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Due to their constrained conformations, cyclic ß2,3-amino acids (cßAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cßAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cßAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cßAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (ß1), (1S,2S)-2-aminocyclohexane carboxylic acid (ß2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one ß2 and two ß1, exhibited potent inhibitory activities with IC50 values of 40 and 20â nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168â h, respectively. Notably, BM3A and BM7A, wherein the cßAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cßAA to the activity and stability of peptides. Overall, our results highlight the potential of cßAA in generating potent and highly stable macrocyclic peptides with drug-like properties.
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The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1-3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2' position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2' contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.
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As we celebrate the 30th anniversary of Structure, we have asked structural biologists about their expectations on how their respective fields are likely to develop in the next ten years in this collection of Voices.
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Biologia Molecular , Biologia Molecular/tendênciasRESUMO
Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.
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The SARS-CoV-2 papain-like protease (PLpro) and main protease (Mpro) are nucleophilic cysteine enzymes that catalyze hydrolysis of the viral polyproteins pp1a/1ab. By contrast with Mpro, PLpro is also a deubiquitinase (DUB) that accepts post-translationally modified human proteins as substrates. Here we report studies on the DUB activity of PLpro using synthetic Nε-lysine-branched oligopeptides as substrates that mimic post-translational protein modifications by ubiquitin (Ub) or Ub-like modifiers (UBLs), such as interferon stimulated gene 15 (ISG15). Mass spectrometry (MS)-based assays confirm the DUB activity of isolated recombinant PLpro. They reveal that the sequence of both the peptide fragment derived from the post-translationally modified protein and that derived from the UBL affects PLpro catalysis; the nature of substrate binding in the S sites appears to be more important for catalytic efficiency than binding in the S' sites. Importantly, the results reflect the reported cellular substrate selectivity of PLpro, i.e. human proteins conjugated to ISG15 are better substrates than those conjugated to Ub or other UBLs. The combined experimental and modelling results imply that PLpro catalysis is affected not only by the identity of the substrate residues binding in the S and S' sites, but also by the substrate fold and the conformational dynamics of the blocking loop 2 of the PLpro:substrate complex. Nε-Lysine-branched oligopeptides thus have potential to help the identification of PLpro substrates. More generally, the results imply that MS-based assays with Nε-lysine-branched oligopeptides have potential to monitor catalysis by human DUBs and hence to inform on their substrate preferences.
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COVID-19 , Lisina , Humanos , Proteínas Virais/metabolismo , SARS-CoV-2 , Ubiquitina/metabolismo , Enzimas Desubiquitinantes , OligopeptídeosRESUMO
Inhibitor discovery for emerging drug-target proteins is challenging, especially when target structure or active molecules are unknown. Here, we experimentally validate the broad utility of a deep generative framework trained at-scale on protein sequences, small molecules, and their mutual interactions-unbiased toward any specific target. We performed a protein sequence-conditioned sampling on the generative foundation model to design small-molecule inhibitors for two dissimilar targets: the spike protein receptor-binding domain (RBD) and the main protease from SARS-CoV-2. Despite using only the target sequence information during the model inference, micromolar-level inhibition was observed in vitro for two candidates out of four synthesized for each target. The most potent spike RBD inhibitor exhibited activity against several variants in live virus neutralization assays. These results establish that a single, broadly deployable generative foundation model for accelerated inhibitor discovery is effective and efficient, even in the absence of target structure or binder information.
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Anticorpos Antivirais , COVID-19 , Humanos , Anticorpos Antivirais/química , SARS-CoV-2/metabolismo , Ligação Proteica , Sequência de AminoácidosRESUMO
γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1' catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.
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Aminoácidos , COVID-19 , Aminoácidos/química , Antivirais/química , Ácidos Carboxílicos , Peptídeos/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Conformação Proteica , SARS-CoV-2/metabolismoRESUMO
Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.
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Antivirais , COVID-19 , Proteases 3C de Coronavírus , Nitrilas , SARS-CoV-2 , Inibidores de Protease Viral , Humanos , Antivirais/farmacologia , Cisteína/química , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Inibidores de Protease Viral/farmacologiaRESUMO
In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals; for example, small-wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipidic mesophases. Whilst the indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. The identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help to optimize the scaling and merging of multiple data sets, a new program, xia2.multiplex, has been developed which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After the description of a number of use cases, the benefit of xia2.multiplex is demonstrated within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment-screening experiments on the SARS-CoV-2 main protease.
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COVID-19 , Cristalografia por Raios X , Análise de Dados , Humanos , Substâncias Macromoleculares/química , SARS-CoV-2RESUMO
The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of ß-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that ß-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl-enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by ß-lactams and related acylating agents.
