Despite effective antiretroviral therapy (ART), latent proviruses can reinitiate viral production upon cell
stimulation or treatment interruption. The viral Tat protein enhances transcript elongation from the HIV-1
promoter, controlling the switch between latency and active viral production. The block-and-lock functional
cure aims at the transcriptional silencing of the viral reservoir rendering suppressed HIV promoters extremely
difficult to reactivate from latency. The Tat inhibitor, didehydro-cortistatin A (dCA) was used to prove this
concept. Combining dCA with ART, inhibits transcription and blocks viral rebound upon treatment interruption,
as the promoter becomes epigenetically repressed. dCA defines a novel class of drugs that can silence and
maintain a transcriptionally inactive HIV promoter, offering a novel approach in the treatment of HIV.
Tat is very attractive target for therapeutic intervention because: 1) is expressed early during virus replication;
2) no cellular homologs; 3) Tat inhibitors block the feedback loop necessary for viral amplification; 4) epigenetic
modifications accumulate at the HIV promoter rendering reactivation less likely. Tat is also known for its role
in neurotoxicity, neurotransmitter modulation, oxidative stress, apoptosis, blood brain barrier disruption, and
neuroinflammation. Thus, the immense interest in the development of Tat inhibitors to complement ART.
The major hurdle towards advancing dCA into clinical trials is the cost of producing large quantities of this
molecule, due to its complex structure. Additional clinical candidates, structurally distinct from dCA, that
embody equivalent bioactivity are needed in the pre-clinical pipeline.
We optimized a cell-based Tat transactivation assay to use in high throughput screening (HTS), with dCA as
control. We combined appropriate counter-screens and a wealth of techniques to quickly ‘weed out’ small
molecules that are not Tat specific. The HTS of 210,240 compounds was completed by Southern Research
(SR), yielding two compounds, SRI-43627 and SRI-43050 with a selectivity index >10 that were further
investigated. This initial success prompted the screen of an additional 369,203 compounds, yielding upon
counter screen 29 hits to be further evaluated. In this application, we propose to perform hit validation and
characterization of these compounds as well as analogs synthesized by SR as part of drug development during
the compound progression pathway. We propose the following aims:
Specific Aim 1. Validate Tat inhibitors based on disruption of Tat HIV-1 LTR transactivation.
Specific Aim 2. Characterize the mechanism of action of selected hits.
At the end of this study we expect to (a) have identified small molecules that will specifically inhibit
Tat in cell-based assays. (b) have adequate metabolic stability and PK properties for future
pharmacological assessment in animal models and eventually in human clinical trials.