RESUMEN
Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteínas Quinasas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular , Línea Celular Tumoral , ADN Nucleotidiltransferasas/genética , Descubrimiento de Drogas , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Genes Reporteros , Humanos , Luciferasas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Fosforilación/efectos de los fármacos , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Estaurosporina/análogos & derivados , Estaurosporina/farmacologíaRESUMEN
The experimenters examine upper limb movement discrimination performance in an arm-raising task for bilateral associations of low-level unilateral performance. On a cue from the experimenter, young adults (n = 23) with no history of shoulder injury raised either their left arm, right arm, or both together in a forward flexion movement until their hand or hands contacted an unseen, adjustable, overhead stop. The participant then judged which of the 5 possible stop positions, in the 12-20 degrees range forward of true vertical, the participant had contacted on the particular trial. Results showed that for the 16 participants whose best performance was in 1 of the unimanual conditions discrimination scores in the bilateral condition were equivalent to those of their worse-performing limb. For the 7 participants whose best discrimination performance was obtained on bimanual arm-raising, scores for the 2 unimanual conditions were equivalently low. Therefore, when a single limb that can perform well operates in conjunction with a limb performing at a lower level, the consequence is lowering the bimanual movement discrimination performance.