Another Necessary preamble

The anti-viral effects of chloroquine, and specifically against coronaviruses, have been known since at least 2005, in research after the original SARS outbreak (caused by the virus called SARS-CoV; the new coronavirus causing “COVID-19” is related genetically and is called SARS-CoV-2. Colloquially Covid-19 is “SARS-2”). 

The anti-viral action is not new and pre-dates even this key paper on action against SARS-CoV-2; in fact chloroquine had been suspected as a "broad-spectrum" anti-viral before 2005. Commentary to the effect that "you wouldn't expect an anti-malarial drug to work against viruses" (given the malarial parasite is a protozoan) is significantly out of date.

This paper shows how CQ is a potent inhibitor as well as an effective treatment against the SARS-CoV virus in vitro (using "Vero" cells i.e. a culture of kidney cells from the African green money).  

CQ was already suspected of having broad spectrum anti-viral action through raising endosomal pH (i.e. making acidic vesicles in the cell more alkaline, making it harder for the virus to take over the chemistry within those vesicles). The paper also examined changes to the ACE2 protein (=”Angiotensin-Converting Enzyme-2”) which had been shown to be the cellular receptor in the cell membrane for the “spike” proteins of the SARS-CoV virus in 2003. This enzyme is found especially in pneumocytes in lung tissues; also in the GI tract. 

Like many proteins (including viral proteins), ACE-2 undergoes “terminal glycosylation” (addition of sugars to the end of the protein molecule) to become functional. This paper showed that at effective concentrations, chloroquine alters this terminal glycosylation, suggesting that it may inhibit attachment of the virus to the cell. 

And there the story stops, because SARS-1 burned itself out, perhaps because of its lethality, and research into treatments cooled off.

The next part of the story doesn’t involve CQ but merely zinc. Zinc mediates many biochemical processes such as the proper folding of proteins, but is usually kept at a low concentration in most living cells.

In 2010 these authors showed how intracellular zinc ions inhibit the "RdRp" enzymes (RdRp = RNA-dependent RNA polymerase) used by many RNA viruses to copy themselves, inside cellular endosomes. The RdRp enzymes are sometimes called “replicase” for this reason.

(Note: most enzymes are called "something-ase" the "something" being the reaction they catalyse. Hence RNA-replicating enzyme is "replicase").

The SARS-CoV virus was one of the RNA viruses studied. Using an ionophore (a substance creating "gateways" in the cell membrane allowing ions to cross) called pyrithione, the zinc concentration within the cell can be increased; the ionophore allows more zinc to cross the cell membrane, inhibiting the viral “replicase” enzyme. 

This paper showed that the effect on virus inhibition was probably a direct interference with the RdRp used by all coronaviruses (and other members of the larger “nidovirus” group) as their central mechanism to sequester the machinery  of the host cell. 

As the authors observe laconically: “an interesting basis for further studies into the use of zinc ionophores as antiviral compounds”. 

If you are not a molecular biologist, this paper is more than slightly technical, but you can find a nice lecture video on the basics, and some of the subsequent papers below. 

Next stage is the discovery that chloroquine is indeed a zinc ionophore – made in an oncology context in 2014, looking for a drug that renders cancer cells susceptible to chemotherapy and radiation. journal.pone.0109180.PDF Chloroquine Is a Zinc Ionophore (2014) Jing Xue et 5 alia PLoS ONE 9(10): e109180. doi:10.1371/journal.pone.0109180

(Please see figure to left)

Chloroquine was known empirically to be helpful in cancer, and as we have seen to elevate the pH (ie make less acidic) in organelles such as lysosomes, accelerating apoptosis (cell death). Zinc also was known to have some anti-cancer activity, in particular by affecting membrane permeability of lysosomes. 

Zinc ionophores were thus a target for drugs concentrating zinc in lysosomes and inducing lysosome-mediated apoptosis (as chloroquine was believed to do). This paper was the ground-breaking work of the interaction of zinc with chloroquine in a biological system, and showed conclusively (in a human cancer cell culture) that chloroquine is a zinc ionophore ie a drug that allows zinc ions to pass cellular membranes.

So the potential modes of action of chloroquine include: 

(1) raising endosomal pH, “constipating” the intra-cellular vesicles where the virus tries to replicate itself, 

(2) the suspected action of altering the terminal glycosylation of ACE2 enzymes in the cell membrane so that the virus spike proteins cannot bind so well, and finally 

(3)  the demonstration that intracellular zinc inhibits the RdRp proteins or “replicase” of all coronaviruses, plus the discovery that chloroquine is a zinc ionophore. 

There are thus at least three credible mechanisms for the anti-viral action of chloroquine, including activity against SARS-CoV, the older relative of SARS-CoV-2 causing COVID-19. 

The effectiveness of chloroquine was demonstrated directly by 4 February 2020 by Chinese virologists at the centre of the Wuhan outbreak in the following paper, in which chloroquine was tested along with a suite of other anti-viral drugs in cell culture, citing the Vincent paper from 2005, and noting

 “Chloroquine is known to block virus infection by increasing endosomal pH required for virus/cell fusion, as well as interfering with the glycosylation of cellular receptors of SARS-CoV” [ie the ACE2 enzyme] (review above mechanisms from 2005 !)

Chloroquine and the anti-viral drug remdesevir were identified as effective against the virus well-below any cytotoxic concentrations; moreover chloroquine (but not remdesevir) was also effective when added before virus infection, suggesting its use as a prophylactic. 

This important prophylactic potential of chloroquine is potentially of great importance given the exposure of primary health-care workers to highly contagious patients, and the critical shortage of masks and other PPE for healthcare workers in many parts of the world.

India is already (end March) issuing HCQ to all front-line healthcare workers (established contraindications excepted), but is then only country so far to adopt this policy

b [shown] Immunofluorescence microscopy of virus infection upon treatment of remdesivir and chloroquine. Virus infection and drug treatment were performed as mentioned above. At 48 h p.i., the infected cells were fixed, and then probed with rabbit sera against the NP of a bat SARS-related CoV as the primary antibody and Alexa 488-labeled goat anti-rabbit IgG (1:500; Abcam) as the secondary antibody, respectively. The nuclei were stained with Hoechst dye. Bars, 100 μm. 

This paper is rightly called a "Breakthrough". 

Cited by the Marseilles group on 11 February when available as pre-publication e-print; 

not actually published until 19 February 2020.  

From Chinese scientists at Qingdao. Multi-centre clinical trials in China were showed success against controls in treating COVID-19 pneumonia, in a group of over 100 patients.

By 14 March 2020, the Marseilles group had tabulated the principal studies on the effectiveness of chloroquine against coronaviruses in vitro; noted the concentrations required, cited the Qingdao clinical trial paper, and noted from their own experience that effective concentrations of CQ could be reached with clinically tolerable doses.