The SARS-CoV-2 pandemic necessitates a review of the molecular mechanisms underlying cellular infection by coronaviruses, in order to identify potential therapeutic targets against the associated new disease (COVID-19). Previous studies on its counterparts prove a complex and concomitant interaction between coronaviruses and autophagy. The precise manipulation of this pathway allows these viruses to exploit the autophagy molecular machinery while avoiding its protective apoptotic drift and cellular innate immune responses. In turn, the maneuverability margins of such hijacking appear to be so narrow that the modulation of the autophagy, regardless of whether using inducers or inhibitors (many of which are FDA-approved for the treatment of other diseases), is usually detrimental to viral replication, including SARS-CoV-2. Recent discoveries indicate that these interactions stretch into the still poorly explored noncanonical autophagy pathway, which might play a substantial role in coronavirus replication. Still, some potential therapeutic targets within this pathway, such as RAB9 and its interacting proteins, look promising considering current knowledge. Thus, the combinatory treatment of COVID-19 with drugs affecting both canonical and noncanonical autophagy pathways may be a turning point in the fight against this and other viral infections, which may also imply beneficial prospects of long-term protection
Autophagy Modulators are Promising Anticoronavirals
The interplay between coronaviruses and autophagy is very complex and not completely understood. During a coronavirus infection, autophagy is both a cellular response mechanism and a viral replication tool. In fact, coronaviruses can both induce and inhibit autophagy with interactions at multiple levels within a narrow action area limited by apoptosis and the IFN response. Other representative examples of this complexity are that, although autophagy activation inhibits TGEV replication, a proviral mitochondria-selective autophagy is induced in TGEV-infected cells, or that PEDV induces autophagy and benefits from it , but it is also inhibited by rapamycin-induced autophagy.
For all this, and despite existing differences between studies that are almost certainly due to the use of distinct experimental systems, the modulation of autophagy usually affects the replication of coronaviruses, and therefore it becomes a promising therapeutic target in the search for anticoronavirals. Table 4 and Table 5 compile the reported effects of autophagy inducers or inhibitors, respectively, on the infection of different coronaviruses in cell cultures. Half of them are already FDA-approved drugs for other diseases/disorders, and several have already shown inhibitory activity against SARS-CoV-2, i.e., ivermectin, (hydroxy-) chloroquine and nitazoxanide. As can be observed, even classic modulators, such as rapamycin, 3-methyladenine (3-MA) or chloroquine, usually exert an effect on coronavirus replication. In general, among all the autophagy modulators tested, independently of being autophagy inducers or inhibitors, the outcome is usually antiviral activity. This fact may reflect not only the precise viral control over the autophagy pathway, but also the difficulty of maintaining such a balance and the detrimental effect on viral replication if there is any dysregulation in this back-and-forth game.
As shown in Table 4, autophagy inducers generally antagonize coronavirus replication. Among the autophagy inhibitors (Table 5), chloroquine (the most tested one) shows broad-spectrum anticoronaviral activity, which is probably because of its multimodal effects. Briefly, chloroquine, apart from disorganizing the Golgi, induces lysosomal alkalinization, which prevents amphisome/autophagosome-lysosome fusion and blocks the vesicle trafficking system, which potentially affects the replication cycle of coronavirus systemically, including their entry, which is mediated by pH-dependent endocytosis and requires a low pH for the S protein to trigger its membrane fusion activity. Nitazoxanide is another late-stage autophagy blocker that shows high anti-SARS-CoV-2 activity in cell cultures (IC50: 2.12 μM), although it should be considered that its main metabolite, tizoxanide, induces autophagy by inhibiting the PI3K-AKT-MTOR pathway. At this moment, the scientific community is focusing efforts in searching, by different approaches, for effective drugs against this pathogen and continuously revealing autophagy modulators.
Outlook and Challenges
As shown here, drugs that target autophagy, as well as those involved in regulating the endocytic pathway could be added to the arsenal of compounds against coronavirus infections (for an extensive list see Zumla et al. (2016)). As a consequence, the discovery of new autophagy regulatory drugs may be a source of new antivirals that is worth testing for this purpose. In this sense, we propose that the alternative autophagy routes are still scarcely explored in this field and can provide unexpected positive outcomes in the fight against viruses, and particularly coronaviruses. In a follow-up prospective effort, we think that interference with RAB9 activity, a key element in these pathways, might be a promising approach. In this sense, the targeting of GDI/RabGDI (GDP dissociation inhibitor), which forms a complex with RAB9 in the cytosol and mediates its activity in the endosome-trans Golgi network, and specific “GDI-displacement factors” such as RABAC1/Yip3 (Rab acceptor 1) are also candidates worth testing for this purpose.
To conclude, we observed that most of the reviewed works tested the anticoronaviral effect of each autophagy modulator individually in order to accurately unravel the mechanisms involved. Thus, having shown that autophagy and coronavirus replication cycles converge in several different stages, treatment strategies including the combination of autophagy-modulating agents might result in synergistic effects that are worth studying. In this vein, among present combinatory treatments, a frequent one is (hydroxy-) chloroquine together with azithromycin, a macrolide antibiotic with extensively reported autophagy-blocking activity, as well as other family members. Another important factor to consider is the scarce number of in vivo studies in this field, which is certainly due to the required and necessary biosafety restrictions. However, these studies are essential to assess the true potential of these drugs for clinical implementation because the outcome within the complex biosystem can be very different from that of in-cell culture tests. In this sense, despite the inhibitory effects observed in vitro, (hydroxy-) chloroquine treatments, either alone or in combination with azithromycin, has shown no benefits against SARS-CoV-2 infection in clinical trials. Besides, the in vivo context allows the identification of not only possible side effects, but also paradoxical issues such as the fact that the virulence of coronaviruses may be different even if showing similar replication levels. Finally, it is important to mention that autophagy also plays a significant role in adaptive immune responses, and in vivo tests are essential for determining the possible implications in this sense when using autophagic modulators in experimental treatments, as they could be either detrimental or beneficial in the long term.
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