RESUMEN
Using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) we have established a general stereoselective detection and screening method of intact racemates which can generate binding affinity information about the individual enantiomers that is also applicable to other ligand isomeric mixtures. FAC-MS has been shown to be a versatile technology utilizing direct binding in screening assays and extending its application toward chiral drug development, especially in the early discovery stages as well as its utility in secondary Structure-activity relationship (SAR) studies allow this platform to make a significant step toward facilitating the demand for pure enantiomeric drugs. Using renin, which is as an important drug target, we show that for detection and screening purposes there is no need to first use timely and costly methods of separating racemates in order to get precise information about the binding affinities of the composite enantiomers.
Asunto(s)
Cromatografía de Afinidad/métodos , Evaluación Preclínica de Medicamentos/métodos , Sustancias Macromoleculares/metabolismo , Espectrometría de Masas/métodos , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Ligandos , Sustancias Macromoleculares/química , Renina/química , Renina/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
High-throughput screening (HTS) efforts to discover "hits" typically rely on the large-scale parallel screening of individual compounds with attempts to screen mixtures of compounds typically and, unfortunately, giving rise to false positives and false negatives due to the nature of the HTS readout (% inhibition/activation above a defined threshold) that makes deconvolution virtually intractable. Bioaffinity screening methods have emerged as an alternative or orthogonal method to classic HTS. One of these methods, frontal affinity chromatography coupled to mass spectrometry detection (FAC-MS), although still a relatively new technique, is turning out to be a viable screening tool. However, to push FAC-MS more to the forefront as a moderate primary HTS system (or a secondary screening assay), automation needs to be addressed. An automated FAC-MS system is described using 2 columns containing immobilized hERbeta, whereby while 1 column is being regenerated, the other is being used. The authors are extrapolating that in a continuous 24-h operation, the number of ligands screened could potentially approach 10,000. In addition, preliminary structure-activity relationship binding information (typically not seen in early primary HTS) can be obtained by observing the rank order of the library members in the various mixtures.
Asunto(s)
Cromatografía de Afinidad/métodos , Receptor beta de Estrógeno/metabolismo , Espectrometría de Masas/métodos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Automatización , Cromatografía de Afinidad/instrumentación , Receptor beta de Estrógeno/química , Receptor beta de Estrógeno/genética , Histidina/química , Humanos , Ligandos , Espectrometría de Masas/instrumentación , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Serina/metabolismoRESUMEN
The emergence of a relatively new technique resulting from a combination of frontal affinity chromatography coupled with MS detection (FAC-MS) has extended the capabilities of MS in drug discovery and development. Its application in a broad range of biological systems, together with its label-free operation, relatively high throughput, ability to rank ligands and determine Kd, makes FAC-MS a universal tool enabling convenient and efficient screening in the identification of new potential drug leads. Here we will highlight FAC-MS screening studies and discuss where it can be applied in evaluating multiple protein-binding sites, protein-protein interactions and inactive proteins, and also in determining selectivity.
Asunto(s)
Cromatografía de Afinidad , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Espectrometría de Masas , Proteínas/química , Sitios de Unión , Ligandos , Modelos Moleculares , Conformación MolecularRESUMEN
We have integrated two complementary methods, high-throughput virtual screening with a "high-content" wet screening technique based on frontal affinity chromatography with mass spectrometry detection (FAC-MS), for identification of hits against the erythropoietin-producing hepatocellular B2 (EphB2) receptor tyrosine kinase domain. Both an EphB2-directed virtual screen combining docking and scoring and a kinase-directed pharmacophore search strategy were used to identify a compound set enriched in bioactive compounds against EphB2. The coupling of virtual screening methodologies with FAC-MS is a unique hybrid approach that can be used to increase the efficacy of both hit discovery and optimization efforts in drug discovery and has successfully identified hits, in particular 19a (36% shift, IC(50) = 5.2 microM, K(d) = 3.3 microM), as inhibitors for EphB2, a potential cancer target.
Asunto(s)
Antineoplásicos/química , Receptor EphB2/antagonistas & inhibidores , Receptor EphB2/química , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Cromatografía de Afinidad , Bases de Datos Factuales , Ensayo de Inmunoadsorción Enzimática , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Humanos , Espectrometría de Masas , Modelos Moleculares , Peso Molecular , Naftoquinonas/química , Naftoquinonas/farmacología , Fosforilación , Estructura Terciaria de Proteína , Relación Estructura-Actividad Cuantitativa , Receptor EphB2/metabolismo , Sulfuros/química , Sulfuros/farmacologíaRESUMEN
Utilizing frontal affinity chromatography with mass spectrometry detection (FAC-MS), we have identified novel applications in the discovery of small-molecule hits to protein targets that are difficult if not impossible to accomplish using traditional assays. We demonstrate for the first time an ability to distinguish between competitive ligands for the ATP and substrate sites of protein kinase C independently in the same experiment and show that ATP competitive ligands using a functionally inactive receptor tyrosine kinase can be identified. This ability of FAC-MS to simultaneously monitor binding at the ATP and substrate binding sites, as well as measure ligand binding to both active and inactive kinases, suggests that FAC-MS can be used as a "global kinase binding assay".
Asunto(s)
Cromatografía de Afinidad/métodos , Evaluación Preclínica de Medicamentos/métodos , Espectrometría de Masas/métodos , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Alcaloides/química , Alcaloides/metabolismo , Animales , Benzofenantridinas/química , Benzofenantridinas/metabolismo , Sitios de Unión , Unión Competitiva , Dominio Catalítico , Humanos , Imidazoles/química , Imidazoles/metabolismo , Ratones , Estructura Molecular , Péptidos/química , Péptidos/metabolismo , Fosforilación , Unión Proteica , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-alfa/química , Proteína Quinasa C-alfa/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Piridinas/química , Piridinas/metabolismo , Quinazolinas/química , Quinazolinas/metabolismo , Receptor EphB2/antagonistas & inhibidores , Receptor EphB2/química , Receptor EphB2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
FAC-MS offers a convenient method for measuring the relative binding strengths of ligands in a mixture and enables a rapid ranking and identification of ligands in the mixture as potential hits against immobilized targets. Using immobilized EphB2 receptor tyrosine kinase as the target and known kinase inhibitors, the results of FAC-MS screening (% shift) have been shown to correlate with the binding constant, K(d), and with IC(50) results from the more traditional ELISA assay. Therefore, since FAC-MS can accommodate a wide variety of target proteins, its applications could play a broad role in drug discovery not only at the hit discovery stage but also during the subsequent more rigorous screening at the hit-to-lead and lead optimization stages.