RESUMEN
Natural products are a valuable resource for the discovery of novel crop protection agents. A series of γ-butyrolactone derivatives, derived from the simplification of podophyllotoxin's structure, were synthesized and assessed for their efficacy against tobacco mosaic virus (TMV). Several derivatives exhibited notable antiviral properties, with compound 3g demonstrating the most potent in vivo anti-TMV activity. At 500 µg/mL, compound 3g achieved an inactivation effect of 87.8%, a protective effect of 71.7%, and a curative effect of 67.7%, surpassing the effectiveness of the commercial plant virucides ningnanmycin and ribavirin. Notably, the syn-diastereomer (syn-3g) exhibited superior antiviral activity compared to the anti-diastereomer (anti-3g). Mechanistic studies revealed that syn-3g could bind to the TMV coat protein and interfere with the self-assembly process of TMV particles. These findings indicate that compound 3g, with its simple chemical structure, could be a potential candidate for the development of novel antiviral agents for crop protection.
Asunto(s)
4-Butirolactona , Antivirales , Podofilotoxina , Virus del Mosaico del Tabaco , Podofilotoxina/química , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Antivirales/síntesis química , Antivirales/farmacología , Virus del Mosaico del Tabaco/efectos de los fármacos , Ensamble de Virus/efectos de los fármacos , Proteínas de la Cápside/metabolismo , Protección de Cultivos , Cristalografía por Rayos X , Relación Estructura-Actividad , Nicotiana/efectos de los fármacos , Nicotiana/metabolismo , Nicotiana/virología , Simulación del Acoplamiento MolecularRESUMEN
High levels of hepatitis B virus (HBV) surface antigen (HBsAg) in the blood of chronic HBV carriers are considered to drive the exhaustion of antigen-specific T and B lymphocytes and thus responsible for the persistence of infection. Accordingly, therapeutic elimination of HBsAg may facilitate the activation of adaptive antiviral immune responses against HBV and achieve a functional cure of chronic hepatitis B. We discovered recently that an amphipathic alpha helix spanning W156 to R169 of HBV small envelope (S) protein plays an essential role in the morphogenesis of subviral particles (SVPs) and metabolism of S protein. We thus hypothesized that pharmacological disruption of SVP morphogenesis may induce intracellular degradation of S protein and reduce HBsAg secretion. To identify inhibitors of SVP biogenesis, we screened 4417 bioactive compounds with a HepG2-derived cell line expressing HBV S protein and efficiently secreting small spherical SVPs. The screen identified 24 compounds that reduced intracellular SVPs and secreted HBsAg in a concentration-dependent manner. However, 18 of those compounds inhibited the secretion of HBsAg and HBeAg in HBV replicon transfected HepG2 cells at similar efficiency, suggesting each of those compounds may disrupt a common cellular function required for the synthesis and/or secretion of these viral proteins. Interestingly, lycorine more efficiently inhibited the secretion of HBsAg in HepG2 cells transfected with HBV replicons, HepG2.2.15 cells and HBV infected - HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). The structure activity relationship and antiviral mechanism of lycorine against HBV have been determined.
Asunto(s)
Antivirales , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Humanos , Virus de la Hepatitis B/efectos de los fármacos , Antivirales/farmacología , Antígenos de Superficie de la Hepatitis B/metabolismo , Células Hep G2 , Ensamble de Virus/efectos de los fármacos , Virión/efectos de los fármacos , Descubrimiento de Drogas , Replicación Viral/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas del Envoltorio Viral/metabolismo , Antígenos e de la Hepatitis B/metabolismoRESUMEN
A series of ferulic acid dimers were designed, synthesized, and evaluated for anti-TMV activity. Biological assays demonstrated that compounds A6, E3, and E5 displayed excellent inactivating against tobacco mosaic virus (TMV) with EC50 values of 62.8, 94.4, and 85.2 µg mL-1, respectively, which were superior to that of ningnanmycin (108.1 µg mL-1). Microscale thermophoresis indicated that compounds A6, E3, and E5 showed strong binding capacity to TMV coat protein with binding affinity values of 1.862, 3.439, and 2.926 µM, respectively. Molecular docking and molecular dynamics simulation revealed that compound A6 could firmly bind to the TMV coat protein through hydrogen and hydrophobic bonds. Transmission electron microscopy and self-assembly experiments indicated that compound A6 obviously destroyed the integrity of the TMV particles and blocked the virus from infecting the host. This study revealed that A6 can be used as a promising leading structure for the development of antiviral agents by inhibiting TMV self-assembly.
