# GS143, an inhibitor of E3 ligase β-TrCP, reverses HIV-1 latency without activating T cells via unconventional activation of NFκB

## TL;DR

GS143 is a new drug that can awaken hidden HIV-1 without overactivating the immune system, offering a promising approach to eliminate the virus.

## Contribution

GS143 is a novel latency reversing agent that reactivates HIV-1 via unconventional NFκB activation without T cell activation.

## Key findings

- GS143 reactivates latent HIV-1 in primary cells and patient samples without T cell activation.
- GS143 activates NFκB p65 through a noncanonical pathway involving NIK and IKK.
- β-catenin supports NF-κB output indirectly, as its inhibitors suppress GS143's reactivation effect.

## Abstract

HIV-1 persists indefinitely in individuals living with HIV-1 even after effective treatment with antiretroviral therapy (ART). Upon cessation of the therapy, latently infected memory CD4+ T cells allow for a rapid rebound of the virus. The development of latency reversing agents (LRAs) to activate latent virus promoting immune recognition and clearance of the infected cells is pivotal for the elimination of the latent arm of the infection. Success of this strategy requires the development of potent highly specific LRAs with fewer off-target effects. LRA activity displayed by proteasome inhibitors although not highly specific opens the possibility of exploiting the high degree of specificity of the ubiquitin-proteasome system to develop targeted LRAs. Here we demonstrate that a small molecule GS143, which inhibits β-TrCP, the substrate recognition subunit of the SCFβ-TrCP E3 ubiquitin protein ligases, exhibits potent LRA activity both in a primary cell model system of latency and cells from aviremic individuals with HIV-1 treated with ART. Furthermore, GS143 reactivates latent HIV-1 without activating T cells, a desirable attribute for LRAs of clinical use. We showed that GS143 acts in a complementary fashion with at least two other classes of LRAs, thereby representing novel drug combinations for targeting HIV-1 latency. Finally, our results suggest that GS143 triggers a novel signaling pathway to reactivate latent HIV-1 that leads to the unconventional activation of NFκB p65, by initiating the noncanonical signaling via NIK, followed by activation of IKK leading to phosphorylation of p65 on S536 and its nuclear translocation. Moreover, we show that β-catenin inhibitors suppress reactivation HIV-1 by GS143, suggesting that β-catenin supports NF-κB output indirectly. Overall, our results suggest that the β-TrCP E3 ligase inhibitor GS143 represents a new type of LRA.

A major barrier to a cure for HIV-1 is the ability of the virus to hide for long periods of time in individuals on antiretroviral therapy. A possible strategy to get rid of latently infected cells is to find drugs that can turn on expression of HIV-1 proteins, leading to destruction of the infected cells by the virus or the immune system, while continued antiviral therapy would prevent new infections. To date, many compounds that can reverse HIV-1 latency lack a high degree of specificity. Compounds that could do so with a high degree of precision would be highly desirable for clinical use, as they would likely have fewer off-target effects and be less toxic than other LRAs. We found that GS143, an inhibitor of a specific protein degradation pathway (ubiquitin proteasome E3 ligase) strongly reactivates latent HIV-1 in primary cells and in patient samples without activating T cells. Furthermore, we show that GS143 reactivates latent HIV-1 via the unconventional activation of the NFκB signaling pathway. Thus, our study provides new insight into the function and mechanism of a new class of latency reversing agent and justifies investigation of GS143 as a possible candidate for further drug development.