RESUMEN
The 3CL protease (3CLpro, Mpro) plays a key role in the replication of the SARS-CoV-2 and was validated as therapeutic target by the development and approval of specific antiviral drugs (nirmatrelvir, ensitrelvir), inhibitors of this protease. Moreover, its high conservation within the coronavirus family renders it an attractive therapeutic target for the development of anti-coronavirus compounds with broad spectrum activity to control COVID-19 and future coronavirus diseases. Here we report on the design, synthesis and structure-activity relationships of a new series of small covalent reversible inhibitors of the SARS-CoV-2 3CLpro. As elucidated thanks to the X-Ray structure of some inhibitors with the 3CLpro, the mode of inhibition involves acylation of the thiol of the catalytic cysteine. The synthesis of 60 analogs led to the identification of compound 56 that inhibits the SARS-CoV-2 3CLpro with high potency (IC50 = 70 nM) and displays antiviral activity in cells (EC50 = 3.1 µM). Notably, compound 56 inhibits the 3CLpro of three other human coronaviruses and exhibit a good selectivity against two human cysteine proteases. These results demonstrate the potential of this electrophilic N-acylbenzimidazole series as a basis for further optimization.
Asunto(s)
Antivirales , Bencimidazoles , Proteasas 3C de Coronavirus , SARS-CoV-2 , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Relación Estructura-Actividad , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Humanos , Bencimidazoles/farmacología , Bencimidazoles/química , Bencimidazoles/síntesis química , Cisteína Endopeptidasas/metabolismo , Acilación , Cisteína/química , Cisteína/farmacología , Estructura Molecular , Relación Dosis-Respuesta a Droga , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Modelos Moleculares , Diseño de Fármacos , Cristalografía por Rayos XRESUMEN
Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.
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Antibacterianos , Proteínas de Escherichia coli , Escherichia coli , Metronidazol , Nitrorreductasas , Proteínas Represoras , Nitrorreductasas/metabolismo , Nitrorreductasas/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/genética , Metronidazol/farmacología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Antibacterianos/farmacología , Antibacterianos/química , Aerobiosis , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
BACKGROUND AND PURPOSE: Nonalcoholic fatty liver disease refers to liver pathologies, ranging from steatosis to steatohepatitis, with fibrosis ultimately leading to cirrhosis and hepatocellular carcinoma. Although several mechanisms have been suggested, including insulin resistance, oxidative stress, and inflammation, its pathophysiology remains imperfectly understood. Over the last decade, a dysfunctional unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress emerged as one of the multiple driving factors. In parallel, growing evidence suggests that insulin-degrading enzyme (IDE), a highly conserved and ubiquitously expressed metallo-endopeptidase originally discovered for its role in insulin decay, may regulate ER stress and UPR. EXPERIMENTAL APPROACH: We investigated, by genetic and pharmacological approaches, in vitro and in vivo, whether IDE modulates ER stress-induced UPR and lipid accumulation in the liver. KEY RESULTS: We found that IDE-deficient mice display higher hepatic triglyceride content along with higher inositol-requiring enzyme 1 (IRE1) pathway activation. Upon induction of ER stress by tunicamycin or palmitate in vitro or in vivo, pharmacological inhibition of IDE, using its inhibitor BDM44768, mainly exacerbated ER stress-induced IRE1 activation and promoted lipid accumulation in hepatocytes, effects that were abolished by the IRE1 inhibitors 4µ8c and KIRA6. Finally, we identified that IDE knockout promotes lipolysis in adipose tissue and increases hepatic CD36 expression, which may contribute to steatosis. CONCLUSION AND IMPLICATIONS: These results unravel a novel role for IDE in the regulation of ER stress and development of hepatic steatosis. These findings pave the way to innovative strategies modulating IDE to treat metabolic diseases.
RESUMEN
Type 2 diabetes (T2D) is a metabolic disorder characterized by loss of pancreatic ß-cell function, decreased insulin secretion and increased insulin resistance, that affects more than 537 million people worldwide. Although several treatments are proposed to patients suffering from T2D, long-term control of glycemia remains a challenge. Therefore, identifying new potential drugs and targets that positively affect ß-cell function and insulin secretion remains crucial. Here, we developed an automated approach to allow the identification of new compounds or genes potentially involved in ß-cell function in a 384-well plate format, using the murine ß-cell model Min6. By using MALDI-TOF mass spectrometry, we implemented a high-throughput screening (HTS) strategy based on the automation of a cellular assay allowing the detection of insulin secretion in response to glucose, i.e., the quantitative detection of insulin, in a miniaturized system. As a proof of concept, we screened siRNA targeting well-know ß-cell genes and 1600 chemical compounds and identified several molecules as potential regulators of insulin secretion and/or synthesis, demonstrating that our approach allows HTS of insulin secretion in vitro.
