Skip to Content
MilliporeSigma
  • SHetA2 interference with mortalin binding to p66shc and p53 identified using drug-conjugated magnetic microspheres.

SHetA2 interference with mortalin binding to p66shc and p53 identified using drug-conjugated magnetic microspheres.

Investigational new drugs (2013-11-21)
Doris Mangiaracina Benbrook, Baskar Nammalwar, Andrew Long, Hiroyuki Matsumoto, Anil Singh, Richard A Bunce, K Darrell Berlin
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Dimethyl sulfoxide, European Pharmacopoeia (EP) Reference Standard
USP
Dimethyl sulfoxide, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Dimethyl sulfoxide, suitable for HPLC, ≥99.7%
Sigma-Aldrich
Formic acid, ACS reagent, ≥96%
Sigma-Aldrich
Formic acid, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥98%
Sigma-Aldrich
Formic acid, reagent grade, ≥95%
Sigma-Aldrich
Dimethyl sulfoxide, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Formic acid, puriss., meets analytical specifications of DAC, FCC, 98.0-100%
Sigma-Aldrich
Dimethyl sulfoxide, ACS reagent, ≥99.9%
Sigma-Aldrich
Dimethyl sulfoxide, puriss. p.a., ACS reagent, ≥99.9% (GC)
Sigma-Aldrich
Dimethyl sulfoxide, Vetec, reagent grade, 99%
Sigma-Aldrich
Dimethyl sulfoxide solution, 50 wt. % in H2O
Supelco
Dimethyl sulfoxide, for inorganic trace analysis, ≥99.99995% (metals basis)
Sigma-Aldrich
Dimethyl sulfoxide, for molecular biology
Sigma-Aldrich
Dimethyl sulfoxide, PCR Reagent
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
Dimethyl sulfoxide, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Dimethyl sulfoxide, meets EP testing specifications, meets USP testing specifications
Sigma-Aldrich
Dimethyl sulfoxide, sterile-filtered, BioPerformance Certified, meets EP, USP testing specifications, suitable for hybridoma
Sigma-Aldrich
Formic acid, ≥95%, FCC, FG
Supelco
Dimethyl sulfoxide, analytical standard
Sigma-Aldrich
Dimethyl sulfoxide, anhydrous, ≥99.9%
Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Dimethyl sulfoxide, puriss. p.a., dried, ≤0.02% water
Sigma-Aldrich
Formic acid, ACS reagent, ≥88%