Hydroxychloroquine - How it works: by raising the pH of your Endosomes
I'm posting a highlighted transcription of this video by JJ Medicine (the parts about how hydroxychloroquine raises your pH), below this video, highlighting the parts I think you may want to see.
This lesson is on chloroquine and hydroxychloroquine, its mechanism of action, more specifically on its antiviral properties against SARS coronavirus - or SARS CoV2 - the novel coronavirus that causes Covid 19. So if you want more information on chloroquine and hydroxychloroquine in general please check out my other lesson on that topic.
So, chloroquine and hydroxychloroquine have multiple effects on cellular and organelle functioning, for one these medications have immunomodulatory effects. So they can actually inhibit certain immune cell functions which is certain immune cell functions which is helpful when we're trying to treat autoimmune conditions. So they can autoimmune conditions so they can actually be used to treat certain actually be used to treat certain rheumatological conditions like lupus and rheumatoid arthritis. I'm not going to talk about these immunomodulatory effects in this lesson but if you want more information please check out my other lesson on chloroquine and hydroxychloroquine.
The second major property that these medications has is that they can alkalinize vacular or organelle pH like the lysosomal pH. So the reason is is that chloroquine is alkaline and it can actually enter into organelles like the lysosome and they can enter into endosomes as well to actually increase the pH of those organelles decreasing those organelle function. So it can be used to treat malaria so malaria is caused by the protozoa of Plasmodium species so it can inhibit protozoal food vacuole functioning by alkalinizing that food vacuole. And as i mentioned before it can actually alkalinize the endosomes, lysosomes and it can actually inhibit in the cytosis and lysosomal fusion and function.
The third property of chloroquine is that it is known to be a zinc ionophore, so being a zinc ionophore, it can actually allow the influx of zinc into cells and into organelles like lysosomes. And there's some question as to allowing the zinc into cells - Does it also have an anti-cancer effect as well? I'm not going to talk about that here, but that's something interesting to note as well.
And the fourth point I want to make here is that chloroquine in hydroxychloroquine are known to bind to sialic acids, and I'll talk about what sialic acids are a little later on. So points 2, 3 & 4 are the points I'm going to talk about in this with regards to antiviral action against SARS CoV2, - or the Coronavirus that causes COVID-19.
So a quick disclaimer before I get into the antiviral properties of these medications this evidence I'm going to show you has been shown in in vitro studies essentially in a cell culture petri dish so keep that in mind before we get into these antiviral mechanisms of action.
So before we get into the mechanism of action I want to lay down the setting as to where all this is taking place.
So here is the plasma membrane of the host cell, and here is the host cell itself. Here is a lysosome - an acidified organelle that is responsible for recycling of nutrients and other waste products and here's the nucleus that contains the genetic code.
The first point I want to talk about here is that chloroquine inhibits endocytosis. We talked about how chloroquine is an alkalinizing agent. Well, we're going to talk about how it blocks endocytosis and there's a paper that has shown that it can block Zika virus infections so this is where we're going to start.
So this paper talks about how it blocks endocytosis. So the SARS CoV2 - virus is here and this virus contains spike or S proteins on its surface. These S proteins allow it to bind to ACE2 - receptors on the host cell. When it does actually bind to the ACE2 - receptor, it can actually be endocytocized into the cell into an endosome so it's brought into the cell in this little endosome.
That endosome will eventually transverse through the cytosol and fuse to a lysosome where the virus can enter into the lysosome and exit the lysosome allowing it to infect the cell. So with regards to chloroquine, chloroquine can block this process through entering into the cell, and it can actually permeate into endosomes and the lysosome allowing it to alkalinize the endosome and lysosome which means that it can increase the pH of the endosome and lysosome, preventing it from being acidified. So those lysosomes and endosomes are not acidic the way they are supposed to be, so they become less functional or dysfunctional.
It may also inhibit a proton pump on the lysosome, I've talked about this in previous lessons that there may be some inhibition of a proton pump on the lysosome known as V ATP ase or vacular ATP ase although this is not entirely known at present.
