Drug repurposing is potentially the fastest available option in the race to identify safe and efficacious drugs that can be used to prevent and/or treat COVID‐19. By describing the life cycle of the newly emergent coronavirus, SARS‐CoV‐2, in light of emerging data on the therapeutic efficacy of various repurposed antimicrobials undergoing testing against the virus, we highlight in this review a possible mechanistic convergence between some of these tested compounds. Specifically, we propose that the lysosomotropic effects of hydroxychloroquine and several other drugs undergoing testing may be responsible for their demonstrated in vitro antiviral activities against COVID‐19. Moreover, we propose that Niemann‐Pick disease type C (NPC), a lysosomal storage disorder, may provide new insights into potential future therapeutic targets for SARS‐CoV‐2, by highlighting key established features of the disorder that together result in an “unfavorable” host cellular environment that may interfere with viral propagation. Our reasoning evolves from previous biochemical and cell biology findings related to NPC, coupled with the rapidly evolving data on COVID‐19. Our overall aim is to suggest that pharmacological interventions targeting lysosomal function in general, and those particularly capable of reversibly inducing transient NPC‐like cellular and biochemical phenotypes, constitute plausible mechanisms that could be used to therapeutically target COVID‐19.
THE LYSOSOMOTROPIC ACTIVITIES OF DIFFERENT DRUGS UNDERGOING TESTING AGAINST COVID‐19
Multiple available drugs are undergoing repurposed testing to evaluate their safety and efficacy against COVID‐19, in an attempt to expedite drug discovery and approval for use in treating patients with COVID‐19.60 In what follows, we discuss a subset of these drugs currently being tested for COVID‐19, highlighting some of their lysosomotropic effects.
5.1 Chloroquine ± Azithromycin
Among the various repurposed drugs currently under investigation for the treatment of COVID‐19, chloroquine and its derivatives, such as hydroxychloroquine, emerged as the first potentially efficacious existing drug for COVID‐19, based on documented pre‐clinical efficacy and expert consensus by several Chinese scientific authorities.As a result, there has been cumulative interest in testing the efficacy of chloroquine and its derivatives, in the treatment of COVID‐19, such that there are now, over 20 different related clinical trials in trial registries.Additionally, two different phase III clinical trials are currently investigating the use of hydroxychloroquine for pre‐ and post‐exposure prophylaxis against COVID‐19 in healthcare workers (NCT04303507 and NCT04328285). However, it remains unclear how an anti‐malarial drug‐like chloroquine could also exert antimicrobial activity against a viral pathogen‐like SARS‐CoV‐2, raising the question of whether the same mechanism underlying chloroquine's antimalarial activity may also be responsible for its antiviral effects.
Importantly, chloroquine has been used for many years in lysosomal storage disease (LSD) research, given its ability to inhibit lysosomal fusion with endosomes, as well as inhibit the activity of various lysosomal enzymes. These properties of chloroquine allowed it to be used in vitro to pharmacologically induce transient LSD‐like cellular pathology.In fact, this lysosomotropic activity of chloroquine is what is thought to be responsible for its anti‐malarial mode of action. Specifically, chloroquine is believed to undergo trafficking into the lysosomes of Plasmodium trophozoites, where it gets protonated and entrapped, thereby disrupting the fusion of these lysosomes with the “food vacuoles” (ie, phagosomes) of the trophozoites, hampering the latter's ability to feed on engulfed red blood cells.However, this same propensity of chloroquine to traffic into, and concentrate within intracellular acidic organelles, also cross‐reacts with mammalian cells, inducing similar disruptions in the functions of their lysosomes as the ones it induces for protozoal food vacuoles, that is, interfering with endo‐lysosomal fusion, elevating intra‐lysosomal pH, and inducing partial permeabilization of lysosomal membranes, which altogether mirror the lysosomal pathology intrinsic to several LSDs.
Such lysosomal “disruptions” are actually intrinsic to NPC in particular, as previously discussed, which further supports the possibility of a lysosome‐mediated antiviral activity for chloroquine against SARS‐CoV‐2, since chloroquine is capable of inducing transient NPC‐like lysosomal abnormalities that may interfere with intracellular viral trafficking and fusion.
