Niharika Choudhury1, Collette Marchesseault2, Dr. John Wise2, and Dr. Pia Vogel21The Hockaday School, Dallas, TX; 2 Department of Biological Sciences, Southern Methodist University, Dallas, TX

High-Throughput Screening System for P-glycoprotein Inhibition

Introduction

While chemotherapeutics have seen some success in eliminating cancerous

cells, they have seen much failure as well. The overexpression of P-

glycoprotein (P-gp) has led to multidrug resistant (MDR) cancer cells that

keep chemotherapeutics from producing effective cytotoxicity in tumor

cells.

The ATP-binding cassette (ABC) transporter P-glycoprotein serves as a

pump that transports cytotoxins across its plasma membrane. In doing so,

P-gp protects the cells in the body from foreign substances and contributes

to the effectiveness of the blood-brain barrier or liver in pumping out toxins

and drugs; however, cancer cells that express P-gp in large amounts are

rendered multidrug resistant, as they do not respond effectively to

chemotherapeutics. Anti-cancer drugs that enter the cells are merely

pumped out by the protein, and higher doses of the chemotherapeutic are

required until the patient can no longer withstand treatment due to the

toxicity.

In response, there has been ongoing research to find drug inhibitors that

keep P-gp from pumping out chemotherapeutics while maintaining

reasonable toxicity levels in the body. Dr. John Wise and Dr. Pia Vogel’s lab

at the Center for Drug Discovery, Design, and Delivery at Southern

Methodist University has been working to find such drugs to inhibit the

activity of P-gp.

In order to screen compounds for inhibition, much time and money has

been spent on purifying the protein from yeast cells. In response, Collette

Marchessault in the Vogel-Wise lab has proposed a high-throughput

screening method to increase the number of compounds that could be

screened and, in turn, provide for a wider set of data to further the search

for effective inhibitors. I have worked with her this summer to introduce

the human protein P-gp, encoded with the MDR1 gene, into a more

versatile bacterial system in order to increase efficiency and reduce cost.

We have worked to get E. coli to take up the plasmid encoded with the

MDR1 gene so that it can express P-gp. Doing so would allow other

undergraduate and graduate students to screen for potential inhibitors of P-

gp in a more effective and efficient manner.

Methodology and Results

1. Ligation of pet24a-glpF and MDR1 (Fig. 1)

AcknowledgmentsI would like to thank Dr. Wise and Dr. Vogel for this wonderful

opportunity and their continuous guidance throughout my time in the lab.

I also would like to thank Collette Marchesseault for taking me under her

wing and letting me contribute to her project. I extend my gratitude to the

rest of the Vogel-Wise lab for teaching me the various lab techniques and

being so welcoming. Finally, I thank Dr. Barbara Fishel from the

Hockaday School for facilitating this research opportunity.

Methodology and Results

2. Transformation of CM2 into BL21 and DH5α, two strains of E. coli

cells using the NEB High Efficiency Transformation Protocol.

3. Plated the cells, Incubated them overnight, and Counted colonies

4. Grew Overnights of the colonies in LB broth

5. Plasmid Preparation [Mini-Prep] on the overnights to isolate

plasmid DNA by means of centrifuging and re-suspending the formed

pellets along with a series of buffers and washes, as indicated by the

Zyppy Plasmid Prep protocol.

6. Polymerase Chain Reaction [PCR] of BL21 and DH5α cells to look

for the 5000bp insert to confirm that MDR1 is in the plasmid. Used

NEB Protocol for PCR Using Q5 High-Fidelity DNA Polymerase

along with T7 and T7term primers, which targeted the DNA region

that included glpF + MDR1 (Fig. 2).

7. DNA Purification: used a series of buffers and centrifuged PCR

products, as indicated by the Zymo DNA Clean and Concentrator Kit,

to isolate and purify DNA.

