1887

Abstract

The shortcomings of current anti-human cytomegalovirus (HCMV) drugs has stimulated a search for anti-HCMV compounds with novel targets. We screened collections of bioactive compounds and identified a range of compounds with the potential to inhibit HCMV replication. Of these compounds, we selected bisbenzimide compound RO-90-7501 for further study. We generated analogues of RO-90-7501 and found that one compound, MRT00210423, had increased anti-HCMV activity compared to RO-90-7501. Using a combination of compound analogues, microscopy and biochemical assays we found RO-90-7501 and MRT00210423 interacted with DNA. In single molecule microscopy experiments we found RO-90-7501, but not MRT00210423, was able to compact DNA, suggesting that compaction of DNA was non-obligatory for anti-HCMV effects. Using bioinformatics analysis, we found that there were many putative bisbenzimide binding sites in the HCMV DNA genome. However, using western blotting, quantitative PCR and electron microscopy, we found that at a concentration able to inhibit HCMV replication our compounds had little or no effect on production of certain HCMV proteins or DNA synthesis, but did have a notable inhibitory effect on HCMV capsid production. We reasoned that these effects may have involved binding of our compounds to the HCMV genome and/or host cell chromatin. Therefore, our data expand our understanding of compounds with anti-HCMV activity and suggest targeting of DNA with bisbenzimide compounds may be a useful anti-HCMV strategy.

Funding
This study was supported by the:
  • National Institute of Allergy and Infectious Diseases (Award R01 AI026077)
  • National Institute of Allergy and Infectious Diseases (Award R01 AI019838)
  • Medical Research Council (Award MR/M016226/1)
    • Principle Award Recipient: BlairL Strang
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001702
2021-12-09
2022-01-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/102/12/jgv001702.html?itemId=/content/journal/jgv/10.1099/jgv.0.001702&mimeType=html&fmt=ahah

