RESUMEN
Historically, natural products have played a major role in the development of antibiotics. Their complex chemical structures and high polarity give them advantages in the drug discovery process. In the broad range of natural products, sesquiterpene lactones are interesting compounds because of their diverse biological activities, their high-polarity, and sp3-carbon-rich chemical structures. Parthenolide (PTL) is a natural compound isolated from Tanacetum parthenium, of the family of germacranolide-type sesquiterpene lactones. In recent years, parthenolide has been studied for its anti-inflammatory, antimigraine, and anticancer properties. Recently, PTL has shown antibacterial activities, especially against Gram-positive bacteria. However, few studies are available on the potential antitubercular activities of parthenolide and its analogs. It has been demonstrated that parthenolide's biological effects are linked to the reactivity of α-exo-methylene-γ-butyrolactone, which reacts with cysteine in targeted proteins via a Michael addition. In this work, we describe the ene reaction of acylnitroso intermediates with parthenolide leading to the regioselective and stereoselective synthesis of new derivatives and their biological evaluation. The addition of hydroxycarbamates and hydroxyureas led to original analogs with higher polarity and solubility than parthenolide. Through this synthetic route, the Michael acceptor motif was preserved and is thus believed to be involved in the selective activity against Mycobacterium tuberculosis.
Asunto(s)
Mycobacterium tuberculosis , Sesquiterpenos , Mycobacterium tuberculosis/metabolismo , Sesquiterpenos/química , Antiinflamatorios , Lactonas/químicaRESUMEN
Since end of 2019, the global and unprecedented outbreak caused by the coronavirus SARS-CoV-2 led to dramatic numbers of infections and deaths worldwide. SARS-CoV-2 produces two large viral polyproteins which are cleaved by two cysteine proteases encoded by the virus, the 3CL protease (3CLpro) and the papain-like protease, to generate non-structural proteins essential for the virus life cycle. Both proteases are recognized as promising drug targets for the development of anti-coronavirus chemotherapy. Aiming at identifying broad spectrum agents for the treatment of COVID-19 but also to fight emergent coronaviruses, we focused on 3CLpro that is well conserved within this viral family. Here we present a high-throughput screening of more than 89,000 small molecules that led to the identification of a new chemotype, potent inhibitor of the SARS-CoV-2 3CLpro. The mechanism of inhibition, the interaction with the protease using NMR and X-Ray, the specificity against host cysteine proteases and promising antiviral properties in cells are reported.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Péptido Hidrolasas , Cisteína Endopeptidasas/metabolismo , Inhibidores de Proteasas/química , Proteasas 3C de Coronavirus , Antivirales/químicaRESUMEN
Insulin degrading enzyme (IDE) is a zinc metalloprotease that cleaves numerous substrates among which amyloid-ß and insulin. It has been linked through genetic studies to the risk of type-2 diabetes (T2D) or Alzheimer's disease (AD). Pharmacological activation of IDE is an attractive therapeutic strategy in AD. While IDE inhibition gave paradoxal activity in glucose homeostasis, recent studies, in particular in the liver suggest that IDE activators could be also of interest in diabetes. Here we describe the discovery of an original series of IDE activators by screening and structure-activity relationships. Early cellular studies show that hit 1 decreases glucose-stimulating insulin secretion. Docking studies revealed it has an unprecedented extended binding to the polyanion-binding site of IDE. These indole-based pharmacological tools are activators of both Aß and insulin hydrolysis by IDE and could be helpful to explore the multiple roles of IDE.
Asunto(s)
Indoles/farmacología , Insulisina/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Indoles/química , Ratones , Modelos Moleculares , Estructura Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.
Asunto(s)
Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antivirales/química , Sitios de Unión , Chlorocebus aethiops , Proteasas 3C de Coronavirus/química , Inhibidores de Cisteína Proteinasa/química , Evaluación Preclínica de Medicamentos , Pruebas de Sensibilidad Microbiana , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Multimerización de Proteína , SARS-CoV-2/química , Bibliotecas de Moléculas Pequeñas/química , Células VeroRESUMEN
Insulin-degrading enzyme, IDE, is a metalloprotease implicated in the metabolism of key peptides such as insulin, glucagon, ß-amyloid peptide. Recent studies have pointed out its broader role in the cell physiology. In order to identify new drug-like inhibitors of IDE with optimal pharmacokinetic properties to probe its multiple roles, we ran a high-throughput drug repurposing screening. Ebselen, cefmetazole and rabeprazole were identified as reversible inhibitors of IDE. Ebselen is the most potent inhibitor (IC50(insulin)â¯=â¯14â¯nM). The molecular mode of action of ebselen was investigated by biophysical methods. We show that ebselen induces the disorder of the IDE catalytic cleft, which significantly differs from the previously reported IDE inhibitors. IDE inhibition by ebselen can explain some of its reported activities in metabolism as well as in neuroprotection.