Nevertheless due to the alkalinization of endosomes and lysosomes, endocytosis - the process of endocytosis - bringing the virus into the cells - is actually inhibited. It's stopped, and the fusion of endosomes to lysosomes is also stopped. So this whole process whereby the virus can enter the cell and get in can be stopped or slowed down.
So the way this happens again is that chloroquine increases endosomal and lysosomal pH, and in doing so decreases endosomal and lysosomal functioning, so it decreases or inhibits endocytosis.
So the second point I want to make regarding chloroquine's antiviral properties is that chloroquine is a zinc ionophore. There's actually a paper published in +1 in 2014 indicating and showing evidence that chloroquine can act as a zinc ionophore. That means that chloroquine can allow the entry of zinc into a cell.
So, how does chloroquine actually do this so without help? Zinc cannot enter into a cell it essentially bounces off of the cell membrane but with chloroquine it can actually attach to chloroquine and chloroquine can actually guide it through the plasma membrane allowing zinc to enter into the cell so why is all of that important?
So the reason that all that is important is that there's a study showing that zinc inhibits coronavirus and artery virus RNA polymerase activity in vitro in zinc ion affords block the replication of these viruses in cell culture and this paper was published in 2010.
So normally, when a virus enter specifically an RNA virus, there's an RNA dependent polymerase or RDRP. That RNA dependent RNA polymerase will essentially make viral RNA. There's a lot of steps involved but I won't get into all of those steps here. Nonetheless, this our RDRP will produce viral RNA, it replicates the virus.
However, when there is chloroquine and zinc around, these can cross over the plasma membrane together. Again, the chloroquine acts as a zinc ionophore, allowing zinc and chloroquine to enter into the cell. We talked about this before, chloroquine has its effects on the endosomes and lysosomes, but this mechanism here more specifically
has to
do with the zinc. So as the chloroquine brings in zinc, zinc concentrations in the cytosol increase. And the zinc can then act to inhibit this RNA dependent RNA polymerase action. So it prevents that polymerase from making viral RNA. So, it essentially stops the virus from replicating its genetic code so it stops making viral RNA.
This study that I just showed actually shows that as zinc concentrations increase, viral RNA decreases. If you want to check out more on that please check out that paper and look at some of their data, they show some interesting data. So again, as zinc concentrations increase viral RNA decreases, that is the second mechanism. And, the third point I want to make about chloroquine and hydroxychloroquine's antiviral properties is based on this newest article that is being published in April of 2020. So, this article is entitled structural and molecular modeling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS CoV 2 infection. So what does this paper show? So it all has to do with the cell membrane around the ACE 2 receptor. There's actually something called sialic acid that is attached to the cell membrane, that is usually pretty close to the ACE2 - receptor, although it can be on much of the cell membrane itself. Salic acid is essentially just a little piece of sugar and the spike protein, the S protein on the SARS CoV 2 virus can actually bind to the silac acid as well. So these silac acid residues actually can act like little receptors for the S proteins on the coronavirus as well. So what this paper shows is that chloroquine and hydroxychloroquine can actually bind to these silac acid residues, preventing the S protein on the SARS CoV 2 virus from binding to the Sialic acid, and essentially preventing the binding of the virus to the ACE 2 receptor as well. So the way that chloroquine binds to the sonic acid it actually prevents proper binding of the SAR COV-2 virus to the ACE 2 receptor. So this is another mechanism by which chloroquine and hydroxychloroquine can inhibit the SAR CoV2 virus from actually infecting a host cell. So again, the three ways that I talked about in this lesson are that chloroquine and hydroxychloroquine can alkalinize endosomes in lysosomes, preventing endocytosis or preventing this process from occurring. Can also allow the influx of zinc into the host cell. Increasing concentrations can lead to inhibition of the RNA dependent RNA polymerase, and preventing viral RNA from being formed. The third point is that chloroquine hydroxychloroquine can bind to silac acid preventing the S protein on SAR CoV 2 from properly binding to the ACE 2 receptor. So these are the few mechanisms by which chloroquine and hydroxychloroquine have been shown to have some antiviral effects, at least in vitro. Again, the big disclaimer here is thatall of this has been shown in vitro essentially in cell culture in a petri dish. So, this is far from being shown in human clinical trials, so keep that in mind when we look at this evidence so far.
