TY - JOUR
T1 - Agents that stabilize mutated von Hippel-Lindau (VHL) protein
T2 - Results of a high-throughput screen to identify compounds that modulate VHL proteostasis
AU - Ding, Zhiyong
AU - German, Peter
AU - Bai, Shanshan
AU - Feng, Zhehui
AU - Gao, Meng
AU - Si, Wendy
AU - Sobieski, Mary M.
AU - Stephan, Clifford C.
AU - Mills, Gordon B.
AU - Jonasch, Eric
N1 - Funding Information:
This research is supported by a grant from NIH through the Common Fund (PN1EY016525).
PY - 2012/6
Y1 - 2012/6
N2 - Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.
AB - Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.
KW - Prestwick
KW - VHL upregulation
KW - high-throughput screen
KW - proteostasis
UR - http://www.scopus.com/inward/record.url?scp=84861818456&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84861818456&partnerID=8YFLogxK
U2 - 10.1177/1087057112436557
DO - 10.1177/1087057112436557
M3 - Article
C2 - 22357874
AN - SCOPUS:84861818456
SN - 1087-0571
VL - 17
SP - 572
EP - 580
JO - Journal of Biomolecular Screening
JF - Journal of Biomolecular Screening
IS - 5
ER -