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Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble

Abstract

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (Ki values of 710 nM–169 μM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC50 23.1 μM).

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Figure 1: Virtually screening RNA dynamic structure ensemble.
Figure 2: Analysis of small-molecule binding specificity.
Figure 3: NMR site-specific characterization of TAR-small molecule binding modes.
Figure 4: Netilmicin specifically inhibits Tat-mediated transactivation and HIV-1 replication.

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Acknowledgements

We thank A.V. Kurochkin for NMR expertise, and we thank the Michigan Economic Development Cooperation and the Michigan Technology Tri-Corridor for support of the purchase of a 600-MHz spectrometer. This work was supported by the US National Institutes of Health (R01 AI066975-01 and R01 CA144043), the US National Science Foundation (NSF Career Award CHE-0918817) and an NSF Graduate Research Fellowship for A.C.S. and A.T.F.

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Contributions

H.M.A.-H. and A.C.S. conceived the docking approach; A.T.F., I.A., A.C.S. and H.M.A.-H. developed the approach for constructing RNA dynamic structure ensembles; A.C.S., with input from A.T.F. and J.D.K., performed the docking simulations; A.C.S., J.D.K. and J.L. preformed the in vitro fluorescence assays and NMR experiments; M.D.S., M.J.G.-H and D.M.M. carried out the transfection and viral-replication assays; H.M.A.-H., A.C.S., A.T.F., J.D.K., I.A. and D.M.M. wrote the paper.

Corresponding author

Correspondence to Hashim M Al-Hashimi.

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Competing interests

H.M.A.-H. is an advisor to and holds an ownership interest in Nymirum, an RNA-based drug-discovery company. A.C.S. completed this work as part of his dissertation and is now employed by Nymirum. The research reported in this article was performed by the University of Michigan faculty and students and was funded by a US National Institutes of Health contract to H.M.A.-H.

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Stelzer, A., Frank, A., Kratz, J. et al. Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble. Nat Chem Biol 7, 553–559 (2011). https://doi.org/10.1038/nchembio.596

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