Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates

Abstract

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA.

NCp7: targeting a multitask protein for next-generation anti-HIV drug development part 2. Noncovalent inhibitors and nucleic acid binders

“…Gatto and colleagues reported a series of anthraquinones symmetrically substituted with aminoacidic side-chains containing terminal amino groups (compounds 29-31 Fig. 4) [54][55][56]. The aromatic scaffold is meant to stack with nucleobases whereas the cationic side-chain can serve to stabilize the interaction via salt bridges with the phosphodiester backbone.…”

Retroviral nucleocapsid proteins and DNA strand transfers

“…However, actinomycin D is not a potent anti-HIV-1 agent in ex vivo cell assays [144] . More recently, 2,6-disubstituted-anthraquinones targeting the TAR RNA and cTAR DNA hairpins have been developed [145] , [146] . These compounds are threading intercalators bearing a polyaromatic nucleus di-substituted at opposite positions with cationic side-chains that stabilize the interaction with the phosphodiester backbone.…”

“…By stabilizing the secondary structures of TAR and cTAR, the anthraquinone derivatives inhibit the NC-mediated TAR-cTAR annealing process. In addition, anthraquinone derivatives with long cationic side-chains compete with NC for the same binding sites on TAR [146] . The antiviral activity of anthraquinone derivatives in ex vivo cell assays has not been reported.…”

Synthesis and Evaluation of Bifunctional Aminothiazoles as Antiretrovirals Targeting the HIV-1 Nucleocapsid Protein

“…1 To date, pharmacological modulation of the NC has been achieved by means of three different strategies: (i) small molecules able to promote the ejection of zinc from the NC and to induce protein unfolding, which are considered rather toxic and poorly specific although some of them have been recently profiled in preclinical studies; 2 (ii) noncovalent NC binders that inhibit the interaction between NC and nucleic acids, which are potentially more specific and less toxic than (i); 1,3−5 (iii) noncovalent binders to nucleic acids that compete with the NC, which are currently more suited as tool compounds than lead candidates. 6,7 Our research has long focused on strategy (ii) with the aim to develop different chemotypes of effective NC inhibitors (NCIs) endowed with antiretroviral activity against wild-type and drug-resistant HIV-1 strains. 8−10 These molecules are designed to bind to a hydrophobic pocket located in the Cterminal zinc finger of NC, which accommodates the critically recognized guanosine of nucleic acid targets through interaction with Gly35, Trp37, Gln45, and Met46, as shown by NMR studies (Figure 1a).…”

“…8 Particularly, compound 1 bearing a two-carbon linker proved to be the most effective NCI of this preliminary series, which guided the design of additional derivatives. Thus, modifications were first introduced within the HPB portion by means of a fluorine (5) or a chlorine (6) substituent in position 4 of the (benzyloxy)benzene ring. Moreover, both aminothiazoles were replaced by aminoxazole rings (7) or a single aminothiazole was replaced by a benzothiazole (8) (Figure 2b).…”