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1.
Viruses ; 13(10)2021 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-34696411

RESUMO

Viral proteases are indispensable for successful virion maturation, thus making them a prominent drug target. Their enzyme activity is tightly spatiotemporally regulated by expression in the precursor form with little or no activity, followed by activation via autoprocessing. These cleavage events are frequently triggered upon transportation to a specific compartment inside the host cell. Typically, precursor oligomerization or the presence of a co-factor is needed for activation. A detailed understanding of these mechanisms will allow ligands with non-canonical mechanisms of action to be designed, which would specifically modulate the initial irreversible steps of viral protease autoactivation. Binding sites exclusive to the precursor, including binding sites beyond the protease domain, can be exploited. Both inhibition and up-regulation of the proteolytic activity of viral proteases can be detrimental for the virus. All these possibilities are discussed using examples of medically relevant viruses including herpesviruses, adenoviruses, retroviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, and coronaviruses.


Assuntos
Antivirais/farmacologia , Inibidores de Protease Viral/farmacologia , Proteases Virais/metabolismo , Viroses/tratamento farmacológico , Adenovírus Humanos/efeitos dos fármacos , Adenovírus Humanos/metabolismo , Flavivirus/efeitos dos fármacos , Flavivirus/metabolismo , HIV-1/efeitos dos fármacos , Herpesviridae/efeitos dos fármacos , Herpesviridae/metabolismo , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Proteases Virais/biossíntese
3.
Nat Commun ; 12(1): 5553, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548480

RESUMO

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/metabolismo , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/tratamento farmacológico , Linhagem Celular , Dipeptídeos/farmacologia , Humanos , Mutação , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Proteólise , Proteômica , RNA Interferente Pequeno/farmacologia , SARS-CoV-2/genética , Proteases Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
4.
Expert Rev Clin Pharmacol ; 14(10): 1305-1315, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34301158

RESUMO

BACKGROUND: The high transmission and pathogenicity of SARS-CoV-2 has led to a pandemic that has halted the world's economy and health. The newly evolved strains and scarcity of vaccines has worsened the situation. The main protease (Mpro) of SARS-CoV-2 can act as a potential target due to its role in viral replication and conservation level. METHODS: In this study, we have enlisted more than 1100 phytochemicals from Asian plants based on deep literature mining. The compounds library was screened against the Mpro of SARS-CoV-2. RESULTS: The selected three ligands, Flemichin, Delta-Oleanolic acid, and Emodin 1-O-beta-D-glucoside had a binding energy of -8.9, -8.9, -8.7 KJ/mol respectively. The compounds bind to the active groove of the main protease at; Cys145, Glu166, His41, Met49, Pro168, Met165, Gln189. The multiple descriptors from the simulation study; root mean square deviation, root mean square fluctuation, radius of gyration, hydrogen bond, solvent accessible surface area confirms the stable nature of the protein-ligand complexes. Furthermore, post-md analysis confirms the rigidness in the docked poses over the simulation trajectories. CONCLUSIONS: Our combinatorial drug design approaches may help researchers to identify suitable drug candidates against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Compostos Fitoquímicos/farmacologia , SARS-CoV-2/enzimologia , Proteases Virais/metabolismo , Antivirais/química , Bases de Dados de Compostos Químicos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Compostos Fitoquímicos/química , Proteases Virais/genética
5.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299333

RESUMO

In the last year, the COVID-19 pandemic has highly affected the lifestyle of the world population, encouraging the scientific community towards a great effort on studying the infection molecular mechanisms. Several vaccine formulations are nowadays available and helping to reach immunity. Nevertheless, there is a growing interest towards the development of novel anti-covid drugs. In this scenario, the main protease (Mpro) represents an appealing target, being the enzyme responsible for the cleavage of polypeptides during the viral genome transcription. With the aim of sharing new insights for the design of novel Mpro inhibitors, our research group developed a machine learning approach using the support vector machine (SVM) classification. Starting from a dataset of two million commercially available compounds, the model was able to classify two hundred novel chemo-types as potentially active against the viral protease. The compounds labelled as actives by SVM were next evaluated through consensus docking studies on two PDB structures and their binding mode was compared to well-known protease inhibitors. The best five compounds selected by consensus docking were then submitted to molecular dynamics to deepen binding interactions stability. Of note, the compounds selected via SVM retrieved all the most important interactions known in the literature.


