Synergistic antiviral effect of hydroxychloroquine and azithromycin in combination against SARS-CoV-2: What molecular dynamics studies of virus-host interactions reveal
Authors: Nouara Yahi
Jacques Fantini
Henri Chahinian
Journal: International Journal of Antimicrobial Agents (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: ACE-2, angiotensin-converting enzyme-2; ATM, azithromycin; Azithromycin; CLQ, chloroquine; CLQ-OH, hydroxychloroquine
Affiliations: INSERM UMR_S 1072, 13015 Marseille, France., Aix-Marseille Université, 13015 Marseille, France.

Abstract:  The emergence of SARS-coronavirus-2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19). Hydroxychloroquine (CLQ-OH)/azithromycin (ATM) combination therapy is currently being tested for the treatment of COVID-19, with promising results. However, the molecular mechanism of action of this combination is not yet established. Using molecular dynamics (MD) simulations, this study shows that the drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells. Unexpected molecular similarity is shown between ATM and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses. Due to this mimicry, ATM interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site shared by ATM and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein. CLQ-OH molecules are shown to saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor, angiotensin-converting enzyme-2 (ACE-2). Taken together, these data show that ATM is directed against the virus, whereas CLQ-OH is directed against cellular attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane. This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where therapeutic intervention is likely to be most efficient. This molecular mechanism may explain the beneficial effects of CLQ-OH/ATM combination therapy in patients with COVID-19. Incidentally, the data also indicate that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies. 

Retracted: No

PubPeer comments:
 There are very deep problems with this study. I m just gonna make a list, because this is very problematic: - Which version of the CHARMM forcefield ? - Where s the validation of the parameters for ATM ? - Manual docking. This is wrong when you don t know in advance where the drug should bind preferentially. - Equilibration isn t defined: we don t know the algorithms used, we just infer it s the equilibration because the temperature goes from 0 to 300K. - Production run follows the same problem: should we assume it used the same algorithms as equilibration ? Berendsen (most probable for equil) all the way ?? Then the protein+membrane part: - The box is EXTREMELY SMALL. 30*30*30A. The final system has only 12 lipids and chain A of the spike protein. - The system uses periodic boundary conditions, though it seems the box only has one side of the membrane (where s the other leaflet ?). Does the top part of the system (protein) touches the bottom part of the lipids ? - Same problem on the sides: the system is very small, does the protein touches itself through the periodic boundary conditions ? - The box has only 1128 water molecules. This very small number gives the previous worries an almost certain ending: everything in the box must be seeing their images through the periodic boundary conditions. This is wrong and no result can be right with this. , 
  A significant viral load reduction was observed in the 20 patients treated with CLQ-OH Quoting Gautret et al. for saying this is a big issue in the introduction, since Gautret et al. did not establish the viral load reduction at all and IJAA refuses to retract the paper while agreeing the results don t show that viral load reduction.  CLQ-OH ATM combination therapy already appears superior to CLQ alone as a firstline treatment for COVID-19. Same remark, the paper does not show this at all. Since preliminary elements are wrong, the whole justification for a docking study seems flawed. Comment #1 shows serious limitations on the docking part, especially manual docking. It lacks controls to assess the probability of random docking. ,