RESUMEN
L-Idose thioglycosides are useful glycosyl donors for the construction of glycosaminoglycan oligosaccharides. When activated with NIS and catalytic TMSOTf in the presence of methanol, the stereoselectivity of O-glycosylation displays an intriguing dependence on the reaction temperature, with an increased preference for formation of the α-glycoside at higher temperatures. Using a combination of vt-NMR spectroscopy and DFT calculations, we show how a simple mechanistic model, based on competing reactions of the iodinated thioglycoside, can explain the main features of the temperature dependence. In this model, the increased selectivity at high temperature is attributed to differences among the entropy and energy terms of the competing reaction pathways. Neighbouring-group participation (giving an intermediate acyloxonium ion) plays an increasingly dominant role as temperature is raised. The general features of this kinetic regime may also apply more broadly to other glycosylations that likewise favour α-glycoside formation at high temperature.
RESUMEN
1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPSâ I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited ß-glucuronidase (ß-GLU). The d-GlcA-configured 9 was a potent inhibitor of ß-GLU and a moderate inhibitor of the endo-ß-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.
Asunto(s)
Iduronidasa , Mucopolisacaridosis I , Humanos , Iduronidasa/química , Iduronidasa/genética , Ácidos Urónicos , Glucuronidasa/química , Mucopolisacaridosis I/genéticaRESUMEN
Wild honeybees (Apis mellifera) are considered extinct in most parts of Europe. The likely causes of their decline include increased parasite burden, lack of high-quality nesting sites and associated depredation pressure, and food scarcity. In Germany, feral honeybees still colonize managed forests, but their survival rate is too low to maintain viable populations. Based on colony observations collected during a monitoring study, data on parasite prevalence, experiments on nest depredation, and analyses of land cover maps, we explored whether parasite pressure, depredation or expected landscape-level food availability explain feral colony winter mortality. Considering the colony-level occurrence of 18 microparasites in the previous summer, colonies that died did not have a higher parasite burden than colonies that survived. Camera traps installed at cavity trees revealed that four woodpecker species, great tits, and pine martens act as nest depredators. In a depredator exclusion experiment, the winter survival rate of colonies in cavities with protected entrances was 50% higher than that of colonies with unmanipulated entrances. Landscapes surrounding surviving colonies contained on average 6.4 percentage points more cropland than landscapes surrounding dying colonies, with cropland being known to disproportionately provide forage for bees in our study system. We conclude that the lack of spacious but well-protected nesting cavities and the shortage of food are currently more important than parasites in limiting populations of wild-living honeybees in German forests. Increasing the density and diversity of large tree cavities and promoting bee forage plants in forests will probably promote wild-living honeybees despite parasite pressure.
Asunto(s)
Parásitos , Animales , Abejas , Bosques , Europa (Continente) , Árboles , AlemaniaRESUMEN
The heparan sulfate (HS) mimetic pixatimod (PG545) is a highly potent inhibitor of angiogenesis, tumor growth, and metastasis currently in clinical trials for cancer. PG545 has also demonstrated potent antiviral activity against numerous HS-dependent viruses, including SARS-CoV-2, and shows promise as an antiviral drug for the treatment of COVID-19. Structurally, PG545 consists of a fully sulfated tetrasaccharide conjugated to the steroid 5α-cholestan-3ß-ol. The reported synthesis of PG545 suffers from a low yield and poor selectivity in the critical glycosylation step. Given its clinical importance, new efficient routes for the synthesis of PG545 and analogues were developed. Particular attention was given to improving the key glycosylation step by using more stable protecting groups and optimized glycosyl donors.
Asunto(s)
COVID-19 , Inhibidores de la Angiogénesis , Línea Celular Tumoral , Heparitina Sulfato , Humanos , Neovascularización PatológicaRESUMEN
Ferrier photobromination enables direct synthetic access to valuable 5-C-bromosugars but has limitations that restrict its broader use. The reaction is typically conducted in CCl4 heated at reflux with irradiation by broad spectrum, energy-inefficient heat lamps. Herein, we demonstrate that the reaction proceeds rapidly and efficiently with PhCF3 as a safe and environmentally benign alternative to CCl4 at mild temperatures (≤40 °C) inside a compact photoreactor fitted with purple light-emitting diodes (LEDs).
