RESUMEN
To discover safe and effective topical skin-lightening agents, we have evaluated alkyl esters of the natural product gentisic acid (GA), which is related to our lead compound methyl gentisate (MG), and four putative tyrosinase inhibitors, utilizing mammalian melanocyte cell cultures and cell-free extracts. Desirable characteristics include the ability to inhibit melanogenesis in cells (IC50 < 100 microg/mL) without cytotoxicity, preferably due to tyrosinase inhibition. Of the six esters synthesized, the smaller esters (e.g. methyl and ethyl) were more effective enzyme inhibitors (IC50 approximately 11 and 20 microg/mL, respectively). For comparison, hydroquinone (HQ), a commercial skin "bleaching" agent, was a less effective enzyme inhibitor (IC50 approximately 72 microg/mL), and was highly cytotoxic to melanocytes in vitro at concentrations substantially lower than the IC50 for enzymatic inhibition. Kojic acid was a potent inhibitor of the mammalian enzyme (IC50 approximately 6 microg/mL), but did not reduce pigmentation in cells. Both arbutin and magnesium ascorbyl phosphate were ineffective in the cell-free and cell-based assays. MG at 100 microg/mL exhibited a minimal inhibitory effect on DHICA oxidase (TRP 1) and no effect on DOPAchrome tautomerase (TRP-2), suggesting that MG inhibits melanogenesis primarily via tyrosinase inhibition. MG and GA were non-mutagenic at the hprt locus in V79 Chinese hamster cells, whereas HQ was highly mutagenic and cytotoxic. The properties of MG in vitro, including (1) pigmentation inhibition in melanocytes, (2) tyrosinase inhibition and selectivity, (3) reduced cytotoxicity relative to HQ, and (4) lack of mutagenic potential in mammalian cells, establish MG as a superior candidate skin-lightening agent.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ésteres/farmacología , Gentisatos , Hidroxibenzoatos/farmacología , Melanocitos/efectos de los fármacos , Glicoproteínas de Membrana , Monofenol Monooxigenasa/antagonistas & inhibidores , Oxidorreductasas , Animales , Línea Celular , Ésteres/síntesis química , Hidroxibenzoatos/toxicidad , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Melaninas/análisis , Melanocitos/enzimología , Ratones , Pruebas de Mutagenicidad , Proteínas/antagonistas & inhibidores , Pigmentación de la Piel/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We report a theoretical characterization of the intermolecular transferred NOESY (inter-TrNOESY) between ligands and receptor macromolecules that bind reversibly, using a COmplete Relaxation and Conformational Exchange MAtrix (CORCEMA) theory developed in our laboratory. We examine the dependence of inter-TrNOESY on the dissociation constant, off-rate, ligand-to-receptor ratio, and distance variations between protons of interacting species within the complex. These factors are analyzed from simulations on two model systems: (i) neuraminidase complexed to a transition-state analogue; and (ii) thermolysin complexed to a leucine-based inhibitor. The latter case utilizes a three-state model of interaction to simulate the effect of hinge-bending motions on the inter-TrNOESY. Our calculations suggest a potential role for inter-TrNOESY (when observable) and CORCEMA analysis in properly docking the ligand within the active site, and in refining the conformation of the ligand-receptor (active-site) complex. These findings have implications on the structure-based design of ligands (e.g., inhibitors) reversibly binding to receptors (e.g., enzymes).
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Modelos Químicos , Receptores de Superficie Celular/química , Programas Informáticos , Sitios de Unión , Simulación por Computador , Diseño Asistido por Computadora , Diseño de Fármacos , Ligandos , Sustancias Macromoleculares , Estructura Molecular , Neuraminidasa/química , Protones , Termolisina/antagonistas & inhibidores , Termolisina/químicaRESUMEN
We present complete 1H NMR assignments for two synthetic glycopeptides representative of the carbohydrate-protein linkage region of serglycin proteoglycans. The peptides are: Ser(Galp-Xylp)-Gly-Ser-Gly-Ser(Galp-Xylp)-Gly and, Ser(Galp-Xylp)-Gly-Ser(Galp-Xylp)-Gly-Ser(Galp-Xylp)-G ly. A number of 2D NMR spectra together with a 3D NOESY-TOCSY spectrum were acquired at 600 MHz to complete the assignments of the glycopeptides dissolved in water with 40% trifluoroethanol. Preliminary analysis of the NMR data suggests folded structures for the glycopeptides.