Asunto(s)
Azoles/farmacología , Reposicionamiento de Medicamentos , Inhibidores Enzimáticos/farmacología , Insulisina/antagonistas & inhibidores , Compuestos de Organoselenio/farmacología , Azoles/química , Biocatálisis , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Insulisina/metabolismo , Isoindoles , Estructura Molecular , Compuestos de Organoselenio/química , Relación Estructura-ActividadRESUMEN
A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC50, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Oxadiazoles/química , Oxadiazoles/farmacología , Animales , Diseño de Fármacos , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/crecimiento & desarrollo , Células RAW 264.7 , Tuberculosis/tratamiento farmacológicoRESUMEN
A new class of Cyclophostin and Cyclipostins (CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC 17 exhibited the best extracellular antitubercular activity (MIC50 = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC 17 may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.
Asunto(s)
Antituberculosos , Macrófagos/microbiología , Mycobacterium tuberculosis/crecimiento & desarrollo , Compuestos Organofosforados , Tuberculosis/tratamiento farmacológico , Antituberculosos/química , Antituberculosos/farmacología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Tuberculosis/metabolismo , Tuberculosis/patologíaRESUMEN
Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-ß. Knockout and genetic studies have linked IDE to Alzheimer's disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.
Asunto(s)
Ácidos Hidroxámicos/síntesis química , Insulisina/antagonistas & inhibidores , Triazoles/síntesis química , Animales , Células CACO-2 , Dominio Catalítico , Diabetes Mellitus/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Prueba de Tolerancia a la Glucosa , Humanos , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos , Terapia Molecular Dirigida , Distribución Aleatoria , Relación Estructura-Actividad , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-â and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer's disease, amyloid-â clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE's role.
Asunto(s)
Carbamatos/farmacología , Ácidos Carboxílicos/química , Inhibidores Enzimáticos/farmacología , Imidazoles/química , Insulisina/antagonistas & inhibidores , Insulisina/metabolismo , Oxadiazoles/farmacología , Carbamatos/síntesis química , Carbamatos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Oxadiazoles/síntesis química , Oxadiazoles/química , Relación Estructura-ActividadRESUMEN
Phenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 µM, Ty38c is bactericidal and active against intracellular bacteria. To investigate its mechanism of action, we isolated mutants resistant to Ty38c and sequenced their genomes. Mutations were found in rv3405c, coding for the transcriptional repressor of the divergently expressed rv3406 gene. Biochemical studies clearly showed that Rv3406 decarboxylates Ty38c into its inactive keto metabolite. The actual target was then identified by isolating Ty38c-resistant mutants of an M. tuberculosis strain lacking rv3406. Here, mutations were found in dprE1, encoding the decaprenylphosphoryl-d-ribose oxidase DprE1, essential for biogenesis of the mycobacterial cell wall. Genetics, biochemical validation, and X-ray crystallography revealed Ty38c to be a noncovalent, noncompetitive DprE1 inhibitor. Structure-activity relationship studies generated a family of DprE1 inhibitors with a range of IC50's and bactericidal activity. Co-crystal structures of DprE1 in complex with eight different quinoxaline analogs provided a high-resolution interaction map of the active site of this extremely vulnerable target in M. tuberculosis.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Quinoxalinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Antituberculosos/síntesis química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Pared Celular/efectos de los fármacos , Pared Celular/enzimología , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Expresión Génica , Enlace de Hidrógeno , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mutación , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Quinoxalinas/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-ActividadRESUMEN
Insulin degrading enzyme (IDE) is a highly conserved zinc metalloprotease that is involved in the clearance of various physiologically peptides like amyloid-beta and insulin. This enzyme has been involved in the physiopathology of diabetes and Alzheimer's disease. We describe here a series of small molecules discovered by screening. Co-crystallization of the compounds with IDE revealed a binding both at the permanent exosite and at the discontinuous, conformational catalytic site. Preliminary structure-activity relationships are described. Selective inhibition of amyloid-beta degradation over insulin hydrolysis was possible. Neuroblastoma cells treated with the optimized compound display a dose-dependent increase in amyloid-beta levels.
