Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 128
Filtrar
1.
Immunity ; 56(9): 2121-2136.e6, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37659412

RESUMO

Genetic association studies have demonstrated the critical involvement of the microglial immune response in Alzheimer's disease (AD) pathogenesis. Phospholipase C-gamma-2 (PLCG2) is selectively expressed by microglia and functions in many immune receptor signaling pathways. In AD, PLCG2 is induced uniquely in plaque-associated microglia. A genetic variant of PLCG2, PLCG2P522R, is a mild hypermorph that attenuates AD risk. Here, we identified a loss-of-function PLCG2 variant, PLCG2M28L, that confers an increased AD risk. PLCG2P522R attenuated disease in an amyloidogenic murine AD model, whereas PLCG2M28L exacerbated the plaque burden associated with altered phagocytosis and Aß clearance. The variants bidirectionally modulated disease pathology by inducing distinct transcriptional programs that identified microglial subpopulations associated with protective or detrimental phenotypes. These findings identify PLCG2M28L as a potential AD risk variant and demonstrate that PLCG2 variants can differentially orchestrate microglial responses in AD pathogenesis that can be therapeutically targeted.


Assuntos
Doença de Alzheimer , Animais , Camundongos , Doença de Alzheimer/genética , Estudos de Associação Genética , Microglia , Fagocitose/genética , Fenótipo , Placa Amiloide , Fosfolipase C gama/metabolismo
2.
Nature ; 586(7827): 113-119, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32707573

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.


Assuntos
Antivirais/análise , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , COVID-19 , Linhagem Celular , Inibidores de Cisteína Proteinase/análise , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazonas , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Morfolinas/análise , Morfolinas/farmacologia , Pandemias , Pirimidinas , Reprodutibilidade dos Testes , SARS-CoV-2 , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Triazinas/análise , Triazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19
4.
Bioorg Med Chem Lett ; 96: 129489, 2023 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-37770002

RESUMO

We report here the synthesis and biological evaluation of a series of small molecule SARS-CoV-2 PLpro inhibitors. We compared the activity of selected compounds in both SARS-CoV-1 and SARS-CoV-2 PLpro inhibitory and antiviral assays. We have synthesized and evaluated several new structural variants of previous leads against SARS-CoV-2 PLpro. The replacement of the carboxamide functionality with sulfonamide derivatives resulted in PLpro inhibitors with potent PLpro inhibitory and antiviral activity in VeroE6 cells similar to GRL0617. To obtain molecular insight, we created an optimized model of a potent sulfonamide derivative in the SARS-CoV-2 PLpro active site.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Antivirais/farmacologia , Antivirais/química , Sulfonamidas/farmacologia
5.
J Infect Dis ; 224(Supplement_1): S1-S21, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111271

RESUMO

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.


Assuntos
Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , COVID-19/virologia , Desenvolvimento de Medicamentos , Humanos , National Institutes of Health (U.S.) , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Estados Unidos , Replicação Viral/efeitos dos fármacos
6.
J Virol ; 94(11)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188728

RESUMO

Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (P < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis.IMPORTANCE Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.


Assuntos
Infecções por Coronavirus/virologia , Vírus da Hepatite Murina/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Interações Hospedeiro-Patógeno , Interferon Tipo I/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Modelos Moleculares , Vírus da Hepatite Murina/patogenicidade , Mutagênese , Conformação Proteica , Relação Estrutura-Atividade , Ubiquitinação , Proteínas Virais/química , Virulência , Replicação Viral
7.
Molecules ; 26(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34641337

