Abstract
Purpose: :
To develop a peptide ligand for an oncoprotein found in uveal melanoma. Recently, oncogenic mutations were identified in more than 80% of uveal melanoma in the genes GNAQ or GNA11, encoding heterotrimeric G-protein α subunits Gαq and Gα11, respectively. These mutations "lock" the proteins in the active, GTP-bound conformation, causing constitutive mitogenic signaling. Selective blockade ofthe function of these oncoproteins offers an appealing route to inhibition of proliferation in uveal melanoma, a chemoresistant cancer currently lacking targeted therapies.
Methods: :
Phage display was used to find peptide ligands selective for the common Q209L mutant of human Gαq. Recombinant wild type and Q209L Gαq-glutathione S-transferase fusions were expressed in E. coli, and folding of the wild type protein was confirmed by binding of BODIPY-GTPγS. Recombinant proteins were used for phage display panning using a 12-mer M13 phage library, and binding confirmed by anti-M13enzyme-linked immunosorbent assays.
Results: :
Multiple phage clones were isolated that showed preferential binding to Gαq Q209L over wild-type protein, while several clones bound preferentially to wild-type Gαq. Sequencing and consensus peptide identification is underway, to be followed by validation with synthetic peptides.
Conclusions: :
We have identified peptide-expressing phage that bind Gαq Q209L. Validated, selective peptide ligands for Gαq Q209L can be used in fluorescence polarization displacement assays to find small-molecule ligands with similar binding modes, tested for cytotoxicity in GNAQ mutant cells, and/or linked to signals for targeted protein degradation. Selective targeting of oncogenic Gαq Q209L will open an importantnew avenue for development of novel therapies for uveal melanoma.
Keywords: melanoma • mutations • proteins encoded by disease genes