the Split-Protein-Based Cell-Cell Fusion Assay
Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro. Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.
In this study, we established a DSP-based cell-cell fusion assay system to measure MERS-S-mediated membrane fusion. We performed a small-scale screening to test whether our DSP assay can be used in a drug screening. This fusion assay did not use live viruses and could be finished within several hours. Since it does not require the use of live viruses, our cell-based DSP assay can be performed in a conventional laboratory. Candidate drugs identified in the DSP assay can be further evaluated in a virus infection assay, since there may be potential differences between cell-cell and virus-cell membrane fusions.
We used the RL mode of the DSP reporter because it could provide a more quantitative reading than the GFP mode without image analyses. Furthermore, the luminescent signals of the RL mode were less likely to suffer from the nonspecific noise of natural color or possible fluorescence emitted from the test drugs in the GFP mode (data not shown). The potential false positivity resulting from nonspecific inhibition of RL itself was easily ruled out by testing of particular drugs in cells coexpressing the two DSPs simultaneously. Our results indicated that the DSP assay was highly stable, and nonspecific inhibition was rarely observed.
In this study, we first utilized 293FT cells constitutively expressing either DSP1-7 or DSP8-11 to determine the necessary components required for the assay system (MERS-S, CD26, and TMPRSS2). Because 293FT cells are easily transfectable, these cells could be conveniently used to express candidate components by transient transfection (data not shown). Consistent with previous studies, we found that both CD26 and TMPRSS2 were necessary in the target cells. However, in contrast to assays of infection by intact viruses, this cell-based assay measured fusion only at the cell surface and could not address the contribution of fusion after endocytosis. While this may be considered a shortcoming, we found it useful to effectively isolate two entry pathways for MERS-CoV and specifically address the contribution of TMPRSS2 at the plasma membrane. Since the proteolytic processing of S may depend on other unidentified cellular proteases, we may need to address this issue in our future study.We also tested the DSP assay by establishing Calu3-based target cells.
Calu-3 cells may be more relevant target cells than 293FT cells; however, the overall patterns in the 293FT-based assays and Calu3-based assays were similar. Interestingly, the fusion was more readily blocked in Calu3 cells than in 293FT cells, which could be explained by differences in the expression levels of CD26, TMPRSS2, and DSP. Notably, we currently cannot rule out the possibility of the involvement of other cellular factors. This suggests that the more sensitive Calu3-based assay may be useful for reducing the concentrations of the test drugs in a much larger scale of screening in the future.
Next, we adapted our DSP assay to an HTS format by establishing stable cell lines expressing the necessary components in effector and target cells. We also established cell lines stably expressing self-reassociated DSPs to exclude nonspecific inhibitors that block the RL activity of the DSPs. Our screening of 1,017 drugs identified nafamostat, a serine protease inhibitor, as a potential hit. Moreover, among the tested inhibitors, we found that nafamostat exhibited the strongest inhibitory activity. Consistent with a previous study, camostat was shown to inhibit membrane fusion; however, its efficacy was weaker than that of nafamostat. In vitro infection assays confirmed these findings.
Because MERS-S requires proteolytic activation by cellular proteases after receptor binding (13), we assumed that the inhibitory mechanism involved suppression of the TMPRSS2 activity. The efficacy of nafamostat in live virus infection was less potent than in our DSP assay. This may have been due to the relatively high MOI used in our assay to facilitate the detection of the internalized viral RNA or to the presence of an alternative endocytic entry mechanism of MERS-CoV that does not rely on TMPRSS2. The alternative endocytic pathway uses other proteases, such as cathepsins. It is hard to estimate the exact contribution of each entry pathway in real infections in vivo; however, our previous results showed that MERS-CoV depends primarily on the direct fusion pathway in vitro. Therefore, TMPRSS2 inhibitors against MERS may be expected to exhibit efficacy in vivo. However, other assay systems are required to screen potential drugs targeted at proteases other than TMPRSS2.
In this study, we screened drugs and reagents already available in clinical use. Such a screening may be relevant to emerging diseases, such as MERS, because screening of chemical leads and development of effective medicines through animal and human trials are highly time-consuming and costly; these factors may prevent prompt responses to the spread of emerging viruses.
As discussed above, nafamostat is expected to manifest its anti-MERS-CoV activity through inhibition of the host protease, TMPRSS2. Targeting host proteins rather than viral proteins has the advantage of avoiding treatment failure due to mutations of the target. However, we cannot rule out possible drug-resistant mutants because RNA viruses are known to generate quasispecies that contain a population of related but distinct mutants, some of which may develop drug resistance against inhibitors targeting the host protein. Targeting of host proteins may cause unwanted side effects during antiviral application, particularly when the antivirals are administered systemically. Unwanted side effects during the treatment of MERS may be reduced by topical introduction into the respiratory tract through a nebulizer. Other than therapeutic use in humans, treatment of potential carrier animals, such as camels or bats, with nafamostat or related compounds may be a possible intervention method.
Although our screening clearly showed the efficacy of the use of clinically available nafamostat against MERS-S-mediated membrane fusion in vitro, further pharmacokinetic or toxicity studies will be required to determine whether nafamostat can be used to treat MERS in human. Nafamostat is currently indicated for systemic use in the treatment of pancreatitis and disseminated intravascular coagulation. The anticoagulatory activity as implicated in the latter indication may cause unwanted side effects when it is applied to MERS. The feasibility of clinical application of nafamostat is beyond the scope of our current study; however, either nafamostat or its derivatives have potential to be used in the treatment of MERS.
TMPRSS2 proteolytically activates a wide range of viral envelope proteins and has been shown to be critical for the in vivo activation of influenza viruses. Therefore, it will be interesting to examine the efficacy of nafamostat against viruses other than MERS-CoV. Further chemical derivatization of compounds using nafamostat as a lead compound is also possible. We expect that these findings may be useful for combatting MERS. Further large-scale screenings using DSP assays are warranted to identify more-effective compounds against MERS-S, and these assays may be applicable to identification of potential drugs to treat other viral diseases.
Reference & source information: https://aac.asm.org/
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