# Identification of HIV Rev-Rev interaction inhibitors by a high throughput, small molecule, cell-free screen

> **NIH NIH R21** · YALE UNIVERSITY · 2024 · $188,438

## Abstract

The HIV pandemic has grown to ~40 million individuals world-wide. Although Highly Active Anti-
Retroviral Therapy (HAART) has been transformative for millions of people, less than half of those infected are
on therapy. And while the current medications are typically well-tolerated and efficacious, because of both
acute and chronic drug toxicities, drug-drug interactions, and virus resistance, there is always room for novel
HIV therapeutics, especially for those considered first-in-class. Rev is an essential regulatory protein of the
virus, which multimerizes on the Rev-response element or RRE to export intron-containing HIV mRNAs from
the nucleus to the cytoplasm. Although there are some compounds in clinical trials that purportedly interfere
with Rev function, there are no FDA-approved agents that specifically target Rev’s known mechanism of
action. Notably, an anti-Rev compound should reduce the amount of both cytoplasmic and virion-associated
unspliced or genomic RNA and also plasma viral loads in the absence of viral replication, an activity that none
of the currently FDA-approved drugs possesses. We have developed a cell-based firefly luciferase (FFLUC)
complementation system that quantifies Rev-Rev interaction but then transitioned over to a cell-free one that
has an excellent Z’ (Z factor) of 0.64 in 384-well format. Here we first wish to further optimize the assay to then
permit testing of compounds in 384-well format, in collaboration with Yale Center for Molecular Discovery
(YCMD), which has multiple collections of compounds and requisite instrumentation to allow fully automated
testing of ~125,000 small molecules. These compounds will initially be screened at a single concentration, and
positives will be retested at a range of concentrations, in replicates. Secondary assays for specificity of
inhibition of Rev-Rev interaction include non-inhibition of FFLUC activity and transcription, non-inhibition of
unrelated protein-protein interaction, and inhibition of Rev function using a variety of assays, including cell-free
multimerization, production of Gag, and both X4 and R5 HIV replication in vitro. Therapeutic index (TI) of any
hits will be determined using a cell-based assay. Based upon these results, the physicochemical properties of
any hits will be assessed and in silico hit expansion along with any necessary synthesis will be achieved by the
commercial entity Life Chemicals. These select hit compounds will be retested in the above assays in order to
identify even more potent and active drug-like compounds. At the end of two years it is hoped that a number of
advanced hit and close-to-lead compounds will have been identified that have high TI (>100) and specifically
prevent Rev-Rev interaction and thus interfere with known Rev function, which will allow future medicinal
chemistry testing and iterative in vitro and in vivo studies to identify lead compounds of enhanced potency,
activity, and specificity. Thus, a long-term goal of this...

## Key facts

- **NIH application ID:** 10923198
- **Project number:** 1R21AI181613-01A1
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Richard Sutton
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $188,438
- **Award type:** 1
- **Project period:** 2024-08-07 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10923198

## Citation

> US National Institutes of Health, RePORTER application 10923198, Identification of HIV Rev-Rev interaction inhibitors by a high throughput, small molecule, cell-free screen (1R21AI181613-01A1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10923198. Licensed CC0.

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