RESUMO

We report the design and synthesis of a series of new 5-chloropyridinyl esters of salicylic acid, ibuprofen, indomethacin, and related aromatic carboxylic acids for evaluation against SARS-CoV-2 3CL protease enzyme. These ester derivatives were synthesized using EDC in the presence of DMAP to provide various esters in good to excellent yields. Compounds are stable and purified by silica gel chromatography and characterized using 1H-NMR, 13C-NMR, and mass spectral analysis. These synthetic derivatives were evaluated in our in vitro SARS-CoV-2 3CLpro inhibition assay using authentic SARS-CoV-2 3CLpro enzyme. Compounds were also evaluated in our in vitro antiviral assay using quantitative VeroE6 cell-based assay with RNAqPCR. A number of compounds exhibited potent SARS-CoV-2 3CLpro inhibitory activity and antiviral activity. Compound 9a was the most potent inhibitor, with an enzyme IC50 value of 160 nM. Compound 13b exhibited an enzyme IC50 value of 4.9 µM. However, it exhibited a potent antiviral EC50 value of 24 µM in VeroE6 cells. Remdesivir, an RdRp inhibitor, exhibited an antiviral EC50 value of 2.4 µM in the same assay. We assessed the mode of inhibition using mass spectral analysis which suggested the formation of a covalent bond with the enzyme. To obtain molecular insight, we have created a model of compound 9a bound to SARS-CoV-2 3CLpro in the active site.


Assuntos
Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacologia , Chlorocebus aethiops , Proteases 3C de Coronavírus/metabolismo , Ésteres/química , Ésteres/farmacologia , Halogenação , Humanos , Ibuprofeno/análogos & derivados , Ibuprofeno/farmacologia , Indometacina/análogos & derivados , Indometacina/farmacologia , Simulação de Acoplamento Molecular , Piridinas/química , Piridinas/farmacologia , SARS-CoV-2/metabolismo , Ácido Salicílico/química , Ácido Salicílico/farmacologia , Células Vero
8.
Biochemistry ; 58(44): 4424-4435, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31549827

RESUMO

BACE1 (Beta-site Amyloid Precursor Protein (APP) Cleaving Enzyme 1) is a promising therapeutic target for Alzheimer's Disease (AD). However, efficient expression, purification, and crystallization systems are not well described or detailed in the literature nor are approaches for treatment of enzyme kinetic data for potent inhibitors well described. We therefore developed a platform for expression and purification of BACE1, including protein refolding from E.coli inclusion bodies, in addition to optimizing a reproducible crystallization procedure of BACE1 bound with inhibitors. We also report a detailed approach to the proper analysis of enzyme kinetic data for compounds that exhibit either rapid-equilibrium or tight-binding mechanisms. Our methods allow for the purification of ∼15 mg of BACE1 enzyme from 1 L of culture which is higher than reported yields in the current literature. To evaluate the data analysis approach developed here, a well-known potent inhibitor and two of its derivatives were tested, analyzed, and compared. The inhibitory constants (Ki) obtained from the kinetic studies are in agreement with dissociation constants (Kd) that were also determined using isothermal titration calorimetry (ITC) experiments. The X-ray structures of these three compounds in complex with BACE1 were readily obtained and provide important insight into the structure and thermodynamics of the BACE1-inhibitor interactions.


Assuntos
Secretases da Proteína Precursora do Amiloide/isolamento & purificação , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/isolamento & purificação , Ácido Aspártico Endopeptidases/metabolismo , Compostos Macrocíclicos/química , Inibidores de Proteases/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Domínio Catalítico , Linhagem Celular Tumoral , Cristalização , Cristalografia por Raios X , Descoberta de Drogas , Ensaios Enzimáticos , Humanos , Cinética , Compostos Macrocíclicos/metabolismo , Inibidores de Proteases/metabolismo , Ligação Proteica , Redobramento de Proteína
9.
Bioorg Med Chem Lett ; 28(15): 2605-2610, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29970308