Asunto(s)
Antivirales , Ácidos Cumáricos , Simulación del Acoplamiento Molecular , Virus del Mosaico del Tabaco , Virus del Mosaico del Tabaco/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacología , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Enfermedades de las Plantas/virología , Ensamble de Virus/efectos de los fármacos , Dimerización , Simulación de Dinámica MolecularRESUMEN
Hepatitis B virus (HBV) displays remarkable self-assembly capabilities that interest the scientific community and biotechnological industries as HBV is leading to an annual mortality of up to 1 million people worldwide (especially in Africa and Southeast Asia). When the ionic strength is increased, hepatitis B virus-like particles (VLPs) can assemble from dimers of the first 149 residues of the HBV capsid protein core assembly domain (Cp149). Using solution small-angle X-ray scattering, we investigated the disassembly of the VLPs by titrating guanidine hydrochloride (GuHCl). Measurements were performed with and without 1 M NaCl, added either before or after titrating GuHCl. Fitting the scattering curves to a linear combination of atomic models of Cp149 dimer (the subunit) and T = 3 and T = 4 icosahedral capsids revealed the mass fraction of the dimer in each structure in all the titration points. Based on the mass fractions, the variation in the dimer-dimer association standard free energy was calculated as a function of added GuHCl, showing a linear relation between the interaction strength and GuHCl concentration. Using the data, we estimated the energy barriers for assembly and disassembly and the critical nucleus size for all of the assembly reactions. Extrapolating the standard free energy to [GuHCl] = 0 showed an evident hysteresis in the assembly process, manifested by differences in the dimer-dimer association standard free energy obtained for the disassembly reactions compared with the equivalent assembly reactions. Similar hysteresis was observed in the energy barriers for assembly and disassembly and the critical nucleus size. The results suggest that above 1.5 M, GuHCl disassembled the capsids by attaching to the protein and adding steric repulsion, thereby weakening the hydrophobic attraction.
Asunto(s)
Cápside , Guanidina , Virus de la Hepatitis B , Guanidina/química , Guanidina/farmacología , Virus de la Hepatitis B/química , Virus de la Hepatitis B/fisiología , Virus de la Hepatitis B/efectos de los fármacos , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Dispersión del Ángulo Pequeño , Multimerización de Proteína , Modelos Moleculares , Ensamble de Virus/efectos de los fármacos , Difracción de Rayos XRESUMEN
Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B Crónica , Hepatocitos , Humanos , Hepatocitos/virología , Hepatocitos/efectos de los fármacos , Animales , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Ratones , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Proteínas del Núcleo Viral/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Antígenos del Núcleo de la Hepatitis B/metabolismo , Cápside/metabolismo , Cápside/efectos de los fármacos , Hígado/virología , Hígado/efectos de los fármacos , Hígado/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Ensamble de Virus/efectos de los fármacos , Apoptosis/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). We show that PAV-104 inhibits >99% of infection with diverse SARS-CoV-2 variants in immortalized AECs, and in primary human AECs cultured at the air-liquid interface (ALI) to represent the lung microenvironment in vivo. Our data demonstrate that PAV-104 inhibits SARS-CoV-2 production without affecting viral entry, mRNA transcription, or protein synthesis. PAV-104 interacts with SARS-CoV-2 nucleocapsid (N) and interferes with its oligomerization, blocking particle assembly. Transcriptomic analysis reveals that PAV-104 reverses SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19 with a mechanism of action that is distinct from existing clinical management approaches.