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Diabetes Mellitus Tipo 2 , Insulina , Humanos , Animales , Ratones , Insulina/metabolismo , Secreción de Insulina , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/farmacología , Glucosa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Ensayos Analíticos de Alto Rendimiento , Insulina Regular Humana/metabolismoRESUMEN
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.
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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
It is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections.
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Mycobacterium tuberculosis , NAD , Animales , Pez Cebra , Antituberculosos/farmacología , NADH NADPH OxidorreductasasRESUMEN
Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.
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Aminopeptidasas , Presentación de Antígeno , Aminopeptidasas/metabolismo , Antígenos de Histocompatibilidad Clase I , Péptidos/metabolismoRESUMEN
Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.
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Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli/efectos de los fármacos , Lipoproteínas/química , Proteínas de Transporte de Membrana/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Piperazinas/farmacología , Piridinas/farmacología , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Antibacterianos/química , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico/efectos de los fármacos , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Lipoproteínas/antagonistas & inhibidores , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Simulación de Dinámica Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Oligopéptidos/química , Oligopéptidos/farmacología , Oxacilina/química , Oxacilina/farmacología , Piperazinas/síntesis química , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Piridinas/síntesis química , Relación Estructura-ActividadRESUMEN
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.
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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
Rare actinomycetes are likely treasure troves for bioactive natural products, and it is therefore important that we enrich our understanding of biosynthetic potential of these relatively understudied bacteria. Dactylosporangium are a genus of such rare Actinobacteria that are known to produce a number of important antibacterial compounds, but for which there are still no fully assembled reference genomes, and where the extent of encoded biosynthetic capacity is not defined. Dactylosporangium vinaceum (NRRL B-16297) is known to readily produce a deep wine red-coloured diffusible pigment of unknown origin, and it was decided to define the chemical identity of this natural product pigment, and in parallel use whole genome sequencing and transcriptional analysis to lay a foundation for understanding the biosynthetic capacity of these bacteria. Results show that the produced pigment is made of various rubrolone conjugates, the spontaneous product of the reactive pre-rubrolone, produced by the bacterium. Genome and transcriptome analysis identified the highly expressed biosynthetic gene cluster (BGC) for pre-rubrolone. Further analysis of the fully assembled genome found it to carry 24 additional BGCs, of which the majority were poorly transcribed, confirming the encoded capacity of this bacterium to produce natural products but also illustrating the main bottleneck to exploiting this capacity. Finally, analysis of the potential environmental role of pre-rubrolone found it to react with a number of amine containing antibiotics, antimicrobial peptides and siderophores pointing to its potential role as a "minesweeper" of xenobiotic molecules in the bacterial environment. KEY POINTS: ⢠D. vinaceum encodes many BGC, but the majority are transcriptionally silent. ⢠Chemical screening identifies molecules that modulate rubrolone production. ⢠Pre-rubrolone is efficient at binding and inactivating many natural antibiotics.
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Actinobacteria , Productos Biológicos , Micromonosporaceae , Actinobacteria/genética , Familia de Multigenes , PiridinasRESUMEN
PEGylation of therapeutic agents is known to improve the pharmacokinetic behavior of macromolecular drugs and nanoparticles. In this work, we performed the conjugation of polyethylene glycols (220-5000 Da) to a series of non-steroidal small agonists of the bile acids receptor TGR5. A suitable anchoring position on the agonist was identified to retain full agonistic potency with the conjugates. We describe herein an extensive structure-properties relationships study allowing us to finely describe the non-linear effects of the PEG length on the physicochemical as well as the in vitro and in vivo pharmacokinetic properties of these compounds. When appending a PEG of suitable length to the TGR5 pharmacophore, we were able to identify either systemic or gut lumen-restricted TGR5 agonists.
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Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Barrera Hematoencefálica/metabolismo , Células CACO-2 , Células HEK293 , Humanos , Hipoglucemiantes/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Polietilenglicoles/química , Receptores Acoplados a Proteínas G/química , Relación Estructura-ActividadRESUMEN
Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments.
