a Microarray-based approach for the development of RNA-binding small molecules with anti-HIV activity

FARE Award Winner

Authors

• FA Abulwerdi
• R van Duyne
• J Sztuba-Solinska
• EO Freed
• SFJ Le Grice
• JS Schneekloth Jr

Abstract

The HIV-1 trans-activation response (TAR) element RNA has long been considered a target for inhibiting virus replication, however most known TAR binders are highly charged compounds or aminoglycosides that lack selectivity. As part of an effort to find drug-like small molecules that bind TAR, we utilized a small molecule microarray screening approach. This work resulted in the identification of a novel thienopyridine scaffold that selectivity binds to TAR. Herein we report a 3-step synthetic route to generate a library of thirty analogs to improve potency and establish a structure-activity relationship. Analogs were evaluated in several complementary biochemical, biophysical and cell-based assays. Assessment of the analogs in a Thermofluor assay showed that the majority of analogs caused a 1-2o decrease in Tm of TAR indicating that binding of this class of compounds causes destabilization of TAR. In addition, evaluation of analogs in a fluorescence intensity assay as well as an AlphaScreen-based competitive assay between a Tat peptide and TAR provided binding affinity and potency for all analogs. Analogs with a 1,3,4-oxadiazole linker bind to TAR and disrupt Tat/TAR complex in mid and low micromolar range respectively. Furthermore, one of the most potent analogs, 116FA, was evaluated in a Tat-mediated HIV-1 LTR-driven luciferase reporter assay and preliminary results suggest inhibition of transcription upon treatment with 10 μM of 116FA. In summary, a novel class of small-molecule TAR binders has been development that binds and destabilizes TAR. The most potent analogs are also able to disrupt Tat/TAR interaction in vitro and in cells.

Scientific Focus Area: Chemical Biology

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