RESUMEN
Gastric cancer is a remarkably heterogeneous tumor. Despite some advances in the diagnosis and treatment of gastric cancer in recent years, the precise treatment and curative outcomes remain unsatisfactory. Poor prognosis continues to pose a major challenge in gastric cancer. Therefore, it is imperative to identify effective targets to improve the treatment and prognosis of gastric cancer patients. It should be noted that glycosylation, a novel form of posttranslational modification, is a process capable of regulating protein function and influencing cellular activities. Currently, numerous studies have shown that glycosylation plays vital roles in the occurrence and progression of gastric cancer. As crucial enzymes that regulate glycan synthesis in glycosylation processes, glycosyltransferases are potential targets for treating GC. Hence, investigating the regulation of glycosyltransferases and the expression of associated proteins in gastric cancer cells is highly important. In this review, the related glycosyltransferases and their related signaling pathways in gastric cancer, as well as the existing inhibitors of glycosyltransferases, provide more possibilities for targeted therapies for gastric cancer.
Asunto(s)
Glicosiltransferasas , Terapia Molecular Dirigida , Neoplasias Gástricas , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/patología , Humanos , Glicosiltransferasas/metabolismo , Glicosiltransferasas/antagonistas & inhibidores , Animales , Glicosilación , Transducción de Señal , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/farmacologíaRESUMEN
Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research.
Asunto(s)
Glicosiltransferasas/antagonistas & inhibidores , Glicosiltransferasas/metabolismo , Mediciones Luminiscentes/métodos , Adenosina Trifosfato/metabolismo , Animales , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glicómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Cinética , Luciferasas de Luciérnaga/metabolismo , Nucleótidos/metabolismo , Especificidad por SustratoRESUMEN
Bacterial cells present a wide diversity of saccharides that decorate the cell surface and help mediate interactions with the environment. Many Gram-negative cells express O-antigens, which are long sugar polymers that makeup the distal portion of lipopolysaccharide (LPS) that constitutes the surface of the outer membrane. This review highlights chemical biology tools that have been developed in recent years to facilitate the modulation of O-antigen synthesis and composition, as well as related bacterial polysaccharide pathways, and the detection of unique glycan sequences. Advances in the biochemistry and structural biology of O-antigen biosynthetic machinery are also described, which provide guidance for the design of novel chemical and biomolecular probes. Many of the tools noted here have not yet been utilized in biological systems and offer researchers the opportunity to investigate the complex sugar architecture of Gram-negative cells.
Asunto(s)
Bacterias Gramnegativas/química , Antígenos O/metabolismo , Inhibidores Enzimáticos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Bacterias Gramnegativas/enzimología , Humanos , Ingeniería Metabólica , Sondas Moleculares/química , Sondas Moleculares/farmacología , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Antígenos O/química , Ingeniería de Proteínas , Especificidad por Sustrato/genéticaRESUMEN
Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N-glycans of the glycocalyx. Glycomic analysis using liquid chromatography-mass spectrometry (LC-MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl-N-acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N-glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell-cell interactions.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glicocálix/efectos de los fármacos , Glicómica , Glicoproteínas/metabolismo , Glicosiltransferasas/antagonistas & inhibidores , Polisacáridos/metabolismo , Células A549 , Alcaloides/química , Alcaloides/farmacología , Células CACO-2 , Cromatografía Liquida , Inhibidores Enzimáticos/química , Fucosa/análogos & derivados , Fucosa/química , Fucosa/farmacología , Glicocálix/enzimología , Glicómica/instrumentación , Glicosilación , Glicosiltransferasas/metabolismo , Humanos , Dispositivos Laboratorio en un Chip , Espectrometría de Masas , Técnicas Analíticas Microfluídicas/instrumentación , Estructura Molecular , Ácidos Neuramínicos/química , Ácidos Neuramínicos/farmacología , Proteómica , Relación Estructura-ActividadRESUMEN
Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.
