Herschel Wade elucidates a mechanism for multiple-drug resistance.
June 2010-Multiple-drug resistance (MDR) is a serious and growing problem. Worldwide, hundreds of thousands of people are infected with strains of tuberculosis resistant to several TB drugs. Gonorrhea, once easily cured by penicillin, is now increasingly resistant to that drug, as well as to other classes of antibiotics, leading some health officials to raise the specter of untreatable gonorrhea. In hospitals, too, multidrug resistance is increasing, including infections caused by the superbug MRSA and, most concerning, strains of gram-negative bacteria that are resistant to all approved antibiotics.
To stem the problem, infectious disease and public health specialists have launched campaigns to promote infection control and urge doctors not to overprescribe antibiotics. In his lab, meanwhile, Johns Hopkins biophysicist Herschel Wade is focusing on MDR through a different lens. To Wade, MDR presents a molecular-scale puzzle. And in the past year, he’s made significant progress in solving one of its parts using a model bacterium called Bacillus subtilus.
“Bacteria such as B. subtilis have evolved the ability to recognize different drugs or harmful chemicals and get rid of them,” says Wade, who received a National Science Foundation Career Award and a Beckman Young Investigators Award supporting his research on MDR. Unfortunately for us, such survival mechanisms also allow bacteria to defeat drugs designed to destroy them.
Biologists have identified two key mechanisms responsible for MDR. In one group are efflux pumps, molecules in the cell membrane that bacteria evolved to pump toxic substances out of their interiors. Normally, such efflux pumps are no match for a therapeutic dose of antibiotics. But over time, as bacteria are exposed to high concentrations of a drug, they may boost the number or activity of their efflux pumps, thus becoming resistant to the drug.
A second, related mechanism, and one that Wade studies, involves proteins inside the cell that control the genes coding for the efflux pumps. When a drug enters the cell, it may bind to one of these gene regulators, changing its shape or chemistry in such a way that it induces the protein to turn on the gene for the efflux pump. The result: more pumps.
As more efflux pumps sprout on the cell surface, less drug is concentrated inside the cell. Scientists have identified a slew of efflux pumps and their regulators in bacteria. They have also found similar mechanisms in cancer cells that have developed resistance to chemotherapeutic drugs.
These systems become more of a puzzle when multiple drugs are involved.
Nature, explains Wade, tends to favor specificity: one “key” to fit one “lock,” one molecule to flip one molecular switch. But multidrug resistance departs from that rule. That’s because a variety of different molecules—drugs—are able to turn the lock, or flip the switch. It’s like having several different keys that can all start the same car.
Simply put, says Wade, “the system is promiscuous.”
Over the years, researchers have proposed different models for how various gene regulators bind and become activated by multiple drugs. Biologists have shown, for example, that a gene regulator called QacR contains several different drug “pockets.” Different drugs fit into the different pockets. The model is something like a lock with multiple keyholes, each responsive to a different key.
In his studies with B. subtilis, Wade, along with graduate student Sharrol Bachas, has focused on a more puzzling gene regulator, a molecule called BmrR.
Unlike other gene regulators, BmrR has only one pocket. Yet Wade has shown, by crystallizing BmrR bound to different drugs, that this single pocket can accommodate an impressively large variety of different drugs. In all, he has demonstrated that BmrR will bind to more than 40 different drugs.
At first, it seemed to Wade that the drugs shared no common motif. Flat rhodamine would bind to BmrR; but so would TPP, a ball-like molecule.
But closer analysis has, in fact, revealed a recurring theme, says Wade. Most of the drugs that bind to BmrR are hydrophobic, or repelled by water.
Wade has also elucidated the structure of the drug-binding pocket itself. The pocket, he says, contains not one, but two chambers. It consists of a long hydrophobic slot that leads to a shallow vestibule. So drugs that are hydrophobic find a home in the pocket. “But the shallow vestibule is not very discriminating,” says Wade. “Like a chameleon, it can change its binding capacity relative to what is in the pocket.”
In other words, as long as a drug is hydrophobic, it can possess any number of other properties and still BmrR will bind it.
Although BmrR is only one gene regulator and B. subtilis only one bacterial species, says Wade, he suspects that the model is repeated in other species and may even help to explain some drug resistance in cancer.
The next step in his experiments, says Wade, is to understand which of the drugs that bind to BmrR also activate it. Just because a drug binds to BmrR, says Wade, doesn’t mean it turns on the gene regulator. Specifically, Wade will study which antibiotics and other substances alter the conformation of BmrR when they bind to it. “If we understand how molecules direct the conformational changes, we can design drugs that block those changes,” he says. “That’s what I’d really like to see.”