3.4  Synergistic antiviral effects of ATM and CLQ-OH
Overall, these molecular modelling studies are consistent with the notion that ATM might inhibit SARS-CoV-2 infection through direct binding to the virus spike and subsequent neutralization of the infection process, which requires spike protein recognition and attachment to gangliosides. This mechanism of action is illustrated in Fig. 8 . Comparing the models in Figs. 8a and 8b shows that both ATM and gangliosides bind to the same site of the spike protein, centred on the QFN triad. Thus, in the presence of ATM, the virus spike would not reach gangliosides on the host plasma membrane (Fig. 8c). To the best of our knowledge, it is the first time that such a mechanism of action is proposed to explain the antiviral effect of ATM.
Fig. 8 CLQ-OH/ATM combination therapy at the molecular level. (a) ATM bound to the SARS-CoV-2 spike protein trimer. (b) Ganglioside dimer (two symmetrically arranged GM1 molecules in a typical chalice-like shape, just like the one observed in lipid raft simulations) bound to SARS-CoV-2 spike protein trimer. Note that both ATM and gangliosides share the same binding region. (c) ATM prevents ganglioside binding to the SARS-CoV-2 spike protein trimer. CLQ-OH, once bound to gangliosides (blue and orange surfaces), also prevents any interaction with the viral spike. (d) 4 CLQ-OH molecules bound to a ganglioside dimer. Each GM1 molecule is blocked by two CLQ-OH molecules (blue and orange surfaces), which wrap around the saccharide part. (e) Detail of the 134-138 SARS-CoV-2 spike protein stretch bound to GM1. Note that the ganglioside interacts with Q-134 and F-135, but not with D-138. (f) Detail of the 134-138 SARS-CoV-2 spike protein stretch bound to ATM. In this case, the binding site includes D-138 in addition to Q-134 and F-135. Note that N-137, which interacts with both ATM and GM1, is not visible in these representations as it is located behind.
This study also looked at CLQ-OH and its interaction with GM1. We recently published a model describing a complex formed by one GM1 and two CLQ-OH molecules [10]. For comparison, the same molecular modelling approaches as for ATM (in the presence of surrounding raft lipids) were applied to this model. A stable complex formed by a ganglioside dimer, each monomer being associated with two CLQ-OH molecules, was obtained (Fig. 8d). The stability of this complex is reinforced by a rearrangement of CLQ-OH molecules that interact with each other as well as interacting with the saccharide part of GM1. Consequently, the surface of the ganglioside is almost completely masked, so that the ganglioside dimer can no longer be recognized by the viral spike (Fig. 8c). In the presence of both ATM and CLQ-OH, virus-ganglioside interactions are efficiently blocked, preventing any close contact between the virus and the plasma membrane of host cells. The molecular details of this synergistic antiviral effect are worth mentioning. As shown in Figs. 5 and 8e and Table S1, the QFN triad of the virus spike protein is predicted to interact with the central region of the ganglioside dimer. If the dimer is compared metaphorically to a butterfly, this region corresponds to the insect's head between the wings. For its part, CLQ-OH binds to the wings (Fig. 8d), whereas ATM neutralizes the QFN triad of the virus spike protein (Fig. 8f). Indeed, all attempts to obtain a stable raft-spike protein complex aborted when GM1 was covered by CLMQ-OH and when ATM was bound to the spike protein. In the case of ATM, these data confirm that the QFN triad is critical for GM1 recognition and that although other residues are involved (Fig. 5 and Table S1), the whole binding process is fully controlled by the primary interaction driven by the QFN triad. In agreement with this notion, mutating the QFN triad with alanine residues resulted in an aborted ATM binding process at the post-docking steps. Taken together, these molecular modelling studies indicate that CLQ-OH and ATM, when bound to their respective targets, totally mask the complementary surfaces provided by the lipid raft and the virus spike (Fig. 8c): CLQ-OH binds to GM1 and covers the wing, ATM binds to the virus spike and prevents any interaction with the central area of the ganglioside dimer. Hence, both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane.