RESUMEN
Affinity technologies have been applied at several stages of the drug discovery process, ranging from target identification and purification to the identification of preclinical candidates. The detection of ligand-macromolecule interactions in lead discovery is the best studied and most powerful of these techniques. Although affinity methods have been in widespread use for about a decade, only recently have many reports emerged on their utility. Primary affinity screens of large libraries of small molecules or fragments have begun to produce results for challenging targets. Furthermore, in secondary assays affinity methods are opening new avenues to tackle important medicinal chemistry tasks.
Asunto(s)
Bioensayo , Técnicas Químicas Combinatorias , Diseño de Fármacos , Animales , Bioensayo/métodos , Bioensayo/tendencias , Técnicas Químicas Combinatorias/métodos , Técnicas Químicas Combinatorias/tendencias , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/tendencias , Humanos , Ligandos , Relación Estructura-ActividadRESUMEN
The application of parallel synthesis is an efficient approach to explore the chemical space and to rapidly develop meaningful structure activity relationship (SAR) data for drug discovery programs. However, the effectiveness of the parallel synthesis requires a high throughput purification workflow that can process a large number of crude samples within a meaningful time frame. This paper describes a high throughput purification platform that has been adopted at Merck's Rahway research site. The platform includes the evaluation of crude samples, mass-directed HPLC purification, fraction analysis, compound registration, final compound purity assessment and assay distribution. Assisting with the sample tracking and the data management is the internally designed laboratory information management system, Light Automation Framework (LAF). Using this process and the tools described herein, the group has successfully achieved purities of 95% or greater for 90% of samples.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Preparaciones Farmacéuticas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Descubrimiento de Drogas/instrumentación , Diseño de Equipo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Preparaciones Farmacéuticas/síntesis química , Preparaciones Farmacéuticas/química , Control de Calidad , Relación Estructura-ActividadRESUMEN
The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.
Asunto(s)
Anemia/tratamiento farmacológico , Compuestos Aza/síntesis química , Hidantoínas/síntesis química , Factor 1 Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Compuestos de Espiro/síntesis química , Animales , Compuestos Aza/farmacocinética , Compuestos Aza/farmacología , Perros , Canal de Potasio ERG1 , Eritropoyetina/biosíntesis , Canales de Potasio Éter-A-Go-Go/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidantoínas/farmacocinética , Hidantoínas/farmacología , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Indoles/síntesis química , Indoles/farmacocinética , Indoles/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Macaca mulatta , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Ratas , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/farmacología , Relación Estructura-Actividad , Regulación hacia ArribaRESUMEN
To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the "straight" parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the "bent" structural isomer (parnafungin B).
Asunto(s)
Oxazolidinonas/química , Oxazolidinonas/metabolismo , Polinucleotido Adenililtransferasa/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Cromatografía Liquida , Hongos/enzimología , Humanos , Isomerismo , Ligandos , Espectrometría de Masas , Oxazolidinonas/análisisRESUMEN
Affinity-based selection strategies have recently emerged as a complement to traditional high throughput screening for the rapid discovery of lead compounds for the large number of protein targets emerging from--omics technologies. Herein, we describe a method for the ranking of mixtures of ligands by affinity selection and apply it to rank order a set of inhibitors for the enzyme dipeptidyl peptidase IV.