Asunto(s)
Glicosiltransferasas/metabolismo , Fenotiazinas/farmacología , Acetatos/metabolismo , Secuencia de Aminoácidos/genética , Proteínas Bacterianas/química , Sitios de Unión/genética , Dominio Catalítico/genética , Pared Celular/metabolismo , Cristalografía por Rayos X/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Glicosiltransferasas/antagonistas & inhibidores , Glicosiltransferasas/efectos de los fármacos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Muramidasa/antagonistas & inhibidores , Muramidasa/metabolismo , Peptidoglicano/metabolismo , Fenotiazinas/metabolismo , Conformación Proteica/efectos de los fármacosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged during the last months of 2019, spreading throughout the world as a highly transmissible infectious illness designated as COVID-19. Vaccines have now appeared, but the challenges in producing sufficient material and distributing them around the world means that effective treatments to limit infection and improve recovery are still urgently needed. This review focuses on the relevance of different glycobiological molecules that could potentially serve as or inspire therapeutic tools during SARS-CoV-2 infection. As such, we highlight the glycobiology of the SARS-CoV-2 infection process, where glycans on viral proteins and on host glycosaminoglycans have critical roles in efficient infection. We also take notice of the glycan-binding proteins involved in the infective capacity of virus and in human defense. In addition, we critically evaluate the glycobiological contribution of candidate drugs for COVID-19 therapy such as glycans for vaccines, anti-glycan antibodies, recombinant lectins, lectin inhibitors, glycosidase inhibitors, polysaccharides, and numerous glycosides, emphasizing some opportunities to repurpose FDA-approved drugs. For the next-generation drugs suggested here, biotechnological engineering of new probes to block the SARS-CoV-2 infection might be based on the essential glycobiological insight on glycosyltransferases, glycans, glycan-binding proteins, and glycosidases related to this pathology.
Asunto(s)
Antivirales/uso terapéutico , COVID-19/prevención & control , Reposicionamiento de Medicamentos , Inhibidores de Glicósido Hidrolasas/uso terapéutico , Glicosiltransferasas/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Anticuerpos Neutralizantes/uso terapéutico , Antivirales/química , COVID-19/epidemiología , COVID-19/inmunología , COVID-19/virología , Diseño de Fármacos , Descubrimiento de Drogas , Expresión Génica , Glicómica/métodos , Glicosaminoglicanos/química , Glicosaminoglicanos/inmunología , Glicosaminoglicanos/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/genética , Glicosiltransferasas/inmunología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Lectinas/química , Lectinas/inmunología , Lectinas/metabolismo , Polisacáridos/química , Polisacáridos/inmunología , Polisacáridos/metabolismo , SARS-CoV-2/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Transducción de Señal , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
The Glycine max xyloglucan endotransglycosylase/hydrolase (EC 2.4.1.207), GmXTH43, has been identified through RNA sequencing of RNA isolated through laser microdissection of Heterodera glycines-parasitized root cells (syncytia) undergoing the process of defense. Experiments reveal that genetically increasing XTH43 transcript abundance in the H. glycines-susceptible genotype G. max[Williams 82/PI 518671] decreases parasitism. Experiments presented here show decreasing XTH43 transcript abundance through RNA interference (RNAi) in the H. glycines-resistant G. max[Peking/PI 548402] increases susceptibility, but it is unclear what role XTH43 performs. The experiments presented here show XTH43 overexpression decreases the relative length of xyloglucan (XyG) chains, however, there is an increase in the amount of those shorter chains. In contrast, XTH43 RNAi increases XyG chain length. The experiments show that XTH43 has the capability to function, when increased in its expression, to limit XyG chain extension. This outcome would likely impair the ability of the cell wall to expand. Consequently, XTH43 could provide an enzymatically-driven capability to the cell that would allow it to limit the ability of parasitic nematodes like H. glycines to develop a feeding structure that, otherwise, would facilitate parasitism. The experiments presented here provide experimentally-based proof that XTHs can function in ways that could be viewed as being able to limit the expansion of the cell wall.
Asunto(s)
Glucanos/metabolismo , Glycine max/parasitología , Glicosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Tylenchida/fisiología , Xilanos/metabolismo , Animales , Cromatografía en Gel , Femenino , Genotipo , Glucanos/química , Glicosiltransferasas/antagonistas & inhibidores , Glicosiltransferasas/genética , Interacciones Huésped-Parásitos , Peso Molecular , Raíces de Plantas/parasitología , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Análisis de Componente Principal , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Glycine max/enzimología , Glycine max/genética , Xilanos/químicaRESUMEN
Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.
