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1.
ACS Nano ; 14(8): 10616-10623, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32806067

RESUMO

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a crucial role in binding the human cell receptor ACE2 that is required for viral entry. Many studies have been conducted to target the structures of RBD-ACE2 binding and to design RBD-targeting vaccines and drugs. Nevertheless, mutations distal from the SARS-CoV-2 RBD also impact its transmissibility and antibody can target non-RBD regions, suggesting the incomplete role of the RBD region in the spike protein-ACE2 binding. Here, in order to elucidate distant binding mechanisms, we analyze complexes of ACE2 with the wild-type spike protein and with key mutants via large-scale all-atom explicit solvent molecular dynamics simulations. We find that though distributed approximately 10 nm away from the RBD, the SARS-CoV-2 polybasic cleavage sites enhance, via electrostatic interactions and hydration, the RBD-ACE2 binding affinity. A negatively charged tetrapeptide (GluGluLeuGlu) is then designed to neutralize the positively charged arginine on the polybasic cleavage sites. We find that the tetrapeptide GluGluLeuGlu binds to one of the three polybasic cleavage sites of the SARS-CoV-2 spike protein lessening by 34% the RBD-ACE2 binding strength. This significant binding energy reduction demonstrates the feasibility to neutralize RBD-ACE2 binding by targeting this specific polybasic cleavage site. Our work enhances understanding of the binding mechanism of SARS-CoV-2 to ACE2, which may aid the design of therapeutics for COVID-19 infection.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/genética , Sítios de Ligação/genética , Desenho de Fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Domínios Proteicos , Receptores Virais/química , Receptores Virais/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
2.
PLoS One ; 15(8): e0234492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790760

RESUMO

Endothelial injury is a common manifestation in IgA nephropathy (IgAN). After the previous identification of the upregulated soluble fms-like tyrosine kinase-1 (sFlt-1) correlated with endothelial injury in IgAN, in the present study, we further explored the role of sFlt-1 in endothelial injury in IgAN. We enrolled 72 patients with IgAN and detected the sFlt-1 levels. The polymeric IgA1 (pIgA1) complexes were isolated from the pooled plasma samples of another 10 patients with IgAN. Apoptosis proteins were detected in cultured human umbilical vein endothelial cells (HUVECs) with the stimulation of recombinant sFlt-1 or the caspase-9 inhibitor Z-LEHD-FMK. We identified there were positive correlations between sFlt-1 and IgA-IgG complex as well as vWF levels in patients with IgAN. The sFlt-1 levels in HUVECs were significantly upregulated by pIgA1 complex derived from IgAN patients in a concentration-dependent manner. The proliferation ability of HUVECs was damaged when stimulated with sFlt-1 protein in a time- and dose- dependent manner. And the apoptosis rate was up-regulated significantly as the stimulation concentrations of sFlt-1 increased. We found sFlt-1 challenge could significantly increase the expression of vWF. In addition, sFlt-1 increased the levels of caspase-9, caspase-3, Bax and mitochondrial membrane potential; facilitated the release of cytochrome C from mitochondria to cytoplasma. In contrast, Z-LEHD-FMK attenuated high sFlt-1-induced HUVECs apoptosis. In conclusion, our study demonstrated that sFlt-1 expression was up-regulated by the challenge of pIgA1 complex derived from patients with IgAN. Furthermore, increased sFlt-1 facilitated human umbilical vein endothelial cells apoptosis via the mitochondrial-dependent pathway.


Assuntos
Endotélio Vascular/fisiopatologia , Glomerulonefrite por IGA/fisiopatologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Adulto , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Caspase 9/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Glomerulonefrite por IGA/sangue , Glomerulonefrite por IGA/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Oligopeptídeos/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangue , Adulto Jovem
3.
PLoS One ; 15(8): e0237110, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790695

