RESUMO
Activating mutations in RAS are found in approximately 30% of human cancers, resulting in the delivery of a persistent signal to critical downstream effectors that drive tumorigenesis. RAS-driven malignancies respond poorly to conventional cancer treatments and inhibitors that target RAS directly are limited; therefore, the identification of new strategies and/or drugs to disrupt RAS signaling in tumor cells remains a pressing therapeutic need. Taking advantage of the live-cell bioluminescence resonance energy transfer (BRET) methodology, we describe the development of a NanoBRET screening platform to identify compounds that modulate binding between activated KRAS and the CRAF kinase, an essential effector of RAS that initiates ERK cascade signaling. Using this strategy, libraries containing synthetic compounds, targeted inhibitors, purified natural products, and natural product extracts were evaluated. These efforts resulted in the identification of compounds that inhibit RAS/RAF binding and in turn suppress RAS-driven ERK activation, but also compounds that have the deleterious effect of enhancing the interaction to upregulate pathway signaling. Among the inhibitor hits identified, the majority were compounds derived from natural products, including ones reported to alter KRAS nanoclustering (ophiobolin A), to impact RAF function (HSP90 inhibitors and ROS inducers) as well as some with unknown targets and activities. These findings demonstrate the potential for this screening platform in natural product drug discovery and in the development of new therapeutic agents to target dysregulated RAS signaling in human disease states such as cancer.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Fibroblastos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas ras/agonistas , Proteínas ras/antagonistas & inibidores , Animais , Fibroblastos/metabolismo , Humanos , Ligantes , Nanotecnologia/métodos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismoRESUMO
The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.
Assuntos
Sítio Alostérico , Compostos Benzidrílicos/química , Disruptores Endócrinos/química , Fenóis/química , Proteínas ras/química , Regulação Alostérica , Compostos Benzidrílicos/farmacologia , Disruptores Endócrinos/farmacologia , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Fenóis/farmacologia , Ligação Proteica , Proteínas ras/agonistas , Proteínas ras/metabolismoRESUMO
The proliferative responses of a human CD4+ T cell clone 29.15.2, reactive with a self-K-ras-derived peptide (3EYKLVVVGAGGVGKSALT20), were tested using a set of X9 combinatorial peptide libraries containing the flanking residues (EYKLVXXXXXXXXXSALT, where X indicates random amino acids). Certain peptide libraries, such as EYKLVXXXXXX