RESUMEN
Herein, we report the synthesis and biological evaluation of a novel series of heparinoid amphiphiles as inhibitors of heparanase and SARS-CoV-2. By employing a tailor-made synthetic strategy, a library of highly sulfated homo-oligosaccharides bearing d-glucose or a C5-epimer (i.e., l-idose or l-iduronic acid) conjugated with various lipophilic groups was synthesized and investigated for antiviral activity. Sulfated higher oligosaccharides of d-glucose or l-idose with lipophilic aglycones displayed potent anti-SARS-CoV-2 and antiheparanse activity, similar to or better than pixatimod (PG545), and were more potent than their isosteric l-iduronic acid congeners. Lipophilic groups such as cholestanol and C18-aliphatic substitution are more advantageous than functional group appended lipophilic moieties. These findings confirm that fine-tuning of higher oligosaccharides, degree of sulfation, and lipophilic groups can yield compounds with potent anti-SARS-CoV-2 activity.
Asunto(s)
Antivirales , SARS-CoV-2 , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , SARS-CoV-2/efectos de los fármacos , Humanos , Oligosacáridos/farmacología , Oligosacáridos/síntesis química , Oligosacáridos/química , Tratamiento Farmacológico de COVID-19 , Animales , Células Vero , Chlorocebus aethiops , Relación Estructura-Actividad , COVID-19/virología , Glucuronidasa , SaponinasRESUMEN
Obesity is an increasingly common disease. While antagonism of the melanin-concentrating hormone-1 receptor (MCH-1R) has been widely reported as a promising therapeutic avenue for obesity treatment, no MCH-1R antagonists have reached the market. Discovery and optimization of new chemical matter targeting MCH-1R is hindered by reduced HTS success rates and a lack of structural information about the MCH-1R binding site. X-ray crystallography and NMR, the major experimental sources of structural information, are very slow processes for membrane proteins and are not currently feasible for every GPCR or GPCR-ligand complex. This situation significantly limits the ability of these methods to impact the drug discovery process for GPCR targets in "real-time", and hence, there is an urgent need for other practical and cost-efficient alternatives. We present here a conceptually pioneering approach that integrates GPCR modeling with design, synthesis, and screening of a diverse library of sugar-based compounds from the VAST technology (versatile assembly on stable templates) to provide structural insights on the MCH-1R binding site. This approach creates a cost-efficient new avenue for structure-based drug discovery (SBDD) against GPCR targets. In our work, a primary VAST hit was used to construct a high-quality MCH-1R model. Following model validation, a structure-based virtual screen yielded a 14% hit rate and 10 novel chemotypes of potent MCH-1R antagonists, including EOAI3367472 (IC50 = 131 nM) and EOAI3367474 (IC50 = 213 nM).
Asunto(s)
Fármacos Antiobesidad/farmacología , Carbohidratos/farmacología , Diseño de Fármacos , Obesidad/tratamiento farmacológico , Receptores de Somatostatina/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Fármacos Antiobesidad/síntesis química , Fármacos Antiobesidad/química , Fármacos Antiobesidad/uso terapéutico , Sitios de Unión , Carbohidratos/síntesis química , Carbohidratos/química , Carbohidratos/uso terapéutico , Evaluación Preclínica de Medicamentos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Somatostatina/química , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Interfaz Usuario-ComputadorRESUMEN
The pyranose scaffold is unique in its ability to position pharmacophore substituents in various ways in 3D space, and unique pharmacophore scanning libraries could be envisaged that focus on scanning topography rather than diversity in the type of substituents. Approaches have been described that make use of amine and acid functionalities on the pyranose scaffolds to append substituents, and this has enabled the generation of libraries of significant structural diversity. Our general aim was to generate libraries of pyranose-based drug-like mimetics, where the substituents are held close to the scaffold, in order to obtain molecules with better defined positions for the pharmacophore substituents. Here we describe the development of a versatile synthetic route toward peptide mimetics build on 2-amino pyranose scaffolds. The method allows introduction of a wide range of substituent types, it is regio- and stereospecific, and the later diversity steps are performed on solid phase. Further, the same process was applied on glucose and allose scaffolds, in the exemplified cases, and is likely adaptable to other pyranose building blocks. The methods developed in this work give access to molecules that position the three selected binding elements in various 3D orientations on a pyranose scaffold and have been applied for the production of a systematically diverse library of several hundred monosaccharide-based mimetics.
