RESUMEN
The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. A crucial viral protein, the non-structural protein 14 (nsp14), catalyzes the methylation of viral RNA and plays a critical role in viral genome replication and transcription. Due to the low mutation rate in the nsp region among various SARS-CoV-2 variants, nsp14 has emerged as a promising therapeutic target. However, discovering potential inhibitors remains a challenge. In this work, we introduce a computational pipeline for the rapid and efficient identification of potential nsp14 inhibitors by leveraging virtual screening and the NCI open compound collection, which contains 250,000 freely available molecules for researchers worldwide. The introduced pipeline provides a cost-effective and efficient approach for early-stage drug discovery by allowing researchers to evaluate promising molecules without incurring synthesis expenses. Our pipeline successfully identified seven promising candidates after experimentally validating only 40 compounds. Notably, we discovered NSC620333, a compound that exhibits a strong binding affinity to nsp14 with a dissociation constant of 427 ± 84 nM. In addition, we gained new insights into the structure and function of this protein through molecular dynamics simulations. We identified new conformational states of the protein and determined that residues Phe367, Tyr368, and Gln354 within the binding pocket serve as stabilizing residues for novel ligand interactions. We also found that metal coordination complexes are crucial for the overall function of the binding pocket. Lastly, we present the solved crystal structure of the nsp14-MTase complexed with SS148 (PDB:8BWU), a potent inhibitor of methyltransferase activity at the nanomolar level (IC50 value of 70 ± 6 nM). Our computational pipeline accurately predicted the binding pose of SS148, demonstrating its effectiveness and potential in accelerating drug discovery efforts against SARS-CoV-2 and other emerging viruses.
RESUMEN
Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.
Asunto(s)
COVID-19 , Virus de la Encefalitis Transmitidos por Garrapatas , Humanos , Pandemias , ARN Polimerasa Dependiente del ARN , SARS-CoV-2RESUMEN
SARS-CoV-2 has caused an extensive pandemic of COVID-19 all around the world. Key viral enzymes are suitable molecular targets for the development of new antivirals against SARS-CoV-2 which could represent potential treatments of the corresponding disease. With respect to its essential role in the replication of viral RNA, RNA-dependent RNA polymerase (RdRp) is one of the prime targets. HeE1-2Tyr and related derivatives were originally discovered as inhibitors of the RdRp of flaviviruses. Here, we present that these pyridobenzothiazole derivatives also significantly inhibit SARS-CoV-2 RdRp, as demonstrated using both polymerase- and cell-based antiviral assays.
Asunto(s)
Antivirales/farmacología , Benzotiazoles/farmacología , ARN Polimerasa Dependiente de ARN de Coronavirus/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Piridonas/farmacología , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , SARS-CoV-2/enzimología , SARS-CoV-2/fisiologíaRESUMEN
A series of novel C4-C7-tethered biscoumarin derivatives (12a-e) linked through piperazine moiety was designed, synthesized, and evaluated biological/therapeutic potential. Biscoumarin 12d was found to be the most effective inhibitor of both acetylcholinesterase (AChE, IC50 = 6.30 µM) and butyrylcholinesterase (BChE, IC50 = 49 µM). Detailed molecular modelling studies compared the accommodation of ensaculin (well-established coumarin derivative tested in phase I of clinical trials) and 12d in the human recombinant AChE (hAChE) active site. The ability of novel compounds to cross the blood-brain barrier (BBB) was predicted with a positive outcome for compound 12e. The antiproliferative effects of newly synthesized biscoumarin derivatives were tested in vitro on human lung carcinoma cell line (A549) and normal colon fibroblast cell line (CCD-18Co). The effect of derivatives on cell proliferation was evaluated by MTT assay, quantification of cell numbers and viability, colony-forming assay, analysis of cell cycle distribution and mitotic activity. Intracellular localization of used derivatives in A549 cells was confirmed by confocal microscopy. Derivatives 12d and 12e showed significant antiproliferative activity in A549 cancer cells without a significant effect on normal CCD-18Co cells. The inhibition of hAChE/human recombinant BChE (hBChE), the antiproliferative activity on cancer cells, and the ability to cross the BBB suggest the high potential of biscoumarin derivatives. Beside the treatment of cancer, 12e might be applicable against disorders such as schizophrenia, and 12d could serve future development as therapeutic agents in the prevention and/or treatment of Alzheimer's disease.