RESUMO

We describe the design, synthesis, X-ray studies, and biological evaluation of novel BACE1 inhibitors containing bicyclic isoxazoline carboxamides as the P3 ligand in combination with methyl cysteine, methylsulfonylalanine and Boc-amino alanine as P2 ligands. Inhibitor 3a displayed a BACE1 Ki value of 10.9 nM and EC50 of 343 nM. The X-ray structure of 3a bound to the active site of BACE1 was determined at 2.85 Šresolution. The structure revealed that the major molecular interactions between BACE1 and the bicyclic tetrahydrofuranyl isoxazoline heterocycle are van der Waals in nature.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Alanina/análogos & derivados , Alanina/química , Amidas/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Compostos Bicíclicos com Pontes/síntese química , Ácidos Carboxílicos/química , Domínio Catalítico , Cristalografia por Raios X , Cisteína/análogos & derivados , Cisteína/química , Humanos , Isoxazóis/química , Estrutura Molecular , Inibidores de Proteases/síntese química
10.
J Mol Recognit ; 30(11)2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28608547

RESUMO

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that poses a major challenge to clinical management. The 3C-like protease (3CLpro ) is essential for viral replication and thus represents a potential target for antiviral drug development. Presently, very few data are available on MERS-CoV 3CLpro inhibition by small molecules. We conducted extensive exploration of the pharmacophoric space of a recently identified set of peptidomimetic inhibitors of the bat HKU4-CoV 3CLpro . HKU4-CoV 3CLpro shares high sequence identity (81%) with the MERS-CoV enzyme and thus represents a potential surrogate model for anti-MERS drug discovery. We used 2 well-established methods: Quantitative structure-activity relationship (QSAR)-guided modeling and docking-based comparative intermolecular contacts analysis. The established pharmacophore models highlight structural features needed for ligand recognition and revealed important binding-pocket regions involved in 3CLpro -ligand interactions. The best models were used as 3D queries to screen the National Cancer Institute database for novel nonpeptidomimetic 3CLpro inhibitors. The identified hits were tested for HKU4-CoV and MERS-CoV 3CLpro inhibition. Two hits, which share the phenylsulfonamide fragment, showed moderate inhibitory activity against the MERS-CoV 3CLpro and represent a potential starting point for the development of novel anti-MERS agents. To the best of our knowledge, this is the first pharmacophore modeling study supported by in vitro validation on the MERS-CoV 3CLpro . HIGHLIGHTS: MERS-CoV is an emerging virus that is closely related to the bat HKU4-CoV. 3CLpro is a potential drug target for coronavirus infection. HKU4-CoV 3CLpro is a useful surrogate model for the identification of MERS-CoV 3CLpro enzyme inhibitors. dbCICA is a very robust modeling method for hit identification. The phenylsulfonamide scaffold represents a potential starting point for MERS coronavirus 3CLpro inhibitors development.


Assuntos
Antivirais/farmacologia , Betacoronavirus/enzimologia , Quirópteros/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas Virais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Simulação por Computador , Ligantes , Modelos Moleculares , Inibidores de Proteases/química , Relação Quantitativa Estrutura-Atividade , Curva ROC , Reprodutibilidade dos Testes , Proteínas Virais/química
11.
Bioorg Med Chem Lett ; 27(11): 2432-2438, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28427814

RESUMO

We report the design and synthesis of a series of BACE1 inhibitors incorporating mono- and bicyclic 6-substituted 2-oxopiperazines as novel P1' and P2' ligands and isophthalamide derivative as P2-P3 ligands. Among mono-substituted 2-oxopiperazines, inhibitor 5a with N-benzyl-2-oxopiperazine and isophthalamide showed potent BACE1 inhibitory activity (Ki=2nM). Inhibitor 5g, with N-benzyl-2-oxopiperazine and substituted indole-derived P2-ligand showed a reduction in potency. The X-ray crystal structure of 5g-bound BACE1 was determined and used to design a set of disubstituted 2-oxopiperazines and bicyclic derivatives that were subsequently investigated. Inhibitor 6j with an oxazolidinone derivative showed a BACE1 inhibitory activity of 23nM and cellular EC50 of 80nM.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácidos Ftálicos/química , Piperazinas/química , Ácidos Ftálicos/síntese química , Piperazinas/síntese química , Relação Estrutura-Atividade
12.
J Biol Chem ; 290(42): 25293-306, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26296883

RESUMO

Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication.