8. Ran PCR Products on a 0.6% Agarose Gel (Fig. 3)

Conclusions

Based on the sequence data, we can conclude that the E. coli did, indeed,

take up our plasmid with the MDR1 gene. In its entirety, the sequence

highlighted some point mutations in the MDR1, but none of them

impacted which amino acid was encoded and were thus deemed

irrelevant. Furthermore, there were 6 cysteine to alanine mutations, all of

which were accounted for since they were engineered to be there. Two

other mutations were merely natural variants in the gene, and the final

one was a glutamic acid to cysteine mutation, engineered to keep the

protein inactive during this stage of experimentation. The sequence data

thus proved that the MDR1 sequence was complete and correct in the

newly engineered bacterial plasmid, CM2.

Discussion

Since we now know that we have the MDR1 gene in the plasmid, the next

step would include activating the gene so that it expresses the protein P-

gp in the bacterial system. Activating the gene would require a

mutagenesis experiment to mutate the cysteine back to glutamic acid in

order to produce active protein. With P-gp in the bacterial system, high-

throughput screening would be possible and prove to be an efficient and

effective means of finding inhibitors for P-gp.

Furthermore, experimentation has begun to induce the E. coli cells

to produce the glpF-MDR1 fusion protein. In order to do so, the bacteria

has to be grown in the presence of IPTG, a molecule that binds to the

repressor that keeps the glpf-MDR1 sequence from being transcribed.

Once the repressor is removed, the bacteria should be able to produce the

fusion protein. P-gp can then be isolated from the bacteria and used for

further experimentation.

This bacterial system is much more versatile and effective model

system than yeast. Once P-gp is expressed and isolated, research on

potential inhibitors can be conducted in an efficient manner.

Figure 1. Plasmid CM1 shows plasmid pet24a with the addition of the glpF gene between the two enzyme cut sites. Plasmid CM2 shows the result of the ligation of pet24a-glpf (CM1) and MDR1.

Figure 2. PCRs use thermal cycling for DNA melting and enzymatic replication of DNA. The Polymerase enzymatically assembles new DNA strands from nucleotides by using DNA strands as templates and DNA primers for DNA synthesis. The DNA primers are complementary to a DNA region that is then targeted for amplification

For further information

Please contact Dr. John Wise ( jwise@smu.edu) at the Southern Methodist University or

visit the website for the Center for Drug Discovery, Design, and Delivery for more

information or to learn more about the ongoing research at SMU.

http://www.smu.edu/Dedman/Academics/InstitutesCenters/CD4Figure 3. For our gel, we filled the first lane with the 1kb DNA ladder, the next three with purified DNA from BL21, and the final three with purified DNA from DH5α. Gel electrophoresis separates DNA fragments by length in order to estimate the DNA size; all samples were at the 5000bp mark, indicating that MDR1 was in the plasmid. Furthermore DH5α-10 yielded the brightest band (and therefore had the highest concentration of DNA), so it was used for the subsequent PCRs.

Methodology and Results

9. Subsequent PCRs on cleaned DH5α-10 resulted in PCR products that

could be used for DNA sequencing. Since the MDR1 gene is almost 4000

nucleotides long, we had to run a series of PCRs in which we used

different forward and reverse primers to target different sections of the

plasmid until we had all of the DNA fragments. For these PCRs, we used

the NEB PCR Protocol for Taq DNA Polymerase with Standard Taq

Buffer.

10. Cleaned the PCR Products with the Zymo DNA Clean and Concentrator

Kit and Ran the products on a 0.6% Agarose Gel. We had estimated the

sizes of the DNA segments earlier based on the location of the primers,

but we ran the gel to confirm the sizes and determine the concentration of

DNA for each segment of the plasmid (Fig. 4).

11. Sequenced the DNA through LoneStar Labs to see if the entire MDR1

gene was in the plasmid (Figs. 5, 6)

Figure 4. Two of the results of the gel electrophoresis on the cleaned PCR products of c10 are shown to the left. “1471” and “1624” refer to the locations of the targeted DNA.

Figure 5. Chromatogram data showing the DNA sequence of a portion of the MDR1 gene. Each color correlates to a different nucleotide, labelled at the top of the graph: Adenine, Guanine, Cytosine or Thymine.

Figure 6. The sequence data (portion shown above) matched up with the genetic sequence of MDR1 and thus allowed us to conclude that MDR1 was, indeed, in our pet24a-glpF-MDR1 plasmid.