RESUMEN
Aided by genome-mining strategies, knowledge of the prevalence and diversity of ribosomally synthesized natural products (RNPs) is rapidly increasing. Among these are the lantipeptides, posttranslationally modified peptides containing characteristic thioether cross-links imperative for bioactivity and stability. Though this family was once thought to be a limited class of antimicrobial compounds produced by gram-positive bacteria, new insights have revealed a much larger diversity of activity, structure, biosynthetic machinery, and producing organisms than previously appreciated. Detailed investigation of the enzymes responsible for installing the posttranslational modifications has resulted in improved in vivo and in vitro engineering systems focusing on enhancement of the therapeutic potential of these compounds. Although dozens of new lantipeptides have been isolated in recent years, bioinformatic analyses indicate that many hundreds more await discovery owing to the widespread frequency of lantipeptide biosynthetic machinery in bacterial genomes.
Asunto(s)
Bacterias/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Bacteriocinas/química , Bacteriocinas/clasificación , Productos Biológicos , Ingeniería Genética , Péptidos/química , Péptidos/clasificación , Péptidos/genética , Péptidos/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
OBJECTIVES: We here assessed whether typical pathogens of laboratory mice affect the development of diet-induced obesity and glucose intolerance, and whether colonization affects the efficacy of the GLP-1R agonist liraglutide and of the GLP-1/GIP co-agonist MAR709 to treat obesity and diabetes. METHODS: Male C57BL/6J mice were experimentally infected with Helicobacter hepaticus, Rodentibacter pneumotropicus and Staphylococcus aureus and compared to a group of uninfected specific and opportunistic pathogen free (SOPF) mice. The development of diet-induced obesity and glucose intolerance was monitored over a period of 26 weeks. To study the influence of pathogens on drug treatment, mice were then subjected for 6 days daily treatment with either the GLP-1 receptor agonist liraglutide or the GLP-1/GIP co-agonist MAR709. RESULTS: Colonized mice did not differ from SOPF controls regarding HFD-induced body weight gain, food intake, body composition, glycemic control, or responsiveness to treatment with liraglutide or the GLP-1/GIP co-agonist MAR709. CONCLUSIONS: We conclude that the occurrence of H. hepaticus, R. pneumotropicus and S. aureus does neither affect the development of diet-induced obesity or type 2 diabetes, nor the efficacy of GLP-1-based drugs to decrease body weight and to improve glucose control in mice.
Asunto(s)
Dieta Alta en Grasa , Intolerancia a la Glucosa , Incretinas , Liraglutida , Ratones Endogámicos C57BL , Obesidad , Staphylococcus aureus , Animales , Dieta Alta en Grasa/efectos adversos , Ratones , Masculino , Staphylococcus aureus/efectos de los fármacos , Incretinas/metabolismo , Obesidad/metabolismo , Liraglutida/farmacología , Intolerancia a la Glucosa/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Organismos Libres de Patógenos Específicos , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/metabolismoRESUMEN
Incretin-based therapies are highly successful in combatting obesity and type 2 diabetes1. Yet both activation and inhibition of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in combination with glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) activation have resulted in similar clinical outcomes, as demonstrated by the GIPR-GLP-1R co-agonist tirzepatide2 and AMG-133 (ref. 3) combining GIPR antagonism with GLP-1R agonism. This underlines the importance of a better understanding of the GIP system. Here we show the necessity of ß-arrestin recruitment for GIPR function, by combining in vitro pharmacological characterization of 47 GIPR variants with burden testing of clinical phenotypes and in vivo studies. Burden testing of variants with distinct ligand-binding capacity, Gs activation (cyclic adenosine monophosphate production) and ß-arrestin 2 recruitment and internalization shows that unlike variants solely impaired in Gs signalling, variants impaired in both Gs and ß-arrestin 2 recruitment contribute to lower adiposity-related traits. Endosomal Gs-mediated signalling of the variants shows a ß-arrestin dependency and genetic ablation of ß-arrestin 2 impairs cyclic adenosine monophosphate production and decreases GIP efficacy on glucose control in male mice. This study highlights a crucial impact of ß-arrestins in regulating GIPR signalling and overall preservation of biological activity that may facilitate new developments in therapeutic targeting of the GIPR system.
