1. Inhibition of Cu/Zn Superoxide Dismutase by Small Organic Molecules Michele Yost, Dr. Jack Summers, Jonathan Markley Department of Chemistry and Physics Western Carolina University, Cullowhee, NC 28723
Abstract: Recently, Cu/Zn superoxide dismutase (SOD) has been proposed as a target for anti-cancer therapeutics. There are studied compounds which inhibit this enzyme. We have studied inhibition of SOD by these compounds and by a variety of chemicals with similar molecular shape. There have been reports that question the use of common SOD assay techniques due to the high reactivities of the species involved in generating and detecting the superoxide ion (O2¯). We have tried to get around this limitation by using the stable anion, fluoride as a superoxide reactivity mimic. We use 19F NMR spectrometry to measure the activity of the enzyme and the effects of each of the potential inhibitors on this activity.
We have begun to use different concentrations of MDHB at pH 10.3 so we can use the natural log of the TFA/F ratios to measure SOD activity/inhibition.
Background: O2·- is a compound that oxides and destroys different human cells. SOD normally protects our cells from O2·- . Dr. Huang and his collogues at the University of Texas have observed that cancer cells have an overabundance of O2·- while showing very low levels of SOD activity. This leads them to believe that cancer cells are SOD dependant. In its absence, O2·- accumulates, causing stress on the cells’ mitochondria, which leads the cell to go through apoptosis. When Dr. Huang used 2ME, the estrogen derivative was specific in destroying lymphoma cells while having no effect on the surrounding healthy lymphocytes.
As of now, we are unable to show that MDHB is able to achieve complete SOD inhibition.
TFA is unaffected by SOD while F’s relaxation changes
Using the NMR, we were unable to show SOD inhibition by 2ME at neither pH 7 nor pH We must now look at why we were unable to reproduce Huang’s results. We will start by observing the inhibition properties of several phytoestrogens with similar structures.
BCI-8 showed immediate inhibition all the way down to 2μM pH10.3; overnight 0.256μM
Phytoestrogens (Provided by Bent Creek Institute)
Compound Active?
BCI-4 NO
BCI-5 NO
BCI-6 NO
Compound Active?
BCI-1 NO
BCI-2 NO
BCI-3 NO
Compound Active?
BCI-7 NO
BCI-8 YES!!
BCI-9 YES!!
Using TFA and Fluoride to measure SOD activity: Because Fluoride relaxation was strongly effected by SOD concentrations and TFA relaxation did not, we are able to measure SOD activity from the ratio of the two integrals at a single delay time.
If a chemical is to have any effect on SOD, it will change the SOD equilibrium.
BCI-8 and BCI-9 were able to show inhibition at pH As a result, we investigated Methyl 3,4- Dihydroxybenzoate. While MDHB is not a phytoestrogens, we have included it in our studies because of its similar structure. BCI-8 has been able to inhibit SOD at lower concentrations as well as at pH 7.
Conclusion: Huang’s studies showed that 2ME2 has IC50~20μM, so we are showing that BCI-8 is a better with IC50~0.6μM. We will test pH 7 as well as filter the MDHB and BCI-8 solutions to see why MDHB has not shown complete inhibition.
Quality Enhancement Plan- Summer Undergraduate Research Fellowship