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Proteínas Bacterianas/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , ortoaminobenzoatos/farmacología , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Ácido Graso Sintasas/metabolismo , Estructura Molecular , Relación Estructura-Actividad , ortoaminobenzoatos/químicaRESUMEN
In the last 5 years, cellular thermal shift assay (CETSA), a technology based on ligand-induced changes in protein thermal stability, has been increasingly used in drug discovery to address the fundamental question of whether drug candidates engage their intended target in a biologically relevant setting. To analyze lysates from cells submitted to increasing temperature, the detection and quantification of the remaining soluble protein can be achieved using quantitative mass spectrometry, Western blotting, or AlphaScreen techniques. Still, these approaches can be time- and cell-consuming. To cope with limitations of throughput and protein amount requirements, we developed a new coupled assay combining the advantages of a nanoacoustic transfer system and reverse-phase protein array technology within CETSA experiments. We validated the technology to assess engagement of inhibitors of insulin-degrading enzyme (IDE), an enzyme involved in diabetes and Alzheimer's disease. CETSA-acoustic reverse-phase protein array (CETSA-aRPPA) allows simultaneous analysis of many conditions and drug-target engagement with a small sample size, in a rapid, cost-effective, and biological material-saving manner.
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Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento/métodos , Análisis por Matrices de Proteínas/métodos , Proteínas/genética , Acústica , Línea Celular Tumoral , Humanos , Ligandos , Preparaciones Farmacéuticas/química , Proteínas/aislamiento & purificaciónRESUMEN
The proximity ligation assay (PLA) allows the detection and subcellular localization of protein-protein interactions with high specificity. We recently developed a high-content screening model based on primary hippocampal neurons cultured in 384-well plates and screened a library of â¼1100 compounds using a PLA between tau and bridging integrator 1, a genetic risk factor for Alzheimer's disease. We developed image-segmentation and spot-detection algorithms to delineate PLA signals in the axonal network, but not in cell bodies, from confocal images acquired via a high-throughput microscope. To compare data generated from different plates and through different experiments, we developed a computational routine to optimize the image analysis parameters for each plate and devised a range of quality-control measures to ultimately identify compounds that consistently increase or decrease our read-out. We provide the following protocols. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Routine culture of rat postnatal hippocampal neurons in 384-well plates Basic Protocol 2: Compound incubation using the high-content screening platform Support Protocol 1: Preparation of intermediate plates for compound screening Support Protocol 2: Preparation of intermediate plates for hit validation (dose-response curves) Basic Protocol 3: Proximity ligation assay in 384-well plates Basic Protocol 4: Image acquisition and analysis Support Protocol 3: Optimization of analysis parameters Basic Protocol 5: Identification of hits Basic Protocol 6: Validation of hits based on dose-response curves.
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Procesamiento de Imagen Asistido por Computador , Neuronas/patología , Mapeo de Interacción de Proteínas , Algoritmos , Animales , Procesamiento de Imagen Asistido por Computador/métodos , Solución de Problemas , Mapeo de Interacción de Proteínas/métodos , RatasRESUMEN
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.
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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
The bridging integrator 1 gene (BIN1) is a major genetic risk factor for Alzheimer's disease (AD). In this report, we investigated how BIN1-dependent pathophysiological processes might be associated with Tau. We first generated a cohort of control and transgenic mice either overexpressing human MAPT (TgMAPT) or both human MAPT and BIN1 (TgMAPT;TgBIN1), which we followed-up from 3 to 15 months. In TgMAPT;TgBIN1 mice short-term memory deficits appeared earlier than in TgMAPT mice; however-unlike TgMAPT mice-TgMAPT;TgBIN1 mice did not exhibit any long-term or spatial memory deficits for at least 15 months. After killing the cohort at 18 months, immunohistochemistry revealed that BIN1 overexpression prevents both Tau mislocalization and somatic inclusion in the hippocampus, where an increase in BIN1-Tau interaction was also observed. We then sought mechanisms controlling the BIN1-Tau interaction. We developed a high-content screening approach to characterize modulators of the BIN1-Tau interaction in an agnostic way (1,126 compounds targeting multiple pathways), and we identified-among others-an inhibitor of calcineurin, a Ser/Thr phosphatase. We determined that calcineurin dephosphorylates BIN1 on a cyclin-dependent kinase phosphorylation site at T348, promoting the open conformation of the neuronal BIN1 isoform. Phosphorylation of this site increases the availability of the BIN1 SH3 domain for Tau interaction, as demonstrated by nuclear magnetic resonance experiments and in primary neurons. Finally, we observed that although the levels of the neuronal BIN1 isoform were unchanged in AD brains, phospho-BIN1(T348):BIN1 ratio was increased, suggesting a compensatory mechanism. In conclusion, our data support the idea that BIN1 modulates the AD risk through an intricate regulation of its interaction with Tau. Alteration in BIN1 expression or activity may disrupt this regulatory balance with Tau and have direct effects on learning and memory.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Trastornos de la Memoria/metabolismo , Memoria a Largo Plazo/fisiología , Proteínas del Tejido Nervioso/metabolismo , Tauopatías/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas tau/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Trastornos de la Memoria/genética , Trastornos de la Memoria/patología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Memoria Espacial/fisiología , Tauopatías/genética , Tauopatías/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis, represents one of the most challenging threat to public health worldwide, and with the increasing resistance to approved TB drugs, it is needed to develop new strategies to address this issue. Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant TB. It is a prodrug that requires activation by mycobacterial monooxygenases to inhibit the enoyl-ACP reductase InhA, which is involved in mycolic acid biosynthesis. Very recently, we identified that inhibition of a transcriptional repressor, termed EthR2, derepresses a new bioactivation pathway that results in the boosting of ethionamide activation. Herein, we describe the identification of potent EthR2 inhibitors using fragment-based screening and structure-based optimization. A target-based screening of a fragment library using thermal shift assay followed by X-ray crystallography identified 5 hits. Rapid optimization of the tropinone chemical series led to compounds with improved in vitro potency.