Asunto(s)
Fenotipo , Receptores de la Hormona Gastrointestinal , beta-Arrestinas , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Animales , Ratones , Humanos , beta-Arrestinas/metabolismo , Variación Genética , Arrestina beta 2/metabolismo , Arrestina beta 2/genética , Transducción de Señal , Polipéptido Inhibidor Gástrico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Obesidad/metabolismo , Obesidad/genética , Masculino , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/genéticaRESUMEN
Lantibiotics are a family of antibacterial peptide natural products characterized by the post-translational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single naturally occurring stereochemical configuration for each of these cross-links was known. The discovery of lantibiotics with alternative lanthionine and methyllanthionine stereochemistry has prompted an investigation of its importance to biological activity. Here, solid-supported chemical synthesis enabled the total synthesis of the lantibiotic lacticin 481 and analogues containing cross-links with non-native stereochemical configurations. Biological evaluation revealed that these alterations abolished the antibacterial activity in all of the analogues, revealing the critical importance of the enzymatically installed stereochemistry for the biological activity of lacticin 481.
Asunto(s)
Antibacterianos/síntesis química , Bacteriocinas/síntesis química , Lactococcus lactis/química , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Bacteriocinas/química , Bacteriocinas/farmacología , Lactococcus lactis/efectos de los fármacos , Datos de Secuencia Molecular , EstereoisomerismoAsunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Descubrimiento de Drogas/métodos , Polipéptido Inhibidor Gástrico/agonistas , Péptido 1 Similar al Glucagón/agonistas , Glucagón/agonistas , Obesidad/tratamiento farmacológico , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Obesidad/metabolismo , Péptidos/química , Péptidos/uso terapéuticoRESUMEN
Our understanding of secondary metabolite production in bacteria has been shaped primarily by studies of attached varieties such as symbionts, pathogens, and soil bacteria. Here we show that a strain of the single-celled, planktonic marine cyanobacterium Prochlorococcus-which conducts a sizable fraction of photosynthesis in the oceans-produces many cyclic, lanthionine-containing peptides (lantipeptides). Remarkably, in Prochlorococcus MIT9313 a single promiscuous enzyme transforms up to 29 different linear ribosomally synthesized peptides into a library of polycyclic, conformationally constrained products with highly diverse ring topologies. Genes encoding this system are found in variable abundances across the oceans-with a hot spot in a Galapagos hypersaline lagoon-suggesting they play a habitat- and/or community-specific role. The extraordinarily efficient pathway for generating structural diversity enables these cyanobacteria to produce as many secondary metabolites as model antibiotic-producing bacteria, but with much smaller genomes.
Asunto(s)
Biocatálisis , Péptidos Cíclicos/biosíntesis , Fitoplancton/metabolismo , Prochlorococcus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Ciclización , Genoma Bacteriano , Datos de Secuencia Molecular , Familia de Multigenes , Fitoplancton/química , Fitoplancton/genética , Prochlorococcus/química , Prochlorococcus/genética , Alineación de SecuenciaRESUMEN
The development of single-molecule co-agonists for the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) is considered a breakthrough in the treatment of obesity and type 2 diabetes. But although GIPR-GLP-1R co-agonism decreases body weight with superior efficacy relative to GLP-1R agonism alone in preclinical1-3 and clinical studies4,5, the role of GIP in regulating energy metabolism remains enigmatic. Increasing evidence suggests that long-acting GIPR agonists act in the brain to decrease body weight through the inhibition of food intake3,6-8; however, the mechanisms and neuronal populations through which GIP affects metabolism remain to be identified. Here, we report that long-acting GIPR agonists and GIPR-GLP-1R co-agonists decrease body weight and food intake via inhibitory GABAergic neurons. We show that acyl-GIP decreases body weight and food intake in male diet-induced obese wild-type mice, but not in mice with deletion of Gipr in Vgat(also known as Slc32a1)-expressing GABAergic neurons (Vgat-Gipr knockout). Whereas the GIPR-GLP-1R co-agonist MAR709 leads, in male diet-induced obese wild-type mice, to greater weight loss and further inhibition of food intake relative to a pharmacokinetically matched acyl-GLP-1 control, this superiority over GLP-1 vanishes in Vgat-Gipr knockout mice. Our data demonstrate that long-acting GIPR agonists crucially depend on GIPR signaling in inhibitory GABAergic neurons to decrease body weight and food intake.