Asunto(s)
Acetatos/farmacología , Péptidos beta-Amiloides/antagonistas & inhibidores , Imidazoles/química , Insulisina/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Acetatos/síntesis química , Acetatos/química , Péptidos beta-Amiloides/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Hidrólisis , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb within host cells.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Mycobacterium tuberculosis/aislamiento & purificación , Células Epiteliales Alveolares/microbiología , Humanos , Microscopía Confocal/métodos , Mycobacterium tuberculosis/genética , Fenotipo , ARN Interferente Pequeño/análisis , ARN Interferente Pequeño/genética , Tuberculosis/microbiologíaRESUMEN
In this Letter we describe the design, synthesis, screening, and optimization of a new family of ADAMTS-5 inhibitors. These inhibitors display an original 1,2,4-triazole-3-thiol scaffold as a putative zinc binding-group. In vitro results are rationalized by in silico docking of the compounds in ADAMTS-5's crystal structure.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Triazoles/síntesis química , Triazoles/farmacología , Proteína ADAMTS5 , Sitios de Unión , Simulación por Computador , Evaluación Preclínica de Medicamentos , Humanos , Indicadores y Reactivos , Modelos Moleculares , Osteoartritis/tratamiento farmacológico , Inhibidores de Proteasas/química , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Triazoles/química , Difracción de Rayos XRESUMEN
We report here the parallel synthesis of 200 compounds based on squaric acid template. These compounds are obtained via a one-step solution-phase procedure starting from three squaric acid N-hydroxylamide esters precursors. The set of diverse reagents qualified (amines, anilines, amino-alcohols and amino-esters) makes this strategy suitable for the search of biologically active compounds. The library was screened on the zinc metalloenzyme ADAMTS-5 and hits with IC(50) in the range of 1-50 microM were identified.
Asunto(s)
Proteínas ADAM/metabolismo , Amidas/síntesis química , Técnicas Químicas Combinatorias , Ciclobutanos/síntesis química , Zinc/metabolismo , Proteína ADAMTS5 , Amidas/química , Ciclobutanos/química , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Cerebral capillary endothelial cells constitute the blood-brain barrier (BBB). In these highly specialized cells, transcellular transports rarely occur, and the presence of tight junctions between them leads to a low paracellular permeability. In order to understand the functions of this barrier, an in vitro model of the BBB has been developed and consists in a co-culture of primary cerebral capillary endothelial cells and glial cells. When these endothelial cells are subjected to an inflammatory agent, such as tumor necrosis factor-alpha (TNF-alpha), in vitro BBB permeability is increased, as indicated by the increase in holotransferrin transcytosis. However, no significant change in the paracellular permeability is observed. In order to understand the molecular mechanisms that underlie these transcytosis processes, we investigated the implication of the mitogen-activated protein kinase (MAPK) signalling pathway, as TNF-alpha is known to activate this kinase family. In the present study, an increase in the activation of p42-44 MAPK is observed after TNF-alpha treatment. Holotransferrin transcytosis as well as p42-44 MAPK activation are inhibited after addition of a p42-44 MAPK pathway inhibitor (UO126) during TNF-alpha challenge. These data suggest that the MAPK pathway is involved in the transcytosis regulation in endothelial cells from an in vitro BBB model.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Actinas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Western Blotting/métodos , Butadienos/farmacología , Bovinos , Células Cultivadas , Técnicas de Cocultivo/métodos , Interacciones Farmacológicas , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica/métodos , Manitol/farmacología , Proteínas de la Membrana/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 11 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Neuroglía/efectos de los fármacos , Nitrilos/farmacología , Ocludina , ARN Mensajero/biosíntesis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
A series of 5-nitro-2-furancarbohydrazides were synthesized. In vitro antitrypanosomal activities of these compounds were determined against the closely related protozoa Trypanosoma cruzi Trypanosoma brucei and discussed in relation to potential targets.
Asunto(s)
Tripanocidas/síntesis química , Tripanocidas/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Humanos , Hidrazinas/síntesis química , Hidrazinas/farmacología , Hidrazinas/toxicidad , Concentración 50 Inhibidora , Leishmania infantum/efectos de los fármacos , Macrófagos Peritoneales/parasitología , Ratones , Relación Estructura-Actividad , Tripanocidas/toxicidad , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Prolyl endopeptidases (PEPs) have been found in numerous species. Inhibitors of human enzyme could correct cognitive deficits in Alzheimer patients while inhibition of Trypanosoma cruzi PEP could prevent invasion phase in Chagas disease. A structure-activity relationship study carried out in a tetrahydroisoquinoline series allowed to obtain potent competitive inhibitors superior to SUAM-1221. Besides, inhibitors expected to act according to an irreversible mechanism revealed to be superior to JPT-4819, for applications linked to human enzyme inhibition.