Hydroxychloroquine and COVID-19: What We Know Right Now | SciShow News (see video further down the page)
From the transcription:
A lysosome’s big job is to gobble up and destroy rogue proteins and other compounds that might be harmful to the cell. And because they have all those bits of rogue protein, they’re able to help some of our immune cells tell other immune system cells what to do.
Basically, the smaller chunks of proteins and other stuff they break down become antigens: compounds that tell the immune system’s soldiers what to go attack. It’s thought that, when a person has an autoimmune condition, the lysosomes inside certain immune cells produce antigens that tell the immune system’s soldiers to attack the wrong things, the body’s own cells. But these antimalarials can interfere with that.
Since lysosomes are slightly acidic, hydroxychloroquine makes its way inside of them and gets stuck there. It ends up taking a lot of hydrogen ions out of the lysosome fluid, making it less acidic. And once the pH of the lysosome gets nice and basic, it can’t function the same way. Which ultimately means there are fewer lysosomes generating antigensthat tell the immune system to attack the person’s own cells.
But, this does not just happen in the cells involved in self-directed attacks. These drugs can get into and mess with all of your immune cells. Plus, they likely have other effects in immune cells that can calm them down. So, they dampen the immune response in general. Now, if it sounds like dampening the immune system would be, y’know, actually very bad for a person who has COVID-19... well, you might be wrong.
Here’s the thing about a lot of deadly viruses, including coronaviruses. It’s not always the virus that actually kills. Don’t get me wrong, these viruses definitely infect cells and kill them. But it isn’t always infected cells dying that kills a person—not directly anyway. When those cells die, your body realizes there’s an invasion taking place. So, it mounts a counter-attack using chemicals called cytokines, which act as a call to arms to immune cells—raising a battle cry that unleashes the full force and fury of the immune response on the infection.
All of the effects of this are collectively called inflammation, and it can do a great job of stamping out an invader The trouble is that massive amounts of inflammation can damage healthy cells, too. So if a viral infection gets really out of control and kills lots of cells, the immune system’s frenzied reaction may damage and kill even more cells, which call in even more immunological attackers,which causes even more inflammation.
If this whole process spirals out of control, it’s called a cytokine storm. It’s basically the immune system equivalent of trying to kill a fly with a hand grenade, and it’s the collateral damage from that that ends up killing the person.
So it’s thought that a drug like hydroxychloroquine, which eases up the immune response, might be able to quell a cytokine storm. At least, that’s one hypothesis. Another idea is that chloroquine and hydroxychloroquine make it harder for viruses, by raising the pH in cellular compartments they need to infiltrate and replicate. Viruses tend to operate best when these compartments are a little on the acidic side, and antimalarial drugs tend to make things nice and basic.
And there is already some promising research in cells and non-human animals that suggests chloroquine can prevent viral replication in SARS and other coronaviruses. Even better, recent in vitro studies — or experiments carried out in cells in a laboratory —suggest it and hydroxychloroquine might work against SARS-CoV-2 specifically, the coronavirus that causes COVID-19.
Based on this, it seems like these drugs could be super helpful — and a super helpful drug is kind of on everybody’s wish list right now.
Safety precautions about Hydroxychloroquine from the video (you may want to watch the whole thing to see what else he says):
"Multiple researchers have now concluded that additional,
well-designed trials are needed before we can really say
whether antimalarials are actually safe and effective in fighting COVID-19.
And, I keep saying safe, because even though these drugs are prescribed regularly for malaria and autoimmune conditions,that doesn’t mean they’re safe to use in treating coronavirus infections. We already know that not everyone can take these drugs safely. Like, people who have a G6PD deficiency
can have a life-threatening reaction to chloroquine.