RESUMO

Serine/threonine phosphatases are responsible for modulating the activities of the protein kinases implicated in the development of several pathologies. Here we identified by a PEP-scan approach a peptide of LRRK2, a Parkinson's disease associated protein, interacting with the phosphatase PP1. In order to study its biological activity, the peptide was fused via its N-terminal to an optimized cell penetrating peptide. We synthesized from the original peptide five interfering peptides and identified two (Mut3DPT-LRRK2-Short and Mut3DPT-LRRK2-Long) able to disrupt the LRRK2/PP1 interaction by competition in anti-LRRK2 immunoprecipitates. Using FITC-labelled peptides, we confirmed their internalization into cell lines as well as into primary cells obtained from healthy or ill human donors. We confirmed by ELISA test the association of Mut3DPT-LRRK2-Long peptide to purified PP1 protein. The peptides Mut3DPT-LRRK2-5 to 8 with either N or C-terminal deletions were not able to disrupt the association LRRK2/PP1 nor to associate with purified PP1 protein. The interfering sequences blocking the PP1/LRRK2 interaction were also fused to a shuttle peptide able to cross the blood brain barrier and showed that the newly generated peptides BBB-LRRK2-Short and BBB-LRRK2-Long were highly resistant to protease degradation. Furthermore, they blocked PP1/LRRK2 interaction and they penetrated into cells. Hence, these newly generated peptides can be employed as new tools in the investigation of the role of the LRRK2/PP1 interaction in normal and pathological conditions.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Oligopeptídeos/química , Proteína Fosfatase 1/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteólise
4.
Artigo em Inglês | MEDLINE | ID: mdl-32707682

RESUMO

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease. Although the lack of dystrophin protein is the primary defect responsible for the development of DMD, secondary disease complications such as persistent inflammation contribute greatly to the pathogenesis and the time-dependent progression of muscle destruction. The immunoproteasome is a potential therapeutic target for conditions or diseases mechanistically linked to inflammation. In this study, we explored the possible effects of ONX-0914 administration, an inhibitor specific for the immunoproteasome subunit LMP7 (ß5i), on motor performance, muscular pathology and protein degradation in 7-week old MDX mice, an age when the dystrophic muscles show extensive degeneration and regeneration. ONX-0914 (10 mg/kg) was injected subcutaneously on Day 2, 4, and 6. The mice were evaluated for physical performance (walking speed and strength) on Day 1 and 8. We show that this short-term treatment of ONX-0914 in MDX mice did not alter strength nor walking speed. The physical performance findings were consistent with no change in muscle inflammatory infiltration, percentage of central nuclei and proteasome content. Taken together, muscle structure and function in the young adult MDX mouse model are not altered with ONX-0914 treatment, indicating the administration of ONX-0914 during this critical time period does not exhibit any detrimental effects and may be an effective treatment of secondary complications of muscular dystrophy after further investigations.


Assuntos
Distrofina , Músculo Esquelético , Distrofia Muscular de Duchenne/tratamento farmacológico , Oligopeptídeos , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fenótipo , Adulto Jovem
5.
Proc Natl Acad Sci U S A ; 117(26): 15363-15373, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554501

RESUMO

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.


Assuntos
Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Oligopeptídeos/farmacologia , Envelhecimento , Animais , Masculino , Camundongos , Modelos Químicos , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Ligação Proteica
6.
Interdiscip Sci ; 12(3): 368-376, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32488835

RESUMO

A novel coronavirus, called 2019-nCoV, was recently found in Wuhan, Hubei Province of China, and now is spreading across China and other parts of the world. Although there are some drugs to treat 2019-nCoV, there is no proper scientific evidence about its activity on the virus. It is of high significance to develop a drug that can combat the virus effectively to save valuable human lives. It usually takes a much longer time to develop a drug using traditional methods. For 2019-nCoV, it is now better to rely on some alternative methods such as deep learning to develop drugs that can combat such a disease effectively since 2019-nCoV is highly homologous to SARS-CoV. In the present work, we first collected virus RNA sequences of 18 patients reported to have 2019-nCoV from the public domain database, translated the RNA into protein sequences, and performed multiple sequence alignment. After a careful literature survey and sequence analysis, 3C-like protease is considered to be a major therapeutic target and we built a protein 3D model of 3C-like protease using homology modeling. Relying on the structural model, we used a pipeline to perform large scale virtual screening by using a deep learning based method to accurately rank/identify protein-ligand interacting pairs developed recently in our group. Our model identified potential drugs for 2019-nCoV 3C-like protease by performing drug screening against four chemical compound databases (Chimdiv, Targetmol-Approved_Drug_Library, Targetmol-Natural_Compound_Library, and Targetmol-Bioactive_Compound_Library) and a database of tripeptides. Through this paper, we provided the list of possible chemical ligands (Meglumine, Vidarabine, Adenosine, D-Sorbitol, D-Mannitol, Sodium_gluconate, Ganciclovir and Chlorobutanol) and peptide drugs (combination of isoleucine, lysine and proline) from the databases to guide the experimental scientists and validate the molecules which can combat the virus in a shorter time.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Aprendizado Profundo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/química , Betacoronavirus/genética , Domínio Catalítico , Infecções por Coronavirus/epidemiologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Produtos Farmacêuticos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricos , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Pandemias , Pneumonia Viral/epidemiologia , Alinhamento de Sequência , Homologia Estrutural de Proteína , Interface Usuário-Computador , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
7.
Int J Mol Sci ; 21(10)2020 May 20.
Artigo em Inglês | MEDLINE | ID: covidwho-324354