Assuntos
COVID-19/tratamento farmacológico , Inibidores de Protease de Coronavírus/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , SARS-CoV-2/efeitos dos fármacos , Máquina de Vetores de Suporte , Antivirais/farmacologia , COVID-19/virologia , Inibidores de Protease de Coronavírus/metabolismo , Bases de Dados de Produtos Farmacêuticos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas , Aprendizado de Máquina Supervisionado , Proteínas não Estruturais Virais/metabolismo , Proteases Virais/metabolismo
6.
J Virol ; 95(18): e0084821, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34232702

RESUMO

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.


Assuntos
DNA/metabolismo , DNA Polimerase Dirigida por RNA/química , RNA/metabolismo , Ribonuclease H/química , Spumavirus/enzimologia , Proteases Virais/química , Proteínas Virais/química , Microscopia Crioeletrônica , DNA/química , Conformação Proteica , RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/metabolismo , Proteases Virais/metabolismo , Proteínas Virais/metabolismo
7.
Chem Biol Drug Des ; 98(4): 604-619, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34148292

RESUMO

3CLpro is essential for SARS-CoV-2 replication and infection; its inhibition using small molecules is a potential therapeutic strategy. In this study, a comprehensive crystallography-guided fragment-based drug discovery approach was employed to design new inhibitors for SARS-CoV-2 3CLpro. All small molecules co-crystallized with SARS-CoV-2 3CLpro with structures deposited in the Protein Data Bank were used as inputs. Fragments sitting in the binding pocket (87) were grouped into eight geographical types. They were interactively coupled using various synthetically reasonable linkers to generate larger molecules with divalent binding modes taking advantage of two different fragments' interactions. In total, 1,251 compounds were proposed, and 7,158 stereoisomers were screened using Glide (standard precision and extra precision), AutoDock Vina, and Prime MMGBSA. The top 22 hits having conformations approaching the linear combination of their constituent fragments were selected for MD simulation on Desmond. MD simulation suggested 15 of these did adopt conformations very close to their constituent pieces with far higher binding affinity than either constituent domain alone. These structures could provide a starting point for the further design of SARS-CoV-2 3CLpro inhibitors with improved binding, and structures are provided.


Assuntos
Antivirais/química , COVID-19/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Inibidores de Protease Viral/química , Proteases Virais/metabolismo , Antivirais/farmacologia , Cristalização , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Análise Multivariada , Ligação Proteica , Conformação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Inibidores de Protease Viral/farmacologia
8.
Biomolecules ; 11(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071582

RESUMO

The urgent need for novel and effective drugs against the SARS-CoV-2 coronavirus pandemic has stimulated research worldwide. The Papain-like protease (PLpro), which is essential for viral replication, shares a similar active site structural architecture to other cysteine proteases. Here, we have used representatives of the Ovarian Tumor Domain deubiquitinase family OTUB1 and OTUB2 along with the PLpro of SARS-CoV-2 to validate and rationalize the binding of inhibitors from previous SARS-CoV candidate compounds. By forming a new chemical bond with the cysteine residue of the catalytic triad, covalent inhibitors irreversibly suppress the protein's activity. Modeling covalent inhibitor binding requires detailed knowledge about the compounds' reactivities and binding. Molecular Dynamics refinement simulations of top poses reveal detailed ligand-protein interactions and show their stability over time. The recently discovered selective OTUB2 covalent inhibitors were used to establish and validate the computational protocol. Structural parameters and ligand dynamics are in excellent agreement with the ligand-bound OTUB2 crystal structures. For SARS-CoV-2 PLpro, recent covalent peptidomimetic inhibitors were simulated and reveal that the ligand-protein interaction is very dynamic. The covalent and non-covalent docking plus subsequent MD refinement of known SARS-CoV inhibitors into DUBs and the SARS-CoV-2 PLpro point out a possible approach to target the PLpro cysteine protease from SARS-CoV-2. The results show that such an approach gives insight into ligand-protein interactions, their dynamic character, and indicates a path for selective ligand design.


