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Targeting Alternative Conformational States of the HIV-1 Rev Response Element Stem IIB using Small Molecules

dc.contributor.advisor Al-Hashimi, Hashim M. CHU, Chia-chieh 2020-06-09T17:59:06Z 2022-05-27T08:17:12Z 2020
dc.description Dissertation
dc.description.abstract <p>RNAs are growing in their importance as regulators in multiple biological processes. A deep understanding of how RNAs function within cells requires an understanding of their dynamic behavior to enable the targeting of RNA in drug discovery as therapeutics. The HIV-1 Rev response element (RRE) RNA which is a known drug target mediates the export of incompletely-spliced viral RNA to express viral proteins required for HIV-1 replication and spread. Rev protein first recognizes the purine-rich region on RRE stem IIB (RREIIB), and then other Rev monomers cooperatively assemble on RRE to form ribonucleoprotein complex. Conformational flexibility at this stem IIB region has been shown to be important for Rev binding. In addition, m6A modifications on HIV-1 RNA has shown their critical roles to HIV-1 replication, and two probably essential m6A sites on purine-rich region on RREIIB was discovered. However, the nature of the flexibility and m6A modification to RREIIB have remained elusive. The work in this thesis is aiming to characterize the conformational dynamics of RRE stem IIB as potential drug targets, and to discover small molecules binding to RRE using computational and experimental drug screening. </p><p>First, nuclear magnetic resonance (NMR) techniques are applied to identify the native and non-native conformations of RREIIB. Including relaxation dispersion NMR and a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger stem II three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of around 20%. The ESs disrupt the Rev binding site by changing local secondary structure and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15 to 80 fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play important roles in the cooperative Rev binding and the development of anti-HIV therapeutics. </p><p>Secondly, m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the RREIIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. We show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev-ARM using optical melting experiments, NMR spectroscopy, and in vitro binding assays. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. </p><p>Next, to confirm the RRE ESs ensemble visualized in vitro can recapitulate in cells, we show that stabilizing ESs using point substitution mutations leads to potent conformation-dependent inhibition of RNA cellular activity. The point substitution mutations with invert the equilibrium so that the ES becomes the dominant (>50% population) conformation and secondary rescue mutations to restore the GS conformation and control for sequence effects. We then demonstrate that the degree to which increasing the population of the ES at the expense of the GS leads to a corresponding decrease in cellular activity. The results also support that stabilizing non-native ESs potentially provides an alternative therapeutic strategy for targeting RNA. </p><p>Finally, we construct atomic resolution ensembles for the RRE ground state using RDC-SAS; perform computational docking against the ensemble of RRE GS ,and validate selected hits using in vitro and cell-based assays. We will also generate the dynamic ensembles of RRE ESs as alternative targets for ensemble-based virtual screening. The final goal is to identify small molecules that stabilize RRE GS or inactive ESs and thereby inhibit Rev-RRE interaction.</p>
dc.subject Biochemistry
dc.subject dynamic ensemble
dc.subject Rev response element
dc.subject RNA drug discovery
dc.subject RNA target
dc.subject RRE
dc.subject virtual screening
dc.title Targeting Alternative Conformational States of the HIV-1 Rev Response Element Stem IIB using Small Molecules
dc.type Dissertation
dc.department Biochemistry
duke.embargo.months 23.572602739726026

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