Cross platform analysis of drug targets and toxicity of bath salts

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Project Summary/Abstract The United States is in the midst of a substance abuse and mental health epidemic. Over the past decade, abuse of psychostimulant (and sometimes hallucinogenic) cathinones, often referred to as “bath salts”, and variants such as ‘flakka’, has become increasing popular in America’s youth. Although there has been substantial research into the mechanisms underlying the addictive and adverse effects of some of these agents, such as 3,4-methylenedioxymethamphetamine (MDMA), a wide variety of variants are available for sale on the internet. Furthermore, these represent only a small fraction of conceivable structures which can be produced on kilograms scale for sale online. Gaining a more complete understanding of the drug targets and toxicity associated with these agents is an important step to treat substance abuse disorders and episodic toxicity. The over-arching goal of our work is to unite whole system level analysis involving the neurobehavioral effects of psychoactive agents with the study of binding partners at the molecular level. The focus of this proposal is to implement a novel in vivo photoaffinity labeling (PAL) approach for cross platform analysis of psychoactive agents, with an emphasis on structures related methylenedioxypyrovalerone (MDPV) and α- pyrrolidinovalerophenone (α-PVP). In Aim 1 we will synthesize a carefully designed set of probes to be cataloged in Aim 2 assays. This includes stereoselective synthesis of PAL probes, structure activity relationship (SAR) controls, and stable isotope labeled standards for LC-MS studies in Aim 2C. The synthesized library will be assayed for 1) receptor binding profile; 2) effect on monoamine uptake and release in rat synaptosomes; and 3) toxicity in multiple cell lines HepG2 (liver), SHSY-5Y (neuronal), AC16 (cardiac myocyte) and PC-12 (neuronal [rat]). In Aim 2C, active compounds (and select inactive controls) will be studied in an adult zebrafish behavioral paradigm utilizing the novel tank test with concurrent in vivo PAL. The concentration of each drug will be measured by LC-MS/MS to correlate target tissue (brain) drug exposure with behavioral responses. Select compounds will be re-examined in the Aim 2 assays using PAL for side-by-side comparison of binding interactome of Aim 2 experimental systems versus the in vivo binding interactome. Chemical biology in Aim 3 will utilize click chemistry to visualize protein labeling via fluoroimaging of SDS PAGE gels. Proteomics will determine protein identification after affinity purification to define the binding interactome of each probe. A variety of competition experiments will be performed to characterize background PAL and recognize bona fide targets. Bioinformatics will be used for network analysis to provide an unbiased comparison of the binding interactomes for each experimental system to propose potential therapeutic targets to treat toxicity and episodic lethality of these agents.