References

  1. Griffiths PD. Burden of disease associated with human cytomegalovirus and prospects for elimination by universal immunisation. Lancet Infect Dis 2012; 12: [View Article] [PubMed]
    [Google Scholar]
  2. Krause PR, Bialek SR, Boppana SB, Griffiths PD, Laughlin CA et al. Priorities for CMV vaccine development. Vaccine 2013; 32:4–10 [View Article] [PubMed]
    [Google Scholar]
  3. Coen DM, Schaffer PA. Antiherpesvirus drugs: a promising spectrum of new drugs and drug targets. Nat Rev Drug Discov 2003; 2:278–288 [View Article] [PubMed]
    [Google Scholar]
  4. Lischka P, Hewlett G, Wunberg T, Baumeister J, Paulsen D et al. In vitro and in vivo activities of the novel anticytomegalovirus compound AIC246. Antimicrob Agents Chemother 2010; 54:1290–1297 [View Article]
    [Google Scholar]
  5. Biron KK, Harvey RJ, Chamberlain SC, Good SS, Smith AA et al. Potent and selective inhibition of human cytomegalovirus replication by 1263W94, a benzimidazole l -riboside with a unique mode of action. Antimicrob Agents Chemother 2002; 46:2365–2372 [View Article]
    [Google Scholar]
  6. Goldner T, Hewlett G, Ettischer N, Ruebsamen-Schaeff H, Zimmermann H et al. The novel anticytomegalovirus compound AIC246 (Letermovir) inhibits human cytomegalovirus replication through a specific antiviral mechanism that involves the viral terminase. J Virol 2011; 85:10884–10893 [View Article] [PubMed]
    [Google Scholar]
  7. Chou S. A third component of the human cytomegalovirus terminase complex is involved in letermovir resistance. Antiviral Res 2017; 148:1–4 [View Article] [PubMed]
    [Google Scholar]
  8. Cherrier L, Nasar A, Goodlet KJ, Nailor MD, Tokman S et al. Emergence of letermovir resistance in a lung transplant recipient with ganciclovir-resistant cytomegalovirus infection. Am J Transplant 2018; 18:3060–3064 [View Article] [PubMed]
    [Google Scholar]
  9. Chou S, Satterwhite LE, Ercolani RJ. New locus of drug resistance in the human cytomegalovirus UL56 gene revealed by in vitro exposure to letermovir and ganciclovir. Antimicrob Agents Chemother 2018; 62: [View Article]
    [Google Scholar]
  10. Chou S. Rapid in vitro evolution of human cytomegalovirus UL56 mutations that confer letermovir resistance. Antimicrob Agents Chemother 2015; 59:6588–6593 [View Article]
    [Google Scholar]
  11. Chou S, Wechel LCV, Marousek GI. Cytomegalovirus UL97 kinase mutations that confer maribavir resistance. J Infect Dis 2007; 196:91–94 [View Article] [PubMed]
    [Google Scholar]
  12. Papanicolaou GA, Silveira FP, Langston AA, Pereira MR, Avery RK et al. Maribavir for refractory or resistant cytomegalovirus infections in hematopoietic-cell or solid-organ transplant recipients: a randomized, dose-ranging, double-blind, phase 2 study. Clin Infect Dis 2019; 68:1255–1264 [View Article] [PubMed]
    [Google Scholar]
  13. Marty FM, Ljungman P, Papanicolaou GA, Winston DJ, Chemaly RF et al. Maribavir prophylaxis for prevention of cytomegalovirus disease in recipients of allogeneic stem-cell transplants: a phase 3, double-blind, placebo-controlled, randomised trial. Lancet Infect Dis 2011; 11:284–292 [View Article] [PubMed]
    [Google Scholar]
  14. Marty FM, Boeckh M. Maribavir and human cytomegalovirus-what happened in the clinical trials and why might the drug have failed?. Curr Opin Virol 2011; 1:555–562 [View Article] [PubMed]
    [Google Scholar]
  15. Mocarski ES, Shenk T, Griffiths PD, Pass RF. Cytomegaloviruses. In Knipe DM, Howley PM. eds Fields Virology, 6th edn. vol. 2 New York, NY: Lippincott, Williams & Wilkins; 2015 pp 1960–2015
    [Google Scholar]
  16. Mercorelli B, Luganini A, Nannetti G, Tabarrini O, Palù G et al. Drug repurposing approach identifies inhibitors of the prototypic viral transcription factor IE2 that block human cytomegalovirus replication. Cell Chem Biol 2016; 23: [View Article] [PubMed]
    [Google Scholar]
  17. Gardner TJ, Cohen T, Redmann V, Lau Z, Felsenfeld D et al. Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle. Antiviral Res 2015; 113:49–61 [View Article] [PubMed]
    [Google Scholar]
  18. Nukui M, O’Connor CM, Murphy EA. The natural flavonoid compound deguelin inhibits HCMV lytic replication within fibroblasts. Viruses 2018; 10:E614 [View Article] [PubMed]
