4  Mode of action of chloroquine
Chloroquine has multiple mechanisms of action that may differ according to the pathogen studied.
Chloroquine can inhibit a pre-entry step of the viral cycle by interfering with viral particles binding to their cellular cell surface receptor. Chloroquine was shown to inhibit quinone reductase 2 [56], a structural neighbour of UDP-N-acetylglucosamine 2-epimerases [57] that are involved in the biosynthesis of sialic acids. The sialic acids are acidic monosaccharides found at the extremity of sugar chains present on cell transmembrane proteins and are critical components of ligand recognition. The possible interference of chloroquine with sialic acid biosynthesis could account for the broad antiviral spectrum of that drug since viruses such as the human coronavirus HCoV-O43 and the orthomyxoviruses use sialic acid moieties as receptors [58]. The potent anti-SARS-CoV-1 effects of chloroquine in vitro were considered attributable to a deficit in the glycosylation of a virus cell surface receptor, the angiotensin-converting enzyme 2 (ACE2) on Vero cells [59].
Chloroquine can also impair another early stage of virus replication by interfering with the pH-dependent endosome-mediated viral entry of enveloped viruses such as Dengue virus or Chikungunya virus [60,61]. Due to the alkalisation of endosomes, chloroquine was an effective in vitro treatment against Chikungunya virus when added to Vero cells prior to virus exposure [30]. The mechanism of inhibition likely involved the prevention of endocytosis and/or rapid elevation of the endosomal pH and abrogation of virus–endosome fusion. A pH-dependant mechanism of entry of coronavirus into target cells was also reported for SARS-CoV-1 after binding of the DC-SIGN receptor [62]. The activation step that occurs in endosomes at acidic pH results in fusion of the viral and endosomal membranes leading to the release of the viral SARS-CoV-1 genome into the cytosol [63]. In the absence of antiviral drug, the virus is targeted to the lysosomal compartment where the low pH, along with the action of enzymes, disrupts the viral particle, thus liberating the infectious nucleic acid and, in several cases, enzymes necessary for its replication [64]. Chloroquine-mediated inhibition of hepatitis A virus was found to be associated with uncoating, thus blocking its entire replication cycle [22].
Chloroquine can also interfere with the post-translational modification of viral proteins. These post-translational modifications, which involve proteases and glycosyltransferases, occur within the endoplasmic reticulum or the trans-Golgi network vesicles and may require a low pH. For HIV, the antiretroviral effect of chloroquine is attributable to a post-transcriptional inhibition of glycosylation of the gp120 envelope glycoprotein, and the neosynthesised virus particles are non-infectious [19,65]. Chloroquine also inhibits the replication Dengue-2 virus by affecting the normal proteolytic processing of the flavivirus prM protein to M protein [32]. As a result, viral infectivity is impaired. In the herpes simplex virus (HSV) model, chloroquine inhibited budding with accumulation of non-infectious HSV-1 particles in the trans-Golgi network [66]. Using non-human coronavirus, it was shown that the intracellular site of coronavirus budding is determined by the localisation of its membrane M proteins that accumulate in the Golgi complex beyond the site of virion budding [67], suggesting a possible action of chloroquine on SARS-CoV-2 at this step of the replication cycle. It was recently reported that the C-terminal domain of the MERS-CoV M protein contains a trans-Golgi network localisation signal [68].
Beside affecting the virus maturation process, pH modulation by chloroquine can impair the proper maturation of viral protein [32] and the recognition of viral antigen by dendritic cells, which occurs through a Toll-like receptor-dependent pathway that requires endosomal acidification [69]. On the contrary, other proposed effects of chloroquine on the immune system include increasing the export of soluble antigens into the cytosol of dendritic cells and the enhancement of human cytotoxic CD8+ T-cell responses against viral antigens [70]. In the influenza virus model, it was reported that chloroquine improve the cross-presentation of non-replicating virus antigen by dendritic cells to CD8+ T-cells recruited to lymph nodes draining the site of infection, eliciting a broadly protective immune response [71].
Chloroquine can also act on the immune system through cell signalling and regulation of pro-inflammatory cytokines. Chloroquine is known to inhibit phosphorylation (activation) of the p38 mitogen-activated protein kinase (MAPK) in THP-1 cells as well as caspase-1 [72]. Activation of cells via MAPK signalling is frequently required by viruses to achieve their replication cycle [73]. In the model of HCoV-229 coronavirus, chloroquine-induced virus inhibition occurs through inhibition of p38 MAPK [44]. Chloroquine is a well-known immunomodulatory agent capable of mediating an anti-inflammatory response [11]. Therefore, there are clinical applications of this drug in inflammatory diseases such as rheumatoid arthritis [74], [75], [76], lupus erythematosus [6,77] and sarcoidosis [78]. Chloroquine inhibits interleukin-1 beta (IL-1β) mRNA expression in THP-1 cells and reduces IL-1β release [72]. Chloroquine-induced reduction of IL-1 and IL-6 cytokines was also found in monocytes/macrophages [79]. Chloroquine-induced inhibition of tumour necrosis factor-alpha (TNFα) production by immune cells was reported to occur either through disruption of cellular iron metabolism [80], blockade of the conversion of pro-TNF into soluble mature TNFα molecules [81] and/or inhibition of TNFα mRNA expression [72,82,83]. Inhibition of the TNFα receptor was also reported in U937 monocytic cells treated with chloroquine [84]. In the Dengue virus model, chloroquine was found to inhibit interferon-alpha (IFNα), IFNβ, IFNγ, TNFα, IL-6 and IL-12 gene expression in U937 cells infected with Dengue-2 virus [33].