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The multi-drug transporter P-glycoprotein (P-gp) detoxifies cells by promiscuously exporting chemically unrelated toxins and drugs. Now, X-ray crystallography has helped US scientists home in on how this protein might also assist cancer cells in developing resistance to chemotherapy agents and so provide a target for reversing that effect. Geoffrey Chang and colleagues at the Scripps Research Institute in La Jolla, California, have obtained the first glimpse of the detailed workings of the protein, P-glycoprotein. P-gp prevents many substances, including anticancer drugs, from entering cells. The protein's presence in cancer cells is one of the reasons that anticancer drugs that appear to work in the laboratory often fail as multidrug resistance becomes apparent in the clinic. The discovery of the detailed structure of P-gp could help medicinal chemists design more effective anticancer drugs by circumventing the attentions of this biomolecular drug trafficker. Writing in the current issue of the journal Science, Chang and his colleagues explain the relevance. "This structure is an important advance and we hope it is just the beginning of more breakthroughs for us," he says. "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, preventing drug resistance. It's very exciting." Researchers first identified P-gp in 1976, recognising that it sits in the membranes of many different types of cell, including those of the gut, kidney, and brain. It acts as an important biochemical gatekeeper blocking the passage of potentially harmful agents into the cell. Unfortunately, P-gp cannot usually distinguish between substances that are generally harmful to our cells and those designed to target cancer cells or cells infected with viruses, such as HIV. "We've long known that P-glycoprotein plays a key role in multidrug resistance in cancer patients," adds team member Jean Chin of the National Institutes of Health's (NIH) National Institute of General Medical Sciences (NIGMS), "this work helps us understand how the protein can act on such a wide range of compounds." Of course, as with countless other membrane proteins, the biggest challenge facing the team was to get enough protein to purify and crystallise it, says Stephen Aller in Chang's laboratory. They were successful with the mouse equivalent of P-gp, which is 87% the same as the human protein. It is shaped like an upside down "V" or a tipi with a large cavity inside. The cavity's interior is lined with amino acids that bind to different substances, holding them in place. The top part of the "tipi" resides inside the cell membrane and has two openings through which alien substances might enter. The bottom portion protrudes into the cell, ending in two dumbbell-shaped arms. A resemblance to a bacterial protein, MsbA, which transports lipids out of bacteria, suggested to the team that P-gp might work 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". Any substance caught inside the protein's cavity would then be spilled out to the exterior. However, it is there that the similarity between the bacterial lipid transporter and the mammalian P-gp ends. "Unlike the bacterial protein, the mammalian P-gp was designed to have a wide range of substrates," says Chang. "The presence of so many hydrophobic and aromatic residues within the cavity explains how this happens." The team also obtained structures of P-gp bound to various substrates, including inhibitors. Chang and Aller worked with Qinghai Zhang to study various compounds designed to block P-gp. These compounds bind inside the P-gp cavity, preventing other substances from entering by slowing or blocking the expulsion process. "They both go in the same cavity and bind to different amino acids, but with some overlap," explains Aller. "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." The new insights into the characteristis of P-gp suggest that medicinal chemists will not have to start from scratch in designing inhibitors or in improving cancer chemotherapy. Instead, they could simply redesign current drugs so that they are no longer trapped in the molecular tipi. "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," adds Chin. Related links:
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