RESUMEN
The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.
Asunto(s)
Monitoreo de Drogas/métodos , Calor , Terapia Molecular Dirigida , Preparaciones Farmacéuticas/metabolismo , Proteínas/metabolismo , Antimetabolitos Antineoplásicos/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Antagonistas del Ácido Fólico/metabolismo , Humanos , Riñón/metabolismo , Ligandos , Hígado/metabolismo , Unión Proteica , Estabilidad Proteica , Quinazolinas/metabolismo , Tiofenos/metabolismo , Distribución TisularRESUMEN
Tankyrases constitute potential drug targets for cancer and myelin-degrading diseases. We have applied a structure- and biophysics-driven fragment-based ligand design strategy to discover a novel family of potent inhibitors for human tankyrases. Biophysical screening based on a thermal shift assay identified highly efficient fragments binding in the nicotinamide-binding site, a local hot spot for fragment binding. Evolution of the fragment hit 4-methyl-1,2-dihydroquinolin-2-one (2) along its 7-vector yields dramatic affinity improvements in the first cycle of expansion. A crystal structure of 7-(2-fluorophenyl)-4-methylquinolin-2(1H)-one (11) reveals that the nonplanar compound extends with its fluorine atom into a pocket, which coincides with a region of the active site where structural differences are seen between tankyrases and other poly(ADP-ribose) polymerase (PARP) family members. A further cycle of optimization yielded compounds with affinities and IC50 values in the low nanomolar range and with good solubility, PARP selectivity, and ligand efficiency.
Asunto(s)
Quinolinas/síntesis química , Tanquirasas/antagonistas & inhibidores , Cristalografía por Rayos X , Bases de Datos Factuales , Estabilidad de Medicamentos , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Quinolinas/química , Solubilidad , Relación Estructura-Actividad , Tanquirasas/química , TermodinámicaRESUMEN
Lipid-linked sugar pyrophosphates, such as GlcNAc-pyrophosphoryl undecaprenol, are important intermediates in the biosynthesis of cell-surface bacterial polysaccharides. It was recently demonstrated that much simpler lipids could substitute for undecaprenol while retaining biological activity, thus making efficient synthetic access to this class of compounds highly desirable. In order to facilitate the synthesis of pure substrates for bacterial glycosyltransferases, we have developed a simple 'two-pot' synthesis which we demonstrate here for GlcNAc-alpha-pyrophosphoryl-decanol (4). GlcNAc pyrophosphate, produced by mild periodate oxidation/beta-elimination of commercial UDP-GlcNAc, is alkylated using 1-iododecane to yield the target compound 4 in 39% yield. Compound 4 is shown to be an efficient acceptor for a bacterial galactosyltransferase.
Asunto(s)
Ácidos Decanoicos/síntesis química , Escherichia coli/enzimología , Galactosiltransferasas/metabolismo , Cromatografía Líquida de Alta Presión , Ácidos Decanoicos/metabolismo , Espectrofotometría Ultravioleta , Especificidad por SustratoRESUMEN
We report an ultrasensitive method for the analysis of glycosphingolipid catabolism. The substrate G(M1) and the set of seven metabolites into which it can be degraded (G(A1), G(M2), G(A2), G(M3), LacCer, GlcCer, and Cer) were labeled with the highly fluorescent dye tetramethylrhodamine. CE with LIF detection was used to assay these compounds with 150 +/- 80 yoctomole mass (1 ymol = 10(-24) mol = 0.6 copies) detection limits and 5 +/- 3 pM concentration detection limits. An alignment algorithm based on migration of two components was employed to correct for drift in the separation. The within-day and between-day precision in peak height was 20%, in peak width 15%, and in adjusted migration time 0.03%. After normalization to total sample injected, the RSD in peak height reduced to 2-6%, which approaches the limit set by molecular shot noise in the number of molecules taken for analysis. PC12 cells were incubated with the labeled G(M1). Fluorescent microscopy demonstrated uptake by the cells. CE was used to separate a cellular homogenate prepared from these cells. A set of peaks was observed, which were tentatively identified based on comigration with the standards. Roughly 120 pL of homogenate was injected, which contained a total of 150 zmol of labeled substrate and products. Metabolite that preserves the fluorescent label can be detected at the yoctomole level, which should allow characterization of this metabolic pathway in single cells.
