February 9, 2012
Most drugs are designed to attach to a protein and either activate or inhibit the protein’s function (usually the latter). One of the measures of a good drug is how tightly it binds to its target protein. Eventually a drug molecule, known as a ligand or signaling molecule, will lose its grip and stop working, a process known as disassociation.
The disassociation rates of drugs have been studied and measured for years, but the mechanism behind disassociation for proteins whose binding sites lie on the surface has not been explored to any great degree. Andrew Lee, PhD, and a team of researchers at the University of North Carolina at Chapel Hill took a close look at a series of eight inhibitors to the enzyme dihydrofolate reductase, or DHFR, using an NMR spectrometer. The binding strength of the inhibitors used varied by six orders of magnitude with the most potent being a million times stronger than the weakest.
The researchers were surprised by what they observed.
“In essence, we saw the enzyme physically shake the ligands off its receptor sites,” says Lee, who is an associate professor at the UNC Eshelman School of Pharmacy. “And the weaker the ligand’s binding strength, the faster the enzyme seemed to ‘shake’ to knock it loose.”
The dance of a protein is too small and usually too fast to be seen directly so the research team used nuclear magnetic resonance spectroscopy to make their observations. Structural fluctuations during the disassociation process picked up by the NMR spectrometer indicate that achieving an excited conformation (a certain structural state) allows the protein to “open a gate” and release the bound ligands, Lee says.
The team’s findings were published online in Nature Chemical Biology.
Lee’s lab at the UNC Eshelman School of Pharmacy focuses on protein dynamics, the way a protein molecule changes its shape and function by modifying its structure. Protein dynamics is a relatively young field, and while a great deal is known about how the structure of a protein relates to its function, the way proteins change their structure is not well understood, Lee says, but there is a growing appreciation for how motion contributes to function, especially when it comes to the activation and inhibition of enzymes.
“You want drugs to bind and typically the battle is to get them to bind tightly enough. We’re still not very good at predicting what molecules are likely to bind strongly,” he says. “We don’t really understand all the factors and forces that set the binding affinity.”
Lee says the team’s finding has a clear relevance to small-molecule drug design because you want to be able to create drug molecules that have a high binding affinity and hold tight to their targets. He points to the high-throughput screens that are currently being used to evaluate potential drug candidates to illustrate the problem.
“We’re literally throwing thousands and thousands—if not hundreds of thousands—of molecules against a target to see what might stick,” he says.
Lee’s coauthors on the study from the UNC Eshelman School of Pharmacy are former graduate student Mary Carroll, PhD; former postdoctoral fellow Anna Gromova, PhD; and Associate Professor Scott F Singleton, PhD. Additional coauthors are Randall Mauldin, a former student in the UNC Department of Biochemistry, and Edward Collins, PhD, of the UNC School of Medicine is an additional coauthor.
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