RESUMO
The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.
Assuntos
Antivirais/farmacologia , Retículo Endoplasmático/enzimologia , Inibidores de Glicosídeo Hidrolases/farmacologia , alfa-Glucosidases/química , Animais , Catálise , Cristalografia por Raios X , Camundongos , Conformação Proteica , Subunidades Proteicas , Espalhamento a Baixo Ângulo , Especificidade por SubstratoRESUMO
Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 "WY" conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 µM binding affinity for CtUGGTGT24in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 µM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.
RESUMO
Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of March 29, 2021, has claimed 2â¯776â¯175 lives worldwide. Vaccine development efforts focus on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralization, and antigen efficacy is influenced by spike glycoprotein production in vivo. Therefore, immunogen integrity is important for glycoprotein-based vaccine candidates. Here, we show how site-specific glycosylation differs between virus-derived spikes, wild-type, non-stabilized spikes expressed from a plasmid with a CMV promoter and tPA signal sequence, and commonly used recombinant, engineered spike glycoproteins. Furthermore, we show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, including shedding of spike monomeric subunits for the non-stabilized wild-type spike tested, which may have implications for the resulting immune response and vaccine design.
RESUMO
Most enveloped viruses rely on the host cell endoplasmic reticulum (ER) quality control (QC) machinery for proper folding of glycoproteins. The key ER α-glucosidases (α-Glu) I and II of the ERQC machinery are attractive targets for developing broad-spectrum antivirals. Iminosugars based on deoxynojirimycin have been extensively studied as ER α-glucosidase inhibitors; however, other glycomimetic compounds are less established. Accordingly, we synthesized a series of N-substituted derivatives of valiolamine, the iminosugar scaffold of type 2 diabetes drug voglibose. To understand the basis for up to 100,000-fold improved inhibitory potency, we determined high-resolution crystal structures of mouse ER α-GluII in complex with valiolamine and 10 derivatives. The structures revealed extensive interactions with all four α-GluII subsites. We further showed that N-substituted valiolamines were active against dengue virus and SARS-CoV-2 in vitro. This study introduces valiolamine-based inhibitors of the ERQC machinery as candidates for developing potential broad-spectrum therapeutics against the existing and emerging viruses.
Assuntos
Antivirais/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Imino Açúcares/farmacologia , Inositol/análogos & derivados , alfa-Glucosidases/metabolismo , Animais , Antivirais/síntese química , Antivirais/metabolismo , Sítios de Ligação , Chlorocebus aethiops , Cristalografia por Raios X , Vírus da Dengue/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/metabolismo , Humanos , Imino Açúcares/síntese química , Imino Açúcares/metabolismo , Inositol/síntese química , Inositol/metabolismo , Inositol/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos , Células Vero , alfa-Glucosidases/químicaRESUMO
In an effort to elucidate the mechanism of movement of nanovehicles on nonconducting surfaces, the synthesis and optical properties of five fluorescently tagged nanocars are reported. The nanocars were specifically designed for studies by single-molecule fluorescence spectroscopy and bear a tetramethylrhodamine isothiocyanate fluorescent tag for excitation at 532 nm. The molecules were designed such that the arrangement of their molecular axles and p-carborane wheels relative to the chassis would be conducive to the control of directionality in the motion of these nanovehicles.
Assuntos
Corantes Fluorescentes/síntese química , Nanoestruturas/química , Corantes Fluorescentes/química , Estrutura Molecular , Rodaminas/química , Espectrometria de Fluorescência , EstereoisomerismoRESUMO
Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.
Assuntos
Dengue/prevenção & controle , Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Influenza Humana/prevenção & controle , alfa-Glucosidases , Animais , Dengue/tratamento farmacológico , Dengue/enzimologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , alfa-Glucosidases/metabolismoRESUMO
We disclose the synthesis of a porphyrin-fullerene pinwheel that was subsequently observed by scanning tunneling microscopy. The molecule was designed to further our understanding of fullerene-surface interactions, directional control, and surface-rolling versus pivoting capabilities of this class of nanomachines. The inner porphyrin provides the square planar configuration that might lead to realization of the pinwheel spiraling motion on surfaces.
