RESUMO
The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.
Assuntos
Ensaios de Triagem em Larga Escala , Maleatos , Receptores Histamínicos H1 , Ligantes , Maleatos/química , Ensaios de Triagem em Larga Escala/métodos , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Humanos , Histidina/química , Animais , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Células CHO , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Antagonistas dos Receptores Histamínicos H1/química , Poliestirenos/química , Cricetulus , Oligopeptídeos/químicaRESUMO
The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and profound immune-escape capacity makes it an urgent need to develop broad-spectrum therapeutics. Nanobodies have recently attracted extensive attentions due to their excellent biochemical and binding properties. Here, we report two high-affinity nanobodies (Nb-015 and Nb-021) that target non-overlapping epitopes in SARS-CoV-2 S-RBD. Both nanobodies could efficiently neutralize diverse viruses of SARS-CoV-2. The neutralizing mechanisms for the two nanobodies are further delineated by high-resolution nanobody/S-RBD complex structures. In addition, an Fc-based tetravalent nanobody format is constructed by combining Nb-015 and Nb-021. The resultant nanobody conjugate, designated as Nb-X2-Fc, exhibits significantly enhanced breadth and potency against all-tested SARS-CoV-2 variants, including Omicron sub-lineages. These data demonstrate that Nb-X2-Fc could serve as an effective drug candidate for the treatment of SARS-CoV-2 infection, deserving further in-vivo evaluations in the future.
Assuntos
COVID-19 , Anticorpos de Domínio Único , Humanos , SARS-CoV-2 , Anticorpos de Domínio Único/farmacologia , Epitopos , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes/farmacologia , Anticorpos AntiviraisRESUMO
Alongshan virus (ALSV), a newly discovered member of unclassified Flaviviridae family, is able to infect humans. ALSV has a multi-segmented genome organization and is evolutionarily distant from canonical mono-segmented flaviviruses. The virus-encoded methyltransferase (MTase) plays an important role in viral replication. Here we show that ALSV MTase readily binds S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) but exhibits significantly lower affinities than canonical flaviviral MTases. Structures of ALSV MTase in the free and SAM/SAH-bound forms reveal that the viral enzyme possesses a unique loop-element lining side-wall of the SAM/SAH-binding pocket. While the equivalent loop in flaviviral MTases half-covers SAM/SAH, contributing multiple hydrogen-bond interactions; the pocket-lining loop of ALSV MTase is of short-length and high-flexibility, devoid of any physical contacts with SAM/SAH. Subsequent mutagenesis data further corroborate such structural difference affecting SAM/SAH-binding. Finally, we also report the structure of ALSV MTase bound with sinefungin, an SAM-analogue MTase inhibitor. These data have delineated the basis for the low-affinity interaction between ALSV MTase and SAM/SAH and should inform on antiviral drug design.
Assuntos
Flavivirus , Metiltransferases , Humanos , Metiltransferases/genética , Flavivirus/genética , Flavivirus/metabolismo , S-Adenosilmetionina/metabolismo , MutagêneseRESUMO
The receptor-binding domain (RBD) of spike recognizing the receptor angiotensin-converting enzyme 2 (ACE2) initiates membrane fusion between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cell membrane. Although the structure of the RBD_ACE2 complex has been well studied, its functional mechanism in membrane fusion is still not fully understood. Here, using an in vitro cell-vesicle content-mixing assay, it is found that the cleavage at the S2' site by thrombin (Thr) protease strongly accelerates membrane fusion, compared to that of cleavage at the S1/S2 site by PreScission (3C) protease. Moreover, mutations at the RBD_ACE2 interface resulted in a positive correlation between binding affinity and fusion probability. In both the cell-vesicle and cell-cell fusion assays, by crosslinking two membranes via the neutravidin (NTV)_biotin interaction or complementary DNA strands, it is found that spike drives membrane fusion in the absence of ACE2, and a suitable distance between two membranes is critical for spike-mediated membrane fusion. Finally, unsuitable membrane crosslinkers significantly inhibited the fusion probability in the presence of ACE2. Taken together, the results suggest that the RBD_ACE2 complex may act as a crosslinker to bridge the viral and cell membranes at a suitable distance, which is critical, but also substitutable for spike-mediated SARS-CoV-2 entry.