Asunto(s)
Aminas/química , Monosacáridos/química , Monosacáridos/síntesis química , Péptidos/química , Péptidos/síntesis química , Azúcares Ácidos/química , Azúcares Ácidos/síntesis química , Técnicas Químicas Combinatorias , Glicosilación , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
A synthetic heparan sulfate disaccharide has been assessed as a fluorogenic heparanase substrate, enabling enzyme turnover and inhibition kinetics measurements despite slow turnover. Crystal structures with human heparanase also provide the first ever observation of a substrate in an activated 1S3 conformation, highlighting previously unknown interactions involved in enzymatic processing. Our data provide insights into the heparanase catalytic mechanism, and will inform the design of improved heparanase substrates and inhibitors.
Asunto(s)
Disacáridos/química , Glucuronidasa/química , Heparitina Sulfato/química , Catálisis , Cristalografía por Rayos X , Humanos , Espectroscopía de Resonancia Magnética , Conformación Proteica , Espectrometría de Masa por Ionización de Electrospray , Especificidad por SustratoRESUMEN
Dengue virus (DENV) is the most prevalent mosquito-borne flavivirus that infects humans. At present, there are no specific antiviral drugs to treat DENV infection and vaccine development has met with challenges. DENV encodes two glycosaminoglycan (GAG) binding proteins; Envelope (E) and non-structural protein 1 (NS1). While previous work has validated the use of GAG analogues as inhibitors of E mediated virus-cell attachment, their potential for antiviral intervention in NS1 protein toxicity has not yet been explored. Here, we investigate the potential of the heparan sulfate mimetic PG545 as a dual purpose compound to target both DENV virion infectivity and NS1 function. In comparison to a non-sulfated analogue, we show that PG545 potently inhibits DENV infectivity with no cytotoxic effect. Against NS1, PG545 completely blocks the induction of cellular activation and abolishes NS1-mediated disruption of endothelial monolayer integrity. Furthermore, PG545 treatment moderately improves survival from lethal DENV challenge in a murine model. At peak disease, PG545-treated mice have lower viremia, circulating NS1 and serum TNF-α. Consistent with anti-NS1 activity, PG545 treatment also reduces systemic vascular leakage caused by DENV infection in vivo. Taken together, these findings demonstrate that the dual targeting of DENV virions and NS1 using GAG analogues offers a new avenue for DENV drug development.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Saponinas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Virión/efectos de los fármacos , Animales , Antivirales/química , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Dengue/tratamiento farmacológico , Dengue/metabolismo , Dengue/virología , Heparitina Sulfato/química , Humanos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Saponinas/química , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Viremia/prevención & controlRESUMEN
Synthesis of four glycolipids with different number of lauroyl groups on glucose or cellobiose as scaffolds is described. Their immunological evaluations either admixed with or covalently linked to J8, a peptide antigen derived from the C-terminus of the antiphagocytic M-protein of group A streptococcus, are also investigated. Administration of mixtures of J8 and glycolipids to B10BR (H-2(k)) mice induced low-levels of J8-specific IgG antibodies. While glycolipopeptides, in which J8 was covalently linked to the synthetic glycolipids, demonstrated high-levels of antibody titers comparable with the co-administration of these glycolipopeptides with complete Freund's adjuvant, suggesting clearly the strong potency of the synthesized glycolipids as self-adjuvanting moieties.
