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1.
Nature ; 593(7859): 424-428, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33767445

RESUMEN

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , COVID-19/virología , Inmunoglobulina G/inmunología , SARS-CoV-2/inmunología , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Peso Corporal , COVID-19/prevención & control , Dependovirus/genética , Modelos Animales de Enfermedad , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Femenino , Humanos , Evasión Inmune/genética , Ratones , Ratones Endogámicos C57BL , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Tratamiento Farmacológico de COVID-19
2.
Nucleic Acids Res ; 52(1): 4-21, 2024 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-37993417

RESUMEN

Several cancer core regulatory circuitries (CRCs) depend on the sustained generation of DNA accessibility by SWI/SNF chromatin remodelers. However, the window when SWI/SNF is acutely essential in these settings has not been identified. Here we used neuroblastoma (NB) cells to model and dissect the relationship between cell-cycle progression and SWI/SNF ATPase activity. We find that SWI/SNF inactivation impairs coordinated occupancy of non-pioneer CRC members at enhancers within 1 hour, rapidly breaking their autoregulation. By precisely timing inhibitor treatment following synchronization, we show that SWI/SNF is dispensable for survival in S and G2/M, but becomes acutely essential only during G1 phase. We furthermore developed a new approach to analyze the oscillating patterns of genome-wide DNA accessibility across the cell cycle, which revealed that SWI/SNF-dependent CRC binding sites are enriched at enhancers with peak accessibility during G1 phase, where they activate genes involved in cell-cycle progression. SWI/SNF inhibition strongly impairs G1-S transition and potentiates the ability of retinoids used clinically to induce cell-cycle exit. Similar cell-cycle effects in diverse SWI/SNF-addicted settings highlight G1-S transition as a common cause of SWI/SNF dependency. Our results illustrate that deeper knowledge of the temporal patterns of enhancer-related dependencies may aid the rational targeting of addicted cancers.


Cancer cells driven by runaway transcription factor networks frequently depend on the cellular machinery that promotes DNA accessibility. For this reason, recently developed small molecules that impair SWI/SNF (or BAF) chromatin remodeling activity have been under active evaluation as anti-cancer agents. However, exactly when SWI/SNF activity is essential in dependent cancers has remained unknown. By combining live-cell imaging and genome-wide profiling in neuroblastoma cells, Cermakova et al. discover that SWI/SNF activity is needed for survival only during G1 phase of the cell cycle. The authors reveal that in several cancer settings, dependency on SWI/SNF arises from the need to reactivate factors involved in G1-S transition. Because of this role, authors find that SWI/SNF inhibition potentiates cell-cycle exit by retinoic acid.


Asunto(s)
Fase G1 , Neoplasias , Factores de Transcripción , Humanos , Ciclo Celular , Cromatina/genética , Ensamble y Desensamble de Cromatina , ADN , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Elementos de Facilitación Genéticos
3.
Bioorg Med Chem Lett ; 112: 129939, 2024 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-39218407

RESUMEN

Autophagy is a catabolic process that was described to play a critical role in advanced stages of cancer, wherein it maintains tumor cell homeostasis and growth by supplying nutrients. Autophagy is also described to support alternative cellular trafficking pathways, providing a non-canonical autophagy-dependent inflammatory cytokine secretion mechanism. Therefore, autophagy inhibitors have high potential in the treatment of cancer and acute inflammation. In our study, we identified compound 1 as an inhibitor of the ATG12-ATG3 protein-protein interaction. We focused on the systematic modification of the original hit 1, a casein kinase 2 (CK2) inhibitor, to find potent disruptors of ATG12-ATG3 protein-protein interaction. A systematic modification of the hit structure led us to a wide plethora of compounds that maintain its ATG12-ATG3 inhibitory activity, which could act as a viable starting point to design new compounds with diverse therapeutic applications.