Asunto(s)
Diabetes Mellitus Tipo 2 , Masculino , Ratones , Animales , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Obesidad/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptores Acoplados a Proteínas G , Glucosa , Neuronas GABAérgicas/metabolismo , Ingestión de AlimentosRESUMEN
Lantibiotics are a large family of antibacterial peptide natural products containing multiple post-translational modifications, including the thioether structures lanthionine and methyllanthionine. Efforts to probe structure-activity relationships and engineer improved pharmacological properties have driven the development of new methods to produce non-natural analogues of these compounds. In this study, solid-supported chemical synthesis was used to produce analogues of the potent lantibiotic epilancin 15X, in order to assess the importance of several N-terminal post-translational modifications for biological activity. Surprisingly, substitution of these moieties, including the unusual N-terminal D-lactyl moiety, resulted in relatively small changes in the antimicrobial activity and pore-forming ability of the peptides.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacteriocinas/química , Bacteriocinas/farmacología , Péptidos/química , Péptidos/farmacología , Técnicas de Síntesis en Fase Sólida/métodos , Staphylococcus epidermidis/química , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Bacteriocinas/síntesis química , Humanos , Datos de Secuencia Molecular , Péptidos/síntesis química , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus/efectos de los fármacosRESUMEN
Ribosomally synthesized and post-translationally modified peptides are a rapidly expanding class of natural products. They are typically biosynthesized by modification of a C-terminal segment of the precursor peptide (the core peptide). The precursor peptide also contains an N-terminal leader peptide that is required to guide the biosynthetic enzymes. For bioengineering purposes, the leader peptide is beneficial because it allows promiscuous activity of the biosynthetic enzymes with respect to modification of the core peptide sequence. However, the leader peptide also presents drawbacks as it needs to be present on the core peptide and then removed in a later step. We show that fusing the leader peptide for the lantibiotic lacticin 481 to its biosynthetic enzyme LctM allows the protein to act on core peptides without a leader peptide. We illustrate the use of this methodology for preparation of improved lacticin 481 analogues containing non-proteinogenic amino acids.
Asunto(s)
Enzimas/metabolismo , Péptidos/metabolismo , Ingeniería de Proteínas , Enzimas/química , Péptidos/síntesis química , Péptidos/químicaRESUMEN
OBJECTIVE: Pharmacological strategies that engage multiple mechanisms-of-action have demonstrated synergistic benefits for metabolic disease in preclinical models. One approach, concurrent activation of the glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and glucagon (Gcg) receptors (i.e. triagonism), combines the anorectic and insulinotropic activities of GLP-1 and GIP with the energy expenditure effect of glucagon. While the efficacy of triagonism in preclinical models is known, the relative contribution of GcgR activation remains unassessed. This work aims to addresses that central question. METHODS: Herein, we detail the design of unimolecular peptide triagonists with an empirically optimized receptor potency ratio. These optimized peptide triagonists employ a protraction strategy permitting once-weekly human dosing. Additionally, we assess the effects of these peptides on weight-reduction, food intake, glucose control, and energy expenditure in an established DIO mouse model compared to clinically relevant GLP-1R agonists (e.g. semaglutide) and dual GLP-1R/GIPR agonists (e.g. tirzepatide). RESULTS: Optimized triagonists normalize body weight in DIO mice and enhance energy expenditure in a manner superior to that of GLP-1R mono-agonists and GLP-1R/GIPR co-agonists. CONCLUSIONS: These pre-clinical data suggest unimolecular poly-pharmacology as an effective means to target multiple mechanisms contributing to obesity and further implicate GcgR activation as the differentiating factor between incretin receptor mono- or dual-agonists and triagonists.