RESUMO
At present, the horse or human rabies immunoglobulin (RIG) used for postexposure prevention of human rabies (PEP) has high cost and limited availability. It is strongly encouraged to replace RIG with equivalent or more effective and safer products. Mouse and human monoclonal antibodies have been shown to protect rodents from lethal rabies virus (RABV) attacks. In this study, we reported a human-mouse chimeric monoclonal antibody, 12-2A12, which showed a strong neutralization potency and a wide breadth against multiple street viruses of RABV in vitro. The antibody binded the viral glycoprotein (G) with nanomolar affinity. The complex structure of 12-2A12 bound to RABV G revealed that the antibody recognizes an epitope that partially overlaps with the recognition region for the nicotinic acetylcholine receptor (nAChR). The antibody therefore would interfere with the nAChR/G interaction to block the viral receptor binding. In addition, comparison of our complex structure with the G structure in the acidic state reveals a clear steric clash, highlighting that the antibody would further prevent the conformational changes of the viral glycoprotein that are essential for membrane fusion. In light of these functional and structural data, we believe that 12-2A12 might be developed to be included in an antibody cocktail for potential use in human rabies PEP.
Assuntos
Vírus da Raiva , Raiva , Humanos , Animais , Camundongos , Cavalos , Raiva/prevenção & controle , Anticorpos Antivirais , Glicoproteínas , Anticorpos Monoclonais , Fatores Imunológicos/metabolismo , ImunossupressoresAssuntos
Orthopoxvirus , Vaccinia virus , Humanos , Vaccinia virus/genética , Orthopoxvirus/genética , Células HeLaRESUMO
The continuous spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) around the world has raised unprecedented challenges to the human society. Antibodies and nanobodies possessing neutralization activity represent promising drug candidates. In this study, we report the identification and characterization of a potent SARS-CoV-2 neutralizing nanobody that targets the viral spike receptor-binding domain (S-RBD). The nanobody, termed as Nb-007, engages SARS-CoV-2 S-RBD with the two-digit picomolar binding affinity and shows outstanding virus entry-inhibition activity. The complex structure of Nb-007 bound to SARS-CoV-2 S-RBD reveals an epitope that is partially overlapping with the binding site for the human receptor of angiotensin-converting enzyme 2 (ACE2). The nanobody therefore exerts neutralization by competing with ACE2 for S-RBD binding, which is further ascertained by our in-vitro biochemical analyses. Finally, we also show that Nb-007 reserves promising, though compromised, neutralization activity against the currently-circulating Delta variant and that fusion of the nanobody with Fc dramatically increases its entry-inhibition capacity. Taken together, these data have paved the way of developing Nb-007 as a drug-reserve for potential treatment of SARS-CoV-2 related diseases.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Receptores Virais/metabolismo , Glicoproteína da Espícula de CoronavírusRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related sarbecoviruses enter host cells by receptor-recognition and membrane-fusion. An indispensable step in fusion is the formation of 6-helix bundle by viral spike heptad repeats 1 and 2 (HR1 and HR2). Here, we report the construction of 5-helix bundle (5HB) proteins for virus infection inhibition. The optimal construct inhibits SARS-CoV-2 pseudovirus entry with sub-micromolar IC50. Unlike HR2-based peptides that cannot bind spike in the pre-fusion conformation, 5HB features with the capability of binding to pre-fusion spike. Furthermore, 5HB binds viral HR2 at both serological- and endosomal-pH, highlighting its entry-inhibition capacity when SARS-CoV-2 enters via either cell membrane fusion or endosomal route. Finally, we show that 5HB could neutralize S-mediated entry of the predominant SARS-CoV-2 variants and a wide spectrum of sarbecoviruses. These data provide proof-of-concept evidence that 5HB might be developed for the prevention and treatment of SARS-CoV-2 and other emerging sarbecovirus infections.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Proteínas do Envelope Viral/metabolismo , Internalização do VírusRESUMO
SARS-CoV-2 recognizes, via its spike receptor-binding domain (S-RBD), human angiotensin-converting enzyme 2 (ACE2) to initiate infection. Ecto-domain protein of ACE2 can therefore function as a decoy. Here we show that mutations of S19W, T27W, and N330Y in ACE2 could individually enhance SARS-CoV-2 S-RBD binding. Y330 could be synergistically combined with either W19 or W27, whereas W19 and W27 are mutually unbeneficial. The structures of SARS-CoV-2 S-RBD bound to the ACE2 mutants reveal that the enhanced binding is mainly contributed by the van der Waals interactions mediated by the aromatic side-chains from W19, W27, and Y330. While Y330 and W19/W27 are distantly located and devoid of any steric interference, W19 and W27 are shown to orient their side-chains toward each other and to cause steric conflicts, explaining their incompatibility. Finally, using pseudotyped SARS-CoV-2 viruses, we demonstrate that these residue substitutions are associated with dramatically improved entry-inhibition efficacy toward both wild-type and antibody-resistant viruses. Taken together, our biochemical and structural data have delineated the basis for the elevated S-RBD binding associated with S19W, T27W, and N330Y mutations in ACE2, paving the way for potential application of these mutants in clinical treatment of COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2/química , COVID-19 , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , SARS-CoV-2/química , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMO
The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1', which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.