Asunto(s)
Técnicas de Química Sintética , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Cumarinas/química , Cumarinas/farmacología , Modelos Moleculares , Células A549 , Enfermedad de Alzheimer/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Colinesterasa/síntesis química , Cumarinas/síntesis química , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A549 human lung carcinoma cell lines were treated with a series of new drugs with both tacrine and coumarin pharmacophores (derivatives 1a-2c) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. The ability of human topoisomerase I (hTOPI) and II to relax supercoiled plasmid DNA in the presence of various concentrations of the tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis. The biological activities of the derivatives were studied using MTT assays, clonogenic assays, cell cycle analysis and quantification of cell number and viability. The content and localization of the derivatives in the cells were analysed using flow cytometry and confocal microscopy. All of the studied compounds were found to have inhibited topoisomerase I activity completely. The effect of the tacrine-coumarin hybrid compounds on cancer cells is likely to be dependent on the length of the chain between the tacrine and coumarin moieties (1c, 1d = tacrine-(CH2)8-9-coumarin). The most active of the tested compounds, derivatives 1c and 1d, both display longer chains.
Asunto(s)
Antineoplásicos/farmacología , Cumarinas/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Tacrina/farmacología , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacología , Células A549 , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Cumarinas/química , ADN-Topoisomerasas de Tipo II/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Tacrina/química , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa II/química , Células Tumorales CultivadasRESUMEN
A series of 7-deazaadenine ribonucleosides bearing alkyl, alkenyl, alkynyl, aryl, or hetaryl groups at position 7 as well as their 5'-O-triphosphates and two types of monophosphate prodrugs (phosphoramidates and S-acylthioethanol esters) were prepared and tested for antiviral activity against selected RNA viruses (Dengue, Zika, tick-borne encephalitis, West Nile, and SARS-CoV-2). The modified triphosphates inhibited the viral RNA-dependent RNA polymerases at micromolar concentrations through the incorporation of the modified nucleotide and stopping a further extension of the RNA chain. 7-Deazaadenosine nucleosides bearing ethynyl or small hetaryl groups at position 7 showed (sub)micromolar antiviral activities but significant cytotoxicity, whereas the nucleosides bearing bulkier heterocycles were still active but less toxic. Unexpectedly, the monophosphate prodrugs were similarly or less active than the corresponding nucleosides in the in vitro antiviral assays, although the bis(S-acylthioethanol) prodrug 14h was transported to the Huh7 cells and efficiently released the nucleoside monophosphate.
Asunto(s)
Antivirales/farmacología , Profármacos/farmacología , Purinas/farmacología , Virus ARN/efectos de los fármacos , Ribonucleósidos/farmacología , COVID-19/virología , Línea Celular Tumoral , Virus del Dengue/efectos de los fármacos , Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Humanos , Fosfatos/farmacología , Nucleósidos de Purina , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2/efectos de los fármacos , Virus del Nilo Occidental/efectos de los fármacos , Virus Zika/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Flavivirus/efectos de los fármacos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Adenosina Trifosfato/química , Adenosina Trifosfato/farmacología , Antivirales/química , Betacoronavirus/efectos de los fármacos , Betacoronavirus/enzimología , COVID-19 , Flavivirus/enzimología , Humanos , Concentración 50 Inhibidora , Pandemias , Virus ARN/efectos de los fármacos , Virus ARN/enzimología , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19RESUMEN
We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 ß-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å2 connects the nsp7 to nsp8 and a second one of approx. 950 Å2 connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.