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Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Represoras/antagonistas & inhibidores , Tropanos/farmacología , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Etionamida/metabolismo , Humanos , Mycobacterium tuberculosis/química , Tropanos/síntesis químicaRESUMEN
The transcriptional repressor EthR from Mycobacterium tuberculosis, a member of the TetR family of prokaryotic homo-dimeric transcription factors, controls the expression of the mycobacterial mono-oxygenase EthA. EthA is responsible for the bio-activation of the second-line tuberculosis pro-drug ethionamide, and consequently EthR inhibitors boost drug efficacy. Here, we present a comprehensive in silico structure-based screening protocol that led to the identification of a number of novel scaffolds of EthR inhibitors in subsequent biophysical screening by thermal shift assay. Growth inhibition assays demonstrated that five of the twenty biophysical hits were capable of boosting ethionamide activity in vitro, with the best novel scaffold displaying an EC50 of 34 µM. In addition, the co-crystal structures of EthR with four new ligands at resolution ranging from 2.1 to 1.4 Å confirm the binding and inactivation mode, and will enable future lead development.
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Antituberculosos/uso terapéutico , Descubrimiento de Drogas , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Antituberculosos/síntesis química , Antituberculosos/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/crecimiento & desarrolloRESUMEN
Hydroxamic acids are outstanding zinc chelating groups that can be used to design potent and selective metalloenzyme inhibitors in various therapeutic areas. Some hydroxamic acids display a high plasma clearance resulting in poor in vivo activity, though they may be very potent compounds in vitro. We designed a 57-member library of hydroxamic acids to explore the structure-plasma stability relationships in these series and to identify which enzyme(s) and which pharmacophores are critical for plasma stability. Arylesterases and carboxylesterases were identified as the main metabolic enzymes for hydroxamic acids. Finally, we suggest structural features to be introduced or removed to improve stability. This work thus provides the first medicinal chemistry toolbox (experimental procedures and structural guidance) to assess and control the plasma stability of hydroxamic acids and realize their full potential as in vivo pharmacological probes and therapeutic agents. This study is particularly relevant to preclinical development as it allows obtaining compounds equally stable in human and rodent models.
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Ácidos Hidroxámicos/química , Plasma/química , Bibliotecas de Moléculas Pequeñas , Animales , Hidrolasas de Éster Carboxílico , Estabilidad de Medicamentos , Humanos , Tasa de Depuración Metabólica , Ratones , Plasma/enzimología , Ratas , Relación Estructura-ActividadRESUMEN
The role of the G-protein-coupled bile acid receptor TGR5 in various organs, tissues, and cell types, specifically in intestinal endocrine L-cells and brown adipose tissue, has made it a promising therapeutical target in several diseases, especially type-2 diabetes and metabolic syndrome. However, recent studies have shown deleterious on-target effects of systemic TGR5 agonists. To avoid these systemic effects while stimulating glucagon-like peptide-1 (GLP-1) secreting enteroendocrine L-cells, we have designed TGR5 agonists with low intestinal permeability. In this article, we describe their synthesis, characterization, and biological evaluation. Among them, compound 24 is a potent GLP-1 secretagogue, has low effect on gallbladder volume, and improves glucose homeostasis in a preclinical murine model of diet-induced obesity and insulin resistance, making the proof of concept of the potential of topical intestinal TGR5 agonists as therapeutic agents in type-2 diabetes.