Asunto(s)
Vacunas Bacterianas/administración & dosificación , Adyuvante de Freund/administración & dosificación , Glucolípidos/administración & dosificación , Glicopéptidos/administración & dosificación , Streptococcus/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/farmacología , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Femenino , Adyuvante de Freund/inmunología , Adyuvante de Freund/farmacología , Glucolípidos/síntesis química , Glucolípidos/farmacología , Glicopéptidos/síntesis química , Glicopéptidos/farmacología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Datos de Secuencia Molecular , Fagocitosis , Streptococcus/crecimiento & desarrollo , Streptococcus/inmunologíaRESUMEN
Arsenic trioxide appears to be effective in the treatment of pro-myelocytic leukaemia. The substituted phenylarsen(III)oxides are highly polar, they have a high tendency to undergo oxidation to As (V) and to form oligomers, to prevent this we protected the As-(OH)(2) group as cyclic dithiaarsanes. To increase the compound's biological stability and passive diffusion we conjugated the compound of interest with lipoamino acids (Laas). Alternatively, we further conjugated the dithiaarsane derivative with a carbohydrate to utilize active transport systems and to target compound. We investigated two novel glyco-lipid arsenicals (III) (compounds 9 and 11) for their ability to initiate MCF-7 breast cancer cell death and characterized the mechanism by which death was initiated. A significant decrease in MCF-7 cell proliferation was observed using 1 microM and 10 microM compound (11) and 10 microM of compound (9). Treatment with compound (11) triggered apoptosis of MFC-7 cells while compound (9) induced inhibition of cellular proliferation was not via rapid induction of apoptosis and more likely reflected necrosis and/or alterations in the cell cycle. Differences in the anti-proliferative potency of the two compounds indicate that structural modifications influence effectiveness.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias de la Mama/patología , Cationes , Muerte Celular/efectos de los fármacos , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Glucolípidos/química , Glucolípidos/farmacología , Humanos , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
1. Our aim was to determine whether lipoamino acid conjugation of peptides that are high-affinity activators of ryanodine receptor (RyR) channels would (a) render the peptides membrane permeable, (b) alter their structure or (a) reduce their activity. The peptides correspond to the A region of the II-III loop of the skeletal dihydropyridine receptor. 2. The lipoamino acid conjugation increased the apparent permeability of the peptide across the Caco-2 cell monolayer by up to approximately 20-fold. 3. Nuclear magnetic resonance showed that the alpha-helical structure of critical basic residues, required for optimal activation of RyRs, was retained after conjugation. 4. The conjugated peptides were more effective in enhancing resting Ca2+ release, Ca2+-induced Ca2+ release and caffeine-induced Ca2+ release from isolated sarcoplasmic reticulum (SR) than their unconjugated counterparts, and significantly enhanced caffeine-induced Ca2+ release from mechanically skinned extensor digitorum longus (EDL) fibres. 5. The effect of both conjugated and unconjugated peptides on Ca2+ release from skeletal SR was 30-fold greater than their effect on either cardiac Ca2+ release or on the Ca2+ Mg2+ ATPase. 6. A small and very low affinity effect of the peptide in slowing Ca2+ uptake by the Ca2+, Mg2+ ATPase was exacerbated by lipoamino acid conjugation in both isolated SR and in skinned EDL fibres. 7. The results show that lipoamino acid conjugation of A region peptides increases their membrane permeability without impairing their structure or efficacy in activating skeletal and cardiac RyRs.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células CACO-2 , Cafeína/farmacología , Calcio/metabolismo , Técnicas de Cultivo de Célula , Permeabilidad de la Membrana Celular/fisiología , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Membrana Dobles de Lípidos , Datos de Secuencia Molecular , Estructura Molecular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Estructura Secundaria de Proteína , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Monoclonal antibodies (mAbs) were obtained after immunization of mice with neoglycoconjugates containing as a ligand the disaccharide Kdo(2-->4)Ko or Ko(2-->4)Kdo, representing structural elements of the core region of lipopolysaccharides (LPSs) from Acinetobacter haemolyticus and Burkholderia cepacia, respectively. One antibody, S67-9, bound with high specificity to Ko(2-->4)Kdo-BSA showing no cross reactivity with Kdo(2-->4)Kdo-BSA and the other antigens tested. A second mAb, S68-12, bound preferentially to Kdo(2-->4)Ko-BSA but cross reacted with Ko(2-->4)Kdo-BSA and Kdo(2-->4)Kdo-BSA. A third mAb, S67-27, was found to bind Kdo monosaccharide. Although mAbs S67-9 and S68-12 did not bind to LPS of Burkholderia or Acinetobacter as expected, the mAbs will be useful tools in studying the biosynthesis of LPS containing Ko.