RESUMO

The COVID-19 global pandemic is caused by SARS-CoV-2, and represents an urgent medical and social issue. Unfortunately, there is still not a single proven effective drug available, and therefore, current therapeutic guidelines recommend supportive care including oxygen administration and treatment with antibiotics. Recently, patients have been also treated with off-label therapies which comprise antiretrovirals, anti-inflammatory compounds, antiparasitic agents and plasma from convalescent patients, all with controversial results. The ubiquitin-proteasome system (UPS) is important for the maintenance of cellular homeostasis, and plays a pivotal role in viral replication processes. In this review, we discuss several aspects of the UPS and the effects of its inhibition with particular regard to the life cycle of the coronaviruses (CoVs). In fact, proteasome inhibition by various chemical compounds, such as MG132, epoxomycin and bortezomib, may reduce the virus entry into the eucariotic cell, the synthesis of RNA, and the subsequent protein expression necessary for CoVs. Importantly, since UPS inhibitors reduce the cytokine storm associated with various inflammatory conditions, it is reasonable to assume that they might be repurposed for SARS-CoV-2, thus providing an additional tool to counteract both virus replication as well as its most deleterious consequences triggered by abnormal immunological response.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Pandemias , Pneumonia Viral/epidemiologia , Inibidores de Proteassoma/farmacologia
8.
Int J Mol Sci ; 21(10)2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32443911

RESUMO

The COVID-19 global pandemic is caused by SARS-CoV-2, and represents an urgent medical and social issue. Unfortunately, there is still not a single proven effective drug available, and therefore, current therapeutic guidelines recommend supportive care including oxygen administration and treatment with antibiotics. Recently, patients have been also treated with off-label therapies which comprise antiretrovirals, anti-inflammatory compounds, antiparasitic agents and plasma from convalescent patients, all with controversial results. The ubiquitin-proteasome system (UPS) is important for the maintenance of cellular homeostasis, and plays a pivotal role in viral replication processes. In this review, we discuss several aspects of the UPS and the effects of its inhibition with particular regard to the life cycle of the coronaviruses (CoVs). In fact, proteasome inhibition by various chemical compounds, such as MG132, epoxomycin and bortezomib, may reduce the virus entry into the eucariotic cell, the synthesis of RNA, and the subsequent protein expression necessary for CoVs. Importantly, since UPS inhibitors reduce the cytokine storm associated with various inflammatory conditions, it is reasonable to assume that they might be repurposed for SARS-CoV-2, thus providing an additional tool to counteract both virus replication as well as its most deleterious consequences triggered by abnormal immunological response.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Pandemias , Pneumonia Viral/epidemiologia , Inibidores de Proteassoma/farmacologia
9.
Am J Physiol Renal Physiol ; 318(6): F1531-F1538, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390514

