RESUMO
Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides extracted from Av. paragallinarum genotype A contained chondroitin. Chondroitin synthase of Av. paragallinarum (ApCS) encoded by one gene within the presumed capsule biosynthesis gene cluster exhibited considerable homology to identified bacterial chondroitin synthases. Herein, we report the identification and characterization of ApCS. This enzyme indeed displays chondroitin synthase activity involved in the biosynthesis of the capsule. ApCS is a bifunctional protein catalyzing the elongation of the chondroitin chain by alternatively transferring the glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues from their nucleotide forms to the non-reducing ends of the saccharide chains. GlcA with a para-nitrophenyl group (pNP) could serve as the acceptor for ApCS; this enzyme shows a stringent donor tolerance when the acceptor is as small as this monosaccharide. Then, UDP-GalNAc and GlcA-pNP were injected sequentially through the chip-immobilized chondroitin synthases, and the surface plasmon resonance data demonstrated that the up-regulated extent caused by the binding of the donor is one possibly essential factor in successful polymerization reaction. This conclusion will, therefore, enhance the understanding of the mode of action of glycosyltransferase. Surprisingly, high activity at near-zero temperature as well as weak temperature dependence of this novel bacterial chondroitin synthase indicate that ApCS was a cold-active enzyme. From all accounts, ApCS becomes the fourth known bacterial chondroitin synthase, and the potential applications in artificial chondroitin sulfate and glycosaminoglycan synthetic approaches make it an attractive glycosyltransferase for further investigation.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Gammaproteobacteria/enzimologia , Gammaproteobacteria/genética , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Especificidade por SubstratoRESUMO
O-Mannose glycans account up to 30 % of total O-glycans in the brain. Previous synthesis and functional studies have only focused on the core M3 O-mannose glycans of α-dystroglycan, which are a causative factor for various muscular diseases. In this study, a highly efficient chemoenzymatic strategy was developed that enabled the first collective synthesis of 63 core M1 and core M2 O-mannose glycans. This chemoenzymatic strategy features the gram-scale chemical synthesis of five judiciously designed core structures, and the diversity-oriented modification of the core structures with three enzyme modules to provide 58 complex O-mannose glycans in a linear sequence that does not exceed four steps. The binding profiles of synthetic O-mannose glycans with a panel of lectins, antibodies, and brain proteins were also explored by using a printed O-mannose glycan array.
Assuntos
Manose/química , Polissacarídeos/química , Animais , Biocatálise , Técnicas de Química Sintética , Distroglicanas/síntese química , Distroglicanas/química , Glicosilação , Glicosiltransferases/química , Humanos , Manose/síntese química , Polissacarídeos/síntese químicaRESUMO
Unlike chemosynthetic drugs designed for specific molecular and disease targets, active small-molecule natural products typically have a wide range of bioactivities and multiple targets, necessitating extensive screening and development. To address this issue, we propose a strategy for the direct in situ microdynamic examination of potential drug candidates to rapidly identify their effects and mechanisms of action. As a proof-of-concept, we investigated the behavior of mussel oligosaccharide (MOS-1) by tracking the subcellular dynamics of fluorescently labeled MOS-1 in cultured cells. We recorded the entire dynamic process of the localization of fluorescein isothiocyanate (FITC)-MOS-1 to the lysosomes and visualized the distribution of the drug within the cell. Remarkably, lysosomes containing FITC-MOS-1 actively recruited lipid droplets, leading to fusion events and increased cellular lipid consumption. These drug behaviors confirmed MOS-1 is a candidate for the treatment of lipid-related diseases. Furthermore, in a high-fat HepG2 cell model and in high-fat diet-fed apolipoprotein E (ApoE) -/- mice, MOS-1 significantly promoted triglyceride degradation, reduced lipid droplet accumulation, lowered serum triglyceride levels, and mitigated liver damage and steatosis. Overall, our work supports the prioritization of in situ visual monitoring of drug location and distribution in subcellular compartments during the drug development phase, as this methodology contributes to the rapid identification of drug indications. Collectively, this methodology is significant for the screening and development of selective small-molecule drugs, and is expected to expedite the identification of candidate molecules with medicinal effects.
RESUMO
Frequently, subcellular-targeted drugs tend to accumulate in lysosomes after cellular absorption, a process termed the lysosomal trap. This accumulation often interferes with the drug's ability to bind to its target, resulting in decreased efficiency. Existing methods for addressing lysosome-induced drug resistance mainly involve improving the structures of small molecules or enveloping drugs in nanomaterials. Nonetheless, these approaches can lead to changes in the drug structure or potentially trigger unexpected reactions within organisms. To address these issues, we introduced a strategy that involves inactivating the lysosome with the use of Ag nanoparticles (Cy3.5@Ag NPs). In this method, the Cy3.5@Ag NPs gradually accumulate inside lysosomes, leading to permeation of the lysosomal membrane and subsequent lysosomal inactivation. In addition, Cy3.5@Ag NPs also significantly affected the motility of lysosomes and induced the occurrence of lysosome passivation. Importantly, coincubating Cy3.5@Ag NPs with various subcellular-targeted drugs was found to significantly increase the efficiency of these treatments. Our strategy illustrates the potential of using lysosomal inactivation to enhance drug efficacy, providing a promising therapeutic strategy for cancer.