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Oligosacáridos/química , Oligosacáridos/inmunología , Animales , Burkholderia cepacia/química , Secuencia de Carbohidratos , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB CRESUMEN
Increasing the aglycone lipophilicity of a series of polysulfated oligosaccharide glycoside heparan sulfate (HS) mimetics via attachment of a steroid or long chain alkyl group resulted in compounds with significantly improved in vitro and ex vivo antiangiogenic activity. The compounds potently inhibited heparanase and HS-binding angiogenic growth factors and displayed improved antitumor and antimetastatic activity in vivo compared with the earlier series. Preliminary pharmacokinetic analyses also revealed significant increases in half-life following iv dosing, ultimately supporting less frequent dosing regimens in preclinical tumor models compared with other HS mimetics. The compounds also displayed only mild anticoagulant activity, a common side effect usually associated with HS mimetics. These efforts led to the identification of 3ß-cholestanyl 2,3,4,6-tetra-O-sulfo-α-d-glucopyranosyl-(1â4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1â4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1â4)-2,3,6-tri-O-sulfo-ß-d-glucopyranoside, tridecasodium salt (PG545, 18) as a clinical candidate. Compound 18 was recently evaluated in a phase I clinical trial in cancer patients.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Glucuronidasa/antagonistas & inhibidores , Heparitina Sulfato/análogos & derivados , Saponinas/uso terapéutico , Inhibidores de la Angiogénesis/síntesis química , Animales , Antineoplásicos/síntesis química , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Patológica/tratamiento farmacológico , Saponinas/síntesis química , Saponinas/farmacocinética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
A class of glycolipopeptides for use as building blocks for a new type of dynamic combinatorial library is reported. The members of the library consist of a variable carbohydrate moiety, coded amino acids, and lipoamino acids in order to convert them into amphiphiles. Glycolipopeptidic amphiphiles interact through non-covalent bonding when mixed together in aqueous phase and form micelles in dynamic close-packed fluid mosaic arrays. The head groups of amphiphiles are exposed on the micelle surface, providing entities which could be screened in biological assays to find the most potent combination of building blocks in order to identify new bioactive carbohydrate ligands.
Asunto(s)
Técnicas Químicas Combinatorias/métodos , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas/síntesis química , Tensoactivos/síntesis química , Aminoácidos/química , Cromatografía Líquida de Alta Presión , Interacciones Farmacológicas , Glicoproteínas/química , Ligandos , Espectroscopía de Resonancia Magnética , Micelas , Biblioteca de Péptidos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Tensoactivos/metabolismoRESUMEN
With the aim of providing compounds suitable for further development as microbicides active against human immunodeficiency virus 1 (HIV-1) a library containing 37 lipophile-conjugated sulfated oligosaccharides was screened for antiviral and virucidal activity against this virus. Four highly active compounds had low drug inhibition concentrations (IC(50)) for HIV-1 and inactivated viral particles, suggestive of virucidal properties. Two of these compounds comprising a sulfated tetrasaccharide linked to a cholestanol group by a glycosidic bond, showed low toxicity and high selectivity indices. The two compounds were active both against CCR5 and dual-tropic CCR5/CXCR4 clinical HIV-1 isolates. Since herpes simplex virus type 2 (HSV-2) may be a cofactor for HIV-1 infection, the virucidal effect of the compounds was demonstrated against both viruses when mixed and incubated together on permissive cells. Incubation of compounds with serum, and to a lesser degree, cervical secretions, reduced the HIV-1 inactivating capacity, which suggests the need for molecular modification to reduce host protein binding. Considering the virucidal effect and low toxicity, these sulfated oligosaccharides with lipophilic tails may offer new possibilities of microbicide development.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , VIH-1/efectos de los fármacos , Oligosacáridos/química , Oligosacáridos/farmacología , Animales , Antiinfecciosos/toxicidad , Células Cultivadas , Colestanol/química , Células Epiteliales/virología , Herpesvirus Humano 2/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Leucocitos Mononucleares/virología , Linfocitos/virología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Oligosacáridos/toxicidad , Sulfatos/químicaRESUMEN