Assuntos
Enzimas Desubiquitinantes/antagonistas & inibidores , Inibidores de Proteases/química , SARS-CoV-2/metabolismo , Proteases Virais/química , Sítios de Ligação , COVID-19/patologia , Domínio Catalítico , Enzimas Desubiquitinantes/metabolismo , Desenho de Fármacos , Feminino , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Inibidores de Proteases/metabolismo , SARS-CoV-2/isolamento & purificação , Proteases Virais/metabolismo
9.
Future Med Chem ; 13(16): 1353-1366, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34169729

RESUMO

Background: The new coronavirus pandemic has had a significant impact worldwide, and therapeutic treatment for this viral infection is being strongly pursued. Efforts have been undertaken by medicinal chemists to discover molecules or known drugs that may be effective in COVID-19 treatment - in particular, targeting the main protease (Mpro) of the virus. Materials & methods: We have employed an innovative strategy - application of ligand- and structure-based virtual screening - using a special compilation of an approved and diverse set of SARS-CoV-2 crystallographic complexes that was recently published. Results and conclusion: We identified seven drugs with different original indications that might act as potential Mpro inhibitors and may be preferable to other drugs that have been repurposed. These drugs will be experimentally tested to confirm their potential Mpro inhibition and thus their effectiveness against COVID-19.


Assuntos
Antivirais/química , COVID-19/tratamento farmacológico , Inibidores de Proteases/química , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Proteases Virais/metabolismo , Antivirais/farmacologia , Bases de Dados de Compostos Químicos , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteases/farmacologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
10.
J Pharmacol Exp Ther ; 378(2): 166-172, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33972366

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to be a global threat since its emergence. Although several COVID-19 vaccines have become available, the prospective timeframe for achieving effective levels of vaccination across global populations remains uncertain. Moreover, the emergence of SARS-CoV-2 variants presents continuing potential challenges for future vaccination planning. Therefore, development of effective antiviral therapies continues to be an urgent unmet need for COVID-19. Successful antiviral regimens for the treatment of human immunodeficiency virus and hepatitis C virus infections have established viral proteases as validated targets for antiviral drug development. In this context, we review protease targets in drug development, currently available antiviral protease inhibitors, and therapeutic development efforts on SARS-CoV-2 main protease and papain-like protease. SIGNIFICANCE STATEMENT: Coronavirus disease 2019 (COVID-19) continues to be a global threat since its emergence. The development of effective antiviral therapeutics for COVID-19 remains an urgent and long-term need. Because viral proteases are validated drug targets, specific severe acute respiratory syndrome coronavirus 2 protease inhibitors are critical therapeutics to be developed for treatment of COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Desenvolvimento de Medicamentos , Inibidores de Proteases/farmacologia , SARS-CoV-2/enzimologia , Proteases Virais/metabolismo , Animais , Antivirais/uso terapêutico , Humanos , Inibidores de Proteases/uso terapêutico , SARS-CoV-2/efeitos dos fármacos
11.
ACS Infect Dis ; 7(6): 1483-1502, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34019767

RESUMO

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6-8 amino acids. Short stretches of homologous host-pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify sequences cleaved by the SARS-CoV-2 papain-like protease (PLpro) in vitro within human MYH7 and MYH6 (two cardiac myosins linked to several cardiomyopathies), FOXP3 (an X-linked Treg cell transcription factor), ErbB4 (HER4), and vitamin-K-dependent plasma protein S (PROS1), an anticoagulation protein that prevents blood clots. Zinc inhibited the cleavage of these host sequences in vitro. Other patterns emerged from multispecies sequence alignments of the cleavage sites, which may have implications for the selection of animal models and zoonosis. SSHHPS/nsP is an example of a sequence-specific post-translational silencing mechanism.