RESUMO

Renal ischemia-reperfusion injury (I/R) usually occurs in renal transplantation and partial nephrectomy, which could lead to acute kidney injury. However, the effective treatment for renal I/R still remains limited. In the present study, we investigated whether inhibition of chromobox 7 (CBX7) could attenuate renal I/R injury in vivo and in vitro as well as the potential mechanisms. Adult male mice were subjected to right renal ischemia and reperfusion for different periods, both with and without the CBX7 inhibitor UNC3866. In addition, human kidney cells (HK-2) were subjected to a hypoxia/reoxygenation (H/R) process for different periods, both with or without the CBX7 inhibitor or siRNA for CBX7. The results showed that expression of CBX7, glucose regulator protein-78 (GRP78), phosphorylated eukaryotic translation initiation factor-2α (p-eIF2α), and C/EBP homologous protein (CHOP) were increased after extension of I/R and H/R periods. Moreover, overexpression of CBX7 could elevate the expression of CBX7, GRP78, p-eIF2α, and CHOP. However, CBX7 inhibition with either UNC3866 or genetic knockdown led to reduced expression of GRP78, p-eIF2α, and CHOP through nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 activation in I/R and H/R injury. Furthermore, ML385, the Nrf2 inhibitor, could elevate endoplasmic reticulum stress levels, abrogating the protective effects of UNC3866 against renal I/R injury. In conclusion, our results demonstrated that CBX7 inhibition alleviated acute kidney injury by preventing endoplasmic reticulum stress via the Nrf2/HO-1 pathway, indicating that CBX7 inhibitor could be a potential therapeutic target for renal I/R injury.


Assuntos
Lesão Renal Aguda/prevenção & controle , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Rim/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oligopeptídeos/farmacologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Lesão Renal Aguda/enzimologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Hipóxia Celular , Linhagem Celular , Heme Oxigenase-1/genética , Humanos , Rim/enzimologia , Rim/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Fator 2 Relacionado a NF-E2/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
10.
Nat Commun ; 11(1): 2019, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332747

RESUMO

Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. In cervical cancer cells, human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7-mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. However, it is not clear why cleavage is required for Rb degradation. Here, we report that the proteasome cannot initiate degradation efficiently on full-length Rb. Calpain cleavage exposes a region that is recognized by the proteasome, leading to rapid proteolysis of Rb. These findings identify a mechanism for regulating protein stability by controlling initiation and provide a better understanding of the molecular mechanism underlying transformation by HPV.


Assuntos
Calpaína/metabolismo , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , Proteínas E7 de Papillomavirus/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias do Colo do Útero/genética , Acrilatos/farmacologia , Calpaína/antagonistas & inibidores , Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ciclopentanos/farmacologia , Fatores de Transcrição E2F/metabolismo , Feminino , Células HEK293 , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Humanos , Proteína NEDD8/antagonistas & inibidores , Proteína NEDD8/metabolismo , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
11.
Mol Pharmacol ; 97(6): 384-391, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32234809

RESUMO

Organic anion transporter 1 (OAT1), expressed at the basolateral membrane of renal proximal tubule epithelial cells, mediates the renal excretion of many clinically important drugs. Previous study in our laboratory demonstrated that ubiquitin conjugation to OAT1 leads to OAT1 internalization from the cell surface and subsequent degradation. The current study showed that the ubiquitinated OAT1 accumulated in the presence of the proteasomal inhibitors MG132 and ALLN rather than the lysosomal inhibitors leupeptin and pepstatin A, suggesting that ubiquitinated OAT1 degrades through proteasomes. Anticancer drugs bortezomib and carfilzomib target the ubiquitin-proteasome pathway. We therefore investigate the roles of bortezomib and carfilzomib in reversing the ubiquitination-induced downregulation of OAT1 expression and transport activity. We showed that bortezomib and carfilzomib extremely increased the ubiquitinated OAT1, which correlated well with an enhanced OAT1-mediated transport of p-aminohippuric acid and an enhanced OAT1 surface expression. The augmented OAT1 expression and transport activity after the treatment with bortezomib and carfilzomib resulted from a reduced rate of OAT1 degradation. Consistent with this, we found decreased 20S proteasomal activity in cells that were exposed to bortezomib and carfilzomib. In conclusion, this study identified the pathway in which ubiquitinated OAT1 degrades and unveiled a novel role of anticancer drugs bortezomib and carfilzomib in their regulation of OAT1 expression and transport activity. SIGNIFICANCE STATEMENT: Bortezomib and carfilzomib are two Food and Drug Administration-approved anticancer drugs, and proteasome is the drug target. In this study, we unveiled a new role of bortezomib and carfilzomib in enhancing OAT1 expression and transport activity by preventing the degradation of ubiquitinated OAT1 in proteasomes. This finding provides a new strategy in regulating OAT1 function that can be used to accelerate the clearance of drugs, metabolites, or toxins and reverse the decreased expression under disease conditions.


