Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1.
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 active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, 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 and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each 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-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.
DeJong
Eric S
ES
Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and the National Institute for Standards and Technology, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Chang
Chia-en
CE
Gilson
Michael K
MK
Marino
John P
JP
D015075:2-Aminopurine / Q000737:chemistry; D000891:Anti-Infective Agents, Local / Q000494:pharmacology*; D000963:Antimetabolites / Q000737:chemistry; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000235:genetics; D019281:Dimerization; D005453:Fluorescence; D015694:Gene Products, rev / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015752:Genes, env / Q000187:drug effects*; D015497:HIV-1; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D011370:Proflavine / Q000494:pharmacology*; D011485:Protein Binding / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D054321:rev Gene Products, Human Immunodeficiency Virus
D000891:Anti-Infective Agents, Local; D000963:Antimetabolites; D015694:Gene Products, rev; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D015075:2-Aminopurine; D011370:Proflavine