Asunto(s)
Proteínas Relacionadas con la Autofagia , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Humanos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/antagonistas & inhibidores , Unión Proteica , Estructura Molecular , Autofagia/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo
4.
Bioorg Med Chem Lett ; 97: 129567, 2024 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-38008339

RESUMEN

In human cells, receptor-interacting protein kinase 2 (RIPK2) is mainly known to mediate downstream enzymatic cascades from the nucleotide-binding oligomerization domain-containing receptors 1 and 2 (NOD1/2), which are regulators of pro-inflammatory signaling. Thus, the targeted inhibition of RIPK2 has been proposed as a pharmacological strategy for the treatment of a variety of pathologies, in particular inflammatory and autoimmune diseases. In this work, we designed and developed novel thieno[2,3d]pyrimidine derivatives, in order to explore their activity and selectivity as RIPK2 inhibitors. Primary in vitro evaluations of the new molecules against purified RIPKs (RIPK1-4) demonstrated outstanding inhibitory potency and selectivity for the enzyme RIPK2. Moreover, investigations for efficacy against the RIPK2-NOD1/2 signaling pathways, conducted in living cells, showed their potency could be tuned towards a low nanomolar range. This could be achieved by solely varying the substitutions at position 6 of the thieno[2,3d]pyrimidine scaffold. A subset of lead inhibitors were ultimately evaluated for selectivity against 58 human kinases other than RIPKs, displaying great specificities. We therefore obtained new inhibitors that might serve as starting point for the preparation of targeted tools, which could be useful to gain a better understanding of biological roles and clinical potential of RIPK2.


Asunto(s)
Inflamación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Transducción de Señal , Humanos , Inflamación/tratamiento farmacológico , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo
5.
Nucleic Acids Res ; 50(2): 635-650, 2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-35018474

RESUMEN

Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.


Asunto(s)
Antivirales/química , Antivirales/farmacología , Coronavirus/efectos de los fármacos , Coronavirus/enzimología , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/química , Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Sitios de Unión , Coronavirus/genética , Descubrimiento de Drogas , Humanos , Metilación , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Unión Proteica , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Relación Estructura-Actividad
6.
Beilstein J Org Chem ; 20: 1029-1036, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38746653

RESUMEN

The RNA-dependent RNA polymerase (RdRp) represents a prominent target in the discovery and development of new antivirotics against RNA viruses, inhibiting the replication process. One of the most targeted RNA viruses of the last years is, without doubt, SARS-CoV-2, the cause of the recent COVID-19 pandemic. HeE1-2Tyr, a known inhibitor of flaviviral RdRp, has been discovered to also have antiviral potency against this coronavirus. In this study, we report three distinct modifications of HeE1-2Tyr: conversion of the core from a benzothiazole to a benzoxazole moiety and two different scaffold simplifications, respectively. We provide a novel synthetic approach and, in addition, evaluate the final molecules in an in vitro polymerase assay for biological activity.

7.
PLoS Pathog ; 16(12): e1009100, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33264373

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.


Asunto(s)
COVID-19 , Simulación del Acoplamiento Molecular , Proteínas de la Nucleocápside/química , Fosfoproteínas/química , ARN Viral/química , SARS-CoV-2/química , Humanos , Espectroscopía de Resonancia Magnética , Proteínas de la Nucleocápside/genética , Fosfoproteínas/genética , ARN Viral/genética , SARS-CoV-2/genética
8.
Bioorg Med Chem Lett ; 76: 129010, 2022 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-36184029

RESUMEN

Novel 4-aminoquinazoline-6-carboxamide derivatives bearing differently substituted aryl or heteroaryl groups at position 7 in the core were rationally designed, synthesized and evaluated for biological activity in vitro as phosphatidylinositol 4-kinase IIα (PI4K2A) inhibitors. The straightforward approach described here enabled the sequential, modular synthesis and broad functionalization of the scaffold in a mere six steps. The SAR investigation reported here is based on detailed structural analysis of the conserved binding mode of ATP and other adenine derivatives to the catalytic site of type II PI4Ks, combined with extensive docking studies. Several compounds exhibited significant activity against PI4K2A. Moreover, we solved a crystal structure of PI4K2B in complex with one of our lead ligand candidates, which validated the ligand binding site and pose predicted by our docking-based ligand model. These discoveries suggest that our structure-based approach may be further developed and employed to synthesize new inhibitors with optimized potency and selectivity for this class of PI4Ks.


Asunto(s)
1-Fosfatidilinositol 4-Quinasa , Adenosina Trifosfato , 1-Fosfatidilinositol 4-Quinasa/química , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Ligandos , Adenosina Trifosfato/metabolismo , Adenina , Relación Estructura-Actividad , Diseño de Fármacos , Simulación del Acoplamiento Molecular
10.
Molecules ; 26(13)2021 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-34206406

RESUMEN

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.