Assuntos
Lisossomos , Nanopartículas Metálicas , Prata , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Prata/química , Prata/farmacologia , Nanopartículas Metálicas/química , Humanos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Nano-MoS2 exhibit oxidoreductase-like activities, and has been shown to effectively eliminate excessive intracellular ROS and inhibit Aß aggregation, thus demonstrating promising potential for anti-Alzheimer's disease (anti-AD) intervention. However, the low water dispersibility and high toxicity of nano-MoS2 limits its further application. In this study, we developed a chondroitin sulphate (CS)-modified MoS2 nanoenzyme (CS@MoS2) by harnessing the excellent biocompatibility of CS and the exceptional activities of nano-MoS2 to explore its potential in anti-AD research. Promisingly, CS@MoS2 significantly inhibited Aß1-40 aggregation and prevented toxic injury in SH-SY5Y cells caused by Aß1-40. In addition, CS@MoS2 protected these cells from oxidative stress damage by regulating ROS production, as well as promoting the activities of SOD and GSH-Px. CS@MoS2 also modulated the intracellular Ca2+ imbalance and downregulated Tau hyperphosphorylation by activating GSK-3ß. CS@MoS2 suppressed p-NF-κB (p65) translocation to the nucleus by inhibiting MAPK phosphorylation, and modulated the expression of downstream anti- and proinflammatory cytokines. Owing to its multifunctional activities, CS@MoS2 effectively improved spatial learning, memory, and anxiety in D-gal/AlCl3-induced AD mice. Taken together, these results indicate that CS@MoS2 has significant potential for improving the therapeutic efficacy of the prevention and treatment of AD, while also presenting a novel framework for the application of nanoenzymes.
Assuntos
Doença de Alzheimer , Sulfatos de Condroitina , Dissulfetos , Molibdênio , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Animais , Camundongos , Humanos , Molibdênio/química , Molibdênio/farmacologia , Dissulfetos/química , Dissulfetos/farmacologia , Peptídeos beta-Amiloides/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular Tumoral , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Masculino , Modelos Animais de DoençasRESUMO
Organelles perform critical biological functions due to their distinct molecular composition and internal environment. Disorders in organelles or their interacting networks have been linked to the incidence of numerous diseases, and the research of pharmacological actions at the organelle level has sparked pharmacists' interest. Currently, cell imaging has evolved into a critical tool for drug delivery, drug discovery, and pharmacological research. The introduction of advanced imaging techniques in recent years has provided researchers with richer biological information for viewing and studying the ultrastructure of organelles, protein interactions, and gene transcription activities, leading to the design and delivery of precision-targeted drugs. Therefore, this reviews the research on organelles-targeted drugs based upon imaging technologies and development of fluorescent molecules for medicinal purposes. We also give a thorough analysis of a number of subcellular-level elements of drug development, including subcellular research instruments and methods, organelle biological event investigation, subcellular target and drug identification, and design of subcellular delivery systems. This review will make it possible to promote drug research from the individual/cellular level to the subcellular level, as well as give a new focus based on newly found organelle activities.
Assuntos
Sistemas de Liberação de Medicamentos , Organelas , Humanos , Preparações Farmacêuticas/química , Desenvolvimento de Medicamentos , Descoberta de DrogasRESUMO
A novel series of piperidine-linked amino-triazine derivatives were designed, synthesized and evaluated for in vitro anti-HIV activity as non-nucleoside reverse transcriptase inhibitors on the basis of our previous work. Screening results indicated that most compounds showed excellent activity against wild-type HIV-1 with EC(50) values in low nanomolar concentration range (especially compound 6b3, EC(50) = 4.61 nM, SI = 5945) and high activity against K103N/Y181C resistant mutant strain of HIV-1 with EC(50) values in low micromolar concentration range. In addition, preliminary structure-activity relationship and molecular modeling of these new analogs were detailed in this manuscript.
Assuntos
Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Aminas/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Transcriptase Reversa do HIV/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Piperidinas/química , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade , Triazinas/químicaRESUMO
The diversity-oriented chemoenzymatic synthesis of α-dystroglycan (α-DG) core M1 O-mannose glycans has been achieved via a three-step sequential one-pot multienzyme (OPME) glycosylation of a chemically prepared disaccharyl serine intermediate. The high flexibility and efficiency of this chemoenzymatic strategy was demonstrated for the synthesis of three more complex core M1 O-mannose glycans for the first time along with three previously reported core M1 structures.