Asunto(s)
Betacoronavirus/química , ADN Primasa/química , Proteínas no Estructurales Virales/química , Sitios de Unión , ARN Polimerasa Dependiente de ARN de Coronavirus , Cristalografía por Rayos X , ADN Primasa/metabolismo , Modelos Moleculares , Complejos Multiproteicos , Conformación Proteica , Multimerización de Proteína , ARN/metabolismo , SARS-CoV-2 , Proteínas no Estructurales Virales/metabolismoRESUMEN
A series of new 3,6,9-trisubstituted acridine derivatives with fluorine substituents on phenyl ring were synthesized and their interaction with calf thymus DNA was investigated. Analysis using UV-Vis absorbance spectra provided valuable information about the formation of the acridine-DNA complex. In addition, compounds 8b and 8d were found to display an increased binding affinity (Kâ¯=â¯2.32 and 2.28â¯×â¯106 M-1, respectively). Topo I/II inhibition mode assays were also performed, and the results verify that the novel compounds display topoisomerase I and II inhibitory activity; compounds 8a, 8b and 8c completely inhibited topoisomerase I activity at a concentration of 60â¯×â¯10-6 M, but only compound 8d showed partial ability to inhibit topoisomerase II at concentrations of 30 and 50â¯×â¯10-6 M. The ability of the derivatives to impair cell proliferation was tested through an analysis of cell cycle distribution, quantification of cell number, viability studies, metabolic activity measurement and clonogenic assay. The content and localization of the derivatives in cells were analyzed using flow cytometry and fluorescence microscopy. The compounds 8b and 8d altered the physiochemical properties and improved antiproliferative activity in A549 human lung carcinoma cells (compound 8d displayed the highest level of activity, 4.25â¯×â¯10-6 M, after 48â¯h).
Asunto(s)
Acridinas/farmacología , Antineoplásicos/farmacología , ADN/efectos de los fármacos , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacología , Células A549 , Acridinas/síntesis química , Acridinas/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Bovinos , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Halogenación , Humanos , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa I/síntesis química , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/químicaRESUMEN
A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA interaction with newly synthesized 7-MEOTA-tacrine thiourea 12-17 and urea heterodimers 18-22, and to measure interference with type I and II topoisomerases. Their biological profile was also inspected in vitro on the HL-60 cell line using different flow cytometric techniques (cell cycle distribution, detection of mitochondrial membrane potential dissipation, and analysis of metabolic activity/viability). The compounds exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topoisomerase at 1 µM concentration). The treatment of HL-60 cells with the studied compounds showed inhibition of cell growth especially with hybrids containing thiourea (14-17) and urea moieties (21 and 22). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate significant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential drugs to target cancer cells.
Asunto(s)
Acridinas/farmacología , Antineoplásicos/farmacología , Tacrina/farmacología , Tiourea/farmacología , Células A549 , Acridinas/síntesis química , Acridinas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Fibroblastos/efectos de los fármacos , Células HL-60 , Humanos , Relación Estructura-Actividad , Tacrina/química , Tiourea/químicaRESUMEN
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs in the world but some NSAIDs such as diclofenac and tolfenamic acid display levels of cytotoxicity, an effect which has been attributed to the presence of diphenylamine contained in their structures. A novel series of diphenylamine derivatives were synthetised and evaluated for their cytotoxic activities and proliferation inhibition. The most active compounds in the cytotoxicity tests were derivative 6g with an IC50 value of 2.5⯱â¯1.1â¯×â¯10-6â¯M and derivative 6f with an IC50 value of 6.0⯱â¯3.0â¯×â¯10-6â¯M (L1210 cell line) after 48â¯h incubation. The results demonstrate that leukemic L1210 cells were much more sensitive to compounds 6f and 6g than the HEK293T cells (IC50â¯=â¯35â¯×â¯10-6â¯M for 6f and IC50â¯>â¯50â¯×â¯10-6â¯M for 6g) and NIH-3T3 (IC50â¯>â¯50â¯×â¯10-6â¯M for both derivatives). The IC50 values show that these substances may selectively kill leukemic cells over non-cancer cells. Cell cycle analysis revealed that a primary trend of the diphenylamine derivatives was to arrest the cells in the G1-phase of the cell cycle within the first 24â¯h. UV-visible, fluorescence spectroscopy and circular dichroism were used in order to study the binding mode of the novel compounds with DNA. The binding constants determined by UV-visible spectroscopy were found to be in the range of 2.1-8.7â¯×â¯104â¯M-1. We suggest that the observed trend for binding constant K is likely to be a result of different binding thermodynamics accompanying the formation of the complexes.