Asunto(s)
Polipéptido Inhibidor Gástrico , Glucagón , Animales , Peso Corporal , Polipéptido Inhibidor Gástrico/metabolismo , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Ratones , Ratones Obesos , Péptidos/farmacología , Receptores de Glucagón/metabolismoRESUMEN
OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) is one of the two major incretin factors that regulate metabolic homeostasis. Genetic ablation of its receptor (GIPR) in mice confers protection against diet-induced obesity (DIO), while GIPR neutralizing antibodies produce additive weight reduction when combined with GLP-1R agonists in preclinical models and clinical trials. Conversely, GIPR agonists have been shown to promote weight loss in rodents, while dual GLP-1R/GIPR agonists have proven superior to GLP-1R monoagonists for weight reduction in clinical trials. We sought to develop a long-acting, specific GIPR peptide antagonist as a tool compound suitable for investigating GIPR pharmacology in both rodent and human systems. METHODS: We report a structure-activity relationship of GIPR peptide antagonists based on the human and mouse GIP sequences with fatty acid-based protraction. We assessed these compounds in vitro, in vivo in DIO mice, and ex vivo in islets from human donors. RESULTS: We report the discovery of a GIP(5-31) palmitoylated analogue, [Nα-Ac, L14, R18, E21] hGIP(5-31)-K11 (γE-C16), which potently inhibits in vitro GIP-mediated cAMP generation at both the hGIPR and mGIPR. In vivo, this peptide effectively blocks GIP-mediated reductions in glycemia in response to exogenous and endogenous GIP and displays a circulating pharmacokinetic profile amenable for once-daily dosing in rodents. Co-administration with the GLP-1R agonist semaglutide and this GIPR peptide antagonist potentiates weight loss compared to semaglutide alone. Finally, this antagonist inhibits GIP- but not GLP-1-stimulated insulin secretion in intact human islets. CONCLUSIONS: Our work demonstrates the discovery of a potent, specific, and long-acting GIPR peptide antagonist that effectively blocks GIP action in vitro, ex vivo in human islets, and in vivo in mice while producing additive weight-loss when combined with a GLP-1R agonist in DIO mice.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón , Receptores de la Hormona Gastrointestinal , Roedores , Animales , Humanos , Ratones , Polipéptido Inhibidor Gástrico/antagonistas & inhibidores , Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Ratones Obesos , Péptidos/farmacología , Péptidos/química , Roedores/metabolismo , Pérdida de Peso , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidoresRESUMEN
Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARÉ/É£) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARÉ/É£ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARÉ£-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography-mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance.
Asunto(s)
Diabetes Mellitus Tipo 2 , PPAR alfa , Alcanosulfonatos , Animales , Peso Corporal , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón/uso terapéutico , Receptor del Péptido 1 Similar al Glucagón , Glucosa , Masculino , Ratones , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , PPAR alfa/agonistas , PPAR alfa/uso terapéutico , FenilpropionatosRESUMEN
OBJECTIVE: We assessed the spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics of GIPR mono-agonists, GLP-1R mono-agonists including semaglutide, and GLP-1/GIP dual-agonists MAR709 and tirzepatide. METHODS: Receptor G protein recruitment and internalization/trafficking dynamics were assessed using bioluminescence resonance energy transfer (BRET)-based technology and live-cell HILO microscopy. RESULTS: Relative to native and acylated GLP-1 agonists, MAR709 and tirzepatide showed preserved maximal cAMP production despite partial Gαs recruitment paralleled by diminished ligand-induced receptor internalization at both target receptors. Despite MAR709's lower internalization rate, GLP-1R co-localization with Rab11-associated recycling endosomes was not different between MAR709 and GLP-1R specific mono-agonists. CONCLUSIONS: Our data indicated that MAR709 and tirzepatide induce unique spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics relative to native peptides, semaglutide, and matched mono-agonist controls. These findings support the hypothesis that the structure of GLP-1/GIP dual-agonists confer a biased agonism that, in addition to its influence on intracellular signaling, uniquely modulates receptor trafficking.
