Methionine Synthase: A target for novel small molecules to inhibit cocaine and methamphetamine induced neuronal death

Purpose: Oxidative stress-induced cell death is involved in the pathology of psychostimulant addiction neuropathies and ischemic stroke. These conditions potentially cause neuronal and functional changes via different mechanisms - epigenetic alterations (DNA hypomethylation) and reactive oxygen species (ROS) accumulation. Current medications for the treatment of psychostimulants (e.g., cocaine and methamphetamine) induced addiction neuropathies are largely ineffective due to the high rate of relapse and marginal alterations of dependency to these diseases. To circumvent this, our laboratory has synthesized novel hybrid antioxidant small molecules: SA-30 and SA-31, with predictive neuroprotective and broad-spectrum reactive oxygen species (ROS) scavenging abilities in mouse hippocampal HT22 neural cells. Our objective here was to test if the compounds increase cell proliferation, superoxide dismutase (SOD) enzyme, as well as methionine synthase (MS) enzyme, a key enzyme largely responsible for DNA methylation, neuronal growth, and survival using human neuroblastoma cells (SH-SY5Y) expressing dopaminergic neurons. Methods: The synthesis and structure characterization of compounds SA-30 and SA-31 were previously synthesized in Acharya lab using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Human neuroblastoma cells SH-SY5Y expressing dopaminergic neurons were purchased (ATCC), cultured, and treated with different concentrations of cocaine hydrochloride or methamphetamine for 8, 24, and 48h for determining EC50 using MTT assay. In the next experiment, either cocaine hydrochloride (1.5mM) or Methamphetamine (METH, 3.5 mM) was added followed by co-treatment with compounds SA-30 and SA-31 and 4-hydroxy tempol (all at 100µM) for 24 hours. Cell viability was assessed using MTT assay. The level of intracellular MS and SOD enzymes was assessed using ELISA. Results: In SHSY5Y cells, the EC50 of cocaine was 1.5 mM, and for METH was 3.5 mM after 24h of treatment. Both compounds SA-30 and SA-31 were not cytotoxic at varying concentrations (0.01, 0.1, 1, 10, 100µM) and rescued cells from both cocaine and METH-induced oxidative stress/cell death at 100µM concentrations. SA-31 at 100µM significantly increased (~1.5 fold) intracellular MS as compared to control. There was a decrease in MS level after METH treatment and treatment with SA-30 and SA-31 increased the level. SOD levels were significantly higher (~3 fold) in METH+SA-31 treated groups than only METH groups. Conclusion: Both novel hybrid small molecules SA-30 and SA-31 are neuroprotective in SH-SY5Y cells from psychostimulants cocaine and METH-induced oxidative stress/neural cell death. Increasing MS and SOD enzyme activities is one of the mechanisms by which neuroprotection was attained. Future studies will address the potential of both SA-30 and SA-31 to progress further in pre-clinical drug development, with a future application for the treatment of substance abuse disorder.