Assuntos
Papaína , Peptídeo Hidrolases , SARS-CoV-2/enzimologia , Proteases Virais/metabolismo , Sequência de Aminoácidos , Miosinas Cardíacas/química , Fatores de Transcrição Forkhead/química , Humanos , Cadeias Pesadas de Miosina/química , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , Proteína S/química , Receptor ErbB-4/química
13.
Pharmacol Rep ; 73(3): 926-938, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33970450

RESUMO

INTRODUCTION: Drug repurposing is the need of the hour considering the medical emergency caused by the COVID-19 pandemic. Recently, cytokine storm by the host immune system has been linked with high viral load, loss of lung function, acute respiratory distress syndrome (ARDS), multiple organ failure, and subsequent fatal outcome. OBJECTIVE: This study aimed to identify potential FDA approved drugs that can be repurposed for COVID-19 treatment using an in-silico analysis. METHODS: In this study, virtual screening of selected FDA approved drugs was performed by targeting the main protease (Mpro) of SARS-CoV-2 and the key molecules involved in the 'Cytokine storm' in COVID-19 patients. Based on our preliminary screening supported by extensive literature search, we selected FDA approved drugs to target the SARS-CoV-2 main protease (Mpro) and the key players of cytokine storm, TNF-α, IL-6, and IL-1ß. These compounds were examined based on systematic docking studies and further validated using a combination of molecular dynamics simulations and molecular mechanic/generalized/Born/Poisson-Boltzmann surface area (MM/G/P/BSA) free energy calculations. RESULTS: Based on the findings, Rifampicin and Letermovir appeared as the most promising drug showing a very good binding affinity with the main protease of SARS-CoV-2 and TNF-α, IL-6, and IL-1ß. However, it is pertinent to mention here that our findings need further validation by in vitro analysis and clinical trials. CONCLUSION: This study provides an insight into the drug repurposing approach in which several FDA approved drugs were examined to inhibit COVID-19 infection by targeting the main protease of SARS-COV-2 and the cytokine storm.


Assuntos
Acetatos/uso terapêutico , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Quinazolinas/uso terapêutico , Rifampina/uso terapêutico , COVID-19/metabolismo , Síndrome da Liberação de Citocina/tratamento farmacológico , Citocinas/metabolismo , Reposicionamento de Medicamentos/métodos , Humanos , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , Proteases Virais/metabolismo
14.
Viruses ; 13(4)2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921849

RESUMO

Various viruses alter nuclear pore complex (NPC) integrity to access the nuclear content favoring their replication. Alteration of the nuclear pore complex has been observed not only in viruses that replicate in the nucleus but also in viruses with a cytoplasmic replicative cycle. In this last case, the alteration of the NPC can reduce the transport of transcription factors involved in the immune response or mRNA maturation, or inhibit the transport of mRNA from the nucleus to the cytoplasm, favoring the translation of viral mRNAs or allowing access to nuclear factors necessary for viral replication. In most cases, the alteration of the NPC is mediated by viral proteins, being the viral proteases, one of the most critical groups of viral proteins that regulate these nucleus-cytoplasmic transport changes. This review focuses on the description and discussion of the role of viral proteases in the modification of nucleus-cytoplasmic transport in viruses with cytoplasmic replicative cycles and its repercussions in viral replication.


Assuntos
Poro Nuclear/metabolismo , Proteases Virais/metabolismo , Replicação Viral , Vírus , Transporte Ativo do Núcleo Celular , Linhagem Celular , Humanos , Vírus/metabolismo , Vírus/patogenicidade
15.
Biomolecules ; 11(3)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808721