Asunto(s)
Carbohidratos/química , Glicopéptidos/química , Proteínas/química , Proteoglicanos/química , Espectroscopía de Resonancia Magnética , Estructura Secundaria de Proteína , Protones , Proteínas de Transporte VesicularRESUMEN
A very general procedure entitled complete relaxation and conformational exchange matrix (CORCEMA) analysis has been developed to analyze the 2D-NOESY spectra of interacting systems undergoing multistate conformational exchange. This is an extension of earlier work from this laboratory on the methodological treatment of multistate conformational exchange [Krishna et al., Biopolymers 19, 2003 (1980)] and the theory of transferred NOESY for finite exchange off-rates [Lee and Krishna, J. Magn. Reson. 98, 36 (1992)]. The current theory is based on generalized rate matrices for relaxation and conformational exchange. The CORCEMA algorithm explicitly incorporates intermolecular dipolar cross relaxation between the molecules when they are complexed. It permits an analysis of NOESY intensities for the intra- as well as intermolecular contacts between the interacting molecules under a variety of binding conditions. Its application is illustrated on two examples of transferred NOESY simulations: (1) a two-state system involving a ligand and an enzyme forming a ligand-enzyme complex, and (2) a three-state system in which the ligand-enzyme complex can undergo a conformational transition from an "open state" to a "closed state," and can include conformational changes in both the complexed ligand and the complexed enzyme, such as hinge-bending motions. Simplifying expressions for generalized matrix analyses are derived for three limiting cases of the three-state system. This three-state example is illustrated using a hypothetical model of the hinge-bending motion in a thermolysin-inhibitor complex. It is shown that: (1) The neglect of cross relaxation between the interacting species in their complexed forms can lead to misleading conclusions on the "bound" conformation of the ligand. (2) If protein-mediated spin diffusion is dominant, caution is needed in analyses based on initial slopes alone due to one's inability to identify the exact range of the initial growth curve under poor signal/noise situations. (3) The neglect of conformational changes upon complexation, e.g., hinge-bending motions of the ligand-enzyme complex, can lead to erroneous results on the nature of "bound" conformations of the ligand. In this case, attempts to analyze the transferred NOESY data with a two-state model will result in a "virtual" conformation for the bound ligand. (4) When the hinge-bending rate is slower than the cross relaxation and enzyme off-rates, the bound conformation of a ligand deduced from the transferred NOESY experiment is more likely to represent nonspecific or weak binding in an open state of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Espectroscopía de Resonancia Magnética , Conformación Proteica , Proteínas/química , Algoritmos , Artefactos , Quelantes/química , Enzimas/metabolismo , Aumento de la Imagen/métodos , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Modelos Químicos , Unión Proteica , Procesamiento de Señales Asistido por Computador , Termolisina/antagonistas & inhibidores , Termolisina/metabolismoRESUMEN
From the original observation that the codons for the hydrophobic and hydrophilic amino acids on one strand of the DNA may be complemented by the codons for the hydrophilic and hydrophobic amino acids, respectively, on the complementary strand, arose the molecular recognition theory which forms the basis for much of the work involving complementary peptides. A number of examples have been documented where peptides with inverted hydropathic profiles have been shown to form complexes in high-affinity chromatography and solid matrix binding assays. Nevertheless, our current understanding of the molecular forces leading to the formation of these complexes is rather rudimentary, and it is highly desirable to have a detailed three-dimensional structure of a complex of interacting complementary peptides. In this article, we provide a brief review of the solution NMR work done by different laboratories in an attempt to study these interactions.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Péptidos/análisis , Proteínas/química , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Péptidos/metabolismoRESUMEN
Ovine interferon tau (IFN tau) is a type I interferon that was originally identified as ovine trophoblast protein and is associated with the maternal recognition of pregnancy in sheep. Additionally, IFN tau possesses potent antiviral and antiproliferative activity without the corresponding toxicity found in known IFN alpha s. Structure-function studies with synthetic peptides have identified three discontinuous functional sites on the protein that are involved in receptor interaction and biological activity. However, the structural relationship of these regions is unknown. Therefore, a model relationship of these regions is unknown. Therefore, a model of the 3-D structure of IFN tau would be useful in interpretation of existing data and the design of future structure-function studies. Combining information from circular dichroism (CD) of both the full length recombinant IFN tau and synthetic peptides representing regions of the IFN tau molecule, with sequence homology of IFN tau to IFN beta, a protein of known 3-D structure, we have constructed a model of IFN tau using distance geometry and energy minimization methods. The most striking feature of this model is that functionally active domains of IFN tau, discontinuous in the primary structure, are localized to one side of the molecule and found to be spatially contiguous. This observation is consistent with multiple binding sites on IFN tau interacting simultaneously with the IFN tau receptor.
Asunto(s)
Interferón Tipo I/química , Modelos Moleculares , Proteínas Gestacionales/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Cristalografía por Rayos X , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ovinos/metabolismo , Especificidad de la EspecieRESUMEN
The solution structure of trypsin modulating oostatic factor (TMOF), a decapeptide (H-YDPAPPPPPP-OH) hormone that signals the termination of trypsin-like biosynthesis in mosquito midgut epithelial cells, was determined by 2-D 1H nuclear magnetic resonance spectroscopy and molecular modeling. The peptide forms a rod-shaped left-handed helix about 30 A long. No evidence was found to support a poly-L-proline beta-turn model. Hydrophobic contacts between the rings of tyrosine 1 and proline 3 may enhance the stability of the N-terminal segment. This peptide provides an interesting exception to the normal chemical shift index (csi) rules. Our results suggest that a sequence of positive csi indices, normally expected for a beta-strand structure, could also describe a left-handed poly-L-proline-like helix.