The secondary structure of nucleic acids containing quartets of guanines, termed G-quadruplexes, is known to regulate the transcription of many genes. Several G-quadruplexes can be formed in the HIV-1 long terminal repeat promoter region and their stabilization results in the inhibition of HIV-1 replication. Here, we identified helquat-based compounds as a new class of anti-HIV-1 inhibitors that inhibit HIV-1 replication at the stage of reverse transcription and provirus expression. Using Taq polymerase stop and FRET melting assays, we have demonstrated their ability to stabilize G-quadruplexes in the HIV-1 long-terminal repeat sequence. Moreover, these compounds were not binding to the general G-rich region, but rather to G-quadruplex-forming regions. Finally, docking and molecular dynamics calculations indicate that the structure of the helquat core greatly affects the binding mode to the individual G-quadruplexes. Our findings can provide useful information for the further rational design of inhibitors targeting G-quadruplexes in HIV-1.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague 160 00 Czech Republic

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Lago S, et al. Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome. Nat. Commun. 2021;12:3885. doi: 10.1038/s41467-021-24198-2. PubMed DOI PMC

Haeusler AR, et al. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature. 2014;507:195–200. doi: 10.1038/nature13124. PubMed DOI PMC

Sket P, et al. Characterization of DNA G-quadruplex species forming from C9ORF72 G4C2-expanded repeats associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Neurobiol. Aging. 2015;36:1091–1096. doi: 10.1016/j.neurobiolaging.2014.09.012. PubMed DOI

Varshney D, Spiegel J, Zyner K, Tannahill D, Balasubramanian S. The regulation and functions of DNA and RNA G-quadruplexes. Nat. Rev. Mol. Cell Biol. 2020;21:459–474. doi: 10.1038/s41580-020-0236-x. PubMed DOI PMC

Perrone R, et al. Mapping and characterization of G-quadruplexes in Mycobacterium tuberculosis gene promoter regions. Sci. Rep. 2017;7:5743. doi: 10.1038/s41598-017-05867-z. PubMed DOI PMC

Zuffo M, et al. Carbohydrate-naphthalene diimide conjugates as potential antiparasitic drugs: Synthesis, evaluation and structure-activity studies. Eur. J. Med. Chem. 2019;163:54–66. doi: 10.1016/j.ejmech.2018.11.043. PubMed DOI

Yadav P, et al. G-quadruplex structures in bacteria: Biological relevance and potential as an antimicrobial target. J. Bacteriol. 2021;203:e0057720. doi: 10.1128/JB.00577-20. PubMed DOI PMC

Lavezzo E, et al. G-quadruplex forming sequences in the genome of all known human viruses: A comprehensive guide. PLoS Comput. Biol. 2018;14:e1006675. doi: 10.1371/journal.pcbi.1006675. PubMed DOI PMC

Metifiot M, Amrane S, Litvak S, Andreola ML. G-quadruplexes in viruses: Function and potential therapeutic applications. Nucleic Acids Res. 2014;42:12352–12366. doi: 10.1093/nar/gku999. PubMed DOI PMC

Murat P, et al. G-quadruplexes regulate Epstein–Barr virus-encoded nuclear antigen 1 mRNA translation. Nat. Chem. Biol. 2014;10:358–364. doi: 10.1038/nchembio.1479. PubMed DOI PMC

Perrone R, et al. A dynamic G-quadruplex region regulates the HIV-1 long terminal repeat promoter. J. Med. Chem. 2013;56:6521–6530. doi: 10.1021/jm400914r. PubMed DOI PMC

Tan J, et al. The SARS-unique domain (SUD) of SARS coronavirus contains two macrodomains that bind G-quadruplexes. PLoS Pathog. 2009;5:e1000428. doi: 10.1371/journal.ppat.1000428. PubMed DOI PMC

Kong JN, et al. Identification and characterization of G-quadruplex formation within the EP0 promoter of pseudorabies virus. Sci. Rep. 2018;8:14029. doi: 10.1038/s41598-018-32222-7. PubMed DOI PMC

