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2.
Nat Commun ; 11(1): 4370, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873792

RESUMO

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sítio Alostérico/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/genética , Simulação de Acoplamento Molecular , Mutação , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/ultraestrutura , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
3.
Anticancer Res ; 40(9): 4929-4935, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878781

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Antineoplásicos/química , Linhagem Celular Tumoral , Descoberta de Drogas , Glutaratos/metabolismo , Humanos , Isocitrato Desidrogenase/metabolismo , Estrutura Molecular , Mutação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
4.
Nat Commun ; 11(1): 4659, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938936

RESUMO

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.


Assuntos
Butiratos/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Integrinas/antagonistas & inibidores , Naftiridinas/farmacologia , Pirazóis/farmacologia , Pirrolidinas/farmacologia , Administração por Inalação , Animais , Antígenos de Neoplasias/metabolismo , Bleomicina/toxicidade , Butiratos/administração & dosagem , Butiratos/metabolismo , Butiratos/farmacocinética , Colágeno/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/patologia , Integrinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Naftiridinas/administração & dosagem , Naftiridinas/metabolismo , Naftiridinas/farmacocinética , Pirazóis/administração & dosagem , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirrolidinas/administração & dosagem , Pirrolidinas/metabolismo , Pirrolidinas/farmacocinética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tomografia Computadorizada de Emissão de Fóton Único , Fator de Crescimento Transformador beta/metabolismo , Pesquisa Médica Translacional
5.
Anal Bioanal Chem ; 412(28): 7685-7699, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32870351

RESUMO

Pathogen-host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane-protein interactions were found to mediate E. coli invasion of the blood-brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)-host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract.


Assuntos
Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , Ensaios de Triagem em Larga Escala/instrumentação , Dispositivos Lab-On-A-Chip , Testes de Sensibilidade Microbiana/instrumentação , Animais , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/imunologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Citocinas/imunologia , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/métodos , Humanos , Testes de Sensibilidade Microbiana/métodos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Bibliotecas de Moléculas Pequenas/farmacologia , Vírion/efeitos dos fármacos , Vírion/imunologia , Viroses/tratamento farmacológico , Viroses/imunologia
6.
Nat Commun ; 11(1): 4761, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958762

RESUMO

Chemical synthesis based on the skeletal variation has been prolifically utilized as an attractive approach for modification of molecular properties. Given the ubiquity of unstrained cyclic amines, the ability to directly alter such motifs would grant an efficient platform to access unique chemical space. Here, we report a highly efficient and practical strategy that enables the selective ring-opening functionalization of unstrained cyclic amines. The use of difluorocarbene leads to a wide variety of multifaceted acyclic architectures, which can be further diversified to a range of distinctive homologative cyclic scaffolds. The virtue of this deconstructive strategy is demonstrated by successful modification of several natural products and pharmaceutical analogues.


Assuntos
Aminas/química , Aminas/síntese química , Hidrocarbonetos Fluorados/química , Técnicas de Química Sintética , Estrutura Molecular , Bibliotecas de Moléculas Pequenas , Estereoisomerismo
7.
J Chem Inf Model ; 60(8): 3910-3934, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32786511

