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1.
Pharm Res ; 37(10): 194, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32918191

RESUMO

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Neoplasias/metabolismo , Farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Dibenzocicloeptenos/farmacologia , Dicetopiperazinas/farmacologia , Cães , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Madin Darby de Rim Canino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prazosina/farmacocinética , Quinidina/farmacocinética , Quinolinas/farmacologia , Especificidade por Substrato , Transfecção
2.
PLoS One ; 15(7): e0236710, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735606

RESUMO

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


Assuntos
Proteínas Argonauta/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Espectroscopia de Ressonância Magnética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ressonância de Plasmônio de Superfície , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Simulação por Computador , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo
3.
Life Sci ; 258: 118228, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781071

RESUMO

AIMS: Cyclin-dependent kinase 9 (CDK9) is a member of the CDK subfamily and plays a major role in the regulation of transcriptional elongation. It has attracted widespread attention as a therapeutic target for cancer. Here, we aimed to explore novel CDK 9 inhibitors by using a hybrid virtual screening strategy. MAIN METHODS: A hybrid virtual screening strategy was constructed with computer-aided drug design (CADD). First, compounds were filtered in accordance with Lipinski's rule of five and adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Second, a 3D-QSAR pharmacophore model was built and used as a 3D query to screen the obtained hit compounds. Third, the hit compounds were subjected to molecular docking studies. Fourth, molecular dynamics (MD) simulations were performed on CDK9 in complex with the final hits to examine the structural stability. Finally, CDK9 kinase biochemical assay was performed to identify the biological activity of the hit compounds. KEY FINDINGS: Seven hit compounds were screened out. These hit compounds showed drug-like properties in accordance with Lipinski's rule of five and ADMET. Complexes involving the six hit compounds bound to CDK9 exhibited good structural stability in the MD simulation. Furthermore, these six hit compounds had strong inhibitory activity against CDK9 kinase. In particular, hit 3 showed the most promising activity with the percentage of 71%. SIGNIFICANCE: The six hit compounds may be promising novel CDK9 inhibitors, and the hybrid virtual screening strategy designed in this study provides an important reference for the design and synthesis of novel CDK9 inhibitors.


Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/metabolismo , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/metabolismo , Quinase 9 Dependente de Ciclina/química , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína
4.
PLoS One ; 15(8): e0235723, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797045

RESUMO

Mixtures of drugs often have greater therapeutic value than any of their constituent drugs alone, and such combination therapies are widely used to treat diseases such as cancer, malaria, and viral infections. However, developing useful drug mixtures is challenging due to complex interactions between drugs. Natural substances can be fruitful sources of useful drug mixtures because secondary metabolites produced by living organisms do not often act in isolation in vivo. In order to facilitate the study of interactions within natural substances, a new analytical method to quantify interactions using data generated in the process of bioassay-guided fractionation is presented here: the extract fractional inhibitory concentration index (EFICI). The EFICI method uses the framework of Loewe additivity to calculate fractional inhibitory concentration values by which interactions can be determined for any combination of fractions that make up a parent extract. The EFICI method was applied to data on the bioassay-guided fractionation of Lechea mucronata and Schinus terebinthifolia for growth inhibition of the pathogenic bacterium Acinetobacter baumannii. The L. mucronata extract contained synergistic interactions (EFICI = 0.4181) and the S. terebinthifolia extract was non-interactive overall (EFICI = 0.9129). Quantifying interactions in the bioassay-guided fractionation of natural substances does not require additional experiments and can be useful to guide the experimental process and to support the development of standardized extracts as botanical drugs.


