A recent study tested the efficacy in a dose–response paradigm of amantadine in vitro on Vero E6 cells infected with SARS-CoV-2 . Amantadine (0.3–300 µM) did not inhibit the binding of the SARS-CoV-2 spike protein to the human ACE2 protein as previously suggested by docking calculations . However, amantadine reproducibly inhibited viral replication with a half-maximal effective concentration (EC50) range of 83–119 µM, which is somewhat higher than therapeutic concentrations after systemic administration.
Recent advances towards the elucidation of the molecular mechanisms implicated in the entry of SARS-CoV-2 into the host cell and the effects of amantadine on protein–protein interactions implicated in these mechanisms have resulted in the formulation of possible mechanisms. The SARS-CoV-2 virus contains four proteins: E and M proteins form part of the viral envelope; N protein binds to the viral genome; and the S (spike) protein binds to the host cell receptor ACE2. Molecular docking studies suggest that amantadine, via interactions with specific amino acids, has the potential to bind to the transmembrane domain of protein E (ETM) .
In a bid to identify underlying mechanisms and candidate drugs for COVID-19, an analysis of differentially expressed genes that co-express with ACE2 indicated by the expression of RNAs isolated from bronchoalveolar lavage fluid cells of patients with COVID-19 by functional enrichment and hub gene cluster analyses were undertaken . Using the connectivity map database with transcriptome profiles of patients with COVID-19, candidate drugs were identified, one of which was amantadine.
These docking calculations suggested that an interaction occurs by hydrogen bridging of amantadine with amino acids PHE26 and ALA22, leading to impaired release of the virus into the host cell and inhibition of its propagation . A bacterial assay suggests that the SARS-CoV-2 E protein may also be inhibited by memantine . The issue relating to the importance of the E protein channel has recently been revisited following elucidation of the structure of the ETM from SARS-CoV-2 , which was found comparable  to that of SARS-CoV-1 in which amantadine significantly inhibited protein E ion channel-mediated activity .
A third potential mechanism focuses on the lysosomotropic action of amantadine [16, 17]. Cleavage of the S protein by host proteases is critical for viral activation and subsequent infection and, given the similarity to SARS-CoV-1, SARS-CoV-2 takes advantage of the endosomal proteases Cathepsin B and L (CTSL, CTSB).
Cathepsin L is a key element of the lysosomal pathway and its disruption is likely to provide a potential therapy for COVID-19. Mechanisms of disruption include decreased expression of the CTSL gene, inhibition of CTSL activity and alterations of its environment following, for example, an increase of lysosomal pH. A simplified schematic of some of the key steps and associated proteins implicated in the invasion of the host cell by SARS CoV-2 is depicted in Fig. 2 where binding to ACE2 is followed by cleavage due to CTSL, resulting in fusion of viral and host cell membranes and release of the viral genome.
Circulating levels of CTSL are increased after SARS-CoV-2 infection where they are positively correlated with disease course and severity  and, importantly, SARS-CoV-2 pseudovirus infection results in increased CTSL expression in human cells in vitro and in human ACE2-transgenic mice in vivo, while CTSL overexpression leads to enhancement of pseudovirus infection in human cells. Moreover, CTSL functionally cleaves the SARS-CoV-2 spike protein leading to enhanced viral entry and amantadine significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection both in vitro and in vivo.
In an independent series of investigations, high-throughput drug screen gene expression analysis identified compounds with the capacity to down-regulate CTSL expression in a well-characterized, retinal pigment epithelial cell line. One of the most potent agents for the down-regulation of the CTSL gene was amantadine (10 µM), which ranked fifth of 466 compounds tested. Furthermore, the lysosomal trapping capacity of amantadine owing to its lipophilicity and increase of lysosomal pH may be indicative of interference with the capacity of the virus to replicate. Together, these findings suggest that amantadine has the potential to decrease the viral load in SARS-CoV-2-infected patients leading to decreased replication and infectivity of the virus .
Damage to the central nervous system (CNS) is well established in COVID-19 and it has been suggested that the neuroinvasive nature of SARS-CoV-2 may contribute to the acute respiratory failure characteristic of COVID-19 . There is evidence to suggest that amantadine and memantine exert their protective effects against COVID-19 via mechanisms involving specific CNS neurotransmitter systems . Both amantadine and memantine are potent antagonists of the NMDA subclass of glutamate receptor and memantine prevents motor incoordination problems in animals infected with the neuroinvasive HCoV-OC43 with concomitant suppression of viral replication in a dose-dependent manner. Rimantadine has inhibitory effects on SARS-CoV-1 and both amantadine and rimantadine have the capacity to block viral entry into vulnerable cell populations. Memantine is also a potent antagonist of the α-7 subtype of the nicotinic acetylcholine receptor, leading to decreased ACE2 receptor expression in respiratory epithelial cells with the potential to inhibit the entry of SARS-CoV-2 .