RESUMO

The huge global expansion of the COVID-19 pandemic caused by the novel SARS-corona virus-2 is an extraordinary public health emergency. The unavailability of specific treatment against SARS-CoV-2 infection necessitates the focus of all scientists in this direction. The reported antiviral activities of guanidine alkaloids encouraged us to run a comprehensive in silico binding affinity of fifteen guanidine alkaloids against five different proteins of SARS-CoV-2, which we investigated. The investigated proteins are COVID-19 main protease (Mpro) (PDB ID: 6lu7), spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and a non-structural protein (nsp10) (PDB ID: 6W4H). The binding energies for all tested compounds indicated promising binding affinities. A noticeable superiority for the pentacyclic alkaloids particularly, crambescidin 786 (5) and crambescidin 826 (13) has been observed. Compound 5 exhibited very good binding affinities against Mpro (ΔG = -8.05 kcal/mol), nucleocapsid phosphoprotein (ΔG = -6.49 kcal/mol), and nsp10 (ΔG = -9.06 kcal/mol). Compound 13 showed promising binding affinities against Mpro (ΔG = -7.99 kcal/mol), spike glycoproteins (ΔG = -6.95 kcal/mol), and nucleocapsid phosphoprotein (ΔG = -8.01 kcal/mol). Such promising activities might be attributed to the long ω-fatty acid chain, which may play a vital role in binding within the active sites. The correlation of c Log P with free binding energies has been calculated. Furthermore, the SAR of the active compounds has been clarified. The Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) studies were carried out in silico for the 15 compounds; most examined compounds showed optimal to good range levels of ADMET aqueous solubility, intestinal absorption and being unable to pass blood brain barrier (BBB), non-inhibitors of CYP2D6, non-hepatotoxic, and bind plasma protein with a percentage less than 90%. The toxicity of the tested compounds was screened in silico against five models (FDA rodent carcinogenicity, carcinogenic potency TD50, rat maximum tolerated dose, rat oral LD50, and rat chronic lowest observed adverse effect level (LOAEL)). All compounds showed expected low toxicity against the tested models. Molecular dynamic (MD) simulations were also carried out to confirm the stable binding interactions of the most promising compounds, 5 and 13, with their targets. In conclusion, the examined 15 alkaloids specially 5 and 13 showed promising docking, ADMET, toxicity and MD results which open the door for further investigations for them against SARS-CoV-2.


Assuntos
Alcaloides/química , Antivirais/química , Proteínas do Nucleocapsídeo de Coronavírus/química , Poríferos/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Animais , Antivirais/farmacologia , Antivirais/toxicidade , Barreira Hematoencefálica , Cristalografia por Raios X , Ligantes , Glicoproteínas de Membrana/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfoproteínas/química , Inibidores de Proteases/química , Ratos , Software , Proteases Virais/química
16.
Methods Mol Biol ; 2266: 227-238, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33759130

RESUMO

Three-dimensional pharmacophore models have been proven extremely valuable in exploring novel chemical space through virtual screening. However, traditional pharmacophore-based approaches need ligand information and rely on static snapshots of highly dynamic systems. In this chapter, we describe PyRod, a novel tool to generate three-dimensional pharmacophore models based on water traces of a molecular dynamics simulation of an apo-protein.The protocol described herein was successfully applied for the discovery of novel drug-like inhibitors of West Nile virus NS2B-NS3 protease. By using this recent example, we highlight the key steps of the generation and validation of PyRod-derived pharmacophore models and their application for virtual screening.


Assuntos
Descoberta de Drogas/métodos , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Software , Proteases Virais/química , Água/química , Sítios de Ligação , Ligantes , Modelos Moleculares , Conformação Molecular , Simulação de Acoplamento Molecular , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Proteínas não Estruturais Virais/química , Vírus do Nilo Ocidental/química
17.
Eur J Pharm Sci ; 160: 105744, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33540040

RESUMO

The current global pandemic outbreak of COVID-19, caused by the SARS-CoV-2, strikes an invincible damage to both daily life and the global economy. WHO guidelines for COVID-19 clinical management includes infection control and prevention, social distancing and supportive care using supplemental oxygen and mechanical ventilator support. Currently, evolving researches and clinical reports regarding infected patients with SARS-CoV-2 suggest a potential list of repurposed drugs that may produce appropriate pharmacological therapeutic efficacies in treating COVID-19 infected patients. In this study, we performed virtual screening and evaluated the obtained results of US-FDA approved small molecular database library (302 drug molecule) against two important different protein targets in COVID-19. Best compounds in molecular docking were used as a training set for generation of two different pharmacophores. The obtained pharmacophores were employed for virtual screening of ChEMBL database. The filtered compounds were clustered using Finger print model to obtain two compounds that will be subjected to molecular docking simulations against the two targets. Compounds complexes with SARS-CoV-2 main protease and S-protein were studied using molecular dynamics (MD) simulation. MD simulation studies suggest the potential inhibitory activity of ChEMBL398869 against SARS-CoV-2 main protease and restress the importance of Gln189 flexibility in inhibitors recognition through increasing S2 subsite plasticity.