Assuntos
Antibacterianos/farmacologia , Produtos Biológicos/farmacologia , Extratos Vegetais/farmacologia , Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Anacardiaceae/química , Antibacterianos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Fracionamento Químico , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Humanos , Extratos Vegetais/isolamento & purificação
5.
Cell Death Dis ; 11(8): 656, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814759

RESUMO

The current epidemic of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for the development of inhibitors of viral replication. Here, we performed a bioinformatic analysis of published and purported SARS-CoV-2 antivirals including imatinib mesylate that we found to suppress SARS-CoV-2 replication on Vero E6 cells and that, according to the published literature on other coronaviruses is likely to act on-target, as a tyrosine kinase inhibitor. We identified a cluster of SARS-CoV-2 antivirals with characteristics of lysosomotropic agents, meaning that they are lipophilic weak bases capable of penetrating into cells. These agents include cepharentine, chloroquine, chlorpromazine, clemastine, cloperastine, emetine, hydroxychloroquine, haloperidol, ML240, PB28, ponatinib, siramesine, and zotatifin (eFT226) all of which are likely to inhibit SARS-CoV-2 replication by non-specific (off-target) effects, meaning that they probably do not act on their 'official' pharmacological targets, but rather interfere with viral replication through non-specific effects on acidophilic organelles including autophagosomes, endosomes, and lysosomes. Imatinib mesylate did not fall into this cluster. In conclusion, we propose a tentative classification of SARS-CoV-2 antivirals into specific (on-target) versus non-specific (off-target) agents based on their physicochemical characteristics.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Morte Celular/efeitos dos fármacos , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Hidroxicloroquina/farmacologia , Mesilato de Imatinib/farmacologia , Lisossomos/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Inibidores de Proteínas Quinases/farmacologia , RNA Viral/efeitos dos fármacos , Células Vero , Carga Viral/efeitos dos fármacos
7.
Cell ; 182(3): 713-721.e9, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32778225

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Avaliação Pré-Clínica de Medicamentos/métodos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas de Produtos Inativados/uso terapêutico , Vacinas Virais/uso terapêutico , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/genética , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Feminino , Cobaias , Imunogenicidade da Vacina , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Pneumonia Viral/virologia , Coelhos , Ratos , Ratos Wistar , Vacinas de Produtos Inativados/efeitos adversos , Células Vero , Vacinas Virais/efeitos adversos
8.
Proc Natl Acad Sci U S A ; 117(31): 18431-18438, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690700

RESUMO

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência/métodos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Influenza Humana/virologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antivirais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Bibliotecas de Moléculas Pequenas/química
9.
Proc Natl Acad Sci U S A ; 117(31): 18477-18488, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32669436

RESUMO

With the recent explosion in the size of libraries available for screening, virtual screening is positioned to assume a more prominent role in early drug discovery's search for active chemical matter. In typical virtual screens, however, only about 12% of the top-scoring compounds actually show activity when tested in biochemical assays. We argue that most scoring functions used for this task have been developed with insufficient thoughtfulness into the datasets on which they are trained and tested, leading to overly simplistic models and/or overtraining. These problems are compounded in the literature because studies reporting new scoring methods have not validated their models prospectively within the same study. Here, we report a strategy for building a training dataset (D-COID) that aims to generate highly compelling decoy complexes that are individually matched to available active complexes. Using this dataset, we train a general-purpose classifier for virtual screening (vScreenML) that is built on the XGBoost framework. In retrospective benchmarks, our classifier shows outstanding performance relative to other scoring functions. In a prospective context, nearly all candidate inhibitors from a screen against acetylcholinesterase show detectable activity; beyond this, 10 of 23 compounds have IC50 better than 50 µM. Without any medicinal chemistry optimization, the most potent hit has IC50 280 nM, corresponding to K i of 173 nM. These results support using the D-COID strategy for training classifiers in other computational biology tasks, and for vScreenML in virtual screening campaigns against other protein targets. Both D-COID and vScreenML are freely distributed to facilitate such efforts.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Aprendizado de Máquina , Bibliotecas de Moléculas Pequenas/farmacologia , Bases de Dados de Proteínas , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/instrumentação , Humanos
10.
Nat Rev Drug Discov ; 19(7): 447-462, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612262