Asunto(s)
Antifúngicos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Policétidos/farmacología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Fermentación , Genes Reporteros , Glicosilfosfatidilinositoles/biosíntesis , Células HCT116 , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Células K562 , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos ICR , Mucorales , Familia de Multigenes , Rhizopus , Saccharomyces cerevisiaeRESUMEN
Cancer is a deadly disease that encompasses numerous cellular modifications. Among them, alterations in glycosylation are a proven reliable hallmark of cancer, with most biomarkers used in the clinic detecting cancer-associated glycans. Despite their clear potential as therapy targets, glycans have been overlooked in drug discovery strategies. The complexity associated with the glycosylation process, and lack of specific methodologies to study it, have long hampered progress. However, recent advances in new methodologies, such as glycoengineering of cells and high-throughput screening (HTS), have opened new avenues of discovery. We envision that glycan-based targeting has the potential to start a new era of cancer therapy. In this article, we discuss the promise of cancer-associated glycosylation for the discovery of effective cancer drugs.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas/tendencias , Glicosiltransferasas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Polisacáridos/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Ingeniería Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos de Selección de Medicamentos Antitumorales/tendencias , Glicosilación/efectos de los fármacos , Glicosiltransferasas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Neoplasias/patología , Polisacáridos/metabolismoRESUMEN
Iminosugars are sugar analogues endowed with a high pharmacological potential. The wide range of biological activities exhibited by these glycomimetics associated with their excellent drug profile make them attractive therapeutic candidates for several medical interventions. The ability of iminosugars to act as inhibitors or enhancers of carbohydrate-processing enzymes suggests their potential use as therapeutics for the treatment of cystic fibrosis (CF). Herein we review the most relevant advances in the field, paying attention to both the chemical synthesis of the iminosugars and their biological evaluations, resulting from in vitro and in vivo assays. Starting from the example of the marketed drug NBDNJ (N-butyl deoxynojirimycin), a variety of iminosugars have exhibited the capacity to rescue the trafficking of F508del-CFTR (deletion of F508 residue in the CF transmembrane conductance regulator), either alone or in combination with other correctors. Interesting results have also been obtained when iminosugars were considered as anti-inflammatory agents in CF lung disease. The data herein reported demonstrate that iminosugars hold considerable potential to be applied for both therapeutic purposes.
Asunto(s)
Fibrosis Quística/tratamiento farmacológico , Compuestos Heterocíclicos con 1 Anillo/uso terapéutico , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/uso terapéutico , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Glicósido Hidrolasas/antagonistas & inhibidores , Glicosiltransferasas/antagonistas & inhibidores , Compuestos Heterocíclicos con 1 Anillo/síntesis química , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Iminopiranosas/química , Iminopiranosas/uso terapéutico , Inflamación , Estructura Molecular , Mutación , Eliminación de Secuencia , Tartratos/química , Tartratos/uso terapéuticoRESUMEN
High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a KD value of 7.2â µm with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches.
Asunto(s)
Clostridioides difficile/enzimología , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Secuenciación de Nucleótidos de Alto Rendimiento , Nucleósidos/farmacología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Glicosiltransferasas/metabolismo , Modelos Moleculares , Nucleósidos/síntesis química , Nucleósidos/químicaRESUMEN
One of the major threats to human life nowadays is widespread antibiotic resistance. Antibiotics are used to treat bacterial infections by targeting their essential pathways, such as the biosynthesis of bacterial cell walls. Bacterial transglycosylase, particularly glycosyltransferase family 51 (GT51), is one critical player in the cell wall biosynthesis and has long been known as a promising yet challenging target for antibiotic development. Here, we review the structural studies of this protein and summarize recent progress in developing its specific inhibitors, including synthetic substrate analogs and novel compounds identified from high-throughput screens. A detailed analysis of the protein-ligand interface has also provided us with valuable insights into the future antibiotic development against the bacterial transglycosylase.