Assuntos
Antivirais/farmacologia , COVID-19/virologia , Bases de Dados de Proteínas , Simulação de Dinâmica Molecular , SARS-CoV-2/enzimologia , Proteases Virais/metabolismo , Substituição de Aminoácidos , Antivirais/química , Humanos , Modelos Químicos , Estrutura Molecular , Conformação Proteica , SARS-CoV-2/genética , Relação Estrutura-Atividade , Inibidores de Protease Viral , Proteases Virais/química , Proteases Virais/genética
18.
J R Soc Interface ; 18(174): 20200591, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33402024

RESUMO

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has no publicly available vaccine or antiviral drugs at the time of writing. An attractive coronavirus drug target is the main protease (Mpro, also known as 3CLpro) because of its vital role in the viral cycle. A significant body of work has been focused on finding inhibitors which bind and block the active site of the main protease, but little has been done to address potential non-competitive inhibition, targeting regions other than the active site, partly because the fundamental biophysics of such allosteric control is still poorly understood. In this work, we construct an elastic network model (ENM) of the SARS-CoV-2 Mpro homodimer protein and analyse its dynamics and thermodynamics. We found a rich and heterogeneous dynamical structure, including allosterically correlated motions between the homodimeric protease's active sites. Exhaustive 1-point and 2-point mutation scans of the ENM and their effect on fluctuation free energies confirm previously experimentally identified bioactive residues, but also suggest several new candidate regions that are distant from the active site, yet control the protease function. Our results suggest new dynamically driven control regions as possible candidates for non-competitive inhibiting binding sites in the protease, which may assist the development of current fragment-based binding screens. The results also provide new insights into the active biophysical research field of protein fluctuation allostery and its underpinning dynamical structure.


Assuntos
COVID-19/virologia , SARS-CoV-2/metabolismo , Proteases Virais/química , Simulação por Computador , Cristalização , Humanos , Modelos Moleculares , Conformação Proteica , SARS-CoV-2/enzimologia , Termodinâmica , Proteases Virais/efeitos dos fármacos , Proteases Virais/metabolismo
19.
Biochimie ; 182: 177-184, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33484784

RESUMO

The main protease (Mpro) of SARS-CoV and SARS-CoV-2 is a key enzyme in viral replication and a promising target for the development of antiviral therapeutics. The understanding of this protein is based on a number of observations derived from earlier x-ray structures, which mostly consider substrates or ligands as the main reason behind modulation of the active site. This lead to the concept of substrate-induced subsite cooperativity as an initial attempt to explain the dual binding specificity of this enzyme in recognizing the cleavage sequences at its N- and C-termini, which are important processing steps in obtaining the mature protease. The presented hypothesis proposes that structural heterogeneity is a property of the enzyme, independent of the presence of a substrate or ligand. Indeed, the analysis of Mpro structures of SARS-CoV and SARS-CoV-2 reveals a conformational diversity for the catalytically competent state in ligand-free structures. Variation in the binding site appears to result from flexibility at residues lining the S1 subpocket and segments incorporating methionine 49 and glutamine 189. The structural evidence introduces "structure-based recognition" as a new paradigm in substrate proteolysis by Mpro. In this concept, the binding space in subpockets of the enzyme varies in a non-cooperative manner, causing distinct conformations, which recognize and process different cleavage sites, as the N- and C-termini. Insights into the recognition basis of the protease provide explanation to the ordered processing of cleavage sites. The hypothesis expands the conformational space of the enzyme and consequently opportunities for drug development and repurposing efforts.


Assuntos
COVID-19/virologia , Conformação Proteica , Vírus da SARS/enzimologia , SARS-CoV-2/enzimologia , Proteases Virais/química , Proteases Virais/metabolismo , Antivirais/química , Antivirais/metabolismo , Domínio Catalítico , Desenho de Fármacos , Humanos , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Especificidade por Substrato
20.
Nat Commun ; 12(1): 668, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510133

RESUMO

Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 µM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.


Assuntos
COVID-19/tratamento farmacológico , Inibidores de Protease de Coronavírus/farmacologia , SARS-CoV-2/efeitos dos fármacos , Proteases Virais/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Humanos , Indóis/farmacologia , Piridinas/farmacologia , Células Vero , Proteases Virais/metabolismo
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