Also, these antimalarials might negatively interact with other drugs a person is on. For example, studies have found that these antimalarials seem to slow the breakdown of the heart medication digoxin, which can cause everything from nausea and vomiting to irregular heart rhythms, which can be fatal.
Plus, even in people who don’t have specific reasons not to take them,
antimalarials aren’t totally harmless.
Since they pretty much ubiquitously get into and mess with cells,
the dose really matters
.
Like, for example, although chloroquine and hydroxychloroquine can be taken safely by many people, higher doses can lead to blindness and heart problems. we don’t know for sure whether this drug could make that specific infection worse, or at what point in the course of the illness it might be best to take it.* In the end, it’s very possible that at least one of these drugs really will be great for treating, or maybe even preventing, COVID-19. There are several clinical trials trying to figure all that out."
The antimalarial agents chloroquine and hydroxychloroquine have been used widely for the treatment of rheumatoid arthritis and systemic lupus erythematosus. These compounds lead to improvement of clinical and laboratory parameters, but their slow onset of action distinguishes them from glucocorticoids and nonsteroidal antiinflammatory agents. Chloroquine and hydroxychloroquine increase pH within intracellular vacuoles and alter processes such as protein degradation by acidic hydrolases in the lysosome, assembly of macromolecules in the endosomes, and posttranslation modification of proteins in the Golgi apparatus. It is proposed that the antirheumatic properties of these compounds results from their interference with "antigen processing" in macrophages and other antigen-presenting cells. Acidic cytoplasmic compartments are required for the antigenic protein to be digested and for the peptides to assemble with the alpha and beta chains of MHC class II proteins. As a result, antimalarials diminish the formation of peptide-MHC protein complexes required to stimulate CD4+ T cells and result in down-regulation of the immune response against autoantigenic peptides. Because this mechanism differs from other antirheumatic drugs, antimalarials are well suited to complement these other compounds in combination drug therapy.
Recently, a novel coronavirus (2019-nCoV), officially known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China. Despite drastic containment measures, the spread of this virus is ongoing. SARS-CoV-2 is the aetiological agent of coronavirus disease 2019 (COVID-19) characterised by pulmonary infection in humans. The efforts of international health authorities have since focused on rapid diagnosis and isolation of patients as well as the search for therapies able to counter the most severe effects of the disease. In the absence of a known efficient therapy and because of the situation of a public-health emergency, it made sense to investigate the possible effect of chloroquine/hydroxychloroquine against SARS-CoV-2 since this molecule was previously described as a potent inhibitor of most coronaviruses, including SARS-CoV-1. Preliminary trials of chloroquine repurposing in the treatment of COVID-19 in China have been encouraging, leading to several new trials. Here we discuss the possible mechanisms of chloroquine interference with the SARS-CoV-2 replication cycle.
Chloroquine is an amine acidotropic form of quinine that was synthesised in Germany by Bayer in 1934 and emerged approximately 70 years ago as an effective substitute for natural quinine [1,2]. Quinine is a compound found in the bark of Cinchona trees native to Peru and was the previous drug of choice against malaria [3]. For decades, chloroquine was a front-line drug for the treatment and prophylaxis of malaria and is one of the most prescribed drugs worldwide [4]. Chloroquine and the 4-aminoquinoline drug hydroxychloroquine belong to the same molecular family. Hydroxychloroquine differs from chloroquine by the presence of a hydroxyl group at the end of the side chain: the N-ethyl substituent is β-hydroxylated. This molecule is available for oral administration in the form of hydroxychloroquine sulfate. Hydroxychloroquine has pharmacokinetics similar to that of chloroquine, with rapid gastrointestinal absorption and renal elimination. However, the clinical indications and toxic doses of these drugs slightly differ. In malaria, the indication for chloroquine was a high dose for a short period of time (due to its toxicity at high doses) or a low dose for a long period of time. Hydroxychloroquine was reported to be as active as chloroquine against Plasmodium falciparum malaria and less toxic, but it is much less active than chloroquine against chloroquine-resistant P. falciparum owing to its physicochemical properties. What is advantageous with hydroxychloroquine is that it can be used in high doses for long periods with very good tolerance. Unfortunately, the efficacy of chloroquine gradually declined due to the continuous emergence of chloroquine-resistant P. falciparum strains [5]. Chloroquine is also utilised in the treatment of autoimmune diseases [6]. Yet the activity of the molecule is not limited to malaria and the control of inflammatory processes, as illustrated by its broad-spectrum activity against a range of bacterial, fungal and viral infections [7], [8], [9], [10]. Indeed, in the mid-1990s, due to its tolerability, rare toxicity reports, inexpensive cost and immunomodulatory properties [11], chloroquine repurposing was explored against human immunodeficiency virus (HIV) and other viruses associated with inflammation and was found to be efficient in inhibiting their replication cycle [12].