RESUMO

A large number of mouse models have been engineered, characterized and used to advance biomedical research in Alzheimer disease (AD). Early models simply damaged the rodent brain through toxins or lesions. Later, the spread of genetic engineering technology enabled investigators to develop models of familial AD by overexpressing human genes such as those encoding amyloid precursor protein (APP) or presenilins (PSEN1 or PSEN2) carrying mutations linked to early-onset AD. Recently, more complex models have sought to explore the impact of multiple genetic risk factors in the context of different biological challenges. Although none of these models has proven to be a fully faithful reproduction of the human disease, models remain essential as tools to improve our understanding of AD biology, conduct thorough pharmacokinetic and pharmacodynamic analyses, discover translatable biomarkers and evaluate specific therapeutic approaches. To realize the full potential of animal models as new technologies and knowledge become available, it is critical to define an optimal strategy for their use. Here, we review progress and challenges in the use of AD mouse models, highlight emerging scientific innovations in model development, and introduce a conceptual framework for use of preclinical models for therapeutic development.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Modelos Animais de Doenças , Desenvolvimento de Medicamentos/métodos , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Animais , Biomarcadores/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Predisposição Genética para Doença , Humanos , Camundongos , Mutação , Fatores de Risco
11.
ACS Chem Neurosci ; 11(15): 2137-2144, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32639711

RESUMO

Now, it has been evidenced that Covid19 (SARS-CoV-2) infects the brain tissues. Along with this, a challenge has been raised for research professionals to find effective drugs for its treatment since the recent spread of this virus from Wuhan, China. Targeting the treatment of brain infection, it has also been a challenge that the clinical drug should have good CNS penetration ability to cross the blood-brain barrier.


Assuntos
Betacoronavirus , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/metabolismo , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/metabolismo , Antivirais/administração & dosagem , Antivirais/metabolismo , Betacoronavirus/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/virologia , Encéfalo/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/administração & dosagem , Fármacos do Sistema Nervoso Central/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Metilprednisolona/administração & dosagem , Metilprednisolona/metabolismo , Pandemias , Resultado do Tratamento
12.
Science ; 369(6502): 403-413, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703874

RESUMO

Excipients, considered "inactive ingredients," are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant K d values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.


Assuntos
Composição de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Excipientes/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Excipientes/efeitos adversos , Humanos , Terapia de Alvo Molecular
13.
Cell ; 182(3): 734-743.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32643603

RESUMO

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Pandemias/prevenção & controle , Pneumonia Viral/patologia , Pneumonia Viral/prevenção & controle , Vacinação , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Organismos Livres de Patógenos Específicos , Transdução Genética , Células Vero , Carga Viral , Replicação Viral
14.
Cell ; 182(3): 685-712.e19, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645325

RESUMO

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/metabolismo , Proteômica/métodos , Células A549 , Animais , Antivirais/farmacologia , Células CACO-2 , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação , Pneumonia Viral/virologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
J Comput Aided Mol Des ; 34(10): 1091-1103, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32601839

RESUMO

Herein, the LASSBio Chemical Library is presented as a valuable source of compounds for screening to identify hits suitable for subsequent hit-to-lead optimization stages. A feature of the LASSBio Chemical Library worth highlighting is the fact that it is a smart library designed by medicinal chemists with pharmacological activity as the main priority. The great majority of the compounds part of this library have shown in vivo activity in animal models, which is an indication that they possess overall favorable bioavailability properties and, hence, adequate pharmacokinetic profiles. This, in turn, is supported by the fact that approximately 85% of the compounds are compliant with Lipinski's rule of five and ca. 95% are compliant with Veber's rules, two important guidelines for oral bioavailability. In this work it is presented a virtual screening methodology combining a pharmacophore-based model and an empirical Gibbs free energy-based model for the ligand-protein interaction to explore the LASSBio Chemical Library as a source of new hits for the inhibition of the phosphatidylinositol 4-kinase IIIß (PI4KIIIß) enzyme, which is related to the development of viral infections (including enteroviruses, SARS coronavirus, and hepatitis C virus), cancers and neurological diseases. The approach resulted in the identification of two hits, LASSBio-1799 (7) and LASSBio-1814 (10), which inhibited the target enzyme with IC50 values of 3.66 µM and IC50 and 6.09 µM, respectively. This study also enabled the determination of the structural requirements for interactions with the active site of PI4KIIIß, demonstrating the importance of both acceptor and donor hydrogen bonding groups for forming interactions with binding site residues Val598 and Lys549.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sítios de Ligação , Domínio Catalítico , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Álcool)/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
16.
PLoS One ; 15(6): e0233860, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479513