Assuntos
Biocatálise , Exorribonucleases/química , Exorribonucleases/metabolismo , SARS-CoV-2/química , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Exorribonucleases/genética , Guanina , Metiltransferases/química , Metiltransferases/deficiência , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Domínios Proteicos/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has led to a pandemic. An effective preventive vaccine against this virus is urgently needed. As an essential step during infection, SARS-CoV-2 uses the receptor-binding domain (RBD) of the spike protein to engage with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells1,2. Here we show that a recombinant vaccine that comprises residues 319-545 of the RBD of the spike protein induces a potent functional antibody response in immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after the injection of a single vaccine dose. The sera from the immunized animals blocked the binding of the RBD to ACE2, which is expressed on the cell surface, and neutralized infection with a SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates to an in vivo challenge with SARS-CoV-2. We found increased levels of RBD-specific antibodies in the sera of patients with COVID-19. We show that several immune pathways and CD4 T lymphocytes are involved in the induction of the vaccine antibody response. Our findings highlight the importance of the RBD domain in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective vaccine through the induction of antibodies against the RBD domain.
Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , COVID-19 , Vacinas contra COVID-19 , Humanos , Macaca mulatta/imunologia , Macaca mulatta/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Modelos Moleculares , Domínios Proteicos , SARS-CoV-2 , Soro/imunologia , Baço/citologia , Baço/imunologia , Linfócitos T/imunologia , VacinaçãoAssuntos
Betacoronavirus/ultraestrutura , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Antivirais/química , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/genética , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Metiltransferases , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/genética , Conformação Proteica/efeitos dos fármacos , S-Adenosilmetionina/química , SARS-CoV-2 , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/ultraestrutura , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/ultraestrutura , tRNA Metiltransferases/antagonistas & inibidores , tRNA Metiltransferases/químicaRESUMO
Bovine herpesvirus 1 (BHV-1) has received increasing attention for its potential oncolytic applications. BHV-1 recognizes nectin-1 for cell entry via viral glycoprotein D (gD) but represents a low-affinity nectin-1 binding virus. The molecular basis underlying this low receptor-binding affinity, however, remains unknown. Here, the crystal structures of BHV-1 gD in the free and nectin-1-bound forms are presented. While showing an overall resembled nectin-1 binding mode to other alphaherpesvirus gDs, BHV-1 gD has a unique G-strand/α2-helix interloop that disturbs gD/nectin-1 interactions. Residue R188 residing in this loop is observed to otherwise cause strong steric hindrance with the bound receptor, making a large conformational change of the loop a prerequisite for nectin-1 engagement. Subsequently, substitution of R188 with glycine markedly enhances the affinity of the BHV-1-gD/nectin-1 interaction (by about fivefold). These structural and functional data delineate the receptor-recognition basis for BHV-1, which might facilitate BHV-1-based oncolytic design in the future.
RESUMO
Rabies virus (RABV), the etiological agent for the lethal disease of rabies, is a deadly zoonotic pathogen. The RABV glycoprotein (RABV-G) is a key factor mediating virus entry and the major target of neutralizing antibodies. Here, we report the crystal structures of RABV-G solved in the free form at â¼pH-8.0 and in the complex form with a neutralizing antibody 523-11 at â¼pH-6.5, respectively. RABV-G has three domains, and the basic-to-acidic pH change results in large domain re-orientations and concomitant domain-linker re-constructions, switching it from a bent hairpin conformation into an extended conformation. During such low-pH-induced structural transitions, residues located in the domain-linker are found to play important roles in glycoprotein-mediated membrane fusion. Finally, the antibody interacts with RABV-G mainly through its heavy chain and binds to a bipartite conformational epitope in the viral protein for neutralization. These structures provide valuable information for vaccine and drug design.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Antígenos Virais/química , Concentração de Íons de Hidrogênio , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Fusão Celular , Linhagem Celular , Cricetinae , Multimerização Proteica , Estrutura Terciária de Proteína , Vírus da RaivaRESUMO
The DcCoV UAE-HKU23 coronavirus is a newly-found betacoronavirus (betaCoV) that can infect human cells. The viral spike protein plays pivotal roles in mediating receptor-recognition and membrane-fusion, and is therefore a key factor involved in viral pathogenesis and inter-species transmission. Here we reported the structural and functional characterization of the spike N-terminal domain (NTD) from DcCoV UAE-HKU23 (HKU23-NTD). Via mucin-binding assays, we showed that HKU23-NTD is able to bind sugars. We further solved the structure of HKU23-NTD, performed structure-guided mutagenesis and successfully located the potential sugar-binding pockets in the structure. Furthermore, via comparison of available betaCoV NTD structures, we demonstrated that betaCoV NTDs contain a conserved ß-sandwich core, but exhibit variant folds in the peripheral elements located in the top-ceiling region and on the lateral side. While showing different compositions and structures, these peripheral elements are topologically equivalent ß-sandwich-core insertions, highlighting a divergent evolution process for betaCoVs to form different lineages.