Success in discovering bioactive peptide mimetics is often limited by the difficulties in correctly transposing known binding elements of the active peptide onto a small and metabolically more stable scaffold while maintaining bioactivity. Here we describe a scanning approach using a library of pyranose-based peptidomimetics that is structurally diverse in a systematic manner, designed to cover all possible conformations of tripeptide motifs containing two aromatic groups and one positive charge. Structural diversity was achieved by efficient selection of various chemoforms, characterized by a choice of pyranose scaffold of defined chirality and substitution pattern. A systematic scanning library of 490 compounds was thus designed, produced, and screened in vitro for activity at the somatostatin (sst(1-5)) and melanin-concentrating hormone (MCH(1)) receptors. Bioactive compounds were found for each target, with specific chemoform preferences identified in each case, which can be used to guide follow-on drug discovery projects without the need for scaffold hopping.
Asunto(s)
Monosacáridos/química , Oligopéptidos/química , Aminoácidos/química , Animales , Unión Competitiva , Células CHO , Cricetinae , Cricetulus , Bases de Datos Factuales , Humanos , Modelos Moleculares , Conformación Molecular , Imitación Molecular , Monosacáridos/farmacología , Oligopéptidos/farmacología , Ensayo de Unión Radioligante , Receptores de Somatostatina/antagonistas & inhibidores , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
In search of new oligodeoxynucleotide (ODN) delivery agents, we evaluated novel peptides derived from core peptide H-GLRILLLKV-OH (CP). CP is a fragment designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence. CP was able to enter cells including T-cells and inhibited interleukin-2 (IL-2) production. To examine the effect of increased lipophilicity on cellular uptake and activity of CP, a lipoamino acid (2-aminododecanoic acid) was incorporated into peptide CP resulting in 2-aminodecanoyl-CP (LP). The toxicity of CP and LP was assessed by measuring the haemolytic activity. Neither compound caused any haemolysis of red blood cells. We have also compared the biological activities of the CP and LP. Using a T-cell antigen presentation assay, the more lipophilic LP caused greater inhibition of IL-2 production than the parent CP in the antigen stimulated T-cells. The LP also showed increased permeability than CP in the Caco-2 cell assay. We utilised the enhanced cell permeability property of LP in oligodeoxynucleotide ODN1 delivery. Isothermal titration calorimetry (ITC) suggested that CP and LP complex with ODN1 in a 12:1 (CP:ODN1) and 15:1 (LP:ODN1) ratio. These complexes were then transfected into human retinal pigment epithelial cells. The level of transfection was measured by the decreased production of the protein human vascular endothelial growth factor (hVEGF). The results revealed greater transfection efficiency for both CP and LP (47%, 55% more inhibition) compared to commercially available transfection agent cytofectin GSV. These results suggested that the CP and particularly its lipophilic analogue LP have the potential to be used as oligodeoxynucleotide delivery systems.
Asunto(s)
Oligonucleótidos/administración & dosificación , Péptidos/administración & dosificación , Secuencia de Bases , Células CACO-2 , Calorimetría , Cationes , Hemólisis/efectos de los fármacos , Humanos , Interleucina-2/biosíntesis , Péptidos/química , Péptidos/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Synthesis of novel polycationic lipophilic peptide core(s) was accomplished and these agents successfully transfected human retinal pigment epithelium cells with ODN1 upon complexation with the oligonucleotide. The level of transfection was indirectly measured by the decreased production of the protein hVEGF (human vascular endothelial growth factor) in comparison to the transfection agent cytofectin GSV.