A new study, released on the bioRxiv* preprint server, shows that rapamycin, an immunosuppressive drug used in some cancers, and its analogs, may have an undesirable effect on viral entry into the host cells. This finding could inform their use to treat symptomatic coronavirus disease 2019 (COVID-19).
There is a serious lack of effective antivirals and other preventive agents capable of counteracting the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for the COVID-19 pandemic now ravaging the world. In response, many older drugs are being tested for their potential usefulness in this condition.
Small molecules that are being evaluated for their activity in COVID-19 should therefore be first studied for their impact on host dependency factors, that is, those cellular factors that must be present for the virus to infect the cell; and host restriction factors, that is, those which can suppress viral replication.
The underlying research identified viral targets suitable for drug inhibition, but also revealed the immune pathways that need to be protected in order to fend off symptomatic illness.
Cancer is a condition associated with an immunocompromised state, and, as such, patients with this condition may be more likely to have prolonged infection, shed the virus at higher levels and for longer periods, and be responsible for super-spreader events.
Rapamycin and its analogs (“rapalogs”) are being used as FDA-approved inhibitors of the mTOR (mammalian targets of rapamycin) kinase, in order to inhibit various cancer pathways, treat autoimmune conditions, mitigate graft-vs-host disease, atherosclerosis and even treat the effects of aging. These compounds act by binding to FKB506-binding protein 12 (FKBP12), forming a complex that physically disrupts mTOR-mediated signaling pathways.
The activation of this pathway is associated with cell growth and proliferation, and with cell survival, as well as the differentiation of inflammatory T cells. For this reason, inhibitors of this molecule are used to prevent lymphocyte proliferation and the cytokine storm.
In severe or critical COVID-19, hyperactive inflammation associated with aberrant immunological responses is thought to be the cause of the serious symptoms and signs. Thus, rapalogs and rapamycin are being tested for their utility in COVID-19.
The drug could also be used to prevent the infection, as reported with the earlier Middle East Respiratory Syndrome coronavirus (MERS-CoV). However, further work will be needed to understand how it affects viral replication in the current case, and at different steps of the viral life cycle.
Activity mediated by IFITM
In influenza A, rapamycin has been shown to promote viral replication and worsen disease features because it induces the breakdown of interferon-inducible transmembrane (IFITM) proteins. These unique proteins are found at the lowest layer in many different tissues.
The importance of IFITM lies in its role in innate cell-level immunity, which keeps each cell guard against viral infection. These earlier findings show that in the presence of rapamycin, the IFITM2 and IFITM3 within the endosomes are more rapidly sorted into lysosomes to be broken down.
This sorting process depends on the presence of ubiquitin, and endosomal complexes required for transport (ESCRT). Thus, this work showed that rapamycin is an unsuspected inhibitor of intrinsic cellular immunity.
The IFITM proteins suppress the entry of many enveloped viruses by increasing the difficulty of cell-virus membrane fusion, both at the plasma membrane and the endosomes. Their inhibition by rapamycin could thus make the cell more vulnerable to multiple viruses.
Again, the researchers earlier showed that IFITM1, IFITM2, and/or IFITM3 can be expressed in cell types that allow SARS-CoV-2 infection when they prevent the virus from infecting the cells.
Some single nucleotide polymorphisms in IFITM3, in fact, worsen the outcome in influenza A, and with severe COVID-19 as well. The current study, therefore, aimed to understand how these compounds affect SARS-CoV-2 infection at various stages.
Rapalogs increase infectious viral titer within cells
When pretreated with rapalogs, cells in culture showed a dose-dependent increase in viral titers. At a dose of 20 µM, followed by SARS-CoV-2 addition four hours later, several rapalogs boosted the yield of infectious virus by 9- and 15-fold, for rapamycin and temsirolimus, respectively, and by everolimus to a similar extent in cells expressing the host cell receptors for the virus, angiotensin-converting enzyme 2 (ACE2) and the host protease TMPRSS2.
Rapalogs, generally increase the cell's susceptibility to SARS-CoV-2 spike-bearing pseudoviruses, with the graph resembling that obtained by the wildtype virus. Thus, these compounds activate cell entry into the cell, increasing it by up to one order of magnitude.
In contrast, tacrolimus, which is almost identical in structure to the rapalogs. Also called (FK506), this compound does not bind to mTOR and therefore does not increase HIV-CoV-2-S-mediated cell entry.
Rapalogs enhance virus-cell fusion
The stage at which rapalogs facilitate cell entry appears to be virus-cell fusion, which is enhanced by these compounds. This mechanism is at work with other coronavirus spike proteins as well, though to a lesser extent.
Rapalogs also promote infection mediated by the influenza A virus hemagglutinin antigen, as seen in pseudoviruses incorporating this antigen.
The host enzyme TMPRSS2 is responsible for cleaving the S2’ site on the target cell surface. Cathepsins B and L also perform the same task within cell endosomes. The outcome is an activation of the viral fusion peptide to bring about membrane fusion.
Entry of the virus is mostly by endosomal cathepsin-dependent fusion pathways. This is enhanced by rapalogs, even when the spike protein is mutated such that the polybasic RRAR furin cleavage site motif is replaced by another that does not support the cleavage of the spike into S1 and S2.
Other rapalogs reduce the levels of all three IFITM proteins to the same degree as rapamycin, without any impact on ACE2 levels, by increasing the extent of proteolysis of IFITMs within the endolysosomes.
In primary human small airway or lung epithelial cells, rapalogs promote the entry of the virus-mediated by its spike, partly by reducing the levels of IFITM3. Among all the rapalogs, ridaforolimus is the least active and shows no particular impact on pseudovirus infection rates, except with nasal tissue.
The researchers postulate that rapalogs trigger microautophagy, a cellular process where IFITM proteins are sorted by ESCRT-mediated mechanisms into endosomal vesicles for degradation by lysosomal hydrolyases. This process is under mTOR regulation.
As a result, rapalog-induced inhibition of the mTOR pathway leads to a higher rate of breakdown of lysosomal membrane proteins, both IFITMs and other selected cell proteins.
The IFITMs are non-specifically degraded by rapalogs, but IFITM3 induced by type I interferons are particular targets. Further research will show how they affect IFITMs in infected cells since IFITM3 is among the genes expressed at the highest levels in primary human lung epithelial cells.
In fact, it is expressed at higher levels at baseline on nasal epithelial cells, compared to other cells, and its knockdown caused the cells to become 20-fold more vulnerable to SARS-CoV-2-spike-mediated viral entry.
What are the implications?
Rapalogs have been used to modulate adaptive immunity where its unregulated expression causes harm to the body. This has led to the exploration of their usefulness in severe COVID-19, marked by hyper-inflammatory respiratory compromise.
These results draw attention to a novel immunosuppressive property of rapamycin and rapalogs that acts on intrinsic immunity and impacts cellular susceptibility to infection by multiple viruses, including SARS-CoV-2.”
Further research should tease out the range of consequences of mTOR inhibition, and develop more selective mTOR inhibitors that do not affect intrinsic immunity.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.