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Nef is an accessory protein encoded by HIV-1, HIV-2, and other primate lentiviruses and is closely related to viral replication, persistent infection, and the development of AIDS. Nef interacts with a variety of host cell proteins and downregulates cell surface molecules such as CD4, MHC-I, and SERINC5 restriction factors, thereby enhancing viral infectivity and promoting immune evasion. In addition, Nef promotes viral transcription and release by binding to and activating non-receptor protein tyrosine kinases and proteins that regulate the actin cytoskeleton. Studies in non-human primates have shown that Nef is closely related to viral pathogenicity and the development of AIDS. Nef-deficient SIV has poor replication ability in rhesus monkeys and can delay the onset of disease. In addition, in individuals infected with Nef-deficient HIV-1, viral loads can be maintained at low levels even without antiretroviral administration. Nef is an attractive antiretroviral drug target because of its critical role in HIV pathogenesis.

However, Nef lacks active sites, making it difficult to adopt traditional strategies based on active site binding for drug development. Recently, the team of Professor Thomas E. Smithgall of the University of Pittsburgh published a research paper titled “PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication” in Cell Chemical Biology, innovatively proposed a Nef-targeting PROTAC strategy, and the designed and synthesized Nef-targeting PROTAC demonstrated therapeutic effects beyond traditional antiretroviral drugs.

Figure 1: Targeted degradation of HIV-1 Nef by CRBN-directed PROTAC.1

In this study, the researchers coupled existing hydroxypyrazole Nef-binding compounds with the CRBN/VHL ligand of the ubiquitin E3 ligase through a flexible linker to construct a bivalent PROTAC compound library. Through cell-based orthogonal experiments to detect Nef ubiquitination, degradation, and inhibitory functions, we discovered a highly active Nef-targeting PROTAC that directs the CRBN E3 ubiquitin ligase pathway.

SPR assay results show that this type of bivalent PROTAC can induce multiple HIV Nef variants and SIV Nef to form a ternary complex with ubiquitin E3 ligase, with broad-spectrum activity. Flow cytometry and quantitative western blot analysis experiments showed that Nef-targeted PROTAC can induce Nef degradation, effectively reverse Nef-mediated down-regulation of MHC-I and CD4, and restore receptors that are critical for immune system recognition of HIV-infected cells. Cell surface expression. Furthermore, because there is a significant correlation between Nef’s ability to downregulate MHC-I and the size of viral reservoirs, targeting Nef protein degradation would also help shrink or eliminate viral reservoirs. Further antiviral test results showed that Nef-targeted PROTAC can effectively inhibit Nef-dependent viral replication and reduce infectivity.

Overall, this study demonstrates the excellent effectiveness of PROTAC in targeting the degradation of HIV-1 Nef protein. By targeting the degradation of Nef, PROTAC not only restores the expression of MHC-I and CD4 on the surface of T cells but also significantly inhibits HIV-1 replication and is expected to enhance host immunity and clear viral reservoirs. This strategy provides new ideas for HIV/AIDS treatment and also shows the strong potential of PROTAC technology in the field of antiviral drug development.

Here is a simple list of ideal targets for PROTAC® technology for your reference.

Reference

Emert-Sedlak, Lori A., et al. “PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication.” Cell Chemical Biology 31.4 (2024): 658-668.