This document summarizes a student research project that studied the enzyme glucose-6-phosphate dehydrogenase (G6PD) for applications in cancer drug discovery. G6PD helps maintain cell energy and is important for some cancer cell growth. The study developed an assay to measure G6PD activity using indicators to detect reaction products over time. Potential enzyme inhibitors were screened by adding them to the G6PD reaction and measuring their ability to decrease enzyme activity. The most potent inhibitors were further tested at different concentrations to determine their half maximal inhibitory concentration (IC50), a measure of inhibitor potency. Inhibiting G6PD could stop cancer cell replication, making it a potential target for new cancer drugs.

1. Enzymology: Inhibition of Glucose-6-Phosphate-Dehydrogenase for Cancer Drug Discovery
Angela Huber, Edwin Ochoa, Huynh (Jason) Tran, Excel High School ‘15
Introduction
Summer Research Program, Vertex Pharmaceuticals August, 2015
1) Add enzyme solution, phosphate buffer, and
developing reagent in tubes labeled A, B, C, D,
and E.
2) Add each reagent to the micro assay plate.
3) Add 30 ul of STOP solution to each of the 40 wells.
(rows A to E, columns 1 to 8)
4) At time zero, add 300 ul of the substrate solutions
to each of the 5 mini tubes (A to E)
5) Starting at 1 minute, take 90 ul from each tube and
add it to a fresh well of the micro plate (column 1)
and mix.
6) Repeat step 4 every 2 minutes until you reach
column 8.
A catalyst is a chemical agent that helps with reaction
rate. Enzymes are protein catalysts that help speed up
the reaction process. Each enzyme in your body is
responsible for one chemical reaction that occurs in
your cells. G6PD is present in all human cells but is
important to blood cells and some cancer cells
because it is required to make NADPH and
glutathione. Indicators can be used to detect the
presence of NADP (blue) and NADPH (red) to
measure the activity rate of G6PD (Figure 1).
An enzyme inhibitor is a molecule that binds to an
enzyme and decrease its activities.
Enzymology is the study of enzymes, their kinetics,
structure, and function. Roughly one third of the drugs
on the market target enzymes. This summer we
experimented with Glucose-6-Phosphate
Dehydrogenase (G6PD) protein, which is an enzyme in
the pentose phosphate pathway. It assists with
maintaining the cell’s energy by regenerating the co-
enzyme NADPH. This pathway is important for some
cancer cells to grow and therefore inhibiting it might lead
to a cancer drug.
Background
Process
Results
Application
Acknowledgements
Thank you to Dr. Carl Reid, Kenny
Bonanno, Dr. Jim Hogan, Erin Sayre, and
most importantly Vertex, and the Learning
Lab
References
Jeanteur, Philippe. "Molecular and Cellular
Enzymology." Google Books. Springer
Science & Business Media, Dec 2012.
Web. 6 Aug. 2015.
Lica, Lorraine. "Glucose-6-phosphate
dehydrogenase deficiency." The Gale
Encyclopedia of Medicine. Ed. Jacqueline
L. Longe. 5th ed. Farmington Hills, MI:
Gale, 2015. Science in Context. Web. 11
Aug. 2015.
RateofEnzymeReaction Inhibitor concentration
(Log uM)
By disrupting the enzyme activity, we can stop
the cancer cells from replicating. As a result,
many inhibitors would be tested for their
capacity of decreasing enzyme activities (in this
case, the G6PD enzyme), this process is called
“screening.” Different potential inhibitors will be
added separately to the micro assay plate
containing enzyme solution, substrate, and
indicator (Figure 3). The wells which are
colored blue have active inhibitors which can
be selected for drug discovery.
In Figure 4, the selected inhibitors are diluted to
different concentrations and tested by the same
method to determine their potency.
For that process, we often utilize the instrument
called “Spectrophotometer” to measure the light
absorbency of different wavelengths of the
solution (in this case, the wave lengths are 610
and 570 nm).
Figure 3: Screening Plate Figure 4: IC50 determination
Figure 1
Figure 2: Enzyme Time Course