RESUMO

Protein-protein interactions (PPIs) are attractive targets for drug design because of their essential role in numerous cellular processes and disease pathways. However, in general, PPIs display exposed binding pockets at the interface, and as such, have been largely unexploited for therapeutic interventions with low-molecular weight compounds. Here, we used docking and various rescoring strategies in an attempt to recover PPI inhibitors from a set of active and inactive molecules for 11 targets collected in ChEMBL and PubChem. Our focus is on the screening power of the various developed protocols and on using fast approaches so as to be able to apply such a strategy to the screening of ultralarge libraries in the future. First, we docked compounds into each target using the fast "pscreen" mode of the structure-based virtual screening (VS) package Surflex. Subsequently, the docking poses were postprocessed to derive a set of 3D topological descriptors: (i) shape similarity and (ii) interaction fingerprint similarity with a co-crystallized inhibitor, (iii) solvent-accessible surface area, and (iv) extent of deviation from the geometric center of a reference inhibitor. The derivatized descriptors, together with descriptor-scaled scoring functions, were utilized to investigate possible impacts on VS performance metrics. Moreover, four standalone scoring functions, RF-Score-VS (machine-learning), DLIGAND2 (knowledge-based), Vinardo (empirical), and X-SCORE (empirical), were employed to rescore the PPI compounds. Collectively, the results indicate that the topological scoring algorithms could be valuable both at a global level, with up to 79% increase in areas under the receiver operating characteristic curve for some targets, and in early stages, with up to a 4-fold increase in enrichment factors at 1% of the screened collections. Outstandingly, DLIGAND2 emerged as the best scoring function on this data set, outperforming all rescoring techniques in terms of VS metrics. The described methodology could help in the rational design of small-molecule PPI inhibitors and has direct applications in many therapeutic areas, including cancer, CNS, and infectious diseases such as COVID-19.


Assuntos
Desenho de Fármacos , Descoberta de Drogas , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Algoritmos , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Bases de Dados de Proteínas , Humanos , Ligantes , Aprendizado de Máquina , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Proteínas/química , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química
8.
Sci Transl Med ; 12(557)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32747425

RESUMO

Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Cristalografia por Raios X , Cisteína Endopeptidases/química , Modelos Animais de Doenças , Humanos , Técnicas In Vitro , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Modelos Moleculares , Pandemias , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas , Especificidade da Espécie , Eletricidade Estática , Pesquisa Médica Translacional , Células Vero , Carga Viral/efeitos dos fármacos , Proteínas não Estruturais Virais/química
9.
J Mol Graph Model ; 100: 107697, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32739642

RESUMO

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.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Antivirais/química , Betacoronavirus/química , Peptidil Dipeptidase A/química , Bibliotecas de Moléculas Pequenas/química , Motivos de Aminoácidos , Betacoronavirus/enzimologia , Carbazóis/química , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Difilina/análogos & derivados , Difilina/química , Interações Hospedeiro-Patógeno , Humanos , Ácidos Hidroxâmicos/química , Ligantes , Simulação de Acoplamento Molecular , Pandemias , Paromomicina/análogos & derivados , Paromomicina/química , Pemetrexede/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Termodinâmica
10.
J Mol Graph Model ; 100: 107710, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32829149

RESUMO

The emergence of SARS-CoV-2 has prompted a worldwide health emergency. There is an urgent need for therapeutics, both through the repurposing of approved drugs and the development of new treatments. In addition to the viral drug targets, a number of human drug targets have been suggested. In theory, targeting human proteins should provide an advantage over targeting viral proteins in terms of drug resistance, which is commonly a problem in treating RNA viruses. This paper focuses on the human protein TMPRSS2, which supports coronavirus life cycles by cleaving viral spike proteins. The three-dimensional structure of TMPRSS2 is not known and so we have generated models of the TMPRSS2 in the apo state as well as in complex with a peptide substrate and putative inhibitors to aid future work. Importantly, many related human proteases have 80% or higher identity with TMPRSS2 in the S1-S1' subsites, with plasminogen and urokinase-type plasminogen activator (uPA) having 95% identity. We highlight 376 approved, investigational or experimental drugs targeting S1A serine proteases that may also inhibit TMPRSS2. Whilst the presence of a relatively uncommon lysine residue in the S2/S3 subsites means that some serine protease inhibitors will not inhibit TMPRSS2, this residue is likely to provide a handle for selective targeting in a focused drug discovery project. We discuss how experimental drugs targeting related serine proteases might be repurposed as TMPRSS2 inhibitors to treat coronaviruses.


Assuntos
Antivirais/química , Betacoronavirus/química , Inibidores de Proteases/química , Serina Endopeptidases/química , Bibliotecas de Moléculas Pequenas/química , Sequência de Aminoácidos , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Interações Hospedeiro-Patógeno , Humanos , Ligantes , Simulação de Dinâmica Molecular , Pandemias , Plasminogênio/antagonistas & inibidores , Plasminogênio/química , Plasminogênio/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Serina Endopeptidases/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Termodinâmica , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
11.
Nat Commun ; 11(1): 3954, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770072

RESUMO

The systematic stabilization of protein-protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.


