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

Significance Statement

Drugs that are effective against diseases in animal models often fail in clinical trials.  The potential of a drug to prove effective in clinical trials can be increased by incorporating knowledge of the drug’s mechanism of action in the trial design.  For instance, some drugs work by repressing specific molecules that are mutated to be overactive thereby causing or worsening a disease.  Patients who do not have the disease mutation targeted by the drug may not have a positive result when treated with the drug.  Therefore, a clinical trial that limits patient enrollment to only those with the targeted mutation has a greater chance of showing a positive drug effect in comparison to a trial that is open to all people with the disease regardless of their mutation status.  A range of biochemical, genetic and computational approaches are used to identify drug targets, however these often are unsuccessful and considered high-risk fishing expeditions. This report is a rare success story of fishing for a drug target.  The lure was our anti-cancer drug called SHetA2 (NSC721689) and the fishing pole was the NanoLink Amino-Magnetic Microsphere (SoluLink, Inc.).

We designed and synthesized a SHetA2 analog that had an attachment which could be used by SoluLink to couple the drug to their magnetic microspheres.  A long linker was included to prevent microsphere interference with protein/drug interactions.  We incubated SHetA2-linked microspheres with protein extracts from cancer cells.  After rinsing away proteins not specifically bound to the linked SHetA2, proteins specifically bound to the drug were eluted from the SHetA2-microspheres using an excess of free SHetA2.  Unconjugated microspheres were processed in parallel as a negative control.  The eluents were evaluated on SDS Page Gels in search of bands that appeared in lanes containing eluents from drug-linked microspheres and not in lanes containing eluents from non-linked microspheres.  Multiple experimental conditions were optimized to increase the intensity of the SHetA2-microspheres band while decreasing the intensity of the unconjugated microspheres band.  Bands from a gel generated under optimized conditions were cut out and evaluated by QStar Mass Spectrometry to identify the eluted proteins.  In another “shot-gun” approach we used Orbitrap Mass Spectrometry to directly analyze the eluents without the gel step.  Both approaches identified mortalin (HSPA9) to be in the eluent from the drug-conjugated microspheres and not in the eluent from the un-conjugated microspheres. Our additional experiments validated that SHetA2 interferes with mortalin function in cancer cells.

 

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

 

 

 

 

 

 

 

 

 

 

Journal Reference

Benbrook DM, Nammalwar B, Long A, Matsumoto H, Singh A, Bunce RA, Berlin KD.

Invest New Drugs. 2014 Jun;32(3):412-23.

Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, 975 NE 10th Street, Room 1372, Oklahoma City, OK, USA, [email protected]

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 unconjugatedmagnetic 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.

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