Assuntos
Antineoplásicos/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Bortezomib/farmacologia , Oligopeptídeos/farmacologia , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Células HEK293 , Humanos , Leupeptinas/farmacologia , Proteólise , Ubiquitinação , Ácido p-Aminoipúrico/metabolismo
12.
Chemistry ; 26(33): 7492-7496, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32227540

RESUMO

The use of multimeric ligands is considered as a promising strategy to improve tumor targeting for diagnosis and therapy. Herein, tetrameric RGD (Arg-Gly-Asp) peptidomimetics were designed to target αv ß3 integrin-expressing tumor cells. These compounds were prepared by an oxime chemoselective assembly of cyclo(DKP-RGD) ligands and a cyclodecapeptide scaffold, which allows a tetrameric presentation. The resulting tetrameric RGD peptidomimetics were shown to improve αv ß3 integrin binding compared with the monomeric form. Interestingly, these compounds were also able to enhance tumor cell endocytosis in the same way as tetrameric RGD peptides. Altogether, the results show the potential of the tetrameric cyclo(DKP-RGD) ligands for in vivo imaging and drug delivery.


Assuntos
Integrina alfaVbeta3/metabolismo , Oligopeptídeos/química , Peptidomiméticos/química , Transporte Biológico , Humanos , Integrina alfaVbeta3/química , Ligantes , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia
13.
Clin Sci (Lond) ; 134(7): 853-869, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32227122

RESUMO

Angiotensin II (Ang II) has been reported to aggravate hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Alamandine (ALA) protects against fibrosis by counteracting Ang II via the MAS-related G-protein coupled (MrgD) receptor, though the effects of alamandine on hepatic fibrosis remain unknown. Autophagy activated by reactive oxygen species (ROS) is a novel mechanism of hepatic fibrosis. However, whether autophagy is involved in the regulation of Ang II-induced hepatic fibrosis still requires investigation. We explored the effect of alamandine on hepatic fibrosis via regulation of autophagy by redox balance modulation. In vivo, alamandine reduced CCl4-induced hepatic fibrosis, hydrogen peroxide (H2O2) content, protein levels of NOX4 and autophagy impairment. In vitro, Ang II treatment elevated NOX4 protein expression and ROS production along with up-regulation of the angiotensin converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1R) axis. These changes resulted in the accumulation of impaired autophagosomes in hepatic stellate cells (HSCs). Treatment with NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine (NAC), and NOX4 small interfering RNA (siRNA) inhibited Ang II-induced autophagy and collagen synthesis. Alamandine shifted the balance of renin-angiotensin system (RAS) toward the angiotensin converting enzyme 2 (ACE2)/alamandine/MrgD axis, and inhibited both Ang II-induced ROS and autophagy activation, leading to attenuation of HSCs migration or collagen synthesis. In summary, alamandine attenuated liver fibrosis by regulating autophagy induced by NOX4-dependent ROS.


Assuntos
Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Células Estreladas do Fígado/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Cirrose Hepática Experimental/prevenção & controle , Fígado/efeitos dos fármacos , NADPH Oxidase 4/metabolismo , Oligopeptídeos/farmacologia , Animais , Tetracloreto de Carbono , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colágeno/metabolismo , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/ultraestrutura , Fígado/enzimologia , Fígado/ultraestrutura , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/enzimologia , Cirrose Hepática Experimental/patologia , Masculino , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais
14.
J Chem Inf Model ; 60(6): 3277-3286, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32315171