Asunto(s)
Antibacterianos/farmacología , Bacterias/enzimología , Glicosiltransferasas/química , Antibacterianos/química , Bacterias/efectos de los fármacos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Humanos , Modelos Moleculares , Conformación Proteica , Relación Estructura-ActividadRESUMEN
A glucansucrase encoding gene was cloned into pET-28a(+) vector and expression in Escherichia coli BL21(DE3). An about 160â¯kDa recombinant glucansucrase was purified with a yield of 50.73% and a 4.02-fold increase in activity. The 1464 amino acid residue enzyme belongs to the GH70 subfamily and shares 90% similarity with Leuconostoc sp. glucansucrase. The optimal temperature and pH were 30⯰C and pHâ¯5.5, and 80% of activity was retained after incubation at 10-30⯰C and pHâ¯5-7. Enzyme activity was strongly activated by Ca2+ and Mn2+ and inhibited by various metal ions and chemical agents, and a high affinity for sucrose (Kmâ¯=â¯11.6â¯mM, Vmaxâ¯=â¯8.1â¯mmol/(mL·min)). Circular dichroism (CD) and Raman spectra collectively indicated a high proportion of random coil structure.
Asunto(s)
Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Leuconostoc mesenteroides/enzimología , Leuconostoc mesenteroides/genética , Biocatálisis , Clonación Molecular , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Vectores Genéticos/genética , Glicosiltransferasas/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Cinética , Metales/farmacología , Análisis de Secuencia , Tensoactivos/farmacología , TemperaturaRESUMEN
Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing 3-deoxy-ß-d- manno-oct-2-ulosonic acid (ß-Kdo). The ß-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive antivirulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for ß-Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This "FP-tag" assay should be readily adaptable to high-throughput screens of other glycosyltransferases.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Química Clic , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Azúcares Ácidos/química , Azúcares Ácidos/farmacologíaRESUMEN
Herein we present the methodology for obtaining glycosyltransferase inhibitors, analogues of natural enzyme substrates of donor-type: UDP-glucose and UDP-galactose. The synthesis concerned glycoconjugates, nucleoside analogues containing an acyclic ribose mimetic linked to a uracil moiety in their structure. The biological activity of the synthesised compounds was determined on the basis of their ability to inhibit the model enzyme action of ß-1,4-galactosyltransferase from bovine milk. The obtained results allowed to expand and supplement the existing library of synthetic compounds that are able to regulate the biological activity of enzymes from the GT class.
Asunto(s)
Glicoconjugados/síntesis química , Glicoconjugados/farmacología , Uridina/síntesis química , Uridina/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glicoconjugados/química , Glicosiltransferasas/antagonistas & inhibidores , Estructura Molecular , Uridina/análogos & derivados , Uridina/químicaRESUMEN
The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here, we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-aminosugar acetyltransferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC50 values ranging from 35 ± 7 µM to 174 ± 21 µM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogs of lead compounds. Finally, the compounds were found to be nontoxic to mammalian cells, further supporting the opportunities for future development.
Asunto(s)
Bacterias/enzimología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Uridina Difosfato/metabolismo , Uridina/análogos & derivados , Uridina/farmacología , Acetiltransferasas/antagonistas & inhibidores , Bacterias/metabolismo , Campylobacter/enzimología , Campylobacter/metabolismo , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/enzimología , Línea Celular , Clostridioides difficile/enzimología , Clostridioides difficile/metabolismo , Inhibidores Enzimáticos/síntesis química , Glicosiltransferasas/antagonistas & inhibidores , Humanos , Modelos Moleculares , Nucleósidos/síntesis química , Nucleósidos/química , Nucleósidos/farmacología , Técnicas de Síntesis en Fase Sólida/métodos , Relación Estructura-Actividad , Uridina/síntesis químicaRESUMEN
Heptosyltransferase I (WaaC) is a highly conserved glycosyltransferase found in Gram-negative bacteria that transfers a heptose residue onto the endotoxin inner core structure (ReLPS) of the outer membrane. Knockouts of WaaC have decreased virulence and increased susceptibility to antibiotics, making WaaC a potential drug target. While previous studies have elucidated the structure of the holoenzyme and a donor analog complex, no information on the binding mode of the acceptor has been available so far. By soaking of a chemically modified functional acceptor, along with a stable donor analog, the crystal structure of a pseudo-ternary complex of WaaC was obtained at 2.3-Å resolution. The acceptor is bound in an unusual horseshoe conformation stabilized by interaction of the anionic carboxylate and phosphate groups at its center and tips with highly conserved Lys and Arg residues. This binding is accompanied by both inter- and intra-domain movements within the protein.
