProteínaRESUMEN
Epidemiological data and in vitro studies on cancer chemoprevention by tea polyphenols have gained attention recently from the scientific community, nutritionists, the pharmaceutical industry, and the public. Despite the several efforts made recently to elucidate the molecular basis for the anticancer activity of these natural products, little correlation has been found thus far between the putative protein targets of compounds found in tea extracts and levels found in plasma after tea consumption. Here, by using a combination of nuclear magnetic resonance binding assays, fluorescence polarization assay, and computational docking studies, we found that certain green tea catechins and black tea theaflavins are very potent inhibitors (K(i) in the nanomolar range) of the antiapoptotic Bcl-2-family proteins, Bcl-x(L) and Bcl-2. These data suggest a strong link between the anticancer activities of these tea polyphenols and their inhibition of a crucial antiapoptotic pathway, which is implicated in the development of many human malignancies.
Asunto(s)
Anticarcinógenos/farmacología , Camellia sinensis/química , Flavonoides/farmacología , Ácido Gálico/análogos & derivados , Fenoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Té , Secuencia de Aminoácidos , Anticarcinógenos/química , Anticarcinógenos/metabolismo , Biflavonoides/química , Biflavonoides/metabolismo , Biflavonoides/farmacología , Sitios de Unión , Unión Competitiva , Catequina/química , Catequina/metabolismo , Catequina/farmacología , Flavonoides/química , Flavonoides/metabolismo , Ácido Gálico/química , Ácido Gálico/metabolismo , Ácido Gálico/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fenoles/química , Fenoles/metabolismo , Polifenoles , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Among the most promising chemopreventive agents, certain natural polyphenols have recently received a great deal of attention because of their demonstrated inhibitory activity against tumorigenesis. In view of their anticancer properties, these compounds also hold great promise as potential chemotherapeutic agents. However, to translate these chemopreventive agents into chemotherapeutic compounds, their exact mechanisms of action must be delineated. By using a multidisciplinary approach guided by modern nuclear magnetic resonance spectroscopy techniques, fluorescence polarization displacement assays, and cell-based assays, we have begun to unravel the mechanisms of actions of certain polyphenols such as Gossypol (a compound from cotton seed extracts) and Purpurogallin (a natural compound extracted from Quercus sp. nutgall) and their derivatives. Our findings suggest that these natural products bind and antagonize the antiapoptotic effects of B-cell lymphocyte/leukemia-2 (Bcl-2) family proteins such as Bcl-x(L). Our in vitro and in vivo data not only open a window of opportunities for the development of novel cancer treatments with these compounds but also provide structural information that can be used for the design and development of novel and more effective analogues.