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1.
Nat Commun ; 12(1): 76, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397953

RESUMEN

Full development of IL-17 producing CD4+ T helper cells (TH17 cells) requires the transcriptional activity of both orphan nuclear receptors RORα and RORγt. However, RORα is considered functionally redundant to RORγt; therefore, the function and therapeutic value of RORα in TH17 cells is unclear. Here, using mouse models of autoimmune and chronic inflammation, we show that expression of RORα is required for TH17 cell pathogenicity. T-cell-specific deletion of RORα reduces the development of experimental autoimmune encephalomyelitis (EAE) and colitis. Reduced inflammation is associated with decreased TH17 cell development, lower expression of tissue-homing chemokine receptors and integrins, and increased frequencies of Foxp3+ T regulatory cells. Importantly, inhibition of RORα with a selective small molecule antagonist mostly phenocopies our genetic data, showing potent suppression of the in vivo development of both chronic/progressive and relapsing/remitting EAE, but with no effect on overall thymic cellularity. Furthermore, use of the RORα antagonist effectively inhibits human TH17 cell differentiation and memory cytokine secretion. Together, these data suggest that RORα functions independent of RORγt in programming TH17 pathogenicity and identifies RORα as a safer and more selective therapeutic target for the treatment of TH17-mediated autoimmunity.


Asunto(s)
Inflamación/inmunología , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Células Th17/inmunología , Animales , Autoinmunidad/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Enfermedad Crónica , Colon/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental , Células HEK293 , Humanos , Inflamación/genética , Ratones Endogámicos C57BL , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Tamaño de los Órganos/efectos de los fármacos , Índice de Severidad de la Enfermedad , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Tiofenos/química , Tiofenos/farmacología
2.
Nat Commun ; 12(1): 102, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397994

RESUMEN

Pro-inflammatory activation of adipose tissue macrophages (ATMs) is causally linked to obesity and obesity-associated disorders. A number of studies have demonstrated the crucial role of mitochondrial metabolism in macrophage activation. However, there is a lack of pharmaceutical agents to target the mitochondrial metabolism of ATMs for the treatment of obesity-related diseases. Here, we characterize a near-infrared fluorophore (IR-61) that preferentially accumulates in the mitochondria of ATMs and has a therapeutic effect on diet-induced obesity as well as obesity-associated insulin resistance and fatty liver. IR-61 inhibits the classical activation of ATMs by increasing mitochondrial complex levels and oxidative phosphorylation via the ROS/Akt/Acly pathway. Taken together, our findings indicate that specific enhancement of ATMs oxidative phosphorylation improves chronic inflammation and obesity-related disorders. IR-61 might be an anti-inflammatory agent useful for the treatment of obesity-related diseases by targeting the mitochondria of ATMs.


Asunto(s)
Tejido Adiposo/metabolismo , Sistemas de Liberación de Medicamentos , Macrófagos/metabolismo , Mitocondrias/metabolismo , Obesidad/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Peso Corporal/efectos de los fármacos , Hígado Graso/genética , Hígado Graso/patología , Inflamación/genética , Inflamación/patología , Resistencia a la Insulina , Hígado/metabolismo , Hígado/patología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Obesidad/genética , Obesidad/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células RAW 264.7 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Pérdida de Peso/efectos de los fármacos
3.
Nat Commun ; 12(1): 582, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33495441

RESUMEN

Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors.


Asunto(s)
Multimerización de Proteína/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/química , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Necrosis Tumoral alfa/química , Algoritmos , Animales , Unión Competitiva/efectos de los fármacos , Humanos , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Factor de Necrosis Tumoral alfa/metabolismo
4.
Nat Commun ; 12(1): 583, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33495445

RESUMEN

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Complejos Multiproteicos/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos Monoclonales/farmacología , Células Cultivadas , Cristalografía por Rayos X , Epítopos/química , Epítopos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Complejos Multiproteicos/química , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/química , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Necrosis Tumoral alfa/química
5.
PLoS One ; 16(1): e0245975, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33493227