    [Google Scholar]
  19. Mukhopadhyay R, Roy S, Venkatadri R, Su Y-P, Ye W et al. Efficacy and mechanism of action of low dose emetine against human cytomegalovirus. PLoS Pathog 2016; 12:e1005717 [View Article] [PubMed]
    [Google Scholar]
  20. Strang BL. RO0504985 is an inhibitor of CMGC kinase proteins and has anti-human cytomegalovirus activity. Antiviral Res 2017; 144:21–26 [View Article] [PubMed]
    [Google Scholar]
  21. Khan AS, Murray MJ, Ho CMK, Zuercher WJ, Reeves MB et al. High-throughput screening of a GlaxoSmithKline protein kinase inhibitor set identifies an inhibitor of human cytomegalovirus replication that prevents CREB and histone H3 post-translational modification. J Gen Virol 2017; 98:754–768 [View Article] [PubMed]
    [Google Scholar]
  22. Beelontally R, Wilkie GS, Lau B, Goodmaker CJ, Ho CMK et al. Identification of compounds with anti-human cytomegalovirus activity that inhibit production of IE2 proteins. Antiviral Res 2017; 138:61–67 [View Article] [PubMed]
    [Google Scholar]
  23. Polachek WS, Moshrif HF, Franti M, Coen DM, Sreenu VB et al. High-throughput small interfering RNA screening identifies phosphatidylinositol 3-kinase class II alpha as important for production of human cytomegalovirus virions. J Virol 2016; 90:8360–8371 [View Article] [PubMed]
    [Google Scholar]
  24. Birmingham A, Selfors LM, Forster T, Wrobel D, Kennedy CJ et al. Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 2009; 6:569–575 [View Article] [PubMed]
    [Google Scholar]
  25. Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999; 4:67–73 [View Article] [PubMed]
    [Google Scholar]
  26. Loregian A, Coen DM. Selective anti-cytomegalovirus compounds discovered by screening for inhibitors of subunit interactions of the viral polymerase. Chem Biol 2006; 13:191–200 [View Article] [PubMed]
    [Google Scholar]
  27. Kim H, Loparo JJ. Observing bacterial chromatin protein-DNA interactions by combining DNA flow-stretching with single-molecule imaging. Methods Mol Biol 2018; 1837:277–299 [View Article] [PubMed]
    [Google Scholar]
  28. Kim H, Loparo JJ. Multistep assembly of DNA condensation clusters by SMC. Nat Commun 2016; 7:10200 [View Article]
    [Google Scholar]
  29. Edelstein AD, Tsuchida MA, Amodaj N, Pinkard H, Vale RD et al. Advanced methods of microscope control using μManager software. J Biol Methods 2014; 1:e10. [View Article] [PubMed]
    [Google Scholar]
  30. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012; 9:671–675 [View Article] [PubMed]
    [Google Scholar]
  31. Strang BL, Stow ND. Circularization of the herpes simplex virus type 1 genome upon lytic infection. J Virol 2005; 79:12487–12494 [View Article] [PubMed]
    [Google Scholar]
  32. Strang BL, Bender BJ, Sharma M, Pesola JM, Sanders RL et al. A mutation deleting sequences encoding the amino terminus of human cytomegalovirus UL84 impairs interaction with UL44 and capsid localization. J Virol 2012; 86:11066–11077 [View Article] [PubMed]
    [Google Scholar]
  33. Strang BL, Boulant S, Kirchhausen T, Coen DM, Sandri-Goldin RM. Host cell nucleolin is required to maintain the architecture of human cytomegalovirus replication compartments. mbio 2012; 3: [View Article]
    [Google Scholar]
  34. Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol 1996; 116:71–76 [View Article] [PubMed]
    [Google Scholar]
  35. Wilkinson GWG, Davison AJ, Tomasec P, Fielding CA, Aicheler R et al. Human cytomegalovirus: taking the strain. Med Microbiol Immunol 2015; 204:273–284 [View Article] [PubMed]
    [Google Scholar]
  36. Britt WJ, Jarvis M, Seo JY, Drummond D, Nelson J. Rapid genetic engineering of human cytomegalovirus by using a lambda phage linear recombination system: demonstration that pp28 (UL99) is essential for production of infectious virus. J Virol 2004; 78:539–543 [View Article] [PubMed]
    [Google Scholar]
  37. Depto AS, Stenberg RM. Functional analysis of the true late human cytomegalovirus pp28 upstream promoter: cis-acting elements and viral trans-acting proteins necessary for promoter activation. J Virol 1992; 66:3241–3246 [View Article] [PubMed]
    [Google Scholar]
  38. De Clercq E, Sakuma T, Baba M, Pauwels R, Balzarini J et al. Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrimidines. Antiviral Res 1987; 8:261–272 [View Article] [PubMed]
