Applications of Surface Plasmon Resonance (SPR) in HIV-1 Nef Inhibitor Discovery and PROTAC Development

The HIV-1 Nef accessory factor promotes viral replication and immune escape of HIV-infected cells, making it an attractive drug target. Our team previously identified small molecules that bind directly to Nef and suppress HIV-1 replication in vitro. These compounds also reverse Nef-mediated MHC-I downregulation in latently infected CD4+ T cells, enabling recognition and killing by autologous cytotoxic T lymphocytes in co-culture. However, Nef lacks enzymatic activity and an active site, working instead through protein-protein interactions with diverse host cell partners. These interactions involve multiple Nef surfaces, complicating medicinal chemistry optimization of occupancy-based drug candidates. To address this issue, we initiated PROTAC development for the targeted degradation of Nef. Using a cell-based BRET assay, we identified 12 PROTAC analogs that significantly enhanced Nef ubiquitylation, all of which were coupled to ligands for the Cereblon (CRBN) E3 ubiquitin ligase. Six of these analogs restored cell surface CD4 and MHC-I expression in T cells by more than 50% while inducing a proportional reduction in Nef levels, with the most active analog yielding a DC50 value of 160 nM. Using SPR, we demonstrated direct binding of the active analogs to eight HIV-1 Nef variants representative of diverse M-group subtypes, SIV Nef, and the CRBN thalidomide binding domain (TBD) with KD values in the nM to low µM range, supporting broad spectrum activity against HIV-1. Size exclusion chromatography showed that active Nef PROTACs induce ternary complex formation with Nef and the CRBN TBD. Finally, Nef PROTACs suppressed Nef-mediated enhancement of HIV-1 infectivity in TZM-bl reporter cells and HIV-1 replication in donor PBMCs without cytotoxicity. These results support the hypothesis that PROTAC-mediated Nef degradation will reverse all Nef functions, promoting latency reversal, restoration of adaptive immunity, suppression of viral rebound, and reservoir reduction.

 

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