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak is a public health emergency of international concern. The spike glycoprotein (S protein) of SARS-CoV-2 is a key target of antiviral drugs. Focusing on the existing S protein structure, molecular docking was used in this study to calculate the binding energy and interaction sites between 14 antiviral molecules with different structures and the SARS-CoV-2 S protein, and the potential drug candidates targeting the SARS-CoV-2 S protein were analyzed. Tizoxanide, dolutegravir, bictegravir, and arbidol were found to have high binding energies, and they effectively bind key sites of the S1 and S2 subunits, inhibiting the virus by causing conformational changes in S1 and S2 during the fusion of the S protein with host cells. Based on the interactions among the drug molecules, the S protein and the amino acid environment around the binding sites, rational structure-based optimization was performed using the molecular connection method and bioisosterism strategy to obtain Ti-2, BD-2, and Ar-3, which have much stronger binding ability to the S protein than the original molecules. This study provides valuable clues for identifying S protein inhibitor binding sites and the mechanism of the anti-SARS-CoV-2 effect as well as useful inspiration and help for the discovery and optimization of small molecule S protein inhibitors.


Asunto(s)
Antivirales/farmacología , Diseño de Fármacos , Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas/farmacología , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Antivirales/química , /virología , Humanos , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas Pequeñas/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos
6.
Nat Commun ; 12(1): 653, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33510147

RESUMEN

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4+ T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.


Asunto(s)
Carcinoma Pulmonar de Lewis/terapia , Inmunoterapia/métodos , Receptores Purinérgicos P2X7/inmunología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/inmunología , Línea Celular Tumoral , Terapia Combinada , Femenino , Células HEK293 , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-18/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Estructura Molecular , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunología
7.
Phys Chem Chem Phys ; 23(2): 1197-1214, 2021 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-33355332

RESUMEN

Uncompetitive antagonists of the N-methyl d-aspartate receptor (NMDAR) have demonstrated therapeutic benefit in the treatment of neurological diseases such as Parkinson's and Alzheimer's, but some also cause dissociative effects that have led to the synthesis of illicit drugs. The ability to generate NMDAR antagonists in silico is therefore desirable for both new medication development and preempting and identifying new designer drugs. Recently, generative deep learning models have been applied to de novo drug design as a means to expand the amount of chemical space that can be explored for potential drug-like compounds. In this study, we assess the application of a generative model to the NMDAR to achieve two primary objectives: (i) the creation and release of a comprehensive library of experimentally validated NMDAR phencyclidine (PCP) site antagonists to assist the drug discovery community and (ii) an analysis of both the advantages conferred by applying such generative artificial intelligence models to drug design and the current limitations of the approach. We apply, and provide source code for, a variety of ligand- and structure-based assessment techniques used in standard drug discovery analyses to the deep learning-generated compounds. We present twelve candidate antagonists that are not available in existing chemical databases to provide an example of what this type of workflow can achieve, though synthesis and experimental validation of these compounds are still required.


Asunto(s)
Aprendizaje Profundo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Animales , Sitios de Unión , Diseño de Fármacos , Ligandos , Ratones , Estructura Molecular , Receptores de N-Metil-D-Aspartato/química , Xenopus laevis
8.
PLoS One ; 15(12): e0243041, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33362250

RESUMEN

Alzheimer disease (AD) is a neurodegenerative disorder characterized by the aberrant production and accumulation of amyloid-ß (Aß) peptides in the brain. Accumulated Aß in soluble oligomer and insoluble plaque forms are considered to be a pathological culprit and biomarker of the disorder. Here, we report a fluorescent universal Aß-indicator YI-13, 5-(4-fluorobenzoyl)-7,8-dihydropyrrolo[1,2-b]isoquinolin-9(6H)-one, which detects Aß monomers, dimers, and plaques. We synthesized a library of 26 fluorescence chemicals with the indolizine core and screen them through a series of in vitro tests utilizing Aß as a target and YI-13 was selected as the final imaging candidate. YI-13 was found to stain and visualize insoluble Aß plaques in the brain tissue, of a transgenic mouse model with five familial AD mutations (5XFAD), by a histochemical approach and to label soluble Aß oligomers within brain lysates of the mouse model under a fluorescence plate reader. Among oligomers aggregated from monomers and synthetic dimers from chemically conjugated monomers, YI-13 preferred the dimeric Aß.


Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Indolizinas/síntesis química , Indolizinas/farmacología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Fluorescencia , Humanos , Indolizinas/química , Ratones , Ratones Transgénicos , Estructura Molecular , Mutación , Multimerización de Proteína , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología
9.
PLoS One ; 15(12): e0243746, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33315900

RESUMEN

Niemann-Pick disease type C is a rare, fatal neurodegenerative disorder characterized by massive intracellular accumulation of cholesterol. In most cases, loss-of-function mutations in the NPC1 gene that encodes lysosomal cholesterol transporter NPC1 are responsible for the disease, and more than half of the mutations are considered to interfere with the biogenesis or folding of the protein. We previously identified a series of oxysterol derivatives and phenanthridine-6-one derivatives as pharmacological chaperones, i.e., small molecules that can rescue folding-defective phenotypes of mutated NPC1, opening up an avenue to develop chaperone therapy for Niemann-Pick disease type C. Here, we present an improved image-based screen for NPC1 chaperones and we describe its application for drug-repurposing screening. We identified some azole antifungals, including itraconazole and posaconazole, and a kinase inhibitor, lapatinib, as probable pharmacological chaperones. A photo-crosslinking study confirmed direct binding of itraconazole to a representative folding-defective mutant protein, NPC1-I1061T. Competitive photo-crosslinking experiments suggested that oxysterol-based chaperones and itraconazole share the same or adjacent binding site(s), and the sensitivity of the crosslinking to P691S mutation in the sterol-sensing domain supports the hypothesis that their binding sites are located near this domain. Although the azoles were less effective in reducing cholesterol accumulation than the oxysterol-derived chaperones or an HDAC inhibitor, LBH-589, our findings should offer new starting points for medicinal chemistry efforts to develop better pharmacological chaperones for NPC1.


Asunto(s)
Descubrimiento de Drogas/métodos , Péptidos y Proteínas de Señalización Intracelular/genética , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Pliegue de Proteína/efectos de los fármacos , Reposicionamiento de Medicamentos/métodos , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Mutación/efectos de los fármacos , Enfermedad de Niemann-Pick Tipo C/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología
10.
PLoS One ; 15(12): e0243901, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33378376

RESUMEN

Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2ß) functions as a partial agonist at ErbB4. NRG2ß/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.


Asunto(s)
Proliferación Celular/genética , Melanoma/genética , Factores de Crecimiento Nervioso/genética , Receptor ErbB-4/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas , Mutación con Ganancia de Función/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/patología , Fosforilación/genética , Receptor ErbB-4/agonistas , Receptor ErbB-4/antagonistas & inhibidores , Transducción de Señal/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología
11.
Proc Natl Acad Sci U S A ; 117(52): 33197-33203, 2020 12 29.
Artículo en Inglés | MEDLINE | ID: mdl-33318191

RESUMEN

RNAs have important functions that are dictated by their structure. Indeed, small molecules that interact with RNA structures can perturb function, serving as chemical probes and lead medicines. Here we describe the development of a fragment-based approach to discover and optimize bioactive small molecules targeting RNA. We extended the target validation method chemical cross-linking and isolation by pull-down (Chem-CLIP) to identify and map the binding sites of low molecular weight fragments that engage RNA or Chem-CLIP fragment mapping (Chem-CLIP-Frag-Map). Using Chem-CLIP-Frag-Map, we identified several fragments that bind the precursor to oncogenic microRNA-21 (pre-miR-21). Assembly of these fragments provided a specific bioactive compound with improved potency that inhibits pre-miR-21 processing, reducing mature miR-21 levels. The compound exerted selective effects on the transcriptome and selectively mitigated a miR-21-associated invasive phenotype in triple-negative breast cancer cells. The Chem-CLIP-Frag-Map approach should prove general to expedite the identification and optimization of small molecules that bind RNA targets.