In support of this hypothesis, previous studies have successfully shown that the antiviral activity of chloroquine against several caliciviridae, another family of RNA viruses, occurs through chloroquine's ability to inhibit cathepsin L.Furthermore, chloroquine also inhibits the transport of cholesterol out of the lysosomes, including to the plasma membrane, which would be expected to reduce the abundance, and alter the composition of membrane rafts,thereby mimicking the raft alterations seen in NPC. Additionally, chloroquine has also been shown to interfere with the trimming of the N‐glycosylated side chain of ACE2, which may affect the internalization of ACE2, and subsequently, viral entry.Interestingly, N‐glycosylation has actually been shown to be altered in NPC,74 further suggesting that NPC cells likely possess inherent chloroquine‐like effects of altered N‐glycosylation modification of ACE2, which potentially further offers these cells with “protection” against SARS‐CoV‐2 infection. In that regard, a small open‐label non‐randomized clinical trial conducted in France (EU CTR 2020‐000890‐25) has recently gained considerable interest, after it showed a statistically significant difference in the rates of SARS‐CoV‐2 viral clearance from the nasal swabs of COVID‐19‐positive patients receiving a combination of hydroxychloroquine and azithromycin, the latter being a macrolide antibiotic, compared with those receiving hydroxychloroquine only (P = .002 at Day 3 post‐inclusion), or no antimicrobial therapy whatsoever (P = .005 at Day 3 post‐inclusion).In this context, it is also important to highlight the lysosomotropic activity of azithromycin itself, the drug combined with hydroxychloroquine in that trial, as an add‐on therapy.Similar to chloroquine or its derivatives, azithromycin also undergoes trafficking to, and accumulation within the lysosomes, where it alkalinizes the luminal pH of these organelles, thereby inhibiting the activity of resident enzymes.In fact, the combination of both drugs, chloroquine and azithromycin, has been previously shown to exhibit synergistic lysosomotropic effects, especially with regards to increasing lysosomal pH.Moreover, chronic azithromycin treatment in patients with cystic fibrosis has been shown to increase susceptibility to mycobacterial infections, which usually rely heavily on adequate phagocytosis and bacterial containment within phagosomes.Azithromycin has been particularly shown to block the lysosome‐mediated acidification of phagosomes containing the mycobacteria, allowing the latter to escape the phagosomes and multiply uncontrollably.However, in contrast to mycobacteria where the intact lysosomal function is required to contain/control the infection, in SARS‐CoV‐2 infections, the intact lysosomal function is actually needed for successful viral fusion and establishment of infection, as discussed earlier. Thus, it is possible that the observed synergistic efficacy of azithromycin combination with hydroxychloroquine, in the treatment of SARS‐CoV‐2, is the result of their similar lysosome‐mediated antiviral activities, that is, their independent inhibition of endosomal‐lysosomal fusion and lysosomal proteases, which are key for successful viral fusion. In addition, azithromycin's tropism toward the lysosomes has also been shown to induce an accumulation of neutral lipids, namely free cholesterol and phospholipids, within these organelles, which phenocopies the “natural” cellular phenotype of NPC cells.
5.2 Remdesivir
Another promising drug with documented pre‐clinical efficacy against COVID‐19, and undergoing testing in multiple different phase III clinical trials, is remdesivir (NCT04292899, NCT04292730, etc), an adenosine analog originally developed to treat Ebola. However, besides its efficacy against the Ebola virus, remdesivir has been shown to possess remarkable in vivo antiviral activity against several members of the coronaviridae family, including the feline coronavirus that is implicated in feline infectious peritonitis type I (FCoV‐I),as well as the middle east respiratory syndrome coronavirus (MERS‐CoV) and SARS‐CoV, two human coronaviruses that belong to the same genus as SARS‐CoV‐2, that is, the beta coronaviruses.
Interestingly, infection by FCoV‐I, the coronavirus species showing the greatest response to remdesivir treatment, was inhibited in host cells pre‐treated with U18666A, a well‐known direct pharmacological inhibitor of the NPC1 protein that is used to induce NPC‐like lysosomal dysfunction, with a resultant lysosomal accumulation of cholesterol and sphingolipids.This raises the possibility of a mechanistic convergence or synergy, albeit being a partial one, between the U18666A‐mediated direct inhibition of NPC1, and remdesivir's demonstrated activity against SARS‐CoV‐2, besides its primary mechanism of action being the inhibition of the viral RNA‐dependent RNA polymerase.We, therefore, propose evaluating the antiviral potential of U18666A against SARS‐CoV‐2, given its capacity to induce an NPC cellular phenocopy, as an easy starting point to support our hypothesis. Although the safety of U18666A has not been established yet in humans, it has been shown to be without major toxicity in at least two animal models, that is, rats and cats.