Assuntos
Betacoronavirus/química , Subunidades Proteicas/química , Glicoproteína da Espícula de Coronavírus/química , Betacoronavirus/genética , Betacoronavirus/crescimento & desenvolvimento , Cristalografia por Raios X , Análise Mutacional de DNA , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
In this study, a cell-penetrating peptide named Acylated Steric acid-9 poly-arginine (r9-SA) was sucessfully synthesized. High performance liquid chromatography (HPLC) and mass spectrometry (MS) were used to characterize the structural formula of r9-SA. Diclofenac sodium was chosen as a model drug, and the transdermal permeation-enhancing effect of r9-SA was estimated in vitro. Skin irritation experiment and histopathological observation of tissue sections with HE and Masson staining were performed to analyze the security of r9-SA. The possible penetration-enhancing mechanism of r9-SA was characterized with laser scanning confocal microscopy (LSCM), differential scanning calorimetry (DSC), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and laser Raman spectroscopy, respectively. The in vitro penetration study showed that r9-SA has a promoting effect for enhancing the transdermal penetration of diclofenac sodium. Skin irritation experiment and histopathological observation results showed that r9-SA has good biocompatibility with skin. The experiments were carried on to characterize the penetration mechanism of r9 -SA. It was found that r9-SA may react with the keratin in the stratum corneum (SC), changing its secondary structure and so that drugs can penetrate through SC. In conclusion, all data showed that the r9-SA could be a safe and effective penetration enhancer for topical delivery of drug.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Administração Cutânea , Arginina , Pele , Absorção Cutânea , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
PA0833 of Pseudomonas aeruginosa is recently identified as an OmpA C-like protein that is able to interact with bacterial peptidoglycan (PGN). In this study, we reported the biochemical and structural characterization of the PGN-binding periplasmic-domain of PA0833 (PA0833-PD). Via mutagenesis, key residues responsible for engaging PGN were identified, which also enables us to localize the PGN-binding pocket in a 2.0â¯Å crystal structure solved in this study. In contrast to its homologous proteins (as represented by AbOmpA-PD of Acinetobacter baumannii) that interact with PGN by directly engaging the DAP (diaminopimelate) moiety, PA0833-PD exhibits an enlarged PGN-binding pocket due to residue insertions and the formation of an extra α-helix in one lateral side of the pocket. Accordingly, single DAP molecule does not show detectable interactions with PA0833-PD in solution, highlighting that other PGN-components, in addition to DAP, are also required to restore the full binding capacity observed between PA0833 and PGN.
Assuntos
Peptidoglicano/metabolismo , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Peptidoglicano/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Alinhamento de SequênciaRESUMO
BACKGROUND: Usutu virus (USUV) is a mosquito-born flavivirus that can infect multiple avian and mammalian species. The viral surface envelope (E) protein functions to initiate the viral infection by recognizing cellular receptors and mediating the subsequent membrane fusion, and is therefore a key virulence factor involved in the pathogenesis of USUV. The structural features of USUV-E, however, remains un-investigated thus far. FINDINGS: Using the crystallographic method, we determined the structure of USUV-E in the pre-fusion state at 2.0 angstrom. As expected, the overall fold of USUV-E, with three ß-barrel domains (DI, DII, and DIII), resembles those of other flaviviral E proteins. In comparison to other pre-fusion E structures, however, USUV-E exhibits an apparently enlarged inter-domain angle between DI and DII, leading to a more extended conformation. Using our structure and other reported pre-fusion E structures, the DI-DII domain-angle difference was analyzed in a pairwise manner. The result shows a much higher degree of variations for USUV-E, indicating the potential for remarkable DI-DII domain angle plasticity among flaviviruses. CONCLUSION: We report the crystal structure of USUV-E and show that its pre-fusion structure has an enlarged DI-DII domain-angle which has not been observed in other reported flaviviral E-structures.