Asunto(s)
Antineoplásicos/química , Descubrimiento de Drogas/métodos , MicroARNs/química , Bibliotecas de Moléculas Pequeñas/química , Antineoplásicos/farmacología , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Ligandos , MicroARNs/metabolismo , Simulación del Acoplamiento Molecular , Motivos de Nucleótidos , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias de la Mama Triple Negativas/metabolismo
12.
J Vis Exp ; (166)2020 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-33346194

RESUMEN

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as "broken relics", of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.


Asunto(s)
Bioprospección , Técnicas Biosensibles/métodos , Biología Computacional , Preparaciones Farmacéuticas/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Técnicas de Visualización de Superficie Celular , Humanos , Ligandos , Biblioteca de Péptidos , Péptidos/química , Preparaciones Farmacéuticas/química , Unión Proteica , Tecnicas de Microbalanza del Cristal de Cuarzo , Receptores de Superficie Celular/metabolismo , Bibliotecas de Moléculas Pequeñas/química
13.
Biomolecules ; 10(11)2020 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-33172012

RESUMEN

Natural products and semi-synthetic compounds continue to be a significant source of drug candidates for a broad range of diseases, including coronavirus disease 2019 (COVID-19), which is causing the current pandemic. Besides being attractive sources of bioactive compounds for further development or optimization, natural products are excellent substrates of unique substructures for fragment-based drug discovery. To this end, fragment libraries should be incorporated into automated drug design pipelines. However, public fragment libraries based on extensive collections of natural products are still limited. Herein, we report the generation and analysis of a fragment library of natural products derived from a database with more than 400,000 compounds. We also report fragment libraries of a large food chemical database and other compound datasets of interest in drug discovery, including compound libraries relevant for COVID-19 drug discovery. The fragment libraries were characterized in terms of content and diversity.


Asunto(s)
Productos Biológicos/química , Descubrimiento de Drogas , Algoritmos , Betacoronavirus/aislamiento & purificación , Productos Biológicos/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Bases de Datos de Compuestos Químicos , Humanos , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico
14.
Nat Commun ; 11(1): 5047, 2020 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-33028810

RESUMEN

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.


Asunto(s)
Betacoronavirus/química , Cisteína Endopeptidasas/química , Fragmentos de Péptidos/química , Proteínas no Estructurales Virales/química , Betacoronavirus/enzimología , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Cisteína Endopeptidasas/metabolismo , Diseño de Fármacos , Espectrometría de Masas , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Electricidad Estática , Proteínas no Estructurales Virales/metabolismo
15.
Anticancer Res ; 40(9): 4929-4935, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32878781

RESUMEN

BACKGROUND: Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are frequently found in various cancer types. IDH1 mutants produce 2-hydroxyglutarate (2-HG), an oncometabolite, from alpha-ketoglutarate (α-KG). This 2-HG plays a key role in tumorigenesis via inhibition of α-KG dependent enzymes. For this reason, IDH1 mutant could be an ideal target for the treatment of cancer. MATERIALS AND METHODS: To find a new IDH1 inhibitor, 8,364 compounds were obtained from Korea Chemical Bank. Using high-throughput screening (HTS) of a chemical library, we unveiled a compound that could inhibit the IDH1 mutant. RESULTS: According to the enzyme assay, our compound (KRC-09) effectively inhibited the activity of IDH1 R132H mutant. In addition, KRC-09 decreased the concentration of intracellular 2-HG in the U-87 MG cell line harboring IDH1 R132H. CONCLUSION: In this article, we present a novel chemical scaffold that suppresses the activity of an IDH1 mutant.


Asunto(s)
Antineoplásicos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/genética , Antineoplásicos/química , Línea Celular Tumoral , Descubrimiento de Drogas , Glutaratos/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Estructura Molecular , Mutación , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología
16.
Nat Commun ; 11(1): 4659, 2020 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-32938936

RESUMEN

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.


