In this study, we have