Asunto(s)
Péptido 1 Similar al Glucagón/análogos & derivados , Péptido 1 Similar al Glucagón/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Transducción de Señal/efectos de los fármacos , Polipéptido Inhibidor Gástrico/farmacología , Células HEK293 , Humanos , Ligandos , Péptidos/farmacologíaRESUMEN
Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.
Asunto(s)
Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Polipéptido Inhibidor Gástrico/farmacología , Receptores de la Hormona Gastrointestinal/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Sistema Nervioso Central/metabolismo , Dieta Alta en Grasa , Polipéptido Inhibidor Gástrico/química , Péptido 1 Similar al Glucagón/farmacología , Humanos , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/metabolismo , Obesidad/patología , Obesidad/prevención & control , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de la Hormona Gastrointestinal/deficiencia , Receptores de la Hormona Gastrointestinal/genéticaRESUMEN
The continued global growth in the prevalence of obesity coupled with the limited number of efficacious and safe treatment options elevates the importance of innovative pharmaceutical approaches. Combinatorial strategies that harness the metabolic benefits of multiple hormonal mechanisms have emerged at the preclinical and more recently clinical stages of drug development. A priority has been anti-obesity unimolecular peptides that function as balanced, high potency poly-agonists at two or all the cellular receptors for the endocrine hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon. This report reviews recent progress in this area, with emphasis on what the initial clinical results demonstrate and what remains to be addressed.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Polipéptido Inhibidor Gástrico/agonistas , Glucagón/metabolismo , Obesidad/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Receptores de la Hormona Gastrointestinal/agonistas , Receptores de Glucagón/agonistas , Animales , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Diseño de Fármacos , Glucagón/química , Humanos , Obesidad/metabolismo , Obesidad/patología , Fragmentos de Péptidos/química , Relación Estructura-ActividadRESUMEN
Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics containing the characteristic thioether cross-links lanthionine and methyllanthionine. To date, no analogues of lantibiotics that contain nonproteinogenic amino acids have been reported. In this study, in vitro-reconstituted lacticin 481 synthetase was used in conjunction with synthetic peptide substrates containing nonproteinogenic amino acids to generate 11 analogues of lacticin 481. These analogues contained sarcosine and aminocyclopropanoic acid in place of Gly5, D-valine at position 6, 4-cyanoaminobutyric acid in place of Glu13, beta(3)-homoarginine at the position of Asn15, N-butylglycine and beta-Ala at Met16, naphthylalanine (Nal) at Trp19, 4-pyridynylalanine (Pal) at Phe21, and homophenylalanine (hPhe) at Phe23. Of these analogues, the Trp19Nal and Phe23hPhe mutants provided zones of inhibition larger than the parent compound in agar diffusion assays against the indicator strains Lactococcus lactis HP and Bacillus subtilis 6633. These two compounds also demonstrated improved MIC values against liquid cultures of L. lactis HP.