Asunto(s)
Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Bacterias Gramnegativas/enzimología , Sitios de Unión , Catálisis/efectos de los fármacos , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Bacterias Gramnegativas/patogenicidad , Modelos Moleculares , Estabilidad Proteica , Estructura Cuaternaria de Proteína , VirulenciaRESUMEN
Lipooligosaccharide (LOS) structures in the outer core of Gram-negative mucosal pathogens such as Neisseria meningitidis and Haemophilus influenzae contain characteristic glycoepitopes that contribute significantly to bacterial virulence. An important example is the digalactoside epitope generated by the retaining α-1,4-galactosyltransferase LgtC. These digalactosides camouflage the pathogen from the host immune system and increase its serum resistance. Small molecular inhibitors of LgtC are therefore sought after as chemical tools to study bacterial virulence, and as potential candidates for anti-virulence drug discovery. We have recently discovered a new class of non-substrate-like inhibitors of LgtC. The new inhibitors act via a covalent mode of action, targeting a non-catalytic cysteine residue in the LgtC active site. Here, we describe, for the first time, structure-activity relationships for this new class of glycosyltransferase inhibitors. We have carried out a detailed analysis of the inhibition kinetics to establish the relative contribution of the non-covalent binding and the covalent inactivation steps for overall inhibitory activity. Selected inhibitors were also evaluated against a serum-resistant strain of Haemophilus influenzae, but did not enhance the killing effect of human serum.
Asunto(s)
Inhibidores Enzimáticos/química , Glicosiltransferasas/antagonistas & inhibidores , Neisseria meningitidis/enzimología , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glicosiltransferasas/metabolismo , Haemophilus influenzae/efectos de los fármacos , Cinética , Neisseria meningitidis/efectos de los fármacos , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacología , Relación Estructura-ActividadRESUMEN
Described here is the asymmetric synthesis of iminosugar 2b, a Lipid II analog, designed to mimic the transition state of transglycosylation catalyzed by the bacterial transglycosylase. The high density of functional groups, together with a rich stereochemistry, represents an extraordinary challenge for chemical synthesis. The key 2,6-anti- stereochemistry of the iminosugar ring was established through an iridium-catalyzed asymmetric allylic amination. The developed synthetic route is suitable for the synthesis of focused libraries to enable the structure-activity relationship study and late-stage modification of iminosugar scaffold with variable lipid, peptide and sugar substituents. Compound 2b showed 70% inhibition of transglycosylase from Acinetobacter baumannii, providing a basis for further improvement.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Acinetobacter baumannii/enzimología , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Glicosiltransferasas/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Molecular , Estereoisomerismo , Relación Estructura-Actividad , Uridina Difosfato Ácido N-Acetilmurámico/síntesis química , Uridina Difosfato Ácido N-Acetilmurámico/química , Uridina Difosfato Ácido N-Acetilmurámico/farmacologíaRESUMEN
Transglycosylase (TGase) is essential to biosynthesis of peptidoglycan for formation of bacterial cell wall. Moenomycin is a potent TGase inhibitor, but not used in clinic treatment due to its poor pharmacokinetics. The E-F disaccharide, phosphoglycerate and lipid tail in moenomycin are crucial elements for TGase inhibition and antibacterial activity. Based on this scaffold, a series of truncated mimics comprising biphenyl, amine linker and 2-alkoxy-3-phosphorylpropanoate moieties were designed to test their TGase inhibitory activity. In this design, the phosphorylpropanoate group is a surrogate of phosphoglycerate with improved stability. A library of lipid tails can be constructed by a straightforward approach using Cu(I)-catalyzed (3 + 2) cycloaddition reactions, and the as-synthesized triazole ring can provide additional hydrogen bonds in the TGase active site. Our molecular docking experiments reveal that the biphenyl group provides π-π and π-cation interactions to act as a simplified alternative of the C-E disaccharide in moenomycin. To play the role of the oxonium transition state in transglycosylation, the amine linker exists as a positively charged species in physiological condition to attain electrostatic interactions with acidic residues. In this study, two biphenyl-linked 2-alkoxy-3-phosphorylpropanoate compounds (8 and 10) are found to exhibit modest inhibitory activity (IC50â¯≈â¯150⯵M) against the TGase of Acinetobacter baumannii and good antibacterial activity against Staphylococcus aureus (MICâ¯=â¯6.3⯵M).