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TSRI Scientists Find Clues to Cancer Drug Failure

LA JOLLA, CA – March 2, 2015 – Cancer patients fear the possibility that one day their cells might start rendering many different chemotherapy regimens ineffective. This phenomenon, called multidrug resistance, leads to tumors that defy treatment.

Now scientists at The Scripps Research Institute (TSRI) have published a pair of studies showing how the primary protein responsible for multidrug chemotherapy resistance changes shape and reacts to therapeutic drugs.

“This information will help us design better molecules to inhibit or evade multidrug resistance,” said TSRI Associate Professor Qinghai Zhang, a senior author of both studies.

The findings were published recently in two papers: a study in the journal Structure co-led by Bridget Carragher, a professor at TSRI with a joint appointment at the New York Structural Biology Center, and a paper in Acta Crystallographica Section D co-led by Geoffrey Chang, professor in the UC San Diego Skaggs School of Pharmacy and Pharmaceutical Sciences.

How a Protein Causes Multidrug Resistance 

The proteins at work in multidrug resistance are V-shaped proteins called ABC transporters. ABC transporters are found in all kingdoms of life—from bacteria to humans. In humans, an important ABC transporter is P-glycoprotein (P-gp), which catches harmful toxins in a “binding pocket” and expels them from cells.

The problem is that in cancer patients, P-gp sometimes begins recognizing chemotherapy drugs and expelling them, too. Over time, more and more cancer cells can develop multidrug resistance, eliminating all possible treatments.

“Virtually all cancer deaths can be attributed to the failure of chemotherapy,” said Zhang.

To design more effective cancer drugs, scientists would benefit from a better understanding of P-gp and how it binds to molecules.

A Better Look at Transporters

For the new Structure study, researchers looked at P-gp under one of TSRI’s powerful electron microscopes. They also looked at MsbA, a similar transporter protein found in bacteria.

The electron microscopy (EM) work—spearheaded by a postdoctoral researcher, Arne Moeller, working in the laboratory of Carragher and former TSRI Professor Clint Potter, co-directors of the National Resource for Automated Molecular Microscopy—solved a major problem in transporter research.

Until recently, researchers could only compare images of crystal structures made from transporter proteins. These crystallography images showed single snapshots of the transporter but didn’t show how the shape of the transporters could change. Using EM, however, a whole range of different conformations of the structures could be visualized, essentially capturing P-pg and MsbA in action. 

The new research was also enabled by the development of new chemical tools. Previous studies were hampered by the fact that outside the cell membranes these transporter proteins turned into an unstructured mash.

“They looked like tofu,” said Sung Chang Lee, a research associate in Zhang’s lab at TSRI and co-first author of the Structure study.

In the study, the researchers used a solution of lipids and peptides to mimic natural conditions in the cell membrane. A novel chemical called beta sheet peptide, developed by the Zhang lab, was used to stabilize the protein and provide enough stability for a new perspective.

Together with EM, this technique enabled the research team to capture a series of images showing how transporter proteins change shape in response to drug and nucleotide binding. They found that transporter proteins have an open binding pocket that constantly switches to face different sides of membranes.

“The transporter goes through many steps—it’s like a machine,” said Zhang.

A Closer Look at Binding 

In the second study, the researchers investigated the drug binding sites of P-gp using higher-resolution X-ray crystallography.

Their findings, published in Acta Crystallographica Section D, show how P-gp interacts with drug-like molecules called ligands. The researchers studied crystals of the transporter bound to four different ligands to see how the transporters reacted.

The researchers found that when certain ligands bind to P-gp, they trigger local conformational changes in the transporter. Binding also increased the rate of ATP hydrolysis, which provides mechanical energy and may be the first step in the binding pocket closing process. 

The team also found that ligands could bind to different areas of the transporter, leaving nearby slots open for other molecules. This suggested that it may be difficult to completely halt the drug expulsion process. 

Zhang said the next step in this research is to develop molecules to evade P-gp binding.

In addition to Zhang, Carragher, Potter, Lee and Moeller, authors of the Structure paper, “Distinct Conformational Spectrum of Homologous Multidrug ABC Transporters,” were Houchao Tao and Jeffrey A. Speir of TSRI; Chang of UC San Diego; and Ina L. Urbatsch of Texas Tech University. For more information, see http://www.cell.com/structure/abstract/S0969-2126%2814%2900422-5

The Structure study was supported by the National Institutes of Health (P50 GM073197, R01 GM098538, P41 GM103310, RGM102928, U54 GM94610 and R01 GM94367).

In addition to Zhang, Chang and Lee, authors of the Acta Crystallographica Section D paper, “Snapshots of Ligand Entry, Malleable Binding, and Induced Helical Movement in P-glycoprotein,” were Paul Szewczyk, Aaron P. McGrath, Mark Villaluz, Steven D. Rees and Rupak Doshi of UC San Diego; Ina L. Urbatsch of Texas Tech University; and Houchao Tao of TSRI. For more information, see http://journals.iucr.org/d/issues/2015/03/00/issconts.html

The Acta Crystallographica Section D study was supported by the National Institutes of Health (R01 GM94367, R01 ES021985, R01 GM098538 and RGM102928) and a National Health and Medical Research Council CJ Martin Postdoctoral Research Fellowship.

About The Scripps Research Institute

The Scripps Research Institute (TSRI) is one of the world's largest independent, not-for-profit organizations focusing on research in the biomedical sciences. TSRI is internationally recognized for its contributions to science and health, including its role in laying the foundation for new treatments for cancer, rheumatoid arthritis, hemophilia, and other diseases. An institution that evolved from the Scripps Metabolic Clinic founded by philanthropist Ellen Browning Scripps in 1924, the institute now employs more than 2,500 people on its campuses in La Jolla, CA, and Jupiter, FL, where its renowned scientists—including two Nobel laureates and 20 members of the National Academy of Science, Engineering or Medicine—work toward their next discoveries. The institute's graduate program, which awards PhD degrees in biology and chemistry, ranks among the top ten of its kind in the nation. For more information, see www.scripps.edu.

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TSRI Associate Professor Qinghai Zhang (right), shown here with Research Associate Sung Chang Lee, was a senior author of both studies. (High-res image)


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