A new theory about the dynamics of protease may lead to treatment options
by Chael Needle
LifeGuide [Treatment Horizons]
A recent study published in Biopolymers presents a theory about a potential way to inhibit protease inhibitor-resistant HIV. While a UCSD Biomedical Sciences grad student, lead author Alex L. Perryman conducted the research as a Howard Hughes Medical Institute predoctoral fellow in the lab of J. Andrew McCammon, an HHMI principal investigator at UCSD. Now an Amgen postdoctoral fellow in the lab of Stephen Mayo, an HHMI investigator at the California Institute of Technology, Perryman will continue this research once he finishes his postdoc and establishes his own lab, in collaboration with coinvestigator Jung-Hsin Lin.
Performing calculations on computers provided by the NSF Center for Theoretical Biological Physics at UCSD, the researchers created and then analyzed computer simulations of the molecular dynamics of HIV-1 protease, particularly looking at the active site of the enzyme to which the drug binds. Perryman and his colleagues selected the V82F/I84V drug-resistant double mutant of HIV-1 protease “because it is one of the worst mutants known.”
Perryman notes that looking at static images of a “V82F/I84V double mutant of HIV-1 protease bound to one of the active site inhibitors provides no clues about the reasons why the drugs no longer work against it. We had to study the dynamic flexibilities (that is, the differences in the motions and the shapes that were sampled) of the wild type and that mutant system before we discovered a potential explanation for why the drugs do not work well when those two mutations are present.”
One of the dynamic flexibilities studied centered on “flaps” of the molecule. “When no substrate or drug is bound to [HIV protease’s] active site, the flaps that guard access to the active site are in a ‘semi-open’ conformation.” For binding to occur, the flaps must fully open. And once binding occurs, the flaps are forced shut until closed and, in the case of HIV protease’s natural substrate, it can continue on with one of the last steps of the viral replication process, cutting components derived from the substrate to produce an infectious viral product. After this happens, he says, “those flaps must reopen and allow the cleaved products to leave before the next cycle of catalysis can occur.” Protease inhibitors work by mimicking the general shape of the substrate in order to bind to and plug the active site, which then interrupts the catalytic process.
The researchers’ earlier study suggested that “enhanced flap motion is likely a source of drug resistance.” The V82F/I84V mutant “probably favors the semi-open conformations of the flaps more than the wild type [normal] version of HIV-1 protease favors them.” If the flaps are open more often and in a more mobile way on this mutated enzyme than on the wild-type version, then protease inhibitors designed for wild-type HIV would have to work much harder to get the flaps to close.
The second study confirmed the earlier conclusions about the presence of enhanced flap motion in the mutant system and about the mechanical relationships that control flap motion. “Previously, we noticed that the peripheral ear-to-cheek region was expanding when the flaps were closing, and conversely, the peripheral distance was compressing when the flaps were opening.” Artificial restraints in the computer program blocked the flaps from opening by expanding the gap between “ear” and “cheek” on the peripheral surface (see illustration). “Our new results showed that when that peripheral distance was pinched slightly, the flaps that guard the active site were allowed to open. More importantly, when that peripheral distance was expanded slightly, we did not observe flap opening behavior. By prying open that peripheral distance by about an Angstrom, we were able to prevent the flaps from being able to open.”
An allosteric inhibitor, then, would bind in one location and cause a structural change that affects the activity of a different site that is located in a different region of a protein target, says Perryman. “If these allosteric flap-closing inhibitors can be developed, then they could prevent the flaps from reopening after catalysis has occurred, which would prevent that protease molecule from going through any further rounds of catalysis. Similarly, those allosteric flap-closing inhibitors could help force the flaps to close, which means that the active site inhibitors would no longer have to pay as much energy during their binding process....[and] those allosteric flap-closing inhibitors might be able to rescue the efficacy of the current drugs...against drug-resistant mutants of HIV protease.”
Perryman says that “further experiments need to be performed before the peripheral surface can actually be validated as a new drug target.” Both Jung-Hsin Lin and Perryman will continue to work on the design of these new allosteric inhibitors. “Since the NIH does not usually fund applied research of this sort....I hope this article reaches someone from the Bill and Melinda Gates Foundation, from the Keck Foundation, from the Arnold and Mabel Beckman Foundation, or from other potential philanthropic donors.”
Chael Needle wrote about integrase inhibitor candidate GS 9137 for the March issue.