# Chemical Biology of HIV-1 Nef

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $752,696

## Abstract

Abstract. Existing antiretroviral drugs do not clear HIV-1 from infected individuals, requiring life-long administra-
tion. The HIV-1 Nef accessory factor is an attractive target for drug development because it supports HIV repli-
cation in vivo and promotes immune escape of HIV-infected cells. Our laboratory has discovered small molecules
that bind directly to Nef and inhibit its ability to enhance HIV-1 replication in cell culture. These inhibitors interfere
with Nef-mediated MHC-I downregulation in latently infected CD4 T cells, enabling recognition and killing by
CTLs. Thus, Nef inhibitors represent a new approach to antiretroviral therapy that may promote eradication of
viral reservoirs. During the previous funding cycle, we developed Proteolysis Targeting Chimeras (PROTACs)
for the targeted destruction of the Nef protein in HIV-infected cells. These PROTAC molecules induce ternary
complexes between Nef and the Cereblon E3 ubiquitin ligase resulting in Nef ubiquitylation and proteolytic deg-
radation. Nef PROTACs efficiently reverse Nef-mediated downregulation of MHC-I and CD4 from the T cell sur-
face and inhibit Nef-mediated enhancement of HIV-1 replication in donor PBMCs. PROTAC-mediated degra-
dation is likely to reverse all Nef functions, suppressing viral rebound while enhancing antiviral immunity
and reservoir reduction. Here we propose to explore the mechanism of action and on-target selectivity of our
Nef-directed PROTACs, their propensity for inducing acquired resistance, and their ability to enhance cellular
antiviral immunity. Our proposal is responsive to NOT-AI-23-049, Using Targeted Degradation of Protein and
non-Protein Targets for the Development of Novel Anti-Infectives, and PAR-23-297, Opportunities for HIV Cure
Strategies at the Time of ART Initiation. The following Specific Aims are proposed: 1) Determine the mecha-
nism of action of Nef-directed PROTACs. This Aim will identify the Nef lysine residues required for the initiation
of ubiquitylation by the PROTACs. In addition, PROTAC selectivity for Nef in T cells will be evaluated by prote-
omics. 2) Test the hypothesis the Nef PROTACs are unlikely to induce drug resistance in HIV-infected
cells. PBMCs will be infected with HIV-1 carrying a Nef mutant library in the presence or absence of ‘occupancy-
based’ Nef inhibitors vs. PROTACs. Resistance mutations will be identified in emergent viruses by RNAseq.
Targeted degraders may be less likely to generate resistance compared to their occupancy-based counterparts,
because elimination of Nef may make infected cells susceptible to apoptosis. 3) Test the hypothesis that tar-
geted degradation of HIV-1 Nef affects latent viral reservoirs. Nef-mediated downregulation of MHC-I and
induction of PD-1 both may serve to promote and stabilize HIV reservoirs. This Aim will examine reservoir cells
from HIV-infected donors to determine whether targeted degradation of Nef influences latency reversal and in-
creases the susceptibility of re...

## Key facts

- **NIH application ID:** 11013977
- **Project number:** 2R01AI152677-05
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Thomas E. Smithgall
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $752,696
- **Award type:** 2
- **Project period:** 2020-09-01 → 2029-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11013977

## Citation

> US National Institutes of Health, RePORTER application 11013977, Chemical Biology of HIV-1 Nef (2R01AI152677-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/11013977. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*