Zahin M, et al. Identification of G-quadruplex forming sequences in three manatee papillomaviruses. PLoS ONE. 2018;13:e0195625. doi: 10.1371/journal.pone.0195625. PubMed DOI PMC

Frasson I, Solda P, Nadai M, Lago S, Richter SN. Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells. Commun. Biol. 2021;4:510. doi: 10.1038/s42003-021-02035-y. PubMed DOI PMC

Ruggiero E, Richter SN. Viral G-quadruplexes: New frontiers in virus pathogenesis and antiviral therapy. Annu. Rep. Med. Chem. 2020;54:101–131. doi: 10.1016/bs.armc.2020.04.001. PubMed DOI PMC

Abiri A, et al. Unlocking G-quadruplexes as antiviral targets. Pharmacol. Rev. 2021;73:897–923. doi: 10.1124/pharmrev.120.000230. PubMed DOI

Ruggiero E, Zanin I, Terreri M, Richter SN. G-quadruplex targeting in the fight against viruses: An update. Int. J. Mol. Sci. 2021 doi: 10.3390/ijms222010984. PubMed DOI PMC

Ruggiero E, Richter SN. Targeting G-quadruplexes to achieve antiviral activity. Bioorg. Med. Chem. Lett. 2023;79:129085. doi: 10.1016/j.bmcl.2022.129085. PubMed DOI PMC

Balasubramanian S, Neidle S. G-quadruplex nucleic acids as therapeutic targets. Curr. Opin. Chem. Biol. 2009;13:345–353. doi: 10.1016/j.cbpa.2009.04.637. PubMed DOI PMC

Balasubramanian S, Hurley LH, Neidle S. Targeting G-quadruplexes in gene promoters: A novel anticancer strategy? Nat. Rev. Drug Discov. 2011;10:261–275. doi: 10.1038/nrd3428. PubMed DOI PMC

Lopergolo A, et al. Targeting of RET oncogene by naphthalene diimide-mediated gene promoter G-quadruplex stabilization exerts anti-tumor activity in oncogene-addicted human medullary thyroid cancer. Oncotarget. 2016;7:49649–49663. doi: 10.18632/oncotarget.10105. PubMed DOI PMC

Savva L, Georgiades SN. Recent developments in small-molecule ligands of medicinal relevance for harnessing the anticancer potential of G-quadruplexes. Molecules. 2021 doi: 10.3390/molecules26040841. PubMed DOI PMC

Kosiol N, Juranek S, Brossart P, Heine A, Paeschke K. G-quadruplexes: A promising target for cancer therapy. Mol. Cancer. 2021;20:40. doi: 10.1186/s12943-021-01328-4. PubMed DOI PMC

Artusi S, et al. The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex: Implications for the antiviral activity of a G-quadruplex ligand. Antiviral Res. 2015;118:123–131. doi: 10.1016/j.antiviral.2015.03.016. PubMed DOI PMC

Callegaro S, et al. A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication. Sci. Rep. 2017;7:2341. doi: 10.1038/s41598-017-02667-3. PubMed DOI PMC

Perrone R, et al. Synthesis, binding and antiviral properties of potent core-extended naphthalene diimides targeting the HIV-1 long terminal repeat promoter G-quadruplexes. J. Med. Chem. 2015;58:9639–9652. doi: 10.1021/acs.jmedchem.5b01283. PubMed DOI PMC

Majee P, et al. Inhibition of Zika virus replication by G-quadruplex-binding ligands. Mol. Ther. Nucleic Acids. 2021;23:691–701. doi: 10.1016/j.omtn.2020.12.030. PubMed DOI PMC

Zou M, et al. G-quadruplex binder pyridostatin as an effective multi-target ZIKV inhibitor. Int. J. Biol. Macromol. 2021;190:178–188. doi: 10.1016/j.ijbiomac.2021.08.121. PubMed DOI