Recently, a novel coronavirus emerged in the Chinese city of Wuhan in December 2019. After human coronavirus 229E (HCoV-229E) (classified in the genus Alphacoronavirus) and HCoV-OC43 (Betacoronavirus lineage 2a member) described in the 1960s, SARS-CoV-1 (Betacoronavirus lineage 2b member) that emerged in March 2003, HCoV-NL63 (Alphacoronavirus lineage 1b member) described in 2004, HCoV-HKU1 (Betacoronavirus lineage 2a member) discovered in 2005, and finally MERS-CoV that emerged in 2012 (classified in Betacoronavirus lineage 2c), the novel coronavirus is the seventh human coronavirus described to date as being responsible for respiratory infection. Evidence was rapidly reported that patients were suffering from an infection with a novel Betacoronavirus tentatively named 2019 novel coronavirus (2019-nCoV) [13,14]. Despite drastic containment measures, the spread of 2019-nCoV, now officially known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is ongoing. Phylogenetic analysis of this virus indicated that it is different (~80% nucleotide identity) but related to SARS-CoV-1 [15]. Because the world is threatened by the possibility of a SARS-CoV-2 pandemic, the broad-spectrum antiviral effects of chloroquine warranted particular attention for repurposing this drug in the therapy of the disease caused by SARS-CoV-2, named coronavirus disease 2019 (COVID-19).
2. Antiviral properties of chloroquine
In vitro, chloroquine appears as a versatile bioactive agent reported to possess antiviral activity against RNA viruses as diverse as rabies virus [16], poliovirus [17], HIV [12,[18], [19], [20], hepatitis A virus [21,22], hepatitis C virus [23], influenza A and B viruses [24], [25], [26], [27], influenza A H5N1 virus [28], Chikungunya virus [29], [30], [31], Dengue virus [32,33], Zika virus [34], Lassa virus [35], Hendra and Nipah viruses [36,37], Crimean–Congo hemorrhagic fever virus [38] and Ebola virus [39], as well as various DNA viruses such as hepatitis B virus [40] and herpes simplex virus [41].The antiviral properties of chloroquine described in vitro have sometimes been confirmed during treatment of virus-infected patients but have not always been reproduced in clinical trials depending on the disease, the concentration of chloroquine used, the duration of treatment and the clinical team in charge of the trial.
Regarding coronaviruses, the potential therapeutic benefits of chloroquine were notably reported for SARS-CoV-1 [11,42]. Chloroquine was also reported to inhibit in vitro the replication of HCoV-229E in epithelial lung cell cultures [43,44]. In 2009, it was reported that lethal infections of newborn mice with the HCoV-O43 coronavirus could be averted by administering chloroquine through the mother's milk. In vitro experiments also showed a strong antiviral effect of chloroquine on a recombinant HCoV-O43 coronavirus [45]. Although chloroquine was reported to be active against Middle East respiratory syndrome coronavirus (MERS-CoV) in vitro [46], this observation remains controversial [47].
3. Potential antiviral effect of chloroquine against SARS-CoV-2
Because of its broad spectrum of action against viruses, including most coronaviruses and particularly its close relative SARS-CoV-1, and because coronavirus cell entry occurs through the endolysosomal pathway [48], it made sense in a situation of a public-health emergency and the absence of any known efficient therapy to investigate the possible effect of chloroquine against SARS-CoV-2. A recent paper reported that both chloroquine and the antiviral drug remdesivir inhibited SARS-CoV-2 in vitro and suggested these drugs be assessed in human patients suffering from COVID-19 [49].
