RESUMO
Ultrafiltration provides a generic method to discover ligands for protein drug targets with millimolar to micromolar K(d), the typical range of fragment-based drug discovery. This method was tailored to a 96-well format, and cocktails of fragment-sized molecules, with molecular masses between 150 and 300 Da, were screened against medical structural genomics target proteins. The validity of the method was confirmed through competitive binding assays in the presence of ligands known to bind the target proteins.
Assuntos
Descoberta de Drogas/métodos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ultrafiltração/métodos , Ligação Competitiva , Escherichia coli/metabolismo , Ligantes , Plasmodium yoelii/metabolismo , Ligação Proteica , Trypanosoma brucei brucei/metabolismoRESUMO
The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 A using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed alpha-helical region at one side of the canonical phospholipid-binding site. Re-examination of previously determined PEBP structures suggests that the P. vivax protein and yeast carboxypeptidase Y inhibitor may represent a structurally distinct subfamily of the diverse PEBP-sequence family.
Assuntos
Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Plasmodium vivax/química , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Dados de Sequência Molecular , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Plasmodium vivax/metabolismo , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Secundária de Proteína/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/fisiologiaRESUMO
The history of fragment-based drug discovery, with an emphasis on crystallographic methods, is sketched, illuminating various contributions, including our own, which preceded the industrial development of the method. Subsequently, the creation of the BMSC fragment cocktails library is described. The BMSC collection currently comprises 68 cocktails of 10 compounds that are shape-wise diverse. The utility of these cocktails for initiating lead discovery in structure-based drug design has been explored by soaking numerous protein crystals obtained by our MSGPP (Medical Structural Genomics of Pathogenic Protozoa) consortium. Details of the fragment selection and cocktail design procedures, as well as examples of the successes obtained are given. The BMSC Fragment Cocktail recipes are available free of charge and are in use in over 20 academic labs.