Molecular Tweezers As Weapons Against Enveloped Viruses

Viruses can cause life-threatening infections in humans and spread rapidly over large areas or even globally. Against most viruses no specific treatment or vaccine exists. Moreover, drug development usually lags behind and if at all, develops medication late after a virus has become a problem. Thus, an agent that targets a variety of viruses could be extremely helpful to limit expansion and immediately treat infected people. In order to develop such a broad-spectrum antiviral, one needs to exploit common features of viruses. We identified a small molecule termed the molecular tweezer CLR01 that selectively destroys viral membranes^1,2.

Many human pathogenic viruses like human immunodeficiency virus (HIV), hepatitis C virus, herpes simplex virus, Ebola virus, or Zika virus (ZIKV) are surrounded by a membrane and are therefore called enveloped. This membrane consists of a lipid bilayer that is derived from the host cell where the virus originates from. Not having the potential to replicate autonomously, viruses exploit cellular machineries. After having infected a cell, new viruses are produced and released by budding from cellular membranes.

Since viruses preferentially bud from sites that are enriched in special lipids such as sphingomyelin and cholesterol, there are significant differences in the exact composition of cellular and viral membranes^3,4. Due to its unique structure, the tweezer includes cationic lipid head groups in its torus-shaped cavity^5,6. Thus, CLR01 is able to interact with certain lipids that are enriched in the viral but not in the cellular membrane and most likely leads to increased surface tension. Upon these interactions, the viral envelope is disrupted and the virus loses its infectivity¹. At the same time, CLR01 does not destroy cellular membranes and has been proven safe in different animal models^8–13. Since CLR01 is broadly active against all enveloped viruses tested so far¹, it represents a potent candidate for a broad-spectrum antiviral agent.

Regarding the severe West African Ebola outbreak 2014–2016 or the recent Zika virus outbreaks, we used Ebola and Zika virus as prototypes of emerging pathogenic enveloped viruses. Till date, 84 countries and territories reported ongoing ZIKV transmission. Although symptoms are usually mild (fever, rash, and headache) or even completely absent, ZIKV can cause severe birth defects if women are infected during pregnancy^14,15. Moreover, the virus can induce neurological disorders, especially Guillain- Barré syndrome¹⁶, a disease where the immune system attacks the peripheral nervous system, resulting in general muscle weakness and paralysis. Thus, the WHO declared a Public Health Emergency of International Concern from February until November 2016. In most cases, ZIKV is transmitted via mosquito bites but there is also evidence of sexual transmission^17,18.

So far, no specific treatment or vaccine against ZIKV is available. In our recent study², we showed that infection of cells of the anogenital tract and the central nervous system is prevented by CLR01. These cells represent potential target cells in terms of sexual transmission and the virus’ ability to cause neurological disorders. The findings detailed above were not only made with lab-adapted prototypic strains but also confirmed for recent clinical ZIKV isolates. Moreover, the molecule retains its activity in the presence of body fluids such as semen, saliva, urine, and liquor. Thus, CLR01 could be used in a real setting in order to prevent person-to-person ZIKV transmission.

Unfortunately, the presence of human serum abolishes the antiviral effect of CLR01. Presumably, the molecule is bound by abundant proteins in serum and hence, the remaining amount of free CLR01 is too low to achieve viral inactivation. Thus, the molecule is not suitable for systemic application as an antiviral agent in the current form. However, if CLR01 is applied onto vulnerable body surfaces, such as the skin or mucous membranes it could protect against transmission of a broad range of enveloped viruses e.g. ZIKV or Ebola virus.

In order to restrict airborne viruses such as the Flu or Respiratory Syncytial Virus, which also belong to the class of enveloped viruses, one could consider administration as spray or drops. Of note, apart from its antiviral effect, CLR01 could also protect against neurodegenerative diseases. In brains of patients with Parkinson’s or Alzheimer’s disease, plaques of aggregated protein are found and are suspected to be the disease-causing agent. In general, the self-assembly of protein can result in toxic oligomers and fibrillar structures, termed amyloid fibrils. CLR01 not only destroys viral membranes but also effectively prevents formation and promotes disaggregation of already formed amyloid fibrils. This effect has been already successfully tested in cells and different animal models^7–11.

Moreover, the tweezer penetrates the blood-brain barrier (BBB) which prevents many drugs from entering the brain and protects mice from Alzheimer’s disease when administered systemically⁸. Mice tolerate CLR01 doses up to 250-times higher than actually required. Together with the fact that the tweezer molecule remains stable in plasma for more than two hours¹⁹, these properties qualify CLR01 as a promising drug candidate. Apart from their role in neurodegenerative diseases, natural and non-toxic amyloid fibrils are also present in the semen of healthy men^20–23.

It is well known that semen is not only a passive carrier but can strongly influence viral infectivity. In the case of HIV-1, seminal amyloid fibrils boost viral infectivity by several orders of magnitude^20,21,24. Since CLR01 not only inactivates the virus itself but also disaggregates the infectivity-promoting fibrils, it serves as a dual-function inhibitor of HIV-1¹. Its combined activities against viruses and amyloid fibrils strongly increase the efficiency of CLR01 as a topical microbicide against sexually transmitted, enveloped viruses.

