Structure‐Based Design of an RNA‐Binding p‐Terphenylene Scaffold that Inhibits HIV‐1 Rev Protein Function

Abstract

Numerous antibiotics bind to ribosomal RNA, and many functional RNA motifs have considerable therapeutic potential. However, the development of small RNA-binding agents has been hampered by the difficulties posed by these structures, which have limited physicochemical diversity and are often flexible.[1] In order for this approach to be successful, it is essential to identify new chemical scaffolds recognizing RNA.The Rev response element (RRE) is a strongly conserved 350-nucleotide structure located in the env gene of human immunodeficiency virus type-1 (HIV-1) RNA. Within subdomain IIB of the RRE, the unusually widened major groove of a 4:6 internal loop forms a high-affinity complex [2] with the arginine-rich a-helix of Rev, a virally encoded 116 amino acid protein that adopts a helix-turn-helix conformation [3] ( Figure 1). This interaction between internal loop IIB and the RNA-binding a-helix of Rev (Rev 34-50 ) is essential for virus viability because it triggers a cascade of events that allow the transport of unspliced or incompletely spliced viral RNA molecules into the cytoplasm of the infected cell in the late phase of the viral infectious cycle. These events include the cooperative incorporation of additional Rev molecules into the complex through interactions with further sites on the RRE and protein-protein contacts, [4] and the tethering of the RRE-Rev ribonucleoprotein to the Crm1 host export factor. In addition to RNA nuclear export, Rev has been shown to enhance translation and packaging [5] and to control the nucleocytoplasmic shuttling of the HIV-1 integrase.[6] Clearly, this protein represents a pivotal target for HIV-1 therapy, but to date, the development of Rev-based inhibitors has remained an elusive goal. Herein we report the structurebased design of new RNA-binding p-terphenyl Rev mimics that inhibit RRE-Rev function and HIV-1 replication.We aimed to generate organic ligands that mimic the three-dimensional distribution of the side chains of Rev 34-50 complexed with internal loop IIB of the RRE [2] (Figure 1 a). Some reports had shown that tris-3,2',2''-substituted p-ter- Figure 1. Structure-based design of RNA-binding p-terphenyls. a) View of the complex formed by internal loop IIB of the RRE and Rev 34-50 . The a-helix (red) is deeply embedded in its RNA receptor (gray). b) Representation of a bilaterally substituted p-terphenyl molecule (green) superposed on an a-helix (red). c) Chemical structure and synthesis strategy of a 3,5,2',6',2'',6''-substituted p-terphenyl molecule. d) Secondary structures of the RNA oligonucleotides utilized in this study:

Substituents Have a Large Effect on Photochemical Generation of Benzyl Cations and DNA Cross‐Linking

“…1 HNMR (300 MHz, CDCl 3 ): d = 7.48-7.34 (m, 3H), 7.20-7.17 (d, J = 9.0 Hz, 2H), 3.87 (s, 3H), 2.08 ppm (s, 6H)( the NMR spectra were in agreement with those reported). [21] 1,3-Bis(bromomethyl)-5-methoxybenzene (3 a):Asolution of 13 (1.0 g, 5.95 mmol) in DCM (40 mL) was cooled to 0 8Cu nder argon, then phosphorus tribromide (3.54 g, 13.09 mmol) was added at 0 8Cu nder argon. Solvent was removed and the residue was purified by column chromatography (hexane/DCM 04, 137.97, 136.56, 134.31, 129.88, 128.37, 127.93, 115.98, 55.48, 31.91 to reflux for 2h,t hen as econd portion of N-bromosuccinimide (0.33 g, 1.86 mmol) and azobisisobutyronitrile (14.5 mg, 0.089 mmol) were added and reflux was continued for another 0.5 h. Solvent was removed, and the residue was extracted with ethyl acetate (3 30 mL), washed with brine, and dried over anhydrous Na 2 SO 4 .S olvent was removed, and the residue was purified by column chromatography (hexane/ethyl acetate = 25:1, R f = 0.4) to obtain 4a as aw hite solid (1.2 g, 3.25 mmol);m .p.…”

“…Methylation of 6 with MeI provided 2,5-dimethoxy-1,3-dimethylbenzene (7), which was convertedi nto 1a by bromination with N-bromosuccinimide (NBS) and azobisisobutyronitrile (AIBN) (Scheme 2A). [21] Bromination of 13 using PBr 3 afforded 3a. Direct bromination of 8 using NBS/AIBN provided 4a (Scheme 2B).…”

The Design of RNA Binders: Targeting the HIV Replication Cycle as a Case Study

“…Although this result is consistent with an inhibition of Rev-RRE interaction, an effect on HIV transcription cannot be ruled out, since compound 26 was also reported to have some affinity for DNA. [82] SL1: The Dimerization Initiation Site Like all retroviruses, HIV-1 encapsidates a dimer of two homologous single stranded (+) RNA that are noncovalently held close to their 5'-ends. Dimerization of the viral genomic RNA is a key step in HIV-1 replication.…”

Nucleic acid recognition and antiviral activity of 1,4-substituted terphenyl compounds mimicking all faces of the HIV-1 Rev protein positively-charged α-helix

“…MT-2 cells were transfected with plasmids containing a luciferase reporter gene whose expression was under the control of the full length proviral HIV-1 (NL4.3-luc), the HIV-1 LTR promoter (pLTR-luc), or the HTLV-1 LTR promoter (pLTR(HTLV)-luc). After transfection, cells were treated with different compound concentrations, and activity quantification was performed 48 h later by determining luciferase activity in cell lysates as described 7,21,22 . 50% effective (EC 50 ) concentrations were calculated with Prism using log(inhibitor) vs response non-linear regression analyses, and the results represent the average of at least three independent experiments.Analysis of HIV-1 RNA splicing.…”

“…and opening up the possibility of mimicking interactions in which the helix is deeply embedded in its receptor ( Fig. 1) 7 . One such interaction is formed between the RNA-binding α-helix of the HIV-1 protein Rev and the virus RNA.…”