In addition, various adenosine analogs, especially the ribose‐modified subtypes such as those used as antivirals or antineoplastic agents, have been shown to possess cross‐binding and activation or inhibition capacities for adenosine receptors,with different affinities toward the A1 and A2a adenosine receptor types.The reason for mentioning this is to highlight that stimulating A2a receptors has been previously shown to rescue the cholesterol entrapment seen in NPC, with that effect being abolished by A2a antagonism.Thus, despite a current lack of data on remdesivir's ability to bind and subsequently, activate or inhibit any of the adenosine receptors, it is worth entertaining the possibility that, given its structural similarity to adenosine, remdesivir may potentially act as an A2a receptor antagonist, causing it to induce a transient NPC‐like cellular environment as part of its antiviral activity against SARS‐CoV‐2. In fact, several adenosine analogs have been previously shown to directly inhibit lysosomal activity in neutrophils, as well as compete with intracellular adenosine on binding adenosine deaminase, an intra‐lysosomal adenosine‐metabolizing enzyme,leading to the accumulation of adenosine within the lysosomal lumen and inducing an LSD‐like cellular pathology with impaired regulation of lysosomal calcium stores.Such features have all been previously shown to be part of the cytopathological findings in NPC.
5.3 Triazoles
Another class of drugs that is being considered for testing against COVID‐19, is the triazole antifungals, namely posaconazole, itraconazole, and their modified derivatives. This is based on previous studies that have demonstrated potent antiviral activities for these compounds, against a wide array of coronaviridae members, including FCoV‐I, human coronavirus‐229E (HCoV‐229E), MERS‐CoV, and SARS‐CoV. While triazoles are widely used as antifungal agents owing to their inhibition of ergosterol synthesis, which is an essential component of fungal cell membranes, they have also been shown to possess antiviral activities that were attributed to their differential inhibitory actions on viral helicases, including coronavirus helicase, thereby interfering with viral replication.Interestingly, however, triazole antifungals are also notable for their cross‐interference with mammalian cholesterol homeostasis, including both cholesterol synthesis and trafficking, due the molecular resemblance between cholesterol and ergosterol.106 In fact, triazoles have been actually shown to inhibit lysosomal cholesterol efflux, through directly binding to, and inhibiting the activity of NPC1 within the lysosomal membrane.Thus, besides their inhibition of viral helicase, which would interfere with SARS‐CoV‐2 replication, the predicted antiviral activities of triazole antifungals against SARS‐CoV‐2 could also be mediated by their direct interference with lysosomal cholesterol egress and its eventual trafficking to the plasma membrane and membrane rafts, via NPC1‐targeted inhibition.
5.4 Glycopeptides
An additional class of drugs undergoing testing against COVID‐19 is the glycopeptide antibiotics, primarily vancomycin, teicoplanin, and their modified derivatives. In vitro studies evaluating the antiviral potentials of these compounds against SARS‐CoV‐2 have successfully demonstrated their efficacy in blocking viral entry into host cells.Speculations of a possible antiviral efficacy exhibited by these compounds against SARS‐CoV‐2 were based on their previously demonstrated activities against several members of the coronaviridae family, including FCoV‐I, SARS‐CoV, and MERS‐CoV.Unlike their established anti‐bacterial activity that is mediated by their inhibition of cell wall synthesis, the antiviral activity of glycopeptide antibiotics is attributed to their various intracellular effects on host cells; these mainly include their direct inhibition of cathepsin L and their trafficking to, and accumulation within the lysosomes, such that they “overload” the latter organelles.Additionally, glycopeptide antibiotics have also been shown to induce increased ROS production within eukaryotic cells, which arguably may not be necessarily relevant for viral killing as they normally are for bacterial killing.However, such findings further support our hypothesis, because NPC cells not only have an already reduced cathepsin L activity and overloaded lysosomes, but they also have elevated baseline levels of ROS intracellularly.These combined intracellular effects of glycopeptides, therefore, create intracellular characteristics that mimic those intrinsic to NPC, which possibly accounts for their demonstrated antiviral activity against SARS‐CoV‐2. It is, however, worth mentioning that within the NPC patient community, several patients receiving glycopeptide antibiotics to treat hospital‐acquired pneumonia were subsequently found to develop focal pyogenic skin abscesses, whose underlying mechanism remains unclear (unpublished data). This anecdotal yet consistent finding could be due to the lysosomal accumulation of glycopeptide antibiotics, and their disruption of lysosomal function,which causes these drugs to further burden the already overloaded lysosomes in NPC cells, including phagocytes, thereby hindering the latter's ability to destroy engulfed debris, resulting in abscess formation.
