Performance of Rilpivirine-Based Hydrophobic Tags and PROTACs Directed Against HIV-1 Reverse Transcriptase

Abstract

We aimed to repurpose the non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI) rilpivirine (RPV) as a targeted protein degrader (TPD) of HIV-1 RT. Structure-guided modeling identified a TPD bifunctional linker exit trajectory from RPV that threads the NNRTI entrance channel. Ten RPV analogs (RPV') with varied warhead-linker attachments retained RT inhibition and antiviral activity, guiding selection of an amide-linked connector for degrader construction. We synthesized Proteolysis Targeting Chimeras (PROTACs) designed to recruit CUL4CRBN and CUL2VHL, alongside adamantyl acetic acid-based Hydrophobic Tags (HyTs). TPDs were evaluated for RT inhibition, virus inhibition, biophysical target engagement, and proteasome-dependent degradation. Some PROTACs showed minimal antiviral activity or limited solubility for our assays, whereas the HyT with a tetraethylene glycol (PEG4) linker (PEG4-Ad) exhibited low nanomolar potency to suppress HIV-1 replication and acceptable solubility. PEG4-Ad reduced RT levels in a proteasome-dependent manner without cytotoxicity but did not exhibit superior potency against RPV resistance mutations. MD simulations suggested that PEG4 linkers maximize episodic exposure of the hydrophobic tag, consistent with PEG4-Ad efficacy. These data highlight the promise - and constraints - of antiviral degraders, indicating that linker-controlled hydrophobic tag exposure and subcellular target accessibility may be critical design parameters for prospective therapeutics.

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BibTeX RIS

BibTeX

@misc{amanor2026performance,
  title = {{Performance of Rilpivirine-Based Hydrophobic Tags and PROTACs Directed Against HIV-1 Reverse Transcriptase}},
  author = {F. K. Amanor and G. D. Clements and R. Khurshid and A. F. Howard and D. M. Soto-Martinez and C. Barkley and Z. Yang and K. Gyawali and J. C. Kappes and R. C. Reynolds and S. C. Schürer and T. S. Snowden and C. Ochsenbauer},
  howpublished = {bioRxiv},
  year = {2026},
  month = sep,
  doi = {10.64898/2026.09.24.753933},
  url = {https://www.biorxiv.org/content/10.64898/2026.09.24.753933v1},
  note = {CC BY-NC-ND},
}

RIS

TY  - UNPB
TI  - Performance of Rilpivirine-Based Hydrophobic Tags and PROTACs Directed Against HIV-1 Reverse Transcriptase
AU  - F. K. Amanor
AU  - G. D. Clements
AU  - R. Khurshid
AU  - A. F. Howard
AU  - D. M. Soto-Martinez
AU  - C. Barkley
AU  - Z. Yang
AU  - K. Gyawali
AU  - J. C. Kappes
AU  - R. C. Reynolds
AU  - S. C. Schürer
AU  - T. S. Snowden
AU  - C. Ochsenbauer
PY  - 2026
DA  - 2026/09/24
PB  - bioRxiv
DO  - 10.64898/2026.09.24.753933
UR  - https://www.biorxiv.org/content/10.64898/2026.09.24.753933v1
AB  - We aimed to repurpose the non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI) rilpivirine (RPV) as a targeted protein degrader (TPD) of HIV-1 RT. Structure-guided modeling identified a TPD bifunctional linker exit trajectory from RPV that threads the NNRTI entrance channel. Ten RPV analogs (RPV') with varied warhead-linker attachments retained RT inhibition and antiviral activity, guiding selection of an amide-linked connector for degrader construction. We synthesized Proteolysis Targeting Chimeras (PROTACs) designed to recruit CUL4CRBN and CUL2VHL, alongside adamantyl acetic acid-based Hydrophobic Tags (HyTs). TPDs were evaluated for RT inhibition, virus inhibition, biophysical target engagement, and proteasome-dependent degradation. Some PROTACs showed minimal antiviral activity or limited solubility for our assays, whereas the HyT with a tetraethylene glycol (PEG4) linker (PEG4-Ad) exhibited low nanomolar potency to suppress HIV-1 replication and acceptable solubility. PEG4-Ad reduced RT levels in a proteasome-dependent manner without cytotoxicity but did not exhibit superior potency against RPV resistance mutations. MD simulations suggested that PEG4 linkers maximize episodic exposure of the hydrophobic tag, consistent with PEG4-Ad efficacy. These data highlight the promise - and constraints - of antiviral degraders, indicating that linker-controlled hydrophobic tag exposure and subcellular target accessibility may be critical design parameters for prospective therapeutics.
ER  -

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