Decoding Chemotherapy Resistance

The structure of P-glycoprotein. Image by Mitchell Duarte.

A gatekeeper's job is to keep out the dangerous-looking riff-raff. This is the work of P-glycoprotein (P-gp), a protein that sits in the membranes of human cells. The problem is that, in addition to keeping out harmful substances, P-gp also keeps out drugs targeted at protecting the body from HIV, cancer, and psychiatric conditions. The result? Drug-resistance.

Believing that a better understanding of P-gp was a critical next step in fighting cancer and other diseases, Scripps Research scientists set out to get a closer look. What they produced was the first glimpse of this powerful protein – a glimpse that helps not only to explain how P-gp works against drugs, but also to open the door to fighting drug-resistance.

"This structure is an important advance and we hope it is just the beginning of more breakthroughs for us," says the study's senior author Geoffrey Chang, an associate professor at Scripps Research. "The structure is a nice tool for understanding how drugs are transported out of cells by P-gp and for designing drugs to evade P-gp therapy preventing drug resistance. It's very exciting."

At the atomic level, the team found a structure in the shape of an upside-down "v" or tipi. The pointy top part of the tipi resides in the cell's membrane and has two openings for substances to enter. The bottom portion sticks out inside the cell, ending in two dumbbell-shaped arms.

When substances enter through the top of the tipi, they find a large cavity (the inside of the tipi), which is lined with amino acids that bind to a variety of substances, holding them in place.

This overall shape is similar to that of another protein, MsbA, that transports lipids out of bacteria. This similarity suggests that P-gp works by bringing the two dumbbell-shaped arms together on the inside of the cell and opening the closed end toward the outside of the cell, essentially reversing the direction of the "v" so any substance caught inside the protein's cavity is ejected from the cell.

"We've long known that P-glycoprotein plays a key role in multidrug resistance in cancer patients, and this work helps us understand how the protein can act on such a wide range of compounds," said Jean Chin, of the National Institutes of Health's National Institute of General Medical Sciences, which partially supported the work. "In the future, scientists may be able to use these crystal structures to design chemicals that block P-glycoprotein's activity and restore sensitivity to chemotherapeutic agents."

Scripps Researchers are already making strides toward this goal. Qinghai Zhang, an assistant professor at Scripps Research who had designed several compounds that can block the function of P-gp, collaborated with Chang's team. Together, the group looked at P-gp when it bound to a handful of molecules, and watched as Zhang's compounds bound inside the P-gp cavity, preventing other substances from entering. Chang's team was then able to obtain the structures of two of Zhang's compounds inside P-gp.

"They both go in the same cavity and bind to different amino acids, but with some overlap," says Stephen Aller, a postdoctoral fellow in Chang's laboratory and first author of the new study. "What this tells us is that there is an extremely important core set of amino acids in P-gp that bind all substances, and there are additional amino acids for fine-tuning the binding to specific drugs."

Knowing what the P-gp binding cavity looks like and precisely where substances bind may allow researchers to design better drugs, for example, by using chemistry to modify portions of that drug so that it can sneak past P-gp to get inside the cell.

"[One advantage of this process is] we don't have to design brand new drugs, but rather re-design existing ones to make them work better," says Chang. "Scripps Research is a perfect place for these kinds of studies because there are great chemistry and biology labs here. We can easily find collaborators."

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