In an interdisciplinary and international project, the multifaceted activities of molecular tweezers are investigated and optimized.

These findings are described in the article entitled The molecular tweezer CLR01 inhibits Ebola and Zika virus infection, recently published in the journal Antiviral Research. This work was conducted by Annika E.Röcker, Janis A. Müller, Mirja Harms, Franziska Krüger, Sina Lippold, Jens von Einem, and Jan Münch from Ulm University Medical Center, Erik Dietzel, Alexandra Kupke, and Stephan Becker from Philipps University of Marburg, Christian Heid, Andrea Sowislok, and Thomas Schrader from the University of Duisburg-Essen, Camilla Frich Riber and Alexander N. Zelikin from Aarhus University, Judith Beer, Bernd Knöll, Jonas Schmidt-Chanasit, and Markus Otto from the University of Ulm, Olli Vapalahti from the University of Helsinki, and Gal Bitan from the University of California, Los Angeles.

References:

1.  Lump, E. et al. A molecular tweezer antagonizes seminal amyloids and HIV infection. Elife 4, e05397 (2015).
2.  Röcker, A. E. et al. The molecular tweezer CLR01 inhibits Ebola and Zika virus infection. Antiviral Res. (2018).
3. Brügger, B. et al. The HIV lipidome: a raft with an unusual composition. Proc. Natl. Acad. Sci. U. S. A. 103, 2641–2646 (2006).
4. Lorizate, M. et al. Comparative lipidomics analysis of HIV-1 particles and their producer cell membrane in different cell lines. Cell. Microbiol. 15, 292–304 (2013).
5. Fokkens, M., Schrader, T. & Klärner, F.-G. A molecular tweezer for lysine and arginine. J. Am. Chem. Soc. 127, 14415–14421 (2005).
6. Klärner, F.-G. & Schrader, T. Aromatic interactions by molecular tweezers and clips in chemical and biological systems. Acc. Chem. Res. 46, 967–978 (2012).
7. Sinha, S. et al. Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc. 133, 16958–16969 (2011).
8. Attar, A. et al. Protection of primary neurons and mouse brain from Alzheimer’s pathology by molecular tweezers. Brain 135, 3735–3748 (2012).
9. Prabhudesai, S. et al. A novel “molecular tweezer” inhibitor of α-synuclein neurotoxicity in vitro and in vivo. Neurotherapeutics 9, 464–476 (2012).
10. Richter, F. et al. A Molecular Tweezer Ameliorates Motor Deficits in Mice Overexpressing α-Synuclein. Neurotherapeutics 1–13 (2017).
11. Lulla, A. Neurotoxicity of the Parkinson’s disease-associated pesticide Ziram is synuclein dependent. (2016).
12. Ferreira, N. et al. Molecular tweezers targeting transthyretin amyloidosis. Neurotherapeutics 11, 450–461 (2014).
13. Fogerson, S. M. et al. Reducing synuclein accumulation improves neuronal survival after spinal cord injury. Exp. Neurol. 278, 105–115 (2016).
14. Mlakar, J. et al. Zika virus associated with microcephaly. N. Engl. J. Med. 374, 951–958 (2016).
15. Rasmussen, S. A., Jamieson, D. J., Honein, M. A. & Petersen, L. R. Zika virus and birth defects—reviewing the evidence for causality. N Engl J Med 2016, 1981–1987 (2016).
16. Cao-Lormeau, V.-M. et al. Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. The Lancet 387, 1531–1539 (2016).
17. Moreira, J., Peixoto, T. M., Siqueira, A. M. de & Lamas, C. C. Sexually acquired Zika virus: a systematic review. Clin. Microbiol. Infect. 23, 296–305 (2017).
18. D’Ortenzio, E. et al. Evidence of sexual transmission of Zika virus. N. Engl. J. Med. 374, 2195–2198 (2016).
19. Attar, A., Chan, W.-T. C., Klärner, F.-G., Schrader, T. & Bitan, G. Safety and pharmacological characterization of the molecular tweezer CLR01–a broad-spectrum inhibitor of amyloid proteins’ toxicity. BMC Pharmacol. Toxicol. 15, 1 (2014).
20. Münch, J. et al. Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131, 1059–1071 (2007).
21. Arnold, F. et al. Naturally occurring fragments from two distinct regions of the prostatic acid phosphatase form amyloidogenic enhancers of HIV infection. J. Virol. 86, 1244–1249 (2012).
22. Roan, N. R. et al. Peptides released by physiological cleavage of semen coagulum proteins form amyloids that enhance HIV infection. Cell Host Microbe 10, 541–550 (2011).
23. Usmani, S. M. et al. Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen. Nat. Commun. 5, (2014).
24. Roan, N. R. et al. The cationic properties of SEVI underlie its ability to enhance human immunodeficiency virus infection. J. Virol. 83, 73–80 (2009).