Assuntos
Proteínas 14-3-3/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Proteínas 14-3-3/antagonistas & inibidores , Proteínas 14-3-3/ultraestrutura , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/ultraestrutura , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
12.
PLoS One ; 15(7): e0236710, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735606

RESUMO

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.


Assuntos
Proteínas Argonauta/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Espectroscopia de Ressonância Magnética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ressonância de Plasmônio de Superfície , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Simulação por Computador , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo
13.
Life Sci ; 258: 118205, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777300

RESUMO

AIMS: Coronavirus disease 2019 (COVID-19) has appeared in Wuhan, China but the fast transmission has led to its widespread prevalence in various countries, which has made it a global concern. Another concern is the lack of definitive treatment for this disease. The researchers tried different treatment options which are not specific. The current study aims to identify potential small molecule inhibitors against the main protease protein of SARS-CoV-2 by the computational approach. MAIN METHODS: In this study, a virtual screening procedure employing docking of the two different datasets from the ZINC database, including 1615 FDA approved drugs and 4266 world approved drugs were used to identify new potential small molecule inhibitors for the newly released crystal structure of main protease protein of SARS-CoV-2. In the following to validate the docking result, molecular dynamics simulations were applied on selected ligands to identify the behavior and stability of them in the binding pocket of the main protease in 150 nanoseconds (ns). Furthermore, binding energy using the MMPBSA approach was also calculated. KEY FINDINGS: The result indicates that simeprevir (Hepatitis C virus NS3/4A protease inhibitor) and pyronaridine (antimalarial agent) could fit well to the binding pocket of the main protease and because of some other beneficial features including broad-spectrum antiviral properties and ADME profile, they might be a promising drug candidate for repurposing to the treatment of COVID-19. SIGNIFICANCE: Simeprevir and pyronaridine were selected by the combination of virtual screening and molecular dynamics simulation approaches as a potential candidate for treatment of COVID-19.


Assuntos
Antimaláricos/farmacologia , Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Naftiridinas/farmacologia , Pneumonia Viral/tratamento farmacológico , Simeprevir/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Reposicionamento de Medicamentos , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Inibidores de Proteases/farmacologia , Serina Proteases , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
14.
Sci Rep ; 10(1): 13093, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753646

RESUMO

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.


Assuntos
Betacoronavirus/fisiologia , Bibliotecas de Moléculas Pequenas/química , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Betacoronavirus/isolamento & purificação , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Humanos , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacos
15.
Nucleic Acids Res ; 48(15): 8576-8590, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32761127

RESUMO

The IGF2 mRNA-binding protein 1 (IGF2BP1) is a non-catalytic post-transcriptional enhancer of tumor growth upregulated and associated with adverse prognosis in solid cancers. However, conserved effector pathway(s) and the feasibility of targeting IGF2BP1 in cancer remained elusive. We reveal that IGF2BP1 is a post-transcriptional enhancer of the E2F-driven hallmark in solid cancers. IGF2BP1 promotes G1/S cell cycle transition by stabilizing mRNAs encoding positive regulators of this checkpoint like E2F1. This IGF2BP1-driven shortening of the G1 cell cycle phase relies on 3'UTR-, miRNA- and m6A-dependent regulation and suggests enhancement of cell cycle progression by m6A-modifications across cancers. In addition to E2F transcription factors, IGF2BP1 also stabilizes E2F-driven transcripts directly indicating post-transcriptional 'super'-enhancer role of the protein in E2F-driven gene expression in cancer. The small molecule BTYNB disrupts this enhancer function by impairing IGF2BP1-RNA association. Consistently, BTYNB interferes with E2F-driven gene expression and tumor growth in experimental mouse tumor models.