Assuntos
Vírus da Hepatite Murina/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/fisiologia
13.
J Biol Chem ; 290(32): 19403-22, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26055715

RESUMO

All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the ß-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CL(pro)) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CL(pro) from other ß-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL(pro) is less efficient at processing a peptide substrate due to MERS-CoV 3CL(pro) being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL(pro) enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CL(pro) is a weakly associated dimer (Kd ∼52 µm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL(pro) were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CL(pro) undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CL(pro) from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CL(pro) dimerization. Activation of MERS-CoV 3CL(pro) through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CL(pro) inhibitors as antiviral agents.


Assuntos
Antivirais/química , Cisteína Endopeptidases/química , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Peptidomiméticos/química , Multimerização Proteica/efeitos dos fármacos , Proteínas Virais/química , Sequência de Aminoácidos , Antivirais/síntese química , Antivirais/farmacologia , Proteases 3C de Coronavírus , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligantes , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptidomiméticos/síntese química , Peptidomiméticos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
J Virol ; 89(9): 4907-17, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25694594

RESUMO

UNLABELLED: Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE: Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.


Assuntos
Vírus da Hepatite Murina/enzimologia , Vírus da Hepatite Murina/fisiologia , Peptídeo Hidrolases/metabolismo , Replicação Viral , Animais , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Análise Mutacional de DNA , Estabilidade Enzimática , Feminino , Hepatite Viral Animal/patologia , Hepatite Viral Animal/virologia , Camundongos Endogâmicos C57BL , Vírus da Hepatite Murina/patogenicidade , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeo Hidrolases/química , Estrutura Terciária de Proteína , Temperatura
15.
Arch Biochem Biophys ; 612: 35-45, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27756680

RESUMO

USP17 is a deubiquitinating enzyme that is upregulated in numerous cancers and therefore a drug target. We developed a robust expression, purification, and assay system for USP17 enabling its enzymatic and structural characterization. USP17 was expressed in E. coli as inclusion bodies and then solubilized, refolded, and purified using affinity and size-exclusion chromatography. Milligram quantities of pure USP17 can be produced that is catalytically more efficient (kcat/Km = 1500 (x103) M-1sec-1) than other human USPs studied to date. Analytical size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering studies suggest that the quaternary structure of USP17 is a monomer. Steady-state kinetic studies show that USP17 efficiently hydrolyzes both ubiquitin-AMC (kcat = 1.5 sec-1 and Km = 1.0 µM) and ubiquitin-rhodamine110 (kcat = 1.8 sec-1 and Km = 2.0 µM) substrates. Ubiquitin chain cleavage assays reveal that USP17 efficiently cleaves di-ubiquitin chains with Lys11, Lys33, Lys48 and Lys63 linkages and tetra-ubiquitin chains with Lys11, Lys48 and Lys63 linkages but is inefficient in cleaving di-ubiquitin chains with Lys6, Lys27, or Lys29 linkages or linear ubiquitin chains. The substrate specificity of USP17 is most similar to that of USP1, where both USPs display higher specificity than other characterized members of the USP family.


Assuntos
Enzimas Desubiquitinantes/química , Endopeptidases/química , Regulação da Expressão Gênica , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Cinética , Luz , Lisina/química , Mutagênese Sítio-Dirigida , Neoplasias/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Espalhamento de Radiação , Ubiquitina/química , Ubiquitinação , Ultracentrifugação , Regulação para Cima
16.
J Virol ; 88(21): 12511-27, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25142582

RESUMO

UNLABELLED: The papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition. IMPORTANCE: Unlocking the secrets of how coronavirus (CoV) papain-like proteases (PLpros) perform their multifunctional roles during viral replication entails a complete mechanistic understanding of their substrate recognition and enzymatic activities. We show that the PLpro domains from the MERS and SARS coronaviruses can recognize and process the same substrates, but with different catalytic efficiencies. The differences in substrate recognition between these closely related PLpros suggest that neither enzyme can be used as a generalized model to explain the kinetic behavior of all CoV PLpros. As a consequence, decoding the mechanisms of PLpro-mediated antagonism of the host innate immune response and the development of anti-CoV PLpro enzyme inhibitors will be a challenging undertaking. The results from this study provide valuable information for understanding how MERS-CoV PLpro-mediated antagonism of the host innate immune response is orchestrated, as well as insight into the design of inhibitors against MERS-CoV PLpro.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Peptídeo Hidrolases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Humanos , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Estrutura Quaternária de Proteína , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
17.
J Virol ; 88(20): 11886-98, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25100843

RESUMO

Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance. Importance: RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East respiratory syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor-resistant mutants have not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.