Asunto(s)
Antivirales/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteasas/farmacología , Virus ARN/enzimología , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Proteasas Similares a la Papaína de Coronavirus/química , Proteasas Similares a la Papaína de Coronavirus/genética , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Evaluación Preclínica de Medicamentos , Virus de la Encefalitis Transmitidos por Garrapatas/enzimología , Colorantes Fluorescentes/química , Humanos , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/química , ARN Helicasas/genética , ARN Helicasas/metabolismo , SARS-CoV-2/enzimología , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Tratamiento Farmacológico de COVID-19
11.
Angew Chem Int Ed Engl ; 60(18): 10172-10178, 2021 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-33616279

RESUMEN

STING (stimulator of interferon genes) is a key regulator of innate immunity that has recently been recognized as a promising drug target. STING is activated by cyclic dinucleotides (CDNs) which eventually leads to expression of type I interferons and other cytokines. Factors underlying the affinity of various CDN analogues are poorly understood. Herein, we correlate structural biology, isothermal calorimetry (ITC) and computational modeling to elucidate factors contributing to binding of six CDNs-three pairs of natural (ribo) and fluorinated (2'-fluororibo) 3',3'-CDNs. X-ray structural analyses of six {STING:CDN} complexes did not offer any explanation for the different affinities of the studied ligands. ITC showed entropy/enthalpy compensation up to 25 kcal mol-1 for this set of similar ligands. The higher affinities of fluorinated analogues are explained with help of computational methods by smaller loss of entropy upon binding and by smaller strain (free) energy.


Asunto(s)
Proteínas de la Membrana/química , Nucleótidos Cíclicos/química , Sitios de Unión , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular
12.
J Struct Biol ; 211(2): 107548, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32535228

RESUMEN

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/metabolismo
13.
J Mol Recognit ; 33(8): e2842, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32212222

RESUMEN

We report on the discovery of norbornyl moiety as a novel structural motif for cyclin-dependent kinase 2 (CDK2) inhibitors which was identified by screening a carbocyclic nucleoside analogue library. Three micromolar hits were expanded by the use of medicinal chemistry methods into a series of 16 novel compounds. They had prevailingly micromolar activities against CDK2 and the best compound of the series attained IC50 of 190 nM. The binding modes were explored in molecular details by modeling and docking. Quantum mechanics-based scoring was used to rationalize the affinities. In conclusion, the discovered 9-hydroxymethylnorbornyl moiety was shown by joint experimental-theoretical efforts to be able to serve as a novel substituent for CDK2 inhibitors. This finding opens door to the exploration of chemical space towards more effective derivatives targeting this important class of protein kinases.


Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Norbornanos/farmacología , Nucleósidos/análogos & derivados , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-Actividad
14.
Bioorg Med Chem Lett ; 30(4): 126897, 2020 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-31882298

RESUMEN

The nucleoside/nucleotide derived antiviral agents have been the most important components of antiviral therapy used in clinics. Recently, the focus of the medicinal chemists within this exciting research field has been affected mainly by the lack of effective therapies for the Hepatitis C virus (HCV) infection and several other "neglected" diseases caused by viruses such as Zika or Dengue. 2'-Methyl modified nucleosides and their monophosphate prodrugs (ProTides) have revolutionized the therapies for HCV in the last few years and, according to the latest research efforts, have also brought a promise for treatment of diseases caused by other members of Flaviviridae family. Here, we report on the design and synthesis of 5'-N and S modified ProTides derived from 2'-methyladenosine. We studied potential applicability of these derivatives as prodrugs of this archetypal antiviral compound.


Asunto(s)
Antivirales/química , Nucleótidos/química , Profármacos/química , Adenosina/análogos & derivados , Adenosina/química , Antivirales/síntesis química , Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Nitrógeno/química , Nucleótidos/metabolismo , Nucleótidos/farmacología , Profármacos/síntesis química , Profármacos/farmacología , Azufre/química , Virus Zika/efectos de los fármacos
15.
J Lipid Res ; 60(3): 683-693, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30626625

RESUMEN

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.


Asunto(s)
1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , 1-Fosfatidilinositol 4-Quinasa/química , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Transporte Biológico , Células COS , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Inhibidores Enzimáticos/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Conformación Proteica
16.
Artículo en Inglés | MEDLINE | ID: mdl-30642926

RESUMEN

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2'-C-methyl- or 4'-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2'-methylated nucleosides, 7-deaza-2'-C-methyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2'-C-methyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4'-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro However, the strong anti-WNV effect of 4'-azidocytidine and 4'-azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2'-C-methylated or 4'-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.