Lavigne M, et al. SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction. Nucleic Acids Res. 2021;49:7695–7712. doi: 10.1093/nar/gkab571. PubMed DOI PMC

Carvalho J, et al. Human papillomavirus G-rich regions as potential antiviral drug targets. Nucleic Acid Ther. 2021;31:68–81. doi: 10.1089/nat.2020.0869. PubMed DOI

Artusi S, et al. Antiviral activity of the G-quadruplex ligand TMPyP4 against herpes simplex virus-1. Viruses. 2021 doi: 10.3390/v13020196. PubMed DOI PMC

Lv L, Cui H, Chen Z, Zhou Y, Zhang L. G-quadruplex ligands inhibit chikungunya virus replication. J. Med. Virol. 2022;94:2519–2527. doi: 10.1002/jmv.27622. PubMed DOI

Bohalova N, et al. Analyses of viral genomes for G-quadruplex forming sequences reveal their correlation with the type of infection. Biochimie. 2021;186:13–27. doi: 10.1016/j.biochi.2021.03.017. PubMed DOI

Harpster C, Boyle E, Musier-Forsyth K, Kankia B. HIV-1 genomic RNA U3 region forms a stable quadruplex-hairpin structure. Biophys. Chem. 2021;272:106567. doi: 10.1016/j.bpc.2021.106567. PubMed DOI PMC

Piekna-Przybylska D, Sullivan MA, Sharma G, Bambara RA. U3 region in the HIV-1 genome adopts a G-quadruplex structure in its RNA and DNA sequence. Biochemistry. 2014;53:2581–2593. doi: 10.1021/bi4016692. PubMed DOI PMC

Amrane S, et al. Topology of a DNA G-quadruplex structure formed in the HIV-1 promoter: A potential target for anti-HIV drug development. J. Am. Chem. Soc. 2014;136:5249–5252. doi: 10.1021/ja501500c. PubMed DOI

Butovskaya E, Heddi B, Bakalar B, Richter SN, Phan AT. Major G-quadruplex form of HIV-1 LTR reveals a (3 + 1) folding topology containing a stem-loop. J. Am. Chem. Soc. 2018;140:13654–13662. doi: 10.1021/jacs.8b05332. PubMed DOI PMC

Ruggiero E, Tassinari M, Perrone R, Nadai M, Richter SN. Stable and conserved G-quadruplexes in the long terminal repeat promoter of retroviruses. ACS Infect. Dis. 2019;5:1150–1159. doi: 10.1021/acsinfecdis.9b00011. PubMed DOI PMC

Perrone R, et al. Anti-HIV-1 activity of the G-quadruplex ligand BRACO-19. J. Antimicrob. Chemother. 2014;69:3248–3258. doi: 10.1093/jac/dku280. PubMed DOI

Tosoni E, et al. Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription. Nucleic Acids Res. 2015;43:8884–8897. doi: 10.1093/nar/gkv897. PubMed DOI PMC

Ajoge HO, et al. G-quadruplex DNA and other non-canonical B-form DNA motifs influence productive and latent HIV-1 integration and reactivation potential. Viruses. 2022 doi: 10.3390/v14112494. PubMed DOI PMC

Amrane S, et al. Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection. Nucleic Acids Res. 2022;50:12328–12343. doi: 10.1093/nar/gkac1030. PubMed DOI PMC

Spiegel J, Adhikari S, Balasubramanian S. The structure and function of DNA G-quadruplexes. Trends Chem. 2020;2:123–136. doi: 10.1016/j.trechm.2019.07.002. PubMed DOI PMC

Kužmová, E. et al.HeliDye1: Helquat Fluorogenic Probe Specific for AT-rich DNA Duplexes Preprint at 10.21203/rs.3.rs-737372/v2 (2022).