RESUMO

The recent outbreak of novel coronavirus disease-19 (COVID-19) calls for and welcomes possible treatment strategies using drugs on the market. It is very efficient to apply computer-aided drug design techniques to quickly identify promising drug repurposing candidates, especially after the detailed 3D structures of key viral proteins are resolved. The virus causing COVID-19 is SARS-CoV-2. Taking advantage of a recently released crystal structure of SARS-CoV-2 main protease in complex with a covalently bonded inhibitor, N3 (Liu et al., 10.2210/pdb6LU7/pdb), I conducted virtual docking screening of approved drugs and drug candidates in clinical trials. For the top docking hits, I then performed molecular dynamics simulations followed by binding free energy calculations using an end point method called MM-PBSA-WSAS (molecular mechanics/Poisson-Boltzmann surface area/weighted solvent-accessible surface area; Wang, Chem. Rev. 2019, 119, 9478; Wang, Curr. Comput.-Aided Drug Des. 2006, 2, 287; Wang; ; Hou J. Chem. Inf. Model., 2012, 52, 1199). Several promising known drugs stand out as potential inhibitors of SARS-CoV-2 main protease, including carfilzomib, eravacycline, valrubicin, lopinavir, and elbasvir. Carfilzomib, an approved anticancer drug acting as a proteasome inhibitor, has the best MM-PBSA-WSAS binding free energy, -13.8 kcal/mol. The second-best repurposing drug candidate, eravacycline, is synthetic halogenated tetracycline class antibiotic. Streptomycin, another antibiotic and a charged molecule, also demonstrates some inhibitory effect, even though the predicted binding free energy of the charged form (-3.8 kcal/mol) is not nearly as low as that of the neutral form (-7.9 kcal/mol). One bioactive, PubChem 23727975, has a binding free energy of -12.9 kcal/mol. Detailed receptor-ligand interactions were analyzed and hot spots for the receptor-ligand binding were identified. I found that one hot spot residue, His41, is a conserved residue across many viruses including SARS-CoV, SARS-CoV-2, MERS-CoV, and hepatitis C virus (HCV). The findings of this study can facilitate rational drug design targeting the SARS-CoV-2 main protease.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Betacoronavirus/química , Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Reposicionamento de Medicamentos/economia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Pandemias , Pneumonia Viral/virologia , Inibidores de Proteases/química , Tetraciclinas/química , Tetraciclinas/farmacologia , Termodinâmica , Fatores de Tempo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(15): 8468-8475, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234780

RESUMO

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.


Assuntos
Materiais Biomiméticos/farmacologia , Membrana Celular/metabolismo , Domínio de Fibronectina Tipo III , Necrose , Oligopeptídeos/farmacologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Cristalografia por Raios X , Humanos , Fosforilação , Conformação Proteica , Proteínas Quinases/química , Multimerização Proteica , Transporte Proteico
16.
Diab Vasc Dis Res ; 17(3): 1479164120907971, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32223319

RESUMO

OBJECTIVE: Diabetes mellitus is a significant risk factor for peripheral artery disease. Diabetes mellitus induces chronic states of oxidative stress and vascular inflammation that increase neutrophil activation and release of myeloperoxidase. The goal of this study is to determine whether inhibiting myeloperoxidase reduces oxidative stress and neutrophil infiltration, increases vascularization, and improves blood flow in a diabetic murine model of hindlimb ischaemia. METHODS: Leptin receptor-deficient (db/db) mice were subjected to hindlimb ischaemia. Ischaemic mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC) to inhibit myeloperoxidase. After ligating the femoral artery, effects of treatments were determined with respect to hindlimb blood flow, neutrophil infiltration, oxidative damage, and the capability of hindlimb extracellular matrix to support human endothelial cell proliferation and migration. RESULTS: KYC treatment improved hindlimb blood flow at 7 and 14 days in db/db mice; decreased the formation of advanced glycation end products, 4-hydroxynonenal, and 3-chlorotyrosine; reduced neutrophil infiltration into the hindlimbs; and improved the ability of hindlimb extracellular matrix from db/db mice to support endothelial cell proliferation and migration. CONCLUSION: These results demonstrate that inhibiting myeloperoxidase reduces oxidative stress in ischaemic hindlimbs of db/db mice, which improves blood flow and reduces neutrophil infiltration such that hindlimb extracellular matrix from db/db mice supports endothelial cell proliferation and migration.


Assuntos
Indutores da Angiogênese/farmacologia , Diabetes Mellitus/metabolismo , Inibidores Enzimáticos/farmacologia , Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Peroxidase/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/enzimologia , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Fluxo Sanguíneo Regional , Transdução de Sinais
18.
J Med Chem ; 63(6): 3359-3369, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32142286

RESUMO

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.


