Glycofullerenes
The emerging of mortal viruses, like Ebola or Zika, and the lack of suitable treatments led the academic and the industrial communities to look for alternative therapeutic routes. Most of these pathogens are RNA enveloped viruses, and they share common infection mechanisms that can be targeted for the preparation of wide spectrum antivirals. The external surface of the envelope of these viruses is covered by glycans that tightly interact with lectin receptors on host cells.64 This strong interaction allows the attachment of the virions to the cells, followed by internalization and infection. Blocking lectin receptors is a general strategy used to stop viral infection at an early stage. Fullerenes have been widely investigated as antiviral molecules, drug carriers, or tissue scaffolds.65 Fullerene applications have been recently extended to the design of mannosylated derivatives to block the entry of viral particles into host cells. Mannose, due to the high affinity with lectin receptors, competes with the virus in the interaction with the host cells. For example, one of the targets is the inhibition of viral particles through the interaction of mannose with the dendritic cell-specific ICAM-grabbing non-integrin (DC-sign). DC-sign receptors mediate the interactions between DCs and T cells.66,67 To exploit these characteristics, mannose was combined with fullerene in the design of the so-called glycofullerenes to study their capacity to inhibit Ebola, Dengue, and other pathogens.
For this purpose, different glycofullerenes were synthesized by changing the number of mannose units (from 12 to 36) and the spacers between the fullerene moieties and by varying steric hindrance in order to obtain a library of molecules.66 The synthetic route is composed of three steps based on “click chemistry”: (1) assembly of glycodendrons by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), (2) synthesis of alkyne-substituted Bingel-Hirsch hexakis-adducts, and (3) the coupling between the last two products again by CuAAC. To increase the number of mannose moieties up to 36, the glycodendron core was changed from malonate to trialkynyl pentaerythritol. In order to compare the different derivatives, in vitro studies were performed. Jurkat cells (lymphocyte T CD4 immortalized cells) expressing DC-sign were used to prove the inhibition capacity of the glycofullerenes on viral infection of Ebola (Figure 6, route A). The study revealed an IC50 in the μM range for the 12 mannose fullerene, a lower efficiency with the 36 mannose fullerene with a short spacer (PEG, with 2 ethylene oxide units), while a nanomolar IC50 was achieved with 36 mannose fullerenes with a longer spacer (PEG, with 3 ethylene oxide units) (Table 3). This first proof-of-concept study was then expanded, aiming to obtain a better antiviral activity by increasing the valence and inserting longer and flexible spacers.68
Figure 6  Chemical design and general scheme to block the viral entry by glycofullerenes. Three different shapes of glycofullerenes can act as inhibitor of viral infection: (A) shape composed of monodisperse fullerene bearing mannose, (B) assembly as “sugar balls” of tridecafullerenes exhibiting mannose on the edges, and (C) supramolecular micellar aggregates of fullerenes bearing mannose.
Table 3  Antiviral Activity of Different Fullerenes Functionalized with Mannose
material  functional groups  core function  number of mannose moieties  virus  number of atoms between fullerene and mannose  IC50  ref
glycofullerene  monosaccharide  malonate  12  Ebola  8  2 μM  (66)
glycofullerene  monosaccharide  trialkynyl pentaerythritol  36  Ebola  22  68 μM
glycofullerene  monosaccharide  trialkynyl pentaerythritol  36  Ebola  28  0.3 μM
trideca-fullerenesa  monosaccharide  malonate  120  Ebola  8  20.375 nM  (69)
trideca-fullerenesb  monosaccharide  malonate  120  Ebola  8  0.667 nM
glycofullerene  disaccharide  trialkynyl pentaerythritol  36  Zika  29  8.35 nM  (70)
Dengue  7.71 nM
trideca-fullerenes  disaccharide  trialkynyl pentaerythritol  120  Zika  29  0.52 nM
Dengue  0.098 nM
trideca-fullerenes  disaccharide  trialkynyl pentaerythritol  360  Zika  48  0.067 nM
Dengue  0.035 nM
micellar glycofullerenes  monosaccharide  trialkynyl pentaerythritol  6  Ebola  23  424 nM  (72)
micellar glycofullerenes  monosaccharide  trialkynyl pentaerythritol  12  Ebola  25  196 nM
a  Small spacer between core C60 and surrounding fullerenes.