Recently, the China National Center for Biotechnology Development indicated that chloroquine is one of the three drugs with a promising profile against the new SARS-CoV-2 coronavirus that causes COVID-19. Chloroquine repurposing was investigated in hospitals in Beijing, in central China's Hunan Province and South China's Guangdong Province. According to preliminary reports [50,51] from the Chinese authorities suggesting that approximately 100 infected patients treated with chloroquine experienced a more rapid decline in fever and improvement of lung computed tomography (CT) images and required a shorter time to recover compared with control groups, with no obvious serious adverse effects, the Chinese medical advisory board has suggested chloroquine inclusion in the SARS-CoV-2 treatment guidelines. As a result, chloroquine is probably the first molecule to be used in China and abroad on the front line for the treatment of severe SARS-CoV-2 infections. Although the long use of this drug in malaria therapy demonstrates the safety of acute chloroquine administration to humans, one cannot ignore the minor risk of macular retinopathy, which depends on the cumulative dose [52], and the existence of some reports on cardiomyopathy as a severe adverse effect caused by chloroquine [53,54]. A survey of SARS-CoV-2-infected patients for adverse effects of chloroquine therapy remains to be performed. However, chloroquine is currently among the best available candidates to impact the severity of SARS-CoV-2 infections in humans. Currently, at least ten clinical trials are testing chloroquine as an anti-COVID-19 therapy [55].
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].
5. Conclusion
Chloroquine has been shown to be capable of inhibiting the in vitro replication of several coronaviruses. Recent publications support the hypothesis that chloroquine can improve the clinical outcome of patients infected by SARS-CoV-2. The multiple molecular mechanisms by which chloroquine can achieve such results remain to be further explored. Since SARS-CoV-2 was found a few days ago to utilise the same cell surface receptor ACE2 (expressed in lung, heart, kidney and intestine) as SARS-CoV-1 [85,86] (Table 1), it may be hypothesised that chloroquine also interferes with ACE2 receptor glycosylation thus preventing SARS-CoV-2 binding to target cells. Wang and Cheng reported that SARS-CoV and MERS-CoV upregulate the expression of ACE2 in lung tissue, a process that could accelerate their replication and spread [85]. Although the binding of SARS-CoV to sialic acids has not been reported so far (it is expected that Betacoronavirus adaptation to humans involves progressive loss of hemagglutinin-esterase lectin activity), if SARS-CoV-2 like other coronaviruses targets sialic acids on some cell subtypes, this interaction will be affected by chloroquine treatment[87,88]. Today, preliminary data indicate that chloroquine interferes with SARS-CoV-2 attempts to acidify the lysosomes and presumably inhibits cathepsins, which require a low pH for optimal cleavage of SARS-CoV-2 spike protein[89], a prerequisite to the formation of the autophagosome [49]. Obviously, it can be hypothesised that SARS-CoV-2 molecular crosstalk with its target cell can be altered by chloroquine through inhibition of kinases such as MAPK. Chloroquine could also interfere with proteolytic processing of the M protein and alter virion assembly and budding (Fig. 1). Finally, in COVID-19 disease this drug could act indirectly through reducing the production of pro-inflammatory cytokines and/or by activating anti-SARS-CoV-2 CD8+ T-cells.
Table 1. Human coronavirus (HCoV) receptors/co-receptors as possible targets for chloroquine-induced inhibition of the virus replication cycle
It is worth noting that different host cell proteases are required to activate the spike (S) protein for coronaviruses, such as SARS-CoV-1 S protein that requires activation by cathepsin L [89], or MERS-CoV that requires furin-mediated activation of the S protein [98].