Assuntos
Cumarínicos/farmacologia , Corantes Fluorescentes/farmacologia , Granzimas/análise , Oligopeptídeos/farmacologia , Inibidores de Serino Proteinase/farmacologia , Linhagem Celular Tumoral , Cumarínicos/síntese química , Cumarínicos/toxicidade , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Granzimas/química , Humanos , Oligopeptídeos/síntese química , Oligopeptídeos/toxicidade , Inibidores de Serino Proteinase/síntese química , Inibidores de Serino Proteinase/toxicidade , Especificidade por Substrato
19.
Chem Pharm Bull (Tokyo) ; 68(3): 201-211, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115527

RESUMO

Auristatins are important payloads used in antibody drug conjugates (ADCs), and the most well-known compound family member, monomethyl auristatin (MMAE), is used in two Food and Drug Administration (FDA)-approved ADCs, Adcetris® and Polivy®. Multiple other auristatin-based ADCs are currently being evaluated in human clinical trials and further studies on this class of molecule are underway by several academic and industrial research groups. Our group's main focus is to investigate the structure-activity relationships (SAR) of novel auristatins with the goal of applying these to next generation ADCs. Modifications of the auristatin backbone scaffold have been widely reported in the chemical literature focusing on the terminal subunits: P1 (N-terminus) and P5 (C-terminus). Our approach was to modulate the activity and hydrophilic character through modifications of the central subunits P2-P3-P4 and thorough SAR study on the P5 subunit. Novel hydrophilic auristatins were observed to have greater potency in vitro and displayed enhanced in vivo antitumor activity when conjugated via protease-cleavable linkers and delivered intracellularly. Analysis of ADC aggregation also indicated that novel hydrophilic payloads enabled the synthesis of high-drug-to-antibody ratio (DAR) ADCs that were resistant to aggregation. Modification of the central peptide subunits also resulted in auristatins with potent cytotoxic activity in vitro and these azide-modified auristatins contain a handle for linker attachment from the central portion of the auristatin backbone.


Assuntos
Aminobenzoatos/química , Antineoplásicos/química , Oligopeptídeos/química , Aminobenzoatos/farmacologia , Animais , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados , Estrutura Molecular , Oligopeptídeos/farmacologia
20.
Chem Biol Interact ; 322: 109059, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32171850

RESUMO

Fatty liver is the earliest and most common response of the liver to consumption of excessive alcohol. Steatosis can predispose the fatty liver to develop progressive liver damage. Chief among the many mechanisms involved in development of hepatic steatosis is dysregulation of insulin-mediated adipose tissue metabolism. Particularly, it is the enhanced adipose lipolysis-derived free fatty acids and their delivery to the liver that ultimately results in hepatic steatosis. The adipose-liver axis is modulated by hormones, particularly insulin and adiponectin. In recent studies, we demonstrated that an alcohol-induced increase in serum ghrelin levels impairs insulin secretion from pancreatic ß-cells. The consequent reduction in circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. Because many tissues, including adipose tissue, express ghrelin receptor we hypothesized that ghrelin may directly affect energy metabolism in adipocytes. We have exciting new preliminary data which shows that treatment of premature 3T3-L1 adipocytes with ghrelin impairs adipocyte differentiation and inhibits lipid accumulation in the tissue designed to store energy in the form of fat. We further observed that ghrelin treatment of differentiated adipocytes significantly inhibited secretion of adiponectin, a hepatoprotective hormone that reduces lipid synthesis and promotes lipid oxidation. These results were corroborated by our observations of a significant increase in serum adiponectin levels in ethanol-fed rats treated with a ghrelin receptor antagonist verses the un-treated ethanol-fed rats. Interestingly, in adipocytes, ghrelin also increases secretion of interleukin-6 (IL-6) and CCL2 (chemokine [C-C motif] ligand 2), cytokines which promote hepatic inflammation and progression of liver disease. To summarize, the alcohol-induced increase in serum ghrelin levels dysregulates adipose-liver interaction and promotes hepatic steatosis by increasing the free fatty acid released from adipose for hepatic uptake, and by altering adiponectin and cytokine secretion. Taken together, our data indicates that targeting the activity of ghrelin may be a powerful treatment strategy.


Assuntos
Tecido Adiposo/metabolismo , Fígado Gorduroso Alcoólico/patologia , Grelina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Células 3T3-L1 , Adipocinas/metabolismo , Adiponectina/sangue , Adiponectina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Etanol/farmacologia , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/veterinária , Interleucina-6/metabolismo , Masculino , Camundongos , Oligopeptídeos/farmacologia , PPAR gama/metabolismo , Ratos , Ratos Wistar
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