Development and characterization of HIV-1 Tat degraders

AbstractDespite 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, 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 discard small molecules
that are not Tat specific. The HTS of 369,203 compounds was completed by Southern Research (SR), yielding
three compounds, TT-44951, TT-44881 and TT-44863, with therapeutic index (TI) varying from 51.2 to 181.1
and good chemical properties. In this application, we propose to perform hit validation and characterization of
chemically synthesized analogs generated by Thimble Therapeutics, during the compound progression pathway
(CPP), to expand our panel of Tat inhibitors to commercialize this novel class of compounds. 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.Project Narrative: Development of novel Tat inhibitors
HIV transcription is regulated by the HIV Tat protein, but Tat inhibitors are not yet part of the clinical antiretroviral
ammunition despite their immense potential at shutting down particle production from infected cells and blocking
Tat toxicity. A high throughput drug screening of a total of 369,203 compounds was performed by Southern
Research yielding three strong leads. In collaboration with Thimble Therapeutics we now propose to validate
and characterize these compounds as well as improved chemically synthesized analogs generated during
compound progression pathway to expand our panel of Tat inhibitors for commercialization of this unique class
of compounds.