RESUMO
Iminosugars have therapeutic potential against a range of diseases, due to their efficacy as glycosidase inhibitors. A major challenge in the development of iminosugar drugs lies in making a compound that is selective for the glycosidase associated with a given disease. We report the synthesis of ToP-DNJ, an antiviral iminosugar-tocopherol conjugate. Tocopherol was incorporated into the design of the iminosugar in order to direct the drug to the liver and immune cells, specific tissues of interest for antiviral therapy. ToP-DNJ inhibits ER α-glucosidase II at low micromolar concentrations and selectively accumulates in the liver in vivo. In cellular assays, the drug showed efficacy exclusively in immune cells of the myeloid lineage. Taken together, these data demonstrate that inclusion of a native metabolite into an iminosugar provides selectivity with respect to target enzyme, target cell, and target tissue.
Assuntos
Antivirais/química , Antivirais/farmacologia , Retículo Endoplasmático/enzimologia , Inibidores de Glicosídeo Hidrolases/farmacologia , 1-Desoxinojirimicina/química , Administração Oral , Animais , Antivirais/síntese química , Vírus da Dengue/efeitos dos fármacos , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Inibidores de Glicosídeo Hidrolases/química , Células HL-60 , Hepacivirus/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Ratos , Distribuição Tecidual , Tocoferóis/química , alfa-Glucosidases/metabolismoRESUMO
Within every living organism, countless reactions occur every second. These reactions typically occur more rapidly and with greater efficiency than would be possible under the same conditions in the chemical laboratory, and while using only the subset of elements that are readily available in nature. Despite these apparent differences between life and the laboratory, biological reactions are governed by the same rules as any other chemical reaction. Thus, a firm understanding of the fundamentals of chemistry is invaluable in biochemistry. There are entire textbooks devoted to the application of chemical principles in biological systems and so it is not possible to cover all of the relevant topics in depth in this short article. The aim is instead to provide a brief overview of those areas in chemistry that are most relevant to biochemistry. We summarize the basic principles, give examples of how these principles are applied in biological systems and suggest further reading on individual topics.
Assuntos
Bioquímica/métodos , Metaboloma , Compostos Orgânicos/química , Fenômenos de Química Orgânica , Animais , Bioquímica/educação , Humanos , Compostos Orgânicos/metabolismoRESUMO
It has long been thought that iminosugar antiviral activity is a function of inhibition of endoplasmic reticulum-resident α-glucosidases, and on this basis, many iminosugars have been investigated as therapeutic agents for treatment of infection by a diverse spectrum of viruses, including dengue virus (DENV). However, iminosugars are glycomimetics possessing a nitrogen atom in place of the endocyclic oxygen atom, and the ubiquity of glycans in host metabolism suggests that multiple pathways can be targeted via iminosugar treatment. Successful treatment of patients with glycolipid processing defects using iminosugars highlights the clinical exploitation of iminosugar inhibition of enzymes other than ER α-glucosidases. Evidence correlating antiviral activity with successful inhibition of ER glucosidases together with the exclusion of alternative mechanisms of action of iminosugars in the context of DENV infection is limited. Celgosivir, a bicyclic iminosugar evaluated in phase Ib clinical trials as a therapeutic for the treatment of DENV infection, was confirmed to be antiviral in a lethal mouse model of antibody-enhanced DENV infection. In this study we provide the first evidence of the antiviral activity of celgosivir in primary human macrophages in vitro, in which it inhibits DENV secretion with an EC50 of 5 µM. We further demonstrate that monocyclic glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. By comparison to bicyclic glucose-mimicking iminosugars which inhibit glycoprotein processing but do not inhibit glycolipid processing and galactose-mimicking iminosugars which do not inhibit glycoprotein processing but do inhibit glycolipid processing, we demonstrate that inhibition of endoplasmic reticulum-resident α-glucosidases, not glycolipid processing, is responsible for iminosugar antiviral activity against DENV. Our data suggest that inhibition of ER α-glucosidases prevents release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.