Asunto(s)
Gangliosidosis GM1/metabolismo , Animales , Electroforesis Capilar/métodos , Colorantes Fluorescentes/química , Células PC12 , Ratas , Rodaminas/químicaRESUMEN
Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The preparation involved acylation of the homogenous C(18)lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the N-hydroxysuccinimide ester of a beta-alanine-tethered 6-TMR derivative, followed by conversion of these labeled products using galactosidase, sialidase, and sialyltransferase enzymes. The TMR-glycolipid analogs produced are detectable on TLC down to the 1 ng level by the naked eye. All eight compounds could be separated within 4 min in capillary electrophoresis where they could be detected at the zeptomole (ca. 1000 molecule) level using LIF.
Asunto(s)
Colorantes Fluorescentes/química , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/metabolismo , Rodaminas/química , Animales , Bovinos , Electroforesis Capilar , Gangliósido G(M1)/aislamiento & purificación , Gangliósido G(M2)/química , Gangliósido G(M2)/metabolismo , Gangliósido G(M3)/aislamiento & purificación , Gangliósidos/aislamiento & purificación , Neuraminidasa/metabolismo , Estándares de ReferenciaRESUMEN
A tetra- and a pentasaccharide were synthesized as analogues to the structure of the Streptococcus pneumoniae type 37 capsular polysaccharide, a homopolymer with a disaccharide-repeating unit of -->3)[beta-D-Glcp-(1-->2)]-beta-D-Glcp-(1-->. Synthesis of the tetrasaccharide employed a beta-(1-->2)-diglycosylation of a beta-(1-->3)-linked disaccharide. Subsequently, the pentasaccharide was synthesized from a suitably protected tetrasaccharide derivative by a beta-(1-->3)-extension at O-3'. Steric crowding was found to be an important factor in the formation of the pentasaccharide.
Asunto(s)
Oligosacáridos/síntesis química , Polisacáridos Bacterianos/química , Streptococcus pneumoniae/química , Streptococcus pneumoniae/clasificación , Secuencia de Carbohidratos , Espectrometría de Masas , Datos de Secuencia Molecular , Oligosacáridos/químicaRESUMEN
The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 396/C-1 has been determined. Sugar and methylation analyses together with 1H and 13C NMR spectroscopy were the main methods used. Inter-residue correlations were determined by 1H,1H-NOESY, 1H,13C-heteronuclear multiple-bond correlation and dipole-dipole cross-correlated relaxation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. Analysis of NMR data reveals that on average the PS consists of approximately 13 repeating units and indicates that the biological repeating unit contains an N-acetylglucosamine residue at its reducing end. This structure is different to that reported for the O-antigen polysaccharide from E. coli O126. Monospecific anti-E. coli O126 rabbit serum from The International Escherichia and Klebsiella Centre did not distinguish between the E. coli strain 396/C-1 and the E. coli O126 reference strain, neither in slide agglutination nor in an indirect enzyme immunoassay. Subsequent successful serotyping of the E. coli strain 396/C-1 showed it to be E. coli O126:K+:H27.
Asunto(s)
Escherichia coli/química , Escherichia coli/clasificación , Antígenos O/química , Antígenos O/inmunología , Secuencia de Carbohidratos , Escherichia coli/inmunología , Espectroscopía de Resonancia Magnética , Datos de Secuencia MolecularRESUMEN
[carbohydrate structure: see text] Hydrogen bond mediated NMR J couplings offer additional structural information. The interpretation of these usually small (h)J couplings are, however, not necessarily straightforward. In the present case of a carbohydrate system, a four-bond classical W coupling, (4)J(HO4,H5), is more reasonable on the basis of, in particular, density functional theory calculations of spin-spin coupling constants at the UB3LYP/6-311G** level of theory.