RESUMEN
Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína SOS1/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Mutación/genética , Unión Proteica/efectos de los fármacos , Conformación Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína SOS1/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Two series of inhibitors of type III phosphatidylinositol-4-kinase were identified by high throughput screening and optimised to derive probe compounds that independently and selectively inhibit the α- and the ß-isoforms with no significant activity towards related kinases in the pathway. In a cellular environment, inhibition of the α- but not the ß-subtype led to a reduction in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate concentration, causing inhibition of inositol-1-phosphate formation and inhibition of proliferation in a panel of cancer cell lines.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Fosfatos de Inositol/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Proliferación Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Fosfatos de Inositol/metabolismo , Modelos Moleculares , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The oxidation of a range of cyclic allylic alcohols and amides with OsO4/TMEDA is presented. Under these conditions, hydrogen bonding control leads to the (contrasteric) formation of the syn isomer in almost every example that was examined. Evidence for the bidentate binding of TMEDA to OsO4 is presented and a plausible mechanism described.