b  Large spacer between core C60 and surrounding fullerenes. Based on these studies, another class of multivalent fullerene dendrimers was then designed. A fast and controlled synthetic route was developed to achieve giant globular multivalent fullerenes, containing hundreds of functional groups. The first study was performed with tridecafullerenes containing 120 mannoses.69 The molecular structure is composed of 12 hexakis C60 surrounding a C60 core (Figure 6, route B). Compared to the previous study, an IC50 3 orders of magnitude lower was measured on the inhibition of Ebola virus (Table 3).66,68 In order to present more carbohydrates at the periphery of the dendrimer, a trialkynyl pentaerythritol derivative allowed to afford a tridecafullerene with 360 carbohydrates. In this case, the molecule was synthesized with a C60 tridecafullerene bearing α(1,2)mannobioside.70 The use of this disaccharide was already investigated, showing an increase of affinity with DC-sign receptors by a factor of 3–4.71 The synthetic strategy exploited also the use of strain-promoted copper-free cycloaddition of azides to alkynes (SPAAC) for the coupling of the core fullerene to the surrounding fullerenes. SPAAC allows an easier purification avoiding the removal of cytotoxic copper ions. The inhibition performance of this molecule was studied in vitro with viral pseudoparticles of Dengue and Zika. The comparison was made between 360 and 120 disaccharides tridecafullerenes and 36 disaccharide monofullerene. The results highlighted a picomolar IC50 inhibition on both Zika and Dengue models for the 360 disaccharide glycofullerene (Table 3). The ability to inhibit other types of viruses allows the use of glycofullerenes as broad spectrum antiviral drugs. Moreover, the negligible toxicity to other cells proved the biocompatibility of these molecules.
Following an alternative strategy, a supramolecular assembly of monodisperse glycofullerenes, leading to the formation of micelles, was achieved and tested.72 These micelles present a uniform and spherical shape (Figure 6, route C). The aggregation synthetic route is faster compared to a controlled synthesis of giant glycofullerenes, but it might suffer from low reproducibility and batch-to-batch differences between each formulation. This self-assembled C60 functionalized with 6 or 12 mannoses exposes a large amount of carbohydrate at the surface, leading to an inhibition of Ebola virus in the nanomolar range (IC50 of 424 nM for six mannoses and 196 nM for 12 mannoses, respectively, Table 3). Further in vitro studies evidenced again a good biocompatibility of these glycofullerenes. While there are no in vivo studies yet, these promising results enlarge the panel of molecules in the fight against new emerging viruses.
Functionalized fullerenes have been used for their ability to compete with viral particles through lectin receptors in host cells. There has been a tremendous advancement in the functionalization of fullerenes leading to the preparation of derivatives with a high amount of mannose, capable to enhance the multivalency effect and thus to increase the therapeutic outcome. First, glycofullerenes do not have an intrinsic virucidal activity. They can reduce the infectivity, but they are not able to completely inactivate the virus. Second, the mechanism of action of glycofullerenes relies on their interaction with host cells and not with viral particles. Thus, for a therapeutic application, they should be injected at different time points, ensuring that the local concentration is therapeutically relevant to prevent the virus from invading the host cells. In addition, glycofullerenes can be internalized into host cells losing their viral “shield” activity. On the other hand, the well-developed surface chemistry of glycofullerenes can be used for other key receptors involved in viral entry. For instance, in the case of the current SARS-Cov-2 pandemic, a similar click chemistry strategy can be used to anchor ligands recognized by human lung ACE2 receptors and so inhibiting viral entry.73