Asunto(s)
Antivirales , Células Epiteliales , SARS-CoV-2 , Replicación Viral , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacos , Células Epiteliales/virología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Antivirales/farmacología , Ensamble de Virus/efectos de los fármacos , COVID-19/virología , Tratamiento Farmacológico de COVID-19RESUMEN
Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) has been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress. We find that classically neutralizing antibodies against hemagglutinin are broadly multifunctional, inhibiting virus assembly and release at concentrations 1-20-fold higher than the concentrations at which they inhibit viral entry. These antibodies are also capable of altering the morphological features of shed virions, reducing the proportion of filamentous particles. We find that antibodies against neuraminidase and M2 also restrict viral egress and that inhibition by anti-neuraminidase mAbs is only partly attributable to a loss in enzymatic activity. In all cases, antigen crosslinking-either on the surface of the infected cell, between the viral and cell membrane, or both-plays a critical role in inhibition, and we are able to distinguish between these modes experimentally and through a structure-based computational model. Together, these results provide a framework for dissecting antibody multifunctionality that could help guide the development of improved therapeutic antibodies or vaccines and that can be extended to other viral families and antibody isotypes.IMPORTANCEAntibodies against influenza A virus provide multifaceted protection against infection. Although sensitive and quantitative assays are widely used to measure inhibition of viral attachment and entry, the ability of diverse antibodies to inhibit viral egress is less clear. We address this challenge by developing an imaging-based approach to measure antibody inhibition of virus release across a panel of monoclonal antibodies targeting the influenza A virus surface proteins. Using this approach, we find that inhibition of viral egress is common and can have similar potency to the ability of an antibody to inhibit viral entry. Insights into this understudied aspect of antibody function may help guide the development of improved countermeasures.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Virus de la Influenza A , Gripe Humana , Ensamble de Virus , Humanos , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Virus de la Influenza A/efectos de los fármacos , Vacunas contra la Influenza , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Proteínas de la Membrana , Neuraminidasa/metabolismo , Ensamble de Virus/efectos de los fármacosRESUMEN
IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.
Asunto(s)
Antivirales , Cápside , Virus de la Hepatitis B , Hepatitis B Crónica , Ensamble de Virus , Animales , Humanos , Ratones , Antivirales/clasificación , Antivirales/farmacología , Antivirales/uso terapéutico , Cápside/química , Cápside/efectos de los fármacos , Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/efectos de los fármacos , Proteínas de la Cápside/metabolismo , Células Cultivadas , Virus de la Hepatitis B/química , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis B/metabolismo , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Técnicas In Vitro , Ensamble de Virus/efectos de los fármacos , Modelos Animales de EnfermedadRESUMEN
Capsid assembly modulators (CAMs) represent a novel class of antiviral agents targeting hepatitis B virus (HBV) capsid to disrupt the assembly process. NVR 3-778 is the first CAM to demonstrate antiviral activity in patients infected with HBV. However, the relatively low aqueous solubility and moderate activity in the human body halted further development of NVR 3-778. To improve the anti-HBV activity and the drug-like properties of NVR 3-778, we designed and synthesized a series of NVR 3-778 derivatives. Notably, phenylboronic acid-bearing compound 7b (EC50 = 0.83 ± 0.33 µM, CC50 = 19.4 ± 5.0 µM) displayed comparable anti-HBV activity to NVR 3-778 (EC50 = 0.73 ± 0.20 µM, CC50 = 23.4 ± 7.0 µM). Besides, 7b showed improved water solubility (328.8 µg/mL, pH 7) compared to NVR 3-778 (35.8 µg/mL, pH 7). Size exclusion chromatography (SEC) and quantification of encapsidated viral RNA were used to demonstrate that 7b behaves as a class II CAM similar to NVR 3-778. Moreover, molecular dynamics (MD) simulations were conducted to rationalize the structure-activity relationships (SARs) of these novel derivatives and to understand their key interactions with the binding pocket, which provide useful indications for guiding the further rational design of more effective anti-HBV drugs.
