RESUMO
In this study, we catalog structure activity relationships (SAR) of several short chain fatty acid (SCFA)-modified hexosamine analogues used in metabolic glycoengineering (MGE) by comparing in silico and experimental measurements of physiochemical properties important in drug design. We then describe the impact of these compounds on selected biological parameters that influence the pharmacological properties and safety of drug candidates by monitoring P-glycoprotein (Pgp) efflux, inhibition of cytochrome P450 3A4 (CYP3A4), hERG channel inhibition, and cardiomyocyte cytotoxicity. These parameters are influenced by length of the SCFAs (e.g., acetate vs n-butyrate), which are added to MGE analogues to increase the efficiency of cellular uptake, the regioisomeric arrangement of the SCFAs on the core sugar, the structure of the core sugar itself, and by the type of N-acyl modification (e.g., N-acetyl vs N-azido). By cataloging the influence of these SAR on pharmacological properties of MGE analogues, this study outlines design considerations for tuning the pharmacological, physiochemical, and the toxicological parameters of this emerging class of small molecule drug candidates.
Assuntos
Inibidores do Citocromo P-450 CYP3A/farmacologia , Desenho de Fármacos , Ácidos Graxos Voláteis/farmacologia , Hexosaminas/farmacologia , Engenharia Metabólica/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/química , Avaliação Pré-Clínica de Medicamentos , Ácidos Graxos Voláteis/química , Hexosaminas/química , Estrutura Molecular , Miócitos Cardíacos/efeitos dos fármacos , Cultura Primária de Células , Ratos , Relação Estrutura-Atividade , Testes de Toxicidade/métodos , Regulador Transcricional ERG/antagonistas & inibidoresRESUMO
This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity. By contrast, hexosamine analogues not targeted to sialic acid biosynthesis (e.g., butanoylated GlcNAc or GalNAc) had minimal impact. Measurement of mRNA and protein confirmed that esterase activity was controlled through glycosylation and not through transcription or translation. Azide-modified ManNAc analogues widely used in MGE also enhanced esterase activity and provided a way to enrich targeted glycoengineered proteins (such as CES2), thereby providing unambiguous evidence that the compounds were converted to sialosides and installed into the glycan structures of esterases as intended. Overall, this study provides a pioneering example of the modulation of intracellular enzyme activity through MGE, which expands the value of this technology from its current status as a labeling strategy and modulator of cell surface biological events.
Assuntos
Carboxilesterase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Células Epiteliais/enzimologia , Engenharia Metabólica/métodos , Processamento de Proteína Pós-Traducional , Ácidos Siálicos/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Acetilgalactosamina/farmacologia , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacologia , Animais , Sítios de Ligação , Ácido Butírico/química , Células CHO , Carboxilesterase/química , Carboxilesterase/genética , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Linhagem Celular Tumoral , Cricetulus , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Glicosilação , Hexosaminas/química , Hexosaminas/metabolismo , Hexosaminas/farmacologia , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ácidos Siálicos/químicaRESUMO
Metabolic glycoengineering is a specialization of metabolic engineering that focuses on using small molecule metabolites to manipulate biosynthetic pathways responsible for oligosaccharide and glycoconjugate production. As outlined in this article, this technique has blossomed in mammalian systems over the past three decades but has made only modest progress in prokaryotes. Nevertheless, a sufficient foundation now exists to support several important applications of metabolic glycoengineering in bacteria based on methods to preferentially direct metabolic intermediates into pathways involved in lipopolysaccharide, peptidoglycan, teichoic acid, or capsule polysaccharide production. An overview of current applications and future prospects for this technology are provided in this report.
Assuntos
Metabolismo dos Carboidratos/genética , Glicoproteínas/genética , Engenharia Metabólica , Proteínas Recombinantes/metabolismo , Animais , Glicoconjugados/química , Glicoconjugados/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Lipopolissacarídeos/química , Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Oligossacarídeos/síntese química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Metastatic human pancreatic cancer cells (the SW1990 line) that are resistant to the EGFR-targeting tyrosine kinase inhibitor drugs (TKI) erlotinib and gefitinib were treated with 1,3,4-O-Bu3ManNAc, a 'metabolic glycoengineering' drug candidate that increased sialylation by â¼2-fold. Consistent with genetic methods previously used to increase EGFR sialylation, this small molecule reduced EGF binding, EGFR transphosphorylation, and downstream STAT activation. Significantly, co-treatment with both the sugar pharmacophore and the existing TKI drugs resulted in strong synergy, in essence re-sensitizing the SW1990 cells to these drugs. Finally, 1,3,4-O-Bu3ManNAz, which is the azido-modified counterpart to 1,3,4-O-Bu3ManNAc, provided a similar benefit thereby establishing a broad-based foundation to extend a 'metabolic glycoengineering' approach to clinical applications.
Assuntos
Cloridrato de Erlotinib/química , Engenharia Metabólica , Inibidores de Proteínas Quinases/química , Quinazolinas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/química , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Gefitinibe , Glicosilação , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Fatores de Transcrição STAT/metabolismoRESUMO
Vertebrate myoblast fusion allows for multinucleated muscle fibers to compound the size and strength of mononucleated cells, but the evolution of this important process is unknown. We investigated the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here, we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed a complex evolutionary history of myoblast fusion. A prevertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Evolutionary comparisons between vertebrate and nonvertebrate Myomaker revealed key structural and mechanistic insights into myoblast fusion. Thus, our findings suggest an evolutionary model of chordate fusogens and illustrate how new genes shape the emergence of novel morphogenetic traits and mechanisms.