Fig. 1. Schematic representation of the possible effects of chloroquine on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication cycle. SARS-CoV2, like other human coronaviruses, harbours three envelope proteins, the spike (S) protein (180–220 kDa), the membrane (M) protein (25–35 kDa) and the envelope (E) protein (10–12 kDa), which are required for entry of infectious virions into target cells. The virion also contains the nucleocapsid (N), capable of binding to viral genomic RNA, and nsp3, a key component of the replicase complex. A subset of betacoronaviruses use a hemagglutinin-esterase (65 kDa) that binds sialic acids at the surface of glycoproteins. The S glycoprotein determines the host tropism. There is indication that SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) expressed on pneumocytes [85,99]. Binding to ACE2 is expected to trigger conformational changes in the S glycoprotein allowing cleavage by the transmembrane protease TMPRSS2 of the S protein and the release of S fragments into the cellular supernatant that inhibit virus neutralisation by antibodies [100]. The virus is then transported into the cell through the early and late endosomes where the host protease cathepsin L further cleaves the S protein at low pH, leading to fusion of the viral envelope and phospholipidic membrane of the endosomes resulting in release of the viral genome into the cell cytoplasm. Replication then starts and the positive-strand viral genomic RNA is transcribed into a negative RNA strand that is used as a template for the synthesis of viral mRNA. Synthesis of the negative RNA strand peaks earlier and falls faster than synthesis of the positive strand. Infected cells contain between 10 and 100 times more positive strands than negative strands. The ribosome machinery of the infected cells is diverted in favour of the virus, which then synthesises its non-structural proteins (NSPs) that assemble into the replicase-transcriptase complex to favour viral subgenomic mRNA synthesis (see the review by Fehr and Perlman for details [101]). Following replication, the envelope proteins are translated and inserted into the endoplasmic reticulum and then move to the Golgi compartment. Viral genomic RNA is packaged into the nucleocapsid and then envelope proteins are incorporated during the budding step to form mature virions. The M protein, which localises to the trans-Golgi network, plays an essential role during viral assembly by interacting with the other proteins of the virus. Following assembly, the newly formed viral particles are transported to the cell surface in vesicles and are released by exocytosis. It is possible that chloroquine interferes with ACE2 receptor glycosylation, thus preventing SARS-CoV-2 binding to target cells. Chloroquine could also possibly limit the biosynthesis of sialic acids that may be required for cell surface binding of SARS-CoV-2. If binding of some viral particles is achieved, chloroquine may modulate the acidification of endosomes thereby inhibiting formation of the autophagosome. Through reduction of cellular mitogen-activated protein (MAP) kinase activation, chloroquine may also inhibit virus replication. Moreover, chloroquine could alter M protein maturation and interfere with virion assembly and budding. With respect to the effect of chloroquine on the immune system, see the elegant review by Savarino et al. [11]. ERGIC, ER-Golgi intermediate compartment.
Already in 2007, some of us emphasised in this journal the possibility of using chloroquine to fight orphan viral infections [10]. The worldwide ongoing trials, including those involving the care of patients in our institute [90], will verify whether the hopes raised by chloroquine in the treatment of COVID-19 can be confirmed.
Acknowledgment
The figure was designed using the Servier Medical Art supply of images available under a Creative Commons CC BY 3.0 license.
We aimed to analyze the interactions of both hydroxychloroquine and chloroquine with SARS-CoV-2 and identify their possible role for the prevention/treatment of COVID-19 by molecular docking studies. Protein crystal structures of SARS-CoV-2 and ACE2, the compounds hydroxychloroquine and chloroquine, and other ligand structures were minimized by OPLS3 force field. Glide Standard Precision and Extra Precision docking are performed and MM-GBSA values are calculated. Molecular docking studies showed that hydroxychloroquine and chloroquine do not interact with SARS-CoV-2 proteins, but bind to the amino acids ASP350, ASP382, ALA348, PHE40 and PHE390 on the ACE2 allosteric site rather than the ACE2 active site. Our results showed that neither hydroxychloroquine and chloroquine bind to the active site of ACE2. However, both molecules prevent the binding of SARS-CoV-2 spike protein to ACE2 by interacting with the allosteric site. This result can help ACE2 inhibitor drug development studies to prevent viruses entering the cell by attaching spike protein to ACE2.
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