RESUMEN
Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.
Asunto(s)
Sitio Alostérico/efectos de los fármacos , Antineoplásicos/farmacología , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Repetición de Anquirina , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Histidina/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/patología , Biblioteca de Péptidos , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/química , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Quinolonas/química , Quinolonas/farmacologíaRESUMEN
SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.