Asunto(s)
Antivirales , Benzamidas , Cápside , Diseño de Fármacos , Virus de la Hepatitis B , Ensamble de Virus , Humanos , Antivirales/síntesis química , Antivirales/química , Antivirales/farmacología , Benzamidas/síntesis química , Benzamidas/química , Benzamidas/farmacología , Cápside/efectos de los fármacos , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Ensamble de Virus/efectos de los fármacosRESUMEN
RNA sequences/motifs dispersed across the genome of Hepatitis B Virus regulate formation of nucleocapsid-like particles (NCPs) by core protein (Cp) in vitro, in an epsilon/polymerase-independent fashion. These multiple RNA Packaging Signals (PSs) can each form stem-loops encompassing a Cp-recognition motif, -RGAG-, in their loops. Drug-like molecules that bind the most important of these PS sites for NCP assembly regulation with nanomolar affinities, were identified by screening an immobilized ligand library with a fluorescently-labelled, RNA oligonucleotide encompassing this sequence. Sixty-six of these "hits", with affinities ranging from low nanomolar to high micromolar, were purchased as non-immobilized versions. Their affinities for PSs and effects on NCP assembly were determined in vitro by Surface Plasmon Resonance. High-affinity ligand binding is dependent on the presence of an -RGAG- motif within the loop of the PS, consistent with ligand cross-binding between PS sites. Simple structure-activity relationships show that it is also dependent on the presence of specific functional groups in these ligands. Some compounds are potent inhibitors of in vitro NCP assembly at nanomolar concentrations. Despite appropriate logP values, these ligands do not inhibit HBV replication in cell culture. However, modelling confirms the potential of using PS-binding ligands to target NCP assembly as a novel anti-viral strategy. This also allows for computational exploration of potential synergic effects between anti-viral ligands directed at distinct molecular targets in vivo. HBV PS-regulated assembly can be dysregulated by novel small molecule RNA-binding ligands opening a novel target for developing directly-acting anti-virals against this major pathogen.
Asunto(s)
Virus de la Hepatitis B , Ensamble de Virus , Antivirales/farmacología , Virus de la Hepatitis B/fisiología , Humanos , Ligandos , Nucleocápside/metabolismo , ARN Viral/metabolismo , Ensamble de Virus/efectos de los fármacos , Replicación ViralRESUMEN
Several strategies have been developed to fight viral infections, not only in humans but also in animals and plants. Some of them are based on the development of efficient vaccines, to target the virus by developed antibodies, others focus on finding antiviral compounds with activities that inhibit selected virus replication steps. Currently, there is an increasing number of antiviral drugs on the market; however, some have unpleasant side effects, are toxic to cells, or the viruses quickly develop resistance to them. As the current situation shows, the combination of multiple antiviral strategies or the combination of the use of various compounds within one strategy is very important. The most desirable are combinations of drugs that inhibit different steps in the virus life cycle. This is an important issue especially for RNA viruses, which replicate their genomes using error-prone RNA polymerases and rapidly develop mutants resistant to applied antiviral compounds. Here, we focus on compounds targeting viral structural capsid proteins, thereby inhibiting virus assembly or disassembly, virus binding to cellular receptors, or acting by inhibiting other virus replication mechanisms. This review is an update of existing papers on a similar topic, by focusing on the most recent advances in the rapidly evolving research of compounds targeting capsid proteins of RNA viruses.
Asunto(s)
Antivirales/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , Infecciones por Virus ARN/tratamiento farmacológico , Virus ARN/efectos de los fármacos , Ensamble de Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antivirales/química , Humanos , Infecciones por Virus ARN/virología , Virus ARN/fisiologíaRESUMEN
As a key element during HBV replication, a nucleocapsid is considered a promising target for the treatment of chronic hepatitis B. The present study aimed to identify small molecules as novel capsid assembly modulators with antiviral activity. Structure-based virtual screening of an integrated compound library led to the identification of several types of HBV inhibitors. Among these inhibitors, N-sulfonylpiperidine-3-carboxamides (SPCs) potently reduced the amount of secreted HBV DNA. Through structure-activity relationship studies, we identified an SPC derivative, namely, C-39, which exhibited the highest antiviral activity without causing cytotoxicity. Mechanism studies showed that C-39 dose-dependently inhibited the formation of HBV capsid, synthesis of cccDNA, e antigen (HBeAg), viral pregenomic RNA (pgRNA), and HBV DNA levels, thereby restraining HBV replication. In summary, SPCs represent a new class of capsid assembly modulators. Further optimization of SPCs is expected to obtain new antiviral drugs against HBV infection.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/virología , Nucleocápside/efectos de los fármacos , Piperidinas/química , Piperidinas/farmacología , Replicación Viral/efectos de los fármacos , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Nucleocápside/genética , Nucleocápside/metabolismo , Ensamble de Virus/efectos de los fármacosRESUMEN
Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B virus (HBV) core protein (Cp) is involved in most stages of the viral life cycle; it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g., Impß) via Cp's C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp allosteric modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Impß and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Impß synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Impß. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV life cycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core protein-directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has the potential to disrupt the virus life cycle and activate the innate immune system.
