The goal of this proposal is to develop a new strategy that combines experiment and theory for rapidly characterizing the binding modes and activity of small molecules that target flexible RNA receptors and to apply this strategy in the discovery of lead compounds that bind and disrupt the activity of two major HIV RNA drug targets. HIV/AIDS is currently the world's most urgent public health challenge. While antiretroviral therapies have significantly improved the prognosis for HIV infection, there is a growing need for antiviral agents that target new viral components and thereby further suppress the rate of replication and resistance. Genomes of all RNA viruses, such as HIV, contain essential RNA structures that can widen drug-targets making it possible to inhibit new steps in the viral life cycle for which we do not currently have druggable protein targets. Efforts to find small molecules that target regulatory RNAs have been hindered by lack of suitable methods for efficiently screening RNAs that lack the necessary readout enzymatic activity. Computational docking methods can, in principle, overcome many of the limitations inherent to experimental methods and can provide the structural and dynamical information needed to assess small molecule activity. However, current docking protocols fail to take into account the very large changes in structure that flexible RNA receptors typically undergo on binding small molecules. There is growing evidence that small molecules bind pre-existing RNA conformers from a dynamical ensemble;thus, if the structures of the unbound RNA dynamical ensembles could be determined at atomic resolution, a major obstacle to computational docking could be overcome. We propose to develop a new method, that combines NMR spectroscopy and enhanced computational MD simulations, to visualize at atomic resolution, unbound RNA structural ensembles in free and protein/metal bound states and to use virtual docking simulations to identity small molecules that bind distinct conformers in the ensemble. The new methodology we will be used to identify small molecules that disrupt (i) viral transcription elongation by targeting the transactivation response element (TAR) RNA and (ii) viral genome dimerization, packaging, and maturation by targeting the dimerization initation site (DIS) RNA. Computational predictions will be experimentally verified and complemented by in vitro NMR, florescence, and reporter gene assays as well as in vivo viral replication assays. This unique blend of experiment and theory will help establish a new paradigm for RNA- targeted HIV drug discovery.

Public Health Relevance

The proposed research will develop a novel hybrid experimental-computational method for rapidly identifying small molecules that bind and disrupt the functions of two essential regulatory RNA elements in the HIV genome. The approach will be used to significantly accelerate the pace of searching and testing for novel therapeutic agents that target new viral components and thereby further suppress the rate of HIV replication and resistance as well as help widen the treatment options available.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI066975-08
Application #
8237062
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Miller, Roger H
Project Start
2005-07-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
8
Fiscal Year
2012
Total Cost
$287,152
Indirect Cost
$72,586
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Bothe, Jameson R; Stein, Zachary W; Al-Hashimi, Hashim M (2014) Evaluating the uncertainty in exchange parameters determined from off-resonance R1? relaxation dispersion for systems in fast exchange. J Magn Reson 244:18-29
Yang, Shan; Salmon, Loïc; Al-Hashimi, Hashim M (2014) Measuring similarity between dynamic ensembles of biomolecules. Nat Methods 11:552-4
Mustoe, Anthony M; Brooks, Charles L; Al-Hashimi, Hashim M (2014) Hierarchy of RNA functional dynamics. Annu Rev Biochem 83:441-66
Sathyamoorthy, Bharathwaj; Lee, Janghyun; Kimsey, Isaac et al. (2014) Development and application of aromatic [(13)C, (1)H] SOFAST-HMQC NMR experiment for nucleic acids. J Biomol NMR 60:77-83
Lee, Janghyun; Dethoff, Elizabeth A; Al-Hashimi, Hashim M (2014) Invisible RNA state dynamically couples distant motifs. Proc Natl Acad Sci U S A 111:9485-90
Salmon, Loic; Yang, Shan; Al-Hashimi, Hashim M (2014) Advances in the determination of nucleic acid conformational ensembles. Annu Rev Phys Chem 65:293-316
Ravera, Enrico; Salmon, Loïc; Fragai, Marco et al. (2014) Insights into domain-domain motions in proteins and RNA from solution NMR. Acc Chem Res 47:3118-26
Dickson, Alex; Mustoe, Anthony M; Salmon, Loïc et al. (2014) Efficient in silico exploration of RNA interhelical conformations using Euler angles and WExplore. Nucleic Acids Res 42:12126-37
Salmon, Loic; Bascom, Gavin; Andricioaei, Ioan et al. (2013) A general method for constructing atomic-resolution RNA ensembles using NMR residual dipolar couplings: the basis for interhelical motions revealed. J Am Chem Soc 135:5457-66
Al-Hashimi, Hashim M (2013) NMR studies of nucleic acid dynamics. J Magn Reson 237:191-204

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