RESUMO

The generation of laminated and light responsive retinal organoids from induced pluripotent stem cells (iPSCs) provides a powerful tool for the study of retinal diseases and drug discovery and a robust platform for cell-based therapies. The aim of this study is to investigate whether retinal organoids can retain their morphological and functional characteristics upon storage at room temperature (RT) conditions and shipment by air using a commercially available container that maintains the environment at ambient temperature. Morphological analysis and measurements of neuroepithelial thickness revealed no differences between control, RT incubated and shipped organoids. Similarly immunohistochemical analysis showed no differences in cell type composition and position within the laminated retinal structure. All groups showed a similar response to light, suggesting that the biological function of retinal organoids was not affected by RT storage or shipment. These findings provide an advance in transport of ready-made retinal organoids, increasing their availability to many research and pharma labs worldwide and facilitating cross-collaborative research.


Assuntos
Organoides/transplante , Serviços Postais , Retina/citologia , Doenças Retinianas/terapia , Diferenciação Celular , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Luz , Organoides/efeitos dos fármacos , Organoides/fisiologia , Organoides/efeitos da radiação , Temperatura
17.
Toxicol Appl Pharmacol ; 401: 115103, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32522582

RESUMO

Small cell lung cancer (SCLC) is a particularly aggressive subset of lung cancer, and identification of new therapeutic options is of significant interest. We recently reported that SCLC cell lines display a specific vulnerability to inhibition of squalene epoxidase (SQLE), an enzyme in the cholesterol biosynthetic pathway that catalyzes the conversion of squalene to 2,3-oxidosqualene. Since it has been reported that SQLE inhibition can result in dermatitis in dogs, we conducted a series of experiments to determine if SQLE inhibitors would be tolerated at exposures predicted to drive maximal efficacy in SCLC tumors. Detailed profiling of the SQLE inhibitor NB-598 showed that dogs did not tolerate predicted efficacious exposures, with dose-limiting toxicity due to gastrointestinal clinical observations, although skin toxicities were also observed. To extend these studies, two SQLE inhibitors, NB-598 and Cmpd-4″, and their structurally inactive analogs, NB-598.ia and Cmpd-4″.ia, were profiled in monkeys. While both active SQLE inhibitors resulted in dose-limiting gastrointestinal toxicity, the structurally similar inactive analogs did not. Collectively, our data demonstrate that significant toxicities arise at exposures well below the predicted levels needed for anti-tumor activity. The on-target nature of the toxicities identified is likely to limit the potential therapeutic utility of SQLE inhibition for the treatment of SCLC.


Assuntos
Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/toxicidade , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Esqualeno Mono-Oxigenase/sangue , Animais , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Macaca fascicularis , Masculino , Pele/efeitos dos fármacos , Pele/enzimologia , Pele/patologia
18.
Nat Protoc ; 15(7): 2203-2229, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541940

RESUMO

Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters. The method is based on dynamic self-assembly of commercially available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivatives assemble with diol-type ligands containing a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to determine the amount of each enantiomer in the mixture with an error of <1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asymmetric reaction products. The procedure comprises high-throughput liquid dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asymmetric transformations and chiral drug development. The procedure takes ~4-6 h and requires 10-20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.


Assuntos
Aminas/química , Amino Álcoois/química , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Estereoisomerismo
20.
Exp Parasitol ; 216: 107940, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32562606

RESUMO

Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.


Assuntos
Alternativas aos Testes com Animais/métodos , Antiprotozoários/toxicidade , Citometria de Fluxo/métodos , Leishmania/efeitos dos fármacos , Doenças Negligenciadas/tratamento farmacológico , Adulto , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Leucócitos/parasitologia , Antimoniato de Meglumina/farmacologia , Antimoniato de Meglumina/uso terapêutico , Antimoniato de Meglumina/toxicidade , Microscopia Confocal , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/parasitologia , Fatores de Tempo , Adulto Jovem
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