1

Kelsi Gelle

Developing a Capillary Electrophoresis Assay to Study Enzyme Inhibition by Naphthoquinone

Analogs

Introduction:

Developing new treatments for various types of cancer is crucial to lower the mortality of

this complex disease. Cancer cells are known for their ability to rewire their metabolism and

energy production to support rapid growth and are notorious for their resistance to cancer

treatments. Glycolysis is the preferred way for cancer cells to derive most of their energy, even in

the abundance of oxygen. In this metabolic pathway, glycolytic enzymes are produced in

abundant amounts and are greatly expressed. The enzymes catalyze the metabolic reactions of

glycolysis and play active roles in promoting cancer survival, metastasis, gene expression

regulation, and other essential cellular processes.

Phosphofructokinase-1 (PFK-1) is an important allosteric enzyme targeted in developing

several medical applications. PFK-1 catalyzes the conversion of adenosine triphosphate (ATP) in

the presence of fructose 6-phosphate (F6P) to form adenosine diphosphate (ADP) and fructose

1,6-bisphosphate (F-1,6-BP). There are several assays that can be used to study the inhibition of

PFK-1. In this proposal, we will use a capillary electrophoresis-based assay to monitor the

enzyme catalyzed conversion of ATP to ADP. Capillary electrophoresis (CE) is a powerful

separation technique in which components in a mixture separate based on their charge to mass

ratio. The CE technique offers the advantages of higher resolution of separated peaks and low

sample consumption. In addition, UV detection will allow the monitoring of ATP to ADP

conversion without interference from other components in the mixture.

 2

Previous studies have reported the use of a CE-based assay to study inhibition of PFK-1

activity by inhibitors. The study used a known inhibitor, aurintricarboxylic acid (ATA), to

demonstrate the potential for CE in PFK-1 inhibition studies. Briefly, the conversion of

magnesium-bound adenosine triphosphate (Mg-ATP) to magnesium-bound adenosine

diphosphate (Mg-ADP) was monitored for PFK-1 catalyzed, rate-limiting step of

phosphorylation of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F-1,6-BP) (Malina,

2014). The addition of magnesium was found to stabilize charges in the multiply charged

adenosine phosphate molecules. Both ATP and ADP have UV absorbance at 262 nm. Although a

simple CE assay with an UV absorption method for detection of the reaction catalyzed by PFK-1

has been described, few studies have developed protocols for application of the method to study

inhibitory effects of novel chemical compounds with potential to regulate PFK-1 activity.

Objectives of the Proposal and Research Methodology:

There are two specific objectives for this proposal:

1. Optimize a Capillary Electrophoresis-based assay at Drury.

A previous study published in 2010 described the reversible high affinity inhibition of

phosphofructokinase-1 by acyl-CoA in which several potential inhibitors of PFK-1 were

examined to see how they acted on catalytic activity. Purified rabbit muscle PFK-1 was used to

demonstrate inhibition by using low concentrations of fatty acyl-CoA and derivatives (Jenkins,

2011). The study identified the direct mechanisms by which PFK-1 is regulated by fatty

acyl-CoA and showed the contribution to pathways during certain metabolic transitions. From

this study, it was anticipated that the regulation of PFK-1 by acyl-CoA would play a role in
 3

manipulating pharmacology to aid in lipid-related diseases (Jenkins, 2011). In this proposal, we

will optimize capillary electrophoresis-based assay to study the inhibition of PFK-1. Capillary

electrophoresis was chosen due to its high separation efficiency, resolution, and low sample

requirement. The enzyme activity and inhibition will be measured and monitored in order to

separate and detect the substrates and products for the PFK-1 catalyzed reaction in our

laboratory. A CE-based assay provides a powerful analytical strategy to monitor PFK-1 activity

by measuring the ratio of Mg-ADP to Mg-ATP.

2. Use the developed assay to study the PFK-1 inhibition by novel naphthoquinone

analogs.

Dr. Manpadi’s research lab has synthesized several naphthoquinone analogs which have

shown the potential to inhibit PFK-1. Naphthoquinones have shown promising cytotoxic

properties against cancerous cells on a variety of immune and metabolic pathways. Despite the

potential for these compounds to regulate PFK-1 activity, the assays could not provide

quantitative comparison for the ability of those compounds to inhibit PFK-1. In this proposal,

we will use selected naphthoquinone analogs and apply the developed CE-method to measure

their PFK-1 inhibitory effects. These measurements will be validated by comparing capillary

electrophoresis with western blotting methods which were used in studying the inhibitory

effects of novel naphthoquinone analogs on the JAK/STAT pathway and PFK-1 activity.
 4

Expected Outcome:

The importance of studying phosphofructokinase-1 inhibitors is relevant and necessary to

obtain more understanding in disease processes such as diabetes, metabolic syndrome, and

cancer. In relation to pharmaceutical uses, there are not many that previous research can be

applied to because of the lack of known PFK-1 inhibitors. The motivation behind this project is

to develop a deeper understanding of what could possibly be the next beneficial step in

developing aid in the treatments for prevalent diseases. We hope to have effective results in

studying PFK-1 activity and inhibition in order to create what could possibly be a buffer to

life-changing developments in particularly common problems of society.

 5

Works Cited

Jenkins, C. M., Yang, J., Sims, H. F., & Gross, R. W. (2011). Reversible high Affinity inhibition

of PHOSPHOFRUCTOKINASE-1 By acyl-coa. Journal of Biological Chemistry,

286(14), 11937-11950. doi:10.1074/jbc.m110.203661

Malina, A., Bryant, S. K., Chang, S. H., Waldrop, G. L., & Gilman, S. D. (2014). Capillary

electrophoresis-based assay of phosphofructokinase-1. Analytical Biochemistry, 447, 1-5.

doi:10.1016/j.ab.2013.10.028