Asunto(s)
Aminoácidos/química , Antiinfecciosos/química , Antiinfecciosos/síntesis química , Bacteriocinas/química , Bacteriocinas/síntesis química , Secuencia de Aminoácidos , Antiinfecciosos/farmacología , Bacteriocinas/farmacología , Lactococcus lactis/efectos de los fármacos , Datos de Secuencia Molecular , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The compstatin family of complement inhibitors has shown promise in various immuno-inflammatory disorders. Although recent analogues show beneficial pharmacokinetics, further extension of the plasma half-life is expected to benefit systemic application of these peptidic inhibitors. We therefore synthesized conjugates of compstatin analogues and albumin-binding molecules (ABM) to increase circulatory residence. Equilibrium dialysis in complement-depleted serum showed a marked increase in plasma protein binding from <8 % to >99 % for a resulting chimera (ABM2-Cp20). Further analysis confirmed interaction with albumin from different species, primarily via siteâ II. Importantly, ABM2-Cp20 bound 20-fold stronger to its target protein C3b (KD =150â pM) than the parent peptide. Kinetic and in silico analysis suggested that ABM2 occupies a secondary site on C3b and improves the dissociation rate via additional contacts. Addition of an ABM modifier thereby not only improved plasma protein binding but also produced the most potent compstatin analogue to date with potential implications for the treatment of systemic complement-related diseases.
Asunto(s)
Albúminas/química , Inactivadores del Complemento/farmacología , Péptidos Cíclicos/farmacología , Secuencia de Aminoácidos , Inactivadores del Complemento/farmacocinética , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos Cíclicos/farmacocinética , Unión ProteicaRESUMEN
Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain the thioether structures lanthionine and methyllanthionine and exert potent antimicrobial activity against Gram-positive bacteria. At present, detailed modes-of-action are only known for a small subset of family members. Lacticin 481, a tricyclic lantibiotic, contains a lipid II binding motif present in related compounds such as mersacidin and nukacin ISK-1. Here, we show that lacticin 481 inhibits PBP1b-catalyzed peptidoglycan formation. Furthermore, we show that changes in potency of analogues of lacticin 481 containing non-proteinogenic amino acids correlate positively with the potency of inhibition of the transglycosylase activity of PBP1b. Thus, lipid II is the likely target of lacticin 481, and use of non-proteinogenic amino acids resulted in stronger inhibition of the target. Additionally, we demonstrate that lacticin 481 does not form pores in the membranes of susceptible bacteria, a common mode-of-action of other lantibiotics.
Asunto(s)
Antibacterianos/farmacología , Bacteriocinas/farmacología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano Glicosiltransferasa/metabolismo , Peptidoglicano/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , Secuencia de Aminoácidos , Antibacterianos/química , Bacillus subtilis/citología , Bacillus subtilis/efectos de los fármacos , Bacteriocinas/química , Escherichia coli/metabolismo , Infecciones por Escherichia coli/tratamiento farmacológico , Glicosilación/efectos de los fármacos , Humanos , Lactococcus lactis/química , Lactococcus lactis/citología , Lactococcus lactis/efectos de los fármacos , Datos de Secuencia Molecular , Permeabilidad/efectos de los fármacosRESUMEN
Disulfide bonds are essential for the structural stability and biological activity of many bioactive peptides. However, these bonds are labile to reducing agents, which can limit the therapeutic utility of such peptides. Substitution of a disulfide bond with a reduction-resistant cystathionine bridge is an attractive means of improving stability while imposing minimal structural perturbation to the peptide. We have applied this approach to the therapeutic complement inhibitor compstatin, a disulfide-containing peptide currently in clinical trials for age-related macular degeneration, in an effort to maintain its potent activity while improving its biological stability. Thioether-containing compstatin analogues were produced via solid-phase peptide synthesis utilizing orthogonally protected cystathionine amino acid building blocks and solid-supported peptide cyclization. Overall, the affinity of these analogues for their biological target and potent inhibition of complement activation were largely maintained when compared to those of the parent disulfide-containing peptides. Thus, the improved stability to reduction conferred by the thioether bond makes this new class of compstatin peptides a promising alternative for therapeutic applications. Additionally, the versatility of this synthesis allows for exploration of disulfide-to-thioether substitution in a variety of other therapeutic peptides.