RESUMEN
In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.
Asunto(s)
Péptido Hidrolasas/química , Talidomida/química , Ubiquitina-Proteína Ligasas/química , Proteínas Adaptadoras Transductoras de Señales , Cristalografía por Rayos X , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Lenalidomida , Modelos Moleculares , Complejos Multiproteicos/agonistas , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/metabolismo , Unión Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Talidomida/análogos & derivados , Talidomida/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Both the tricyclic and specific serotonin reuptake inhibitor classes of antidepressants act primarily by inhibiting the reuptake of released serotonin by the human serotonin reuptake transporter (hSERT). In this article, the authors describe the use of a fluorescent substrate of the transporter (4-(4-(dimethylamino)-styrl)-N-methylpyridinium, ASP) to develop a microplate-based high-throughput screen for hSERT function. The assay is sensitive to known inhibitors of serotonin uptake, including fluoxetine (Prozac), with the correct rank order of potency and IC(50) values close to those reported in the literature for tritiated serotonin uptake. The authors also describe the validation of the assay for natural product screening using a test set of 2400 pure phyto-chemicals and 80 plant extracts. The mean Z of the screened plates was 0.53. Hit rates, confirmation rates, and validation of the hits in a "classical" assay for serotonin uptake are all reported. The assay can also be read in "high-content" mode using a subcellular imaging device, which allows direct detection of possible assay interference by acutely cytotoxic compounds. Among the compounds identified were several previously reported inhibitors of the hSERT, as well as compounds having structural similarity to the tricyclic antidepressant drugs.