PROTAC Drug Candidates Could Offer Up Strategy for Eliminating HIV Reservoir

University of Pittsburgh School of Medicine scientists have developed drug candidates that early tests suggest could reverse HIV’s ability to escape detection by the immune system. The team’s research was centered on developing drugs known as proteolysis targeting chimeras (PROTACs) for the targeted degradation of HIV Nef, a viral protein expressed at high levels after HIV infection. Their work uncovered a potential strategy for eliminating the viral reservoir that cannot be cleared with existing antiretroviral drugs. It’s this reservoir that prevents people from being completely cured of HIV even when their viral load is undetectable in standard tests.

“We have excellent antiretroviral drugs that suppress HIV, but unfortunately, none of them clear the virus. If someone with HIV stops taking their medication, the infection will rebound,” said Thomas Smithgall, PhD, the William S. McEllroy Professor and former chair of the Pitt School of Medicine’s department of microbiology and molecular genetics. “HIV establishes a reservoir of infected cells that lay dormant even in the presence of antiretroviral therapy, hiding from immune system detection. We think we’ve uncovered a key to unmasking that reservoir.

Smithgall is senior author of the researchers’ published paper in Cell Chemical Biology, titled “PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication,” in which they concluded “Here we describe the synthesis and evaluation of heterobifunctional PROTAC molecules that induce the targeted degradation of the HIV-1 Nef virulence factor.”

Over the past three decades, Smithgall’s team and others have shown that Nef can prevent the immune system from destroying infected cells by blocking or “hiding” signs of the virus on the cell surface. “The HIV-1 Nef accessory factor enhances the viral life cycle in vivo, promotes immune escape of HIV-infected cells, and represents an attractive antiretroviral drug target,” they team noted. “Nef is expressed at high levels soon after infection and interacts with diverse host cell proteins to enhance viral infectivity and promote immune escape.”

But unlike other HIV proteins that are expressed during infection, Nef is a challenging target for drug design. “Nef lacks enzymatic activity and an active site, complicating traditional occupancy-based drug development,” the investigators continued. First author Lori Emert-Sedlak, PhD, research associate professor of microbiology and molecular genetics at Pitt, added, “A protein like Nef is often considered undruggable because it doesn’t have a defined binding site for drug action. Our existing Nef inhibitors, which only bind to Nef, block some Nef functions extremely well, but don’t touch other functions, many of which are critical for HIV infection.”

For their reported research the team took a different approach. Rather than pursuing a drug molecule that might only block one or two Nef functions, they looked for a compound that would mark the protein for degradation in infected cells. Degrading Nef would block all of its functions, including HIV infection and replication. In addition, HIV antigens would be restored to the surface of infected cells, revealing them to the immune system for destruction.

Working in collaboration with co-author Colin Tice, PhD, a senior research chemist at the biotechnology company Fox Chase Therapeutics Discovery, the team first identified small molecules that bind to the Nef protein. These Nef binders were then coupled to a second molecule that marks the Nef protein for destruction by a natural cellular process. “A major advantage of the PROTAC approach is that it requires only a selective binder of the target protein (Nef in this case) and not a functional inhibitor per se,” they explained. In tests, the researchers confirmed that the resulting PROTAC drug candidates triggered Nef degradation, which suppressed HIV replication in target cells and showed signs that the immune response could be restored.

The PROTAC candidates “… demonstrate potent antiretroviral activity in HIV-infected primary cells, supporting further development for testing in the context of HIV-1 reservoir reduction in vivo,” the team stated. “These molecules may have clinical utility in restoring immune recognition of viral reservoir cells and preventing viral rebound following traditional antiretroviral drug withdrawal.”

“In general, the PROTAC approach has generated a great deal of excitement at drug companies, but they’ve been almost entirely targeted to proteins involved in cancers,” Smithgall said. “Our Nef-directed PROTACs are one of the first examples targeting infectious diseases. In theory, this approach should be applicable to proteins from other viruses that serve functions similar to HIV Nef.” The authors further suggested, “Targeted degradation using PROTACs is anticipated to reverse all Nef functions, potentially promoting latency reversal, restoration of adaptive immunity, suppression of viral replication and ultimately reservoir reduction … Targeted degradation of the HIV-1 Nef protein demonstrates the broader potential of antimicrobials based on PROTACs, expanding the landscape of microbial proteins for therapeutic exploitation.”

While Smithgall said he is excited for the potential of Nef PROTACs to someday treat HIV infections, he cautioned that several major steps are still required before they can be tested in people. “Finding a small molecule that bound selectively to Nef, that was the hardest part,” Smithgall noted. “Now we have to keep going with medicinal chemistry optimization and see how well it works against the HIV reservoir in an animal model.”

The Smithgall team will simultaneously pursue preclinical testing in animal models, mapping of the Nef signaling pathways to learn exactly how the PROTACs are working in cells and using advanced crystallography tools to determine the structure of the Nef protein in complex with the PROTAC molecule and the cellular machinery responsible for its degradation. “… more work is required to explore the effect of PROTAC treatment on the kinetics of Nef protein turnover in the context of HIV-infected primary cells as well as reversal of Nef-mediated phenotypic effects,” the team further noted in their paper.

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