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1.
Cell ; 184(12): 3192-3204.e16, 2021 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-33974910

RESUMEN

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.


Asunto(s)
Anticuerpos Neutralizantes/química , Células Gigantes/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Sitios de Unión , Células CHO , COVID-19/patología , COVID-19/virología , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Células Gigantes/citología , Humanos , Fusión de Membrana , Biblioteca de Péptidos , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo
2.
J Am Chem Soc ; 142(51): 21310-21321, 2020 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-33301681

RESUMEN

Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkers for prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract of E. coli to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads. We next designed conjugates consisting of (1) an N-terminal siderophore to facilitate uptake, (2) a protease-cleavable linker, and (3) an amine-containing antibiotic. Using this strategy, we converted daptomycin-which by itself is active only against Gram-positive bacteria-into an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophore-facilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform's utility for development of protease-activated prodrugs, including Trojan horse antibiotics.


Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Péptido Hidrolasas/metabolismo , Sideróforos/química , Acinetobacter/efectos de los fármacos , Antibacterianos/química , Descubrimiento de Drogas , Escherichia coli/efectos de los fármacos , Periplasma/microbiología
3.
Biomacromolecules ; 21(12): 4781-4794, 2020 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-33170649

RESUMEN

Self-assembling peptide-based hydrogels are a class of tunable soft materials that have been shown to be highly useful for a number of biomedical applications. The dynamic formation of the supramolecular fibrils that compose these materials has heretofore remained poorly characterized. A better understanding of this process would provide important insights into the behavior of these systems and could aid in the rational design of new peptide hydrogels. Here, we report the determination of the microscopic steps that underpin the self-assembly of a hydrogel-forming peptide, SgI37-49. Using theoretical models of linear polymerization to analyze the kinetic self-assembly data, we show that SgI37-49 fibril formation is driven by fibril-catalyzed secondary nucleation and that all the microscopic processes involved in SgI37-49 self-assembly display an enzyme-like saturation behavior. Moreover, this analysis allows us to quantify the rates of the underlying processes at different peptide concentrations and to calculate the time evolution of these reaction rates over the time course of self-assembly. We demonstrate here a new mechanistic approach for the study of self-assembling hydrogel-forming peptides, which is complementary to commonly used materials science characterization techniques.


Asunto(s)
Hidrogeles , Péptidos , Cinética
4.
Fungal Genet Biol ; 89: 18-28, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26808821

RESUMEN

Microorganisms produce a wide range of natural products (NPs) with clinically and agriculturally relevant biological activities. In bacteria and fungi, genes encoding successive steps in a biosynthetic pathway tend to be clustered on the chromosome as biosynthetic gene clusters (BGCs). Historically, "activity-guided" approaches to NP discovery have focused on bioactivity screening of NPs produced by culturable microbes. In contrast, recent "genome mining" approaches first identify candidate BGCs, express these biosynthetic genes using synthetic biology methods, and finally test for the production of NPs. Fungal genome mining efforts and the exploration of novel sequence and NP space are limited, however, by the lack of a comprehensive catalog of BGCs encoding experimentally-validated products. In this study, we generated a comprehensive reference set of fungal NPs whose biosynthetic gene clusters are described in the published literature. To generate this dataset, we first identified NCBI records that included both a peer-reviewed article and an associated nucleotide record. We filtered these records by text and homology criteria to identify putative NP-related articles and BGCs. Next, we manually curated the resulting articles, chemical structures, and protein sequences. The resulting catalog contains 197 unique NP compounds covering several major classes of fungal NPs, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids. The distribution of articles published per compound shows a bias toward the study of certain popular compounds, such as the aflatoxins. Phylogenetic analysis of biosynthetic genes suggests that much chemical and enzymatic diversity remains to be discovered in fungi. Our catalog was incorporated into the recently launched Minimum Information about Biosynthetic Gene cluster (MIBiG) repository to create the largest known set of fungal BGCs and associated NPs, a resource that we anticipate will guide future genome mining and synthetic biology efforts toward discovering novel fungal enzymes and metabolites.


Asunto(s)
Productos Biológicos , Vías Biosintéticas/genética , Genes Fúngicos , Genoma Fúngico , Familia de Multigenes , Alcaloides , Secuencia de Aminoácidos , Biología Computacional , Curaduría de Datos , Hongos/genética , Filogenia , Policétidos , Terpenos
5.
Protein Sci ; 32(8): e4712, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37354015

RESUMEN

Antiviral therapeutics to treat SARS-CoV-2 are needed to diminish the morbidity of the ongoing COVID-19 pandemic. A well-precedented drug target is the main viral protease (MPro ), which is targeted by an approved drug and by several investigational drugs. Emerging viral resistance has made new inhibitor chemotypes more pressing. Adopting a structure-based approach, we docked 1.2 billion non-covalent lead-like molecules and a new library of 6.5 million electrophiles against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 and 20 µM, respectively. Several series were optimized, resulting in low micromolar inhibitors. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. While the new chemotypes may aid further optimization of MPro inhibitors for SARS-CoV-2, the modest success rate also reveals weaknesses in our approach for challenging targets like MPro versus other targets where it has been more successful, and versus other structure-based techniques against MPro itself.


Asunto(s)
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Pandemias , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Simulación del Acoplamiento Molecular , Proteínas no Estructurales Virales/química , Antivirales/farmacología , Antivirales/química
6.
Biol Open ; 7(7)2018 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-30037883

RESUMEN

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

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