Assuntos
Fatores de Transcrição E2F/genética , Neoplasias/genética , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Fator de Transcrição E2F1/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Neoplasias/patologia , Proteínas de Ligação a RNA/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia
16.
Proc Natl Acad Sci U S A ; 117(33): 19854-19865, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759214

RESUMO

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Claudina-5/genética , Claudina-5/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Edição de Genes , Genoma , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(29): 17195-17203, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32606248

RESUMO

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.


Assuntos
Actinobacteria/genética , Genoma Bacteriano , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Sirolimo/química , Sirolimo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(29): 16782-16789, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641511

RESUMO

DNA-encoded chemical libraries are collections of compounds individually coupled to unique DNA tags serving as amplifiable identification barcodes. By bridging split-and-pool combinatorial synthesis with the ligation of unique encoding DNA oligomers, million- to billion-member libraries can be synthesized for use in hundreds of healthcare target screens. Although structural diversity and desirable molecular property ranges generally guide DNA-encoded chemical library design, recent reports have highlighted the utility of focused DNA-encoded chemical libraries that are structurally biased for a class of protein targets. Herein, a protease-focused DNA-encoded chemical library was designed that utilizes chemotypes known to engage conserved catalytic protease residues. The three-cycle library features functional moieties such as guanidine, which interacts strongly with aspartate of the protease catalytic triad, as well as mild electrophiles such as sulfonamide, urea, and carbamate. We developed a DNA-compatible method for guanidinylation of amines and reduction of nitriles. Employing these optimized reactions, we constructed a 9.8-million-membered DNA-encoded chemical library. Affinity selection of the library with thrombin, a common protease, revealed a number of enriched features which ultimately led to the discovery of a 1 nM inhibitor of thrombin. Thus, structurally focused DNA-encoded chemical libraries have tremendous potential to find clinically useful high-affinity hits for the rapid discovery of drugs for targets (e.g., proteases) with essential functions in infectious diseases (e.g., severe acute respiratory syndrome coronavirus 2) and relevant healthcare conditions (e.g., male contraception).


Assuntos
DNA/química , DNA/metabolismo , Descoberta de Drogas , Biblioteca Gênica , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Trombina/antagonistas & inibidores , Técnicas de Química Combinatória , Humanos , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas/química
19.
Cancer Treat Rev ; 89: 102065, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32653806

RESUMO

To date, much progress has been made in early-stage extranodal NK/T-cell lymphoma (ENKTCL), and risk-adapted therapy with radiotherapy (RT) alone for the low-risk group and RT combined with asparaginase-based chemotherapy (CT) for the high-risk group yields favorable outcomes. However, optimal treatment strategies have not been defined yet for advanced-stage ENKTCL. Historically, ENKTCL responded poorly to conventional anthracycline-based chemotherapy probably because of inherent multidrug resistance (MDR). The fact that ENKTCL cells lack asparagine synthetase (ASNS) activity warranted the use of L-asparaginase or pegaspargase as frontline chemotherapies. Even though, due to high mortality of the disease, approximately 50% patients failing the frontline therapy arrived at dismal clinical outcomes with a median progression-free survival (PFS) less than 8 months. As distinctive molecular and biological subgroups are increasingly discovered within the disease entity of ENKTCL, novel targeted therapies and immunotherapy are of the urgent need for those heterogeneous subgroups. In this review, we sought to summarize the preclinical and clinical results of 6 categories of promising targeted therapy and immunotherapy for the treatment of ENKTCL, including monoclonal antibodies, immune checkpoint inhibitors, small-molecular inhibitors, epigenetic therapy, immunomodulatory drugs, and adoptive T-cell therapy, and these might change the landscape of treatment for ENKTCL in the near future.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linfoma Extranodal de Células T-NK/tratamento farmacológico , Linfoma Extranodal de Células T-NK/terapia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/farmacologia , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/farmacologia , Imunoterapia/métodos , Imunoterapia Adotiva , Terapia de Alvo Molecular/métodos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacologia
20.
Biomolecules ; 10(7)2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32679775

RESUMO

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600-1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Amidas/antagonistas & inibidores , Amidas/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Mutação , Pandemias , Proteoma/antagonistas & inibidores , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/química , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo
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