RESUMEN
We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.
Asunto(s)
Diseño de Fármacos , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacocinética , Péptidos/metabolismo , Péptidos/farmacocinética , Albúmina Sérica/metabolismo , Secuencia de Aminoácidos , Animales , Disponibilidad Biológica , Glucemia/metabolismo , Ácidos Grasos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Masculino , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Unión Proteica , Especificidad por SustratoRESUMEN
Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst2 and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst2 in the nM range with best results obtained with peptide 2, that is, IC50 = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC50 = 60 nM.
Asunto(s)
Compuestos Azo/química , Cistina/química , Disulfuros/química , Péptido Natriurético Encefálico/análogos & derivados , Somatostatina/análogos & derivados , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Simulación de Dinámica Molecular , Péptido Natriurético Encefálico/síntesis química , Péptido Natriurético Encefálico/metabolismo , Unión Proteica , Ratas , Somatostatina/síntesis química , Somatostatina/metabolismoRESUMEN
We synthesized two carminic acid (7-alpha-d-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracene carboxlic acid, CA)-GnRH conjugates to be used as a model for potential photoactive targeted compounds. CA was conjugated to the epsilon-amino group of [d-Lys(6)]GnRH through its carboxylic moiety or via a beta-alanine spacer (beta-ala). Redox potentials of CA and its conjugates were determined. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of CA and its CA-GnRH conjugates. Upon irradiation, the compounds stimulated the formation of reactive oxygen species (ROS), that is, singlet oxygen ((1)O(2)) and oxygen radicals (O(2)(-*) and OH(*)). Both conjugates exhibited higher ROS production than the non-conjugated CA. The bioactivity properties of the CA conjugates and the parent peptide, [d-Lys(6)]GnRH, were tested on primary rat pituitary cells. We found that the conjugates preserved the bioactivity of GnRH as illustrated by their capability to induce ERK phosphorylation and LH release.
Asunto(s)
Carmín/análogos & derivados , Hormona Liberadora de Gonadotropina/química , Hipófisis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Carmín/química , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Radicales Libres , Hormona Luteinizante/metabolismo , Oxidación-Reducción , Fosforilación , Fotoquímica , Hipófisis/citología , Hipófisis/metabolismo , Ratas , Oxígeno SingleteRESUMEN
Several important pharmacological features can be integrated into injected drugs to enhance their therapeutic efficacy following administration. Short-lived peptide/protein drugs should be converted into long-lived species in vivo to avoid multiple injections. Circulating levels of anticancer agents need to be maintained within a narrow therapeutic range for prolonged period. Water-insoluble drugs must be turned into soluble species and blood-brain barrier-impermeable agents need to be modified to cross it following peripheral administrations. The derivatization requiring for achieving those desirable pharmacological features typically result in biologically/pharmacologically inactive products, unless those derivatizations can be carried out in a reversible fashion.
Asunto(s)
Péptidos/administración & dosificación , Proteínas/administración & dosificación , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Péptidos/química , Proteínas/químicaRESUMEN
In an attempt to develop efficient chemotherapeutic agents targeted at malignant cells that express receptors, we synthesized five new emodin derivatives and their gonadotropin-releasing hormone (GnRH) conjugates to be used as potential photoactive conjugates. Emodin was modified at its hydroxy groups and included different spacers for conjugation of the peptide. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of the emodin derivatives and their GnRH conjugates. Upon irradiation, all new emodin derivatives and their conjugates stimulated the formation of singlet oxygen, that is, (1)O(2), and oxygen radicals, that is, O(2)(-)(*) and OH(*). However, substantial differences were found between the tested derivatives as to the efficacy of reactive oxygen species (ROS) production. Because of its superior ROS production properties, [d-Lys(6)(MeoEmo)]GnRH was selected as a leading conjugate. En-route to evaluate its targeting capacity, this potentially cytotoxic conjugate was tested in vitro to determine its hormonal activity and binding affinity to GnRH receptors.