Reyes-Gutierrez PE, et al. Functional helquats: Helical cationic dyes with marked, switchable chiroptical properties in the visible region. Chem. Commun. 2015;51:1583–1586. doi: 10.1039/c4cc08967g. PubMed DOI

Wilkinson RA, et al. Novel compounds containing multiple guanide groups that bind the HIV coreceptor CXCR4. Antimicrob. Agents Chemother. 2011;55:255–263. doi: 10.1128/AAC.00709-10. PubMed DOI PMC

Adriaenssens L, et al. Helquats: A facile, modular, scalable route to novel helical dications. Chem.-Eur. J. 2009;15:1072–1076. doi: 10.1002/chem.200801904. PubMed DOI

Chilka P, Desai N, Datta B. Small molecule fluorescent probes for G-quadruplex visualization as potential cancer theranostic agents. Molecules. 2019 doi: 10.3390/molecules24040752. PubMed DOI PMC

De Nicola B, et al. Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome. Nucleic Acids Res. 2016;44:6442–6451. doi: 10.1093/nar/gkw432. PubMed DOI PMC

Sundquist WI, Heaphy S. Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA. Proc. Natl. Acad. Sci. USA. 1993;90:3393–3397. doi: 10.1073/pnas.90.8.3393. PubMed DOI PMC

Ruggiero E, et al. Fused in liposarcoma protein, a new player in the regulation of HIV-1 transcription, binds to known and newly identified LTR G-quadruplexes. ACS Infect. Dis. 2022;8:958–968. doi: 10.1021/acsinfecdis.1c00508. PubMed DOI PMC

Haider SM, Neidle S, Parkinson GN. A structural analysis of G-quadruplex/ligand interactions. Biochimie. 2011;93:1239–1251. doi: 10.1016/j.biochi.2011.05.012. PubMed DOI

Islam B, Stadlbauer P, Neidle S, Haider S, Sponer J. Can we execute reliable MM-PBSA free energy computations of relative stabilities of different guanine quadruplex folds? J. Phys. Chem. B. 2016;120:2899–2912. doi: 10.1021/acs.jpcb.6b01059. PubMed DOI

Scalabrin M, et al. The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes. Sci. Rep. 2017;7:45244. doi: 10.1038/srep45244. PubMed DOI PMC

Raiber EA, Kranaster R, Lam E, Nikan M, Balasubramanian S. A non-canonical DNA structure is a binding motif for the transcription factor SP1 in vitro. Nucleic Acids Res. 2012;40:1499–1508. doi: 10.1093/nar/gkr882. PubMed DOI PMC

Kim N. The interplay between G-quadruplex and transcription. Curr. Med. Chem. 2019;26:2898–2917. doi: 10.2174/0929867325666171229132619. PubMed DOI PMC

Jiang G, Espeseth A, Hazuda DJ, Margolis DM. c-Myc and Sp1 contribute to proviral latency by recruiting histone deacetylase 1 to the human immunodeficiency virus type 1 promoter. J. Virol. 2007;81:10914–10923. doi: 10.1128/JVI.01208-07. PubMed DOI PMC

Bedrat A, Lacroix L, Mergny JL. Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 2016;44:1746–1759. doi: 10.1093/nar/gkw006. PubMed DOI PMC

Lightfoot HL, Hagen T, Tatum NJ, Hall J. The diverse structural landscape of quadruplexes. FEBS Lett. 2019;593:2083–2102. doi: 10.1002/1873-3468.13547. PubMed DOI

Hodek J, et al. Protective hybrid coating containing silver, copper and zinc cations effective against human immunodeficiency virus and other enveloped viruses. BMC Microbiol. 2016;16(Suppl 1):56. doi: 10.1186/s12866-016-0675-x. PubMed DOI PMC

Friesner RA, et al. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 2004;47:1739–1749. doi: 10.1021/jm0306430. PubMed DOI

Miller BR, III, et al. MMPBSA.py: An efficient program for end-state free energy calculations. J. Chem. Theory Comput. 2012;8:3314–3321. doi: 10.1021/ct300418h. PubMed DOI

Asymmetric distribution of G-quadruplex forming sequences in genomes of retroviruses