Bis-3-Chloropiperidines Targeting TAR RNA as A Novel Strategy to Impair the HIV-1 Nucleocapsid Protein

Abstract

Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem–bulge–loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs’ recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro.

In vitro Evaluation of Bis-3-chloropiperidines as RNA Modulators Targeting TAR and TAR-protein Interaction

“…In previous studies, we demonstrated that these compounds possessed the exquisite ability to form bifunctional crosslinks that bridged across opposing RNA strands 8 . We demonstrated also that the crosslinks produced by B-CeP 1 across key guanines in the TAR hairpin prevented both thermal and NC-mediated melting of the double-stranded stem 9 . We have now investigated the effects of lengthening the aliphatic linker of the B-CePs series 1 -3 by comparing the positions of the nucleotides involved in the bridging conjugates.…”

“…We further demonstrated the B-CePs' ability to cross-link base-paired regions within TAR RNA 9 , a stem-loop structure of great pharmacological relevance for the development of antiviral drugs. Follow-up studies aimed at elucidating the structure determinants of B-CePs activity towards RNA indicated that compound 1 -3, which are characterized by progressively longer alkyl linkers between reactive moieties (Figure 1A), were capable of stabilizing TAR secondary structure, as well as affecting the functions of TAR-processing proteins in vitro 9 . The fact that such effects were less prominent for the lysine ester compounds 4 and 5 (Figure 1A) implied that linker size and composition could be determinant in the formation of pertinent TAR-protein complexes.…”

“…At the same time, it has become clear that progress in the identification of new inhibitors is severely limited by the dearth of technologies and biophysical tools for the analysis and characterization of the interactions established by RNA, which still lag far behind those available for other types of biopolymers, most notably proteins. In this context, we have demonstrated that the concerted application of electrophoretic and mass spectrometry (MS)-based techniques is capable of supporting the characterization of multiple covalent modifications introduced by small molecules onto RNA, such as bis-3-chloropiperidines (B-CePs), and the elucidation of their inhibitory activities on specific RNA interactions [8][9] . B-CePs are a class of nucleic acid alkylating agents able to fast react with nucleophilic bases producing stable covalent adducts [8][9] .…”

“…In this context, we have demonstrated that the concerted application of electrophoretic and mass spectrometry (MS)-based techniques is capable of supporting the characterization of multiple covalent modifications introduced by small molecules onto RNA, such as bis-3-chloropiperidines (B-CePs), and the elucidation of their inhibitory activities on specific RNA interactions [8][9] . B-CePs are a class of nucleic acid alkylating agents able to fast react with nucleophilic bases producing stable covalent adducts [8][9] . The comprehensive characterization of reaction products obtained from selected RNA constructs revealed the formation of distinctive inter-molecular cross-links between guanines in double stranded sequences [8][9] , which had not been observed for any DNA construct previously examined [10][11][12][13] .…”

“…B-CePs are a class of nucleic acid alkylating agents able to fast react with nucleophilic bases producing stable covalent adducts [8][9] . The comprehensive characterization of reaction products obtained from selected RNA constructs revealed the formation of distinctive inter-molecular cross-links between guanines in double stranded sequences [8][9] , which had not been observed for any DNA construct previously examined [10][11][12][13] . We leveraged this unique feature to verify the location of guanines involved in long-range quaternary interactions in highly structured RNA systems, such as the kissing loop (KL) complex formed by two copies of the dimerization initiation site (DIS) of the HIV-1 genome 8 .…”