Evaluating the potency of HIV‐1 protease inhibitors in blocking the initial step of HIV gagpol processing

Inhibitors of HIV protease have proven to be important drugs in combination anti‐HIV therapy. These inhibitors target mature protease and prevent viral particle maturation by blocking Gag‐Pol processing. Currently there is little data on the ability of these protease inhibitors to block the initial steps of autoproteolytic processing of Gag‐Pol by the embedded protease. We developed a plasmid encoding a modified form of Gag‐Pol, which can only undergo autocleavage at the initial cleavage site. Using an in vitro translation system, we assessed the ability of protease inhibitors to block the first step of this autocleavage. Seven inhibitors were tested: darunavir, indinavir, nelfinavir, ritonavir, saquinavir, and timpranavir. Of these inhibitors, saquinavir and darunavir were the most potent at blocking the initial processing step. Parallel results were seen when we evaluated the effect of the inhibitors on Gag‐Pol autocleavage in HIV‐infected H9 cells using immunoblot analysis to measure full‐length Gag‐Pol accumulation. We have thus identified two protease inhibitors that also have substantial activity against the immature protease, and their structures could provide a starting point for development of more targeted inhibitors of embedded Gag‐Pol protease that would block viral maturation at the earliest stage of the process. This research is funded by the National Institutes of Health.