    [Google Scholar]
  39. Elion GB. Mechanism of action and selectivity of acyclovir. Am J Med 1982; 73:7–13 [View Article] [PubMed]
    [Google Scholar]
  40. Teng MK, Usman N, Frederick CA, Wang AH. The molecular structure of the complex of Hoechst 33258 and the DNA dodecamer d(CGCGAATTCGCG). Nucleic Acids Res 1988; 16:2671–2690 [View Article] [PubMed]
    [Google Scholar]
  41. Yakimovich A, Huttunen M, Zehnder B, Coulter LJ, Gould V et al. Inhibition of poxvirus gene expression and genome replication by bisbenzimide derivatives. J Virol 2017; 91:18 [View Article]
    [Google Scholar]
  42. Olive PL, Chaplin DJ, Durand RE. Pharmacokinetics, binding and distribution of Hoechst 33342 in spheroids and murine tumours. Br J Cancer 1985; 52:739–746 [View Article] [PubMed]
    [Google Scholar]
  43. Patel SR, Kvols LK, Rubin J, O’Connell MJ, Edmonson JH et al. Phase I-II study of pibenzimol hydrochloride (NSC 322921) in advanced pancreatic carcinoma. Invest New Drugs 1991; 9:53–57 [View Article] [PubMed]
    [Google Scholar]
  44. Faulds D, Heel RC. Ganciclovir. A review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in cytomegalovirus infections. Drugs 1990; 39:597–638 [View Article] [PubMed]
    [Google Scholar]
  45. Harshman KD, Dervan PB. Molecular recognition of B-DNA by Hoechst 33258. Nucleic Acids Res 1985; 13:4825–4835 [View Article] [PubMed]
    [Google Scholar]
  46. Hampshire AJ, Fox KR. The effects of local DNA sequence on the interaction of ligands with their preferred binding sites. Biochimie 2008; 90:988–998 [View Article] [PubMed]
    [Google Scholar]
  47. Saito M, Kobayashi M, Iwabuchi S, Morita Y, Takamura Y et al. DNA condensation monitoring after interaction with hoechst 33258 by atomic force microscopy and fluorescence spectroscopy. J Biochem 2004; 136:813–823 [View Article] [PubMed]
    [Google Scholar]
  48. Strang BL, Boulant S, Chang L, Knipe DM, Kirchhausen T et al. Human cytomegalovirus UL44 concentrates at the periphery of replication compartments, the site of viral DNA synthesis. J Virol 2012; 86:2089–2095 [View Article] [PubMed]
    [Google Scholar]
  49. Guo F, Mead J, Aliya N, Wang L, Cuconati A et al. RO 90-7501 enhances TLR3 and RLR agonist induced antiviral response. PloS one 2012; 7:e42583 [View Article]
    [Google Scholar]
  50. Bathini Y, Rao KE, Shea RG, Lown JW. Molecular recognition between ligands and nucleic acids: novel pyridine- and benzoxazole-containing agents related to Hoechst 33258 that exhibit altered DNA sequence specificity deduced from footprinting analysis and spectroscopic studies. Chem Res Toxicol 1990; 3:268–280 [View Article] [PubMed]
    [Google Scholar]
  51. Guan LL, Zhao R, Lown JW. Enhanced DNA alkylation activities of Hoechst 33258 analogues designed for bioreductive activation. Biochem Biophys Res Commun 1997; 231:94–98 [View Article] [PubMed]
    [Google Scholar]
  52. Gupta R, Wang H, Huang L, Lown JW. Design, synthesis, DNA sequence preferential alkylation and biological evaluation of N-mustard derivatives of Hoechst 33258 analogues. Anticancer Drug Des 1995; 10:25–41 [PubMed]
    [Google Scholar]
  53. Kumar S, Yadagiri B, Zimmermann J, Pon RT, Lown JW. Sequence specific molecular recognition and binding by a GC recognizing Hoechst 33258 analogue to the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies. J Biomol Struct Dyn 1990; 8:331–357 [View Article] [PubMed]
    [Google Scholar]
  54. Rao KE, Lown JW. Molecular recognition between ligands and nucleic acids: DNA binding characteristics of analogues of Hoechst 33258 designed to exhibit altered base and sequence recognition. Chem Res Toxicol 1991; 4:661–669 [View Article] [PubMed]
    [Google Scholar]
  55. Singh MP, Joseph T, Kumar S, Bathini Y, Lown JW. Synthesis and sequence-specific DNA binding of a topoisomerase inhibitory analog of Hoechst 33258 designed for altered base and sequence recognition. Chem Res Toxicol 1992; 5:597–607 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001702
Loading
/content/journal/jgv/10.1099/jgv.0.001702
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

EXCEL

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error