5.5 Cepharanthine
Finally, another repurposed drug being tested against COVID‐19 is cepharanthine, a plant‐derived alkaloid with prominent anti‐inflammatory effects.In fact, cepharanthine demonstrated the highest potency among 2406 different clinically approved drugs that were screened against COVID‐19, with preclinical data suggesting it targets the entry of SARS‐CoV‐2.118 Interestingly, cepharanthine has also been shown to undergo intracellular trafficking to the lysosomes, where it physically interacts with and inhibits the NPC1 protein, resulting in lysosomal cholesterol accumulation and elevated intra‐lysosomal pH.It is, therefore, possible that cepharanthine's exhibited activity against SARS‐CoV‐2 is mediated, at least partially, by its lysosomotropic effects of directly inhibiting the NPC1 protein and inducing a cellular phenocopy of NPC.
CONCLUSION
To summarize, this report raises the hypothesis that the intracellular biochemical abnormalities inherent to LSDs in general and NPC in particular, may pose an “unfavorable” host cell environment for the entry, trafficking, and fusion of SARS‐CoV‐2. Specifically, we postulate that the altered composition of the plasma membrane and lipid rafts in NPC may affect the trafficking of ACE2, the primary host cell membrane receptor responsible for viral docking, thereby interfering with viral infection. Moreover, the increased levels of ADAM17 in the plasma membrane of NPC cells promote ACE2 shedding, thereby inhibiting viral docking at the plasma membrane of host cells. Additionally, the NPC‐related lysosomal membrane permeabilization, which leads to cathepsin L leakage, and the increased intra‐lysosomal pH seen in NPC, impair the activity of cathepsin L, a key protease required for the successful fusion of SARS‐CoV‐2.
Furthermore, we highlight how two key oxysterols whose levels are notably elevated in NPC, 25‐HC, and 7‐KC, possess potent antiviral activities, which further grants NPC cells the characteristic of being an unfavorable host cell environment for successful SARS‐CoV‐2 infectivity. We also discuss how the different repurposed drugs demonstrating preliminary efficacy in the treatment of COVID‐19 (chloroquine, azithromycin, remdesivir, triazoles, glycopeptide antibiotics, and cepharanthine) possess lysosomotropic activities, which we propose as being the unifying mechanism underlying their demonstrated and shared antiviral activity against SARS‐CoV‐2. Overall, we propose that pharmacologically targeting one or more of the metabolic facets that comprise the NPC cellular phenotype, may prove beneficial in identifying and rapidly developing treatments for COVID‐19 (Figure 1).
Finally, it is important to note that most of the evidence we present here in support of a role of the lysosomes in SARS‐CoV‐2 infectivity is mainly circumstantial and inferred from studies originally designed to test other hypotheses. Thus, going forward, it will be important to test some of our proposed connections between LSD‐related lysosomal dysfunction, especially those pertaining to NPC, and reduced SARS‐CoV‐2 infectivity, for example, by comparing the rates of successful infection of human NPC vs wild‐type cells, following incubation with labeled SARS‐CoV‐2 pseudovirions. Alternatively, it would also be interesting to compare infection rates in wild‐type pneumocytes before and after the transient induction of an NPC cellular phenotype, for example, via U18666A treatment or treatment with other lysosomotropic agents. It would also be important to establish the safe yet efficacious dosage ranges for the different lysosomotropic compounds discussed here, that would be capable of sufficiently preventing SARS‐CoV‐2 infection. Such information will be key for determining the precise mechanisms of action underlying the demonstrated antiviral activities of the different agents undergoing testing against COVID‐19, which may expedite drug design and development for proper therapeutic targeting of this pandemic. Ultimately, however, large‐sized, randomized, and double‐blinded controlled clinical trials are needed to determine the safety and efficacy of any drug that may be used in the future for COVID‐19, including for the various agents/compounds we described here.
Reference & source information: https://faseb.onlinelibrary.wiley.com/
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