Coronaviruses (CoVs) are a group of enveloped, single-stranded positive genomic RNA viruses and some of them are known to cause severe respiratory diseases in human, including Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the ongoing coronavirus disease-19 (COVID-19). One key element in viral infection is the process of viral entry into the host cells. In the last two decades, there is increasing understanding on the importance of the endocytic pathway and the autophagy process in viral entry and replication. As a result, the endocytic pathway including endosome and lysosome has become important targets for development of therapeutic strategies in combating diseases caused by CoVs. In this mini-review, we will focus on the importance of the endocytic pathway as well as the autophagy process in viral infection of several pathogenic CoVs inclusive of SARS-CoV, MERS-CoV and the new CoV named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and discuss the development of therapeutic agents by targeting these processes. Such knowledge will provide important clues for control of the ongoing epidemic of SARS-CoV-2 infection and treatment of COVID-19.
Lysosomotropic agents targeting endosomal/lysosomal pH
CQ, a well-known anti-malarial drug, is probably the most well-studied lysosomotropic agent that accumulates in the acidic organelles such as endosomes and lysosomes and neutralizes their pH. At present, it has been well studied that CQ has a wide-spectrum of anti-viral effects including anti-CoVs, anti-HIV, and anti-type A and B influenza viruses. The anti-viral effects of CQ and its analogs have been reviewed elsewhere. Here we would like to focus on the effect of CQ on CoVs, as summarized. For instance, CQ has been shown to inhibit MERS-CoV replication in vitro via a screening of an FDA-approved compound library. Treatment with CQ at a clinically relevant concentration, either before or after SARS-CoV infection into the Vero E6 cells, was found to be effective in suppressing viral infection, indicating its application in both prophylactic and therapeutic conditions. Similar results were also found in another lysosomotropic agent, ammonium chloride (NH4Cl). It is known that the cleavage of the Spike Glycoprotein (S protein) by proteases is required for the SARS-CoV entry to the cells via a pH-dependent manner. Mechanistically, it is believed that the neutralization of endo-lysosomal pH by CQ inhibits the protease activities and prevents the cleavage of S protein and subsequently impairs the viral entry into the host cell. Interestingly, Wang et al showed that in cells treated with CQ, NH4Cl or Bafilomycin A1 (an endo/lysosomal V-ATPase inhibitor), the viral receptor ACE2 was trapped within perinuclear vacuoles, suggesting that these lysosomotropic agents may also affect the function of ACE2. Since ACE2 serves as the viral receptor for both SARS-CoV and SARS-CoV-2, such observations further support the notion for the potent anti-viral activity of those lysosomotropic agents. Indeed, a very recent study showed that CQ inhibits the SARS-CoV-2 infection at both entry and post-entry stages in Vero E6 cells.
In addition to its direct effects on CoVs, there is evidence of the combinational activity of CQ with other therapeutic agents for treatment of SARS, MERS and possibly COVID-19. For instance, He et al reported that CQ has synergistic effect on glucocorticoid signaling by stabilizing glucocorticoid receptor. Since glucocorticoid is one of the recommended therapy for severe SARS patients, it is possible that CQ may can be used for treatment of COVID-19 in combination of glucocorticoids and clinical trials are thus needed to test the efficacy of such combined therapy.
At present, CQ phosphate has been listed as a new therapeutic in the sixth version of “Guidelines for the Prevention, Diagnosis, and Treatment of COVID-19” issued by the National Health Commission of the People's Republic of China. And a number of clinical trials with CQ have been initiated in China. The current suggested dosage of CQ for COVID-19 is as high as 500 mg, with treatment not exceeding 10 days. However, the usage of CQ phosphate should be evaluated carefully as it may also have side effects such as retinopathy and cautions should be taken for close monitoring of the potential side effect throughout the whole treatment period. The dosage should be reduced or stopped if reduction in haemoglobin concentration, lymphocyte count and platelet count, or the eyesight are observed. In addition, since CQ is the substrate of cytochrome P450 (CYP) enzymes which are responsible for the metabolism of multiple drugs, it might interfere with other medications such as digitoxin (a cardiac glycoside) and tamoxifen (used for treatment of breast cancer). More details of the toxicity and precautious of CQ can be referred elsewhere.
Endosomal-lysosomal protease inhibitors
Cathepsins are endosomal and lysosomal cysteine proteases that play important roles in protein degradation in various cellular processes including both the endocytic pathway and autophagy. The role of cathepsins in viral infection was first identified by Huang et al and they found that one cysteine proteases inhibitor E64d and a specific cathepsin L inhibitor Z-FY(t-Bu)-DMK are able to block the SARS-CoV infection. K11777 is another cysteine protease inhibitor that has been reported to block the entry of SARS-CoV at the sub-nanomolar range. In addition, teicoplanin, a glycopeptide antibiotic and its derivatives inhibit the entry of MERS-CoV and SARS-CoV by inhibition of cathepsin L activity. Interestingly, a serine protease inhibitor camostat was known to inhibit transmembrane protease serine 2 (TMPRSS2) and effectively protected the mice against death caused by SARS-CoV infection. More importantly, a very recent study showed that camostat can also block the entry of SARS-CoV-2 by inhibiting ACE2 and TMPRSS2. Since camostat is already in clinical use for the treatment of chronic pancreatitis, suggesting its therapeutic potential for treatment of COVID-19.
Inhibitors for clathrin-mediated endocytosis
As discussed earlier, clathrin-mediated endocytosis is one of the key mechanisms for viral entry into the host cells, including MHV, SARS-CoV, and MERS-CoV. Therefore, chlorpromazine, an inhibitor for clathrin-dependent endocytosis, have been consistently found to possess significant inhibitory effect on the entry of those CoVs. In fact, chlorpromazine is a FDA-approved drug widely used for treatment of psychotic disorders such as schizophrenia. Importantly, it has been well established for its antivirus function for many types of viruses, including SARS-CoV and MERS-CoVs, as summarized. At present, the clinical application of chlorpromazine in treatment of SARS and MERS has not been reported and it would be of interest to conduct clinical trials for testing the therapeutic effect of chlorpromazine on COVID-19.
In addition, two cardiotonic steroids ouabain and bufalin, selective inhibitors of the plasma membrane Na+/K+-ATPase, have been shown to inhibit the MERS-CoV infection at nanomolar concentrations via affecting the clathrin-mediated endocytosis pathway. Since both of them are also FDA-approved drugs and clinically available, it would be of interest to test them clinically for treatment of COVID-19.
Summary and perspectives
The current ongoing epidemic of SARS-CoV-2 and COVID-19 worldwide has emerged as a significant global public health threat. While urgent regulatory measures in control of the rapid spread of this virus are essential, scientists around the world have quickly engaged in this battle by studying the molecular mechanisms and searching for effective therapeutic strategies against this deadly disease. At present, while the exact role of autophagy remains debatable, there is overwhelming evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many CoVs including SARS-CoVs, MERS-CoVs and possibly SARS-CoV-2. As a result, several inhibitors targeting the endocytic pathway appear to have the therapeutic potential in treatment of COVID-19, including a lysosomotropic agent CQ and a clathrin-mediated endocytosis inhibitor chlorpromazine. Since both are FDA-approved and clinically available, clinical trials either as a single therapy or in combination with other anti-viral drugs are much needed.
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