Asunto(s)
Antivirales/uso terapéutico , Tubercidina/análogos & derivados , Fiebre del Nilo Occidental/tratamiento farmacológico , Virus del Nilo Occidental/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Porcinos , Tubercidina/uso terapéutico , Células Vero , Viremia/tratamiento farmacológico , Replicación Viral/efectos de los fármacos , Fiebre del Nilo Occidental/patología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/genética
17.
Nucleic Acids Res ; 45(9): 5231-5242, 2017 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-28334993

RESUMEN

The excision of 8-oxoguanine (oxoG) by the human 8-oxoguanine DNA glycosylase 1 (hOGG1) base-excision repair enzyme was studied by using the QM/MM (M06-2X/6-31G(d,p):OPLS2005) calculation method and nuclear magnetic resonance (NMR) spectroscopy. The calculated glycosylase reaction included excision of the oxoG base, formation of Lys249-ribose enzyme-substrate covalent adduct and formation of a Schiff base. The formation of a Schiff base with ΔG# = 17.7 kcal/mol was the rate-limiting step of the reaction. The excision of the oxoG base with ΔG# = 16.1 kcal/mol proceeded via substitution of the C1΄-N9 N-glycosidic bond with an H-N9 bond where the negative charge on the oxoG base and the positive charge on the ribose were compensated in a concerted manner by NH3+(Lys249) and CO2-(Asp268), respectively. The effect of Asp268 on the oxoG excision was demonstrated with 1H NMR for WT hOGG1 and the hOGG1(D268N) mutant: the excision of oxoG was notably suppressed when Asp268 was mutated to Asn. The loss of the base-excision function was rationalized with QM/MM calculations and Asp268 was confirmed as the electrostatic stabilizer of ribose oxocarbenium through the initial base-excision step of DNA repair. The NMR experiments and QM/MM calculations consistently illustrated the base-excision reaction operated by hOGG1.


Asunto(s)
Ácido Aspártico/metabolismo , ADN Glicosilasas/química , ADN Glicosilasas/metabolismo , Reparación del ADN , Guanina/análogos & derivados , Lisina/metabolismo , Biocatálisis , Guanina/metabolismo , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Relación Estructura-Actividad , Termodinámica
18.
Angew Chem Int Ed Engl ; 58(46): 16617-16628, 2019 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-31454140

RESUMEN

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIß (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.


Asunto(s)
Antineoplásicos/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Antígenos de Histocompatibilidad Menor/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal/efectos de los fármacos , Tiofenos/farmacología , Animales , Antineoplásicos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cilios/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Antígenos de Histocompatibilidad Menor/genética , Morfolinas/farmacología , Osteogénesis/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Purinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Relación Estructura-Actividad , Tiofenos/química
19.
J Virol ; 91(21)2017 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-28814513

RESUMEN

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

20.
Org Biomol Chem ; 16(11): 1903-1911, 2018 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-29484333

RESUMEN

We report here the synthetic route of two constrained dinucleotides and the determination of the sugar puckering by NMR analyses of the starting nucleosides. Enzymatic ligation to microhelix-RNAs provide access to tRNA analogues containing a 3' terminal A76 locked in South conformation. Biological evaluation of our tRNA analogues has been performed using amino-acyl tRNA-dependent transferase FemXWv, which mediates non-ribosomal incorporation of amino acids into the bacterial cell wall. We have shown that our tRNA analogues inhibited the aminoacyl transfer reaction catalyzed by FemXWv with IC50s of 10 and 8 µM. These results indicate that FemXWv displays a moderate preference for tRNAs containing a terminal A76 locked in the South conformation and that a South to North switch in the conformation of the terminal ribose might contribute to the release of the uncharged tRNAAla product of the aminoacyl transfer reaction catalyzed by FemXwv.


Asunto(s)
Técnicas de Química Sintética/métodos , ARN de Transferencia/química , Ribonucleótidos/química , Ribosa/análogos & derivados , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , ARN de Transferencia/síntesis química , ARN de Transferencia/metabolismo , Ribonucleótidos/síntesis química , Ribonucleótidos/metabolismo , Ribosa/síntesis química , Ribosa/metabolismo , Weissella/enzimología , Weissella/metabolismo
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