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Tratamento Farmacológico da COVID-19 , Cisteína , Antivirais/química , Antivirais/farmacologia , Proteases 3C de Coronavírus , Cisteína Endopeptidases/química , Humanos , Penicilinas , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2 , beta-LactamasRESUMO
The two SARS-CoV-2 proteases, i. e. the main protease (Mpro ) and the papain-like protease (PLpro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PLpro catalysis inâ vitro. The assay was applied to investigate the effect of reported small-molecule PLpro inhibitors and selected Mpro inhibitors on PLpro catalysis. The results reveal that some, but not all, PLpro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing Mpro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PLpro . Less selective Mpro inhibitors, e. g. auranofin, inhibit PLpro , highlighting the potential for dual PLpro /Mpro inhibition. MS-based PLpro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.
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COVID-19 , SARS-CoV-2 , Antivirais/química , Proteases Semelhantes à Papaína de Coronavírus , Humanos , Lactamas , Leucina , Espectrometria de Massas , Nitrilas , Peptídeo Hidrolases , Prolina , Inibidores de Proteases/farmacologiaRESUMO
PAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico. The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture. IMPORTANCE To adjust to a variety of life conditions, bacteria typically use multidomain proteins, where the modular structure allows functional differentiation. Proteins responding to environmental cues and regulating physiological responses are found in chemotaxis pathways that respond to a wide range of stimuli to affect movement. Environmental cues also regulate intracellular levels of cyclic-di-GMP, a universal bacterial secondary messenger that is a key determinant of bacterial lifestyle and virulence. We study Pseudomonas aeruginosa, an organism known to colonize a broad range of environments that can switch lifestyle between the sessile biofilm and the planktonic swimming form. We have investigated the PAS and Cache domains, of which we identified 101 in 70 Pseudomonas aeruginosa PAO1 proteins, and have grouped these by phylogeny with domains of known structure. The resulting data set integrates sequence analysis and structure prediction to infer ligand or cofactor binding. With this data set, functional predictions for PAS and Cache domain-containing proteins are made.
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Adaptação Fisiológica/fisiologia , Proteínas de Bactérias/metabolismo , Domínios Proteicos/fisiologia , Pseudomonas aeruginosa/metabolismo , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Filogenia , Ligação Proteica/fisiologia , Conformação Proteica , Domínios Proteicos/genética , Proteoma/genética , Proteômica , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genéticaRESUMO
The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective 'peptibitors' inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.
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Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 µM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 µM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/metabolismo , Haemophilus influenzae/metabolismo , Heme/metabolismo , RNA de Cadeia Dupla/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Proteínas de Transporte/genética , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Otite Média/microbiologia , Otite Média/patologia , Conformação Proteica , Transporte Proteico/fisiologia , RNA de Cadeia Dupla/genética , Motivos de Ligação ao RNA/genética , Fatores de Virulência/metabolismoRESUMO
Chlamydiae are strict intracellular pathogens residing within a specialised membrane-bound compartment called the inclusion. Therefore, each infected cell can, be considered as a single entity where bacteria form a community within the inclusion. It remains unclear as to how the population of bacteria within the inclusion influences individual bacterium. The life cycle of Chlamydia involves transitioning between the invasive elementary bodies (EBs) and replicative reticulate bodies (RBs). We have used cryo-soft X-ray tomography to observe individual inclusions, an approach that combines 40 nm spatial resolution and large volume imaging (up to 16 µm). Using semi-automated segmentation pipeline, we considered each inclusion as an individual bacterial niche. Within each inclusion, we identifyed and classified different forms of the bacteria and confirmed the recent finding that RBs have a variety of volumes (small, large and abnormal). We demonstrate that the proportions of these different RB forms depend on the bacterial concentration in the inclusion. We conclude that each inclusion operates as an autonomous community that influences the characteristics of individual bacteria within the inclusion.
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Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.
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Antivirais/química , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Reposicionamento de Medicamentos , Oligopeptídeos/química , Linhagem Celular , Humanos , Serpinas/química , Proteínas Virais/químicaRESUMO
Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found â¼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 µM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.
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Desenho de Fármacos , Inibidores de Proteínas Quinases/química , SARS-CoV-2/enzimologia , Proteínas da Matriz Viral/antagonistas & inibidores , Acrilamida/química , Acrilamida/metabolismo , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Biologia Computacional/métodos , Bases de Dados de Proteínas , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/metabolismo , SARS-CoV-2/isolamento & purificação , Proteínas da Matriz Viral/metabolismoRESUMO
The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some ß-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.
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Antivirais/farmacologia , Cisteína Endopeptidases/efeitos dos fármacos , Espectrometria de Massas/métodos , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , beta-Lactamas/farmacologia , Acilação , Antivirais/química , COVID-19/virologia , Domínio Catalítico , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , beta-Lactamas/químicaRESUMO
The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.