Asunto(s)
Butiratos/farmacología , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Integrinas/antagonistas & inhibidores , Naftiridinas/farmacología , Pirazoles/farmacología , Pirrolidinas/farmacología , Administración por Inhalación , Animales , Antígenos de Neoplasias/metabolismo , Bleomicina/toxicidad , Butiratos/administración & dosificación , Butiratos/metabolismo , Butiratos/farmacocinética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Humanos , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/patología , Integrinas/metabolismo , Masculino , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Naftiridinas/administración & dosificación , Naftiridinas/metabolismo , Naftiridinas/farmacocinética , Pirazoles/administración & dosificación , Pirazoles/metabolismo , Pirazoles/farmacocinética , Pirrolidinas/administración & dosificación , Pirrolidinas/metabolismo , Pirrolidinas/farmacocinética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tomografía Computarizada de Emisión de Fotón Único , Factor de Crecimiento Transformador beta/metabolismo , Investigación en Medicina Traslacional
17.
J Mol Biol ; 432(21): 5843-5847, 2020 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-32920049

RESUMEN

SARS-CoV-2 uses -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins. Because modulating -1 PRF can attenuate the virus, ligands binding to the RNA pseudoknot that stimulates -1 PRF may have therapeutic potential. Mutations in the pseudoknot have occurred during the pandemic, but how they affect -1 PRF efficiency and ligand activity is unknown. Studying a panel of six mutations in key regions of the pseudoknot, we found that most did not change -1 PRF levels, even when base-pairing was disrupted, but one led to a striking 3-fold decrease, suggesting SARS-CoV-2 may be less sensitive to -1 PRF modulation than expected. Examining the effects of a small-molecule -1 PRF inhibitor active against SARS-CoV-2, it had a similar effect on all mutants tested, regardless of basal -1 PRF efficiency, indicating that anti-frameshifting activity can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of -1 PRF.


Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Sistema de Lectura Ribosómico/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Neumonía Viral/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Antivirales/química , Betacoronavirus/genética , Infecciones por Coronavirus/virología , Humanos , Ligandos , Mutación/efectos de los fármacos , Pandemias , Neumonía Viral/virología , ARN Mensajero/genética , ARN Viral/genética , Bibliotecas de Moléculas Pequeñas/química , Proteínas Virales/genética
18.
J Mol Graph Model ; 100: 107697, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32739642

RESUMEN

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound zinc metallopeptidase that generates the vasodilatory peptide angiotensin 1-7 and thus performs a protective role in heart disease. It is considered an important therapeutic target in controlling the COVID-19 outbreak, since SARS-CoV-2 enters permissive cells via an ACE2-mediated mechanism. The present in silico study attempted to repurpose existing drugs for use as prospective viral-entry inhibitors targeting human ACE2. Initially, a clinically approved drug library of 7,173 ligands was screened against the receptor using molecular docking, followed by energy minimization and rescoring of docked ligands. Finally, potential binders were inspected to ensure molecules with different scaffolds were engaged in favorable contacts with both the metal cofactor and the critical residues lining the receptor's active site. The results of the calculations suggest that lividomycin, burixafor, quisinostat, fluprofylline, pemetrexed, spirofylline, edotecarin, and diniprofylline emerge as promising repositionable drug candidates for stabilizing the closed (substrate/inhibitor-bound) conformation of ACE2, thereby shifting the relative positions of the receptor's critical exterior residues recognized by SARS-CoV-2. This study is among the rare ones in the relevant scientific literature to search for potential ACE2 inhibitors. In practical terms, the drugs, unmodified as they are, may be introduced into the therapeutic armamentarium of the ongoing fight against COVID-19 now, or their scaffolds may serve as rich skeletons for designing novel ACE2 inhibitors in the near future.


Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Antivirales/química , Betacoronavirus/química , Peptidil-Dipeptidasa A/química , Bibliotecas de Moléculas Pequeñas/química , Secuencias de Aminoácidos , Betacoronavirus/enzimología , Carbazoles/química , Dominio Catalítico , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Reposicionamiento de Medicamentos , Difilina/análogos & derivados , Difilina/química , Interacciones Huésped-Patógeno , Humanos , Ácidos Hidroxámicos/química , Ligandos , Simulación del Acoplamiento Molecular , Pandemias , Paromomicina/análogos & derivados , Paromomicina/química , Pemetrexed/química , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Termodinámica
19.
PLoS One ; 15(7): e0236710, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32735606

RESUMEN

Argonaute (AGO) proteins are the key component of the RNA interference machinery that suppresses gene expression by forming an RNA-induced silencing complex (RISC) with microRNAs (miRNAs). Each miRNA is involved in various cellular processes, such as development, differentiation, tumorigenesis, and viral infection. Thus, molecules that regulate miRNA function are expected to have therapeutic potential. In addition, the biogenesis of miRNA is a multistep process involving various proteins, although the complete pathway remains to be elucidated. Therefore, identification of molecules that can specifically modulate each step will help understand the mechanism of gene suppression. To date, several AGO2 inhibitors have been identified. However, these molecules were identified through a single screening method, and no studies have specifically evaluated a combinatorial strategy. Here, we demonstrated a combinatorial screening (SCR) approach comprising an in silico molecular docking study, surface plasmon resonance (SPR) analysis, and nuclear magnetic resonance (NMR) analysis, focusing on the strong binding between the 5'-terminal phosphate of RNA and the AGO2 middle (MID) domain. By combining SPR and NMR, we identified binding modes of amino acid residues binding to AGO2. First, using a large chemical library (over 6,000,000 compounds), 171 compounds with acidic functional groups were screened using in silico SCR. Next, we constructed an SPR inhibition system that could analyze only the 5'-terminal binding site of RNA, and nine molecules that strongly bound to the AGO2 MID domain were selected. Finally, using NMR, three molecules that bound to the desired site were identified. The RISC inhibitory ability of the "hit" compounds was analyzed in human cell lysate, and all three hit compounds strongly inhibited the binding between double-stranded RNA and AGO2.


Asunto(s)
Proteínas Argonauta/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Espectroscopía de Resonancia Magnética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Resonancia por Plasmón de Superficie , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Simulación por Computador , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Conformación Proteica , Bibliotecas de Moléculas Pequeñas/metabolismo
20.
Sci Rep ; 10(1): 13093, 2020 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-32753646

RESUMEN

A novel coronavirus, named SARS-CoV-2, emerged in 2019 in China and rapidly spread worldwide. As no approved therapeutics exists to treat COVID-19, the disease associated to SARS-Cov-2, there is an urgent need to propose molecules that could quickly enter into clinics. Repurposing of approved drugs is a strategy that can bypass the time-consuming stages of drug development. In this study, we screened the PRESTWICK CHEMICAL LIBRARY composed of 1,520 approved drugs in an infected cell-based assay. The robustness of the screen was assessed by the identification of drugs that already demonstrated in vitro antiviral effect against SARS-CoV-2. Thereby, 90 compounds were identified as positive hits from the screen and were grouped according to their chemical composition and their known therapeutic effect. Then EC50 and CC50 were determined for a subset of 15 compounds from a panel of 23 selected drugs covering the different groups. Eleven compounds such as macrolides antibiotics, proton pump inhibitors, antiarrhythmic agents or CNS drugs emerged showing antiviral potency with 2 < EC50 ≤ 20 µM. By providing new information on molecules inhibiting SARS-CoV-2 replication in vitro, this study provides information for the selection of drugs to be further validated in vivo. Disclaimer: This study corresponds to the early stages of antiviral development and the results do not support by themselves the use of the selected drugs to treat SARS-CoV-2 infection.


Asunto(s)
Betacoronavirus/fisiología , Bibliotecas de Moléculas Pequeñas/química , Animales , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Betacoronavirus/aislamiento & purificación , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Aprobación de Drogas , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Humanos , Pandemias , Neumonía Viral/patología , Neumonía Viral/virología , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Células Vero , Replicación Viral/efectos de los fármacos
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