SHetA2 is a small molecule flexible heteroarotinoid (Flex-Het) with promising
cancer prevention and therapeutic activity. Extensive preclinical testing documented lack of
SHetA2 toxicity at doses 25 to 150 fold above effective doses. Knowledge of the
SHetA2 molecular target(s) that mediate(s) the mechanism of
SHetA2 action is critical to appropriate design of clinical trials and improved analogs. The aim of this study was to develop a method to identify
SHetA2 binding proteins in
cancer cells. A known metabolite of
SHetA2 that has a
hydroxyl group available for attachment was synthesized and conjugated to a linker for attachment to a magnetic
microsphere. SHetA2-conjugated magnetic
microspheres and unconjugated magnetic
microspheres were separately incubated with aliquots of a whole cell
protein extract from the A2780 human
ovarian cancer cell line. After washing away non-specifically bound
proteins with the
protein extraction
buffer, SHetA2-binding
proteins were eluted with an excess of free
SHetA2. In two independent experiments, an SDS gel band of about 72 kDa was present at differential levels in wells of eluent from SHetA2-microspheres in comparison to wells of eluent from unconjugated
microspheres. Mass spectrometry analysis of the bands (QStar) and straight eluents (Orbitrap) identified
mortalin (HSPA9) to be present in the eluent from SHetA2-microspheres and not in eluent from unconjugated
microspheres. Co-immunoprecipitation experiments demonstrated that
SHetA2 interfered with
mortalin binding to p53 and p66 Src homologous-
collagen homologue (p66shc) inside
cancer cells.
Mortalin and
SHetA2 conflictingly regulate the same molecules involved in mitochondria-mediated intrinsic apoptosis. The results validate the power of this protocol for revealing
drug targets.