Assuntos
Antivirais/metabolismo , Vírus da Dengue/crescimento & desenvolvimento , Retículo Endoplasmático/enzimologia , Inibidores Enzimáticos/metabolismo , Imino Açúcares/metabolismo , Indolizinas/metabolismo , alfa-Glucosidases/metabolismo , Animais , Células Cultivadas , Vírus da Dengue/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/química , Humanos , Imino Açúcares/química , Indolizinas/química , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Modelos Moleculares , Estrutura Molecular , Liberação de Vírus/efeitos dos fármacosRESUMO
The antiviral activity of UV-4 was previously demonstrated against dengue virus serotype 2 (DENV2) in multiple mouse models. Herein, step-wise minimal effective dose and therapeutic window of efficacy studies of UV-4B (UV-4 hydrochloride salt) were conducted in an antibody-dependent enhancement (ADE) mouse model of severe DENV2 infection in AG129 mice lacking types I and II interferon receptors. Significant survival benefit was demonstrated with 10-20 mg/kg of UV-4B administered thrice daily (TID) for seven days with initiation of treatment up to 48 h after infection. UV-4B also reduced infectious virus production in in vitro antiviral activity assays against all four DENV serotypes, including clinical isolates. A set of purified enzyme, in vitro, and in vivo studies demonstrated that inhibition of endoplasmic reticulum (ER) α-glucosidases and not the glycosphingolipid pathway appears to be responsible for the antiviral activity of UV-4B against DENV. Along with a comprehensive safety package, these and previously published data provided support for an Investigational New Drug (IND) filing and Phases 1 and 2 clinical trials for UV-4B with an indication of acute dengue disease.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Inibidores de Glicosídeo Hidrolases/farmacologia , Dengue Grave/tratamento farmacológico , alfa-Glucosidases/metabolismo , 1-Desoxinojirimicina/administração & dosagem , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/uso terapêutico , Animais , Anticorpos Antivirais/sangue , Anticorpos Facilitadores/efeitos dos fármacos , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Células Cultivadas , Chlorocebus aethiops , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Drogas em Investigação , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/uso terapêutico , Humanos , Concentração Inibidora 50 , Camundongos , Monócitos/virologia , Receptores de Interferon/deficiência , Sorogrupo , Dengue Grave/virologia , Células VeroRESUMO
We report the isolation and structural determination of fourteen iminosugars, containing five pyrrolizidines and five indolizidines, from Castanospermum australe. The structure of a new alkaloid was elucidated by spectroscopic methods as 6,8-diepi-castanospermine (13). Our side-by-side comparison between bicyclic and corresponding monocyclic iminosugars revealed that inhibition potency and spectrum against each enzyme are clearly changed by their core structures. Castanospermine (10) and 1-deoxynojirimycin (DNJ) have a common d-gluco configuration, and they showed the expected similar inhibition potency and spectrum. In sharp contrast, 6-epi-castanospermine (12) and 1-deoxymannojirimycin (manno-DNJ) both have the d-manno configuration but the α-mannosidase inhibition of 6-epi-castanospermine (12) was much better than that of manno-DNJ. 6,8-Diepi-castanospermine (13) could be regarded as a bicyclic derivative of talo-DNJ, but it showed a complete loss of α-galactosidase A inhibition. This behavior against α-galactosidase A is similar to that observed for 1-epi-australine (6) and altro-DMDP.
Assuntos
Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Castanospermum/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Açúcares/isolamento & purificação , Imino Açúcares/farmacologia , Indolizinas/isolamento & purificação , Indolizinas/farmacologia , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/farmacologia , Alcaloides/química , Inibidores Enzimáticos/química , Glucosamina/análogos & derivados , Glucosamina/química , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Imino Açúcares/química , Indolizinas/química , Piperidinas/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A synthetic route to the carboline disaccharide domain (2) of shishijimicin A (1) has been developed. The convergent synthesis relies on a novel application of the Reetz-Müller-Starke reaction to form the central, sulfur-bearing quaternary carbon center and addition of the carboline structural motif as a dianion to a disaccharide aldehyde fragment.