Asunto(s)
Antivirales/farmacología , Cápside/efectos de los fármacos , Antígenos del Núcleo de la Hepatitis B/química , Virus de la Hepatitis B/efectos de los fármacos , beta Carioferinas/farmacología , Antivirales/química , Cápside/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Antígenos del Núcleo de la Hepatitis B/metabolismo , Unión Proteica , Proteolisis , Ensamble de Virus/efectos de los fármacos , beta Carioferinas/metabolismoRESUMEN
AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC50 of 0.077 µM, CC50 > 25 µM) and in vivo (HBV mouse model: â¼3.0 log10 reductions in serum HBV DNA compared to the vehicle control). Binding of AB-506 to HBV core protein accelerates capsid assembly and inhibits HBV pgRNA encapsidation. Furthermore, AB-506 blocks cccDNA establishment in HBV-infected HepG2-hNTCP-C4 cells and primary human hepatocytes, leading to inhibition of viral RNA, HBsAg, and HBeAg production (EC50 from 0.64 µM to 1.92 µM). AB-506 demonstrated activity across HBV genotypes A-H and maintains antiviral activity against nucleos(t)ide analog-resistant variants in vitro. Evaluation of AB-506 against a panel of core variants showed that T33N/Q substitutions results in >200-fold increase in EC50 values, while L30F, L37Q, and I105T substitutions showed an 8 to 20-fold increase in EC50 values in comparison to the wild-type. In vitro combinations of AB-506 with NAs or an RNAi agent were additive to moderately synergistic. AB-506 exhibits good oral bioavailability, systemic exposure, and higher liver to plasma ratios in rodents, a pharmacokinetic profile supporting clinical development for chronic hepatitis B.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Proteínas del Núcleo Viral/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacocinética , Células Cultivadas , Evaluación Preclínica de Medicamentos , Femenino , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Ratones , Ratas , Ensamble de Virus/efectos de los fármacosRESUMEN
Hepatitis B virus (HBV) is a major causative agent of human hepatitis. Its viral genome comprises partially double-stranded DNA, which is complexed with viral polymerase within an icosahedral capsid consisting of a dimeric core protein. Here, we describe the effects of capsid assembly modulators (CAMs) on the geometric or kinetic disruption of capsid construction and the virus life cycle. We highlight classical, early-generation CAMs such as heteroaryldihydropyrimidines, phenylpropenamides or sulfamoylbenzamides, and focus on the chemical structure and antiviral efficacy of recently identified non-classical CAMs, which consist of carboxamides, aryl ureas, bithiazoles, hydrazones, benzylpyridazinones, pyrimidines, quinolines, dyes, and antimicrobial compounds. We summarize the therapeutic efficacy of four representative classical compounds with data from clinical phase 1 studies in chronic HBV patients. Most of these compounds are in phase 2 trials, either as monotherapy or in combination with approved nucleos(t)ides drugs or other immunostimulatory molecules. As followers of the early CAMs, the therapeutic efficacy of several non-classical CAMs has been evaluated in humanized mouse models of HBV infection. It is expected that these next-generation HBV CAMs will be promising candidates for a series of extended human clinical trials.
Asunto(s)
Antivirales/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , Desarrollo de Medicamentos , Virus de la Hepatitis B/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Proteínas de la Cápside/metabolismo , Ensamble de Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.
Asunto(s)
Gránulos de Ribonucleoproteínas Citoplasmáticas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Rotavirus/genética , Proteínas no Estructurales Virales/metabolismo , Animales , Bovinos , Línea Celular , Gránulos de Ribonucleoproteínas Citoplasmáticas/efectos de los fármacos , Gránulos de Ribonucleoproteínas Citoplasmáticas/ultraestructura , Gránulos de Ribonucleoproteínas Citoplasmáticas/virología , Regulación Viral de la Expresión Génica , Genes Reporteros , Glicoles/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Haplorrinos , Interacciones Huésped-Patógeno/genética , Humanos , Concentración Osmolar , Fosforilación , Propilenglicol/farmacología , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Rotavirus/efectos de los fármacos , Rotavirus/crecimiento & desarrollo , Rotavirus/ultraestructura , Transducción de Señal , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Ensamble de Virus/efectos de los fármacos , Ensamble de Virus/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.
Asunto(s)
Amilorida/farmacología , Tratamiento Farmacológico de COVID-19 , Proteínas de la Envoltura de Coronavirus/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Amilorida/farmacocinética , Animales , Antivirales/farmacología , Sitios de Unión/efectos de los fármacos , COVID-19/virología , Chlorocebus aethiops , Proteínas de la Envoltura de Coronavirus/química , Humanos , Canales Iónicos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Dominios Proteicos , Células Vero , Ensamble de Virus/efectos de los fármacosRESUMEN
Hepatitis B virus (HBV) capsid assembly modulators (CpAMs) have shown promise as potent anti-HBV agents in both preclinical and clinical studies. Herein, we report our efforts in identifying novel CpAM hits via a structure-based virtual screening against a small molecule protein-protein interaction (PPI) library, and pharmacophore-guided compound design and synthesis. Curated compounds were first assessed in a thermal shift assay (TSA), and the TSA hits were further evaluated in an antiviral assay. These efforts led to the discovery of two structurally distinct scaffolds, ZW-1841 and ZW-1847, as novel HBV CpAM hits, both inhibiting HBV in single-digit µM concentrations without cytotoxicity at 100 µM. In ADME assays, both hits displayed extraordinary plasma and microsomal stability. Molecular modeling suggests that these hits bind to the Cp dimer interfaces in a mode well aligned with known CpAMs.
Asunto(s)
Antivirales/farmacología , Proteínas de la Cápside/química , Cápside/química , Descubrimiento de Drogas , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Ensamble de Virus/efectos de los fármacos , Antivirales/química , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Descubrimiento de Drogas/métodos , Estabilidad de Medicamentos , Hepatitis B/tratamiento farmacológico , Humanos , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Mapas de Interacción de Proteínas , Solubilidad , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
The concerning increase in HIV-1 resistance argues for prioritizing the development of host-targeting antiviral drugs because such drugs can offer high genetic barriers to the selection of drug-resistant viral variants. Targeting host proteins could also yield drugs that act on viral life cycle events that have proven elusive to inhibition, such as intracellular events of HIV-1 immature capsid assembly. Here, we review small molecule inhibitors identified primarily through HIV-1 self-assembly screens and describe how all act either narrowly post-entry or broadly on early and late events of the HIV-1 life cycle. We propose that a different screening approach could identify compounds that specifically inhibit HIV-1 Gag assembly, as was observed when a potent rabies virus inhibitor was identified using a host-catalyzed rabies assembly screen. As an example of this possibility, we discuss an antiretroviral small molecule recently identified using a screen that recapitulates the host-catalyzed HIV-1 capsid assembly pathway. This chemotype potently blocks HIV-1 replication in T cells by specifically inhibiting immature HIV-1 capsid assembly but fails to select for resistant viral variants over 37 passages, suggesting a host protein target. Development of such small molecules could yield novel host-targeting antiretroviral drugs and provide insight into chronic diseases resulting from dysregulation of host machinery targeted by these drugs.
Asunto(s)
Antirretrovirales/farmacología , Farmacorresistencia Viral , VIH-1/efectos de los fármacos , Interacciones Microbiota-Huesped/efectos de los fármacos , Ensamble de Virus/efectos de los fármacos , Antirretrovirales/aislamiento & purificación , Cápside/metabolismo , Seropositividad para VIH , VIH-1/fisiología , Humanos , Linfocitos T/efectos de los fármacos , Linfocitos T/virologíaRESUMEN
Core assembly modulators of viral capsid proteins have been developed as an effective treatment of chronic hepatitis B virus (HBV) infection. In this study, we synthesized novel potent pyrimidine derivatives as core assembly modulators, and their antiviral effects were evaluated in in vitro and in vivo biological experiments. One of the synthesized derivatives, compound 23h (R1 = MeSO2, R2 = 1-piperidin-4-amine, R3 = 3-Cl-4-F-aniline) displayed potent inhibitory effects in the in vitro assays (52% inhibition in the protein-based assay at 100 nM and an IC50 value of 181 nM in the serum HBV DNA quantification assay). Moreover, treatment with compound 23h for 5 weeks significantly decreased serum levels of HBV DNA levels (3.35 log reduction) in a human liver-chimeric uPA/SCID mouse model, and these effects were significantly increased when 23h was combined with tenofovir, a nucleotide analogue inhibitor of reverse transcriptase used for the treatment of HBV infection.