RESUMEN
Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96â h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology inâ vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.
Asunto(s)
Antineoplásicos/farmacología , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Proteolisis/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Benzamidas/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Dipéptidos/química , Dipéptidos/farmacocinética , Dipéptidos/farmacología , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Ratones , Estructura Molecular , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chlâ¯d, Chlâ¯f or bacteriochlorophyll (BChl) b to replace native BChlâ¯a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg-10 of the LH2 ß polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6â¯ps, energy transfer from Chlâ¯a to B850 BChlâ¯a remained highly efficient. We measured faster energy-transfer time constants for Chlâ¯d (3.5â¯ps) and Chlâ¯f (2.7â¯ps), which have red-shifted absorption maxima compared to Chlâ¯a. BChlâ¯b, red-shifted from the native BChlâ¯a, gave extremely rapid (≤0.1â¯ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range.