Assuntos
Coronavirus/fisiologia , Inibidores de Proteases/farmacologia , Replicação Viral , Animais , Linhagem Celular , Linhagem Celular Tumoral , Coronavirus/efeitos dos fármacos , Coronavirus/genética , Cricetinae , Farmacorresistência Viral , Humanos , Camundongos , Camundongos Endogâmicos C57BL
18.
J Virol ; 88(20): 11825-33, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25100850

RESUMO

To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR(-/-)) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities. Importance: Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the in vivo efficacy of an inhibitor of the papain-like protease of severe acute respiratory syndrome coronavirus. Furthermore, we demonstrate that the chimeric-virus system can be adapted to study the proteases of emerging human pathogens, such as Middle East respiratory syndrome coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens, as well as other enzymes capable of removing ISG15 from cellular proteins.


Assuntos
Coronavirus/fisiologia , Modelos Animais de Doenças , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Chlorocebus aethiops , Coronavirus/enzimologia , Cricetinae , Camundongos , Células Vero
19.
Bioorg Med Chem Lett ; 25(22): 5072-7, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26592814

RESUMO

Feline infectious peritonitis (FIP) is a deadly disease that effects both domestic and wild cats and is caused by a mutation in feline coronavirus (FCoV) that allows the virus to replicate in macrophages. Currently, there are no treatments or vaccines available for the treatment of FIP even though it kills approximately 5% of cats in multi-cat households per year. In an effort to develop small molecule drugs targeting FIP for the treatment of cats, we screened a small set of designed peptidomimetic inhibitors for inhibition of FIPV-3CL(pro), identifying two compounds with low to sub-micromolar inhibition, compound 6 (IC50=0.59±0.06 µM) and compound 7 (IC50=1.3±0.1 µM). We determined the first X-ray crystal structure of FIPV-3CL(pro) in complex with the best inhibitor identified, compound 6, to a resolution of 2.10 Å to better understand the structural basis for inhibitor specificity. Our study provides important insights into the structural requirements for the inhibition of FIPV-3CL(pro) by peptidomimetic inhibitors and expands the current structural knowledge of coronaviral 3CL(pro) architecture.


Assuntos
Antivirais/síntese química , Coronavirus Felino/enzimologia , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Oligopeptídeos/síntese química , Peptidomiméticos/síntese química , Antivirais/química , Domínio Catalítico , Proteases 3C de Coronavírus , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Desenho de Fármacos , Escherichia coli , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Oligopeptídeos/química , Peptidomiméticos/química
20.
Bioorg Med Chem Lett ; 25(3): 668-72, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25537272

RESUMO

We describe structure-based design, synthesis, and biological evaluation of a series of novel inhibitors bearing a pyrazole (compounds 3a-h) or a thiazole moiety (compounds 4a-e) as the P3 ligand. We have also explored Boc-ß-amino-l-alanine as a novel P2 ligand. A number of inhibitors have displayed ß-secretase inhibitory potency. Inhibitor 4c has shown potent BACE1 inhibitory activity, Ki=0.25nM, cellular EC50 of 194nM, and displayed good selectivity over BACE2. A model of 4c was created based upon the X-ray structure of 2-bound ß-secretase which revealed critical interactions in the active site.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Desenho de Fármacos , Inibidores de Proteases/síntese química , Pirazóis/química , Tiazóis/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cinética , Ligantes , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ligação Proteica , Pirazóis/síntese química , Pirazóis/metabolismo , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA