Proflavine Acts as a Rev Inhibitor by Targeting the High-Affinity Rev Binding Site of the Rev Responsive Element of HIV-1
详细信息 查看全文
- 作者：Eric S. DeJong ; Chia-en Chang ; Michael K. Gilson ; and John P. Marino
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：July 8, 2003
- 年：2003
- 卷：42
- 期：26
- 页码：8035 - 8046
- 全文大小：189K
- 年卷期：v.42,no.26(July 8, 2003)
- ISSN：1520-4995
文摘
Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE)within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and activeviral replication. Previously, we have shown that selective incorporation of the fluorescent probe2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of anarginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescencemethods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has beenidentified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct andcompetitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine bindingsites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (KD ~0.1 ± 0.05 
M) and a weaker binding site(s) (KD ~ 1.1 ± 0.05 M). Titrations of RRE-IIB with proflavine,monitored using 1H NMR, demonstrate that the high-affinity proflavine binding interaction occurs witha 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1proflavine·RRE-IIB complex indicate that the two proflavine molecules bind specifically and close toeach other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine·RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIBin a manner analogous to what has been observed in the Rev peptide·RRE-IIB complex. The observationthat proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinitysite opens the possibility for rational drug design based on linking and modifying it and related compounds.
M) and a weaker binding site(s) (KD ~ 1.1 ± 0.05 M). Titrations of RRE-IIB with proflavine,monitored using 1H NMR, demonstrate that the high-affinity proflavine binding interaction occurs witha 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1proflavine·RRE-IIB complex indicate that the two proflavine molecules bind specifically and close toeach other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine·RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIBin a manner analogous to what has been observed in the Rev peptide·RRE-IIB complex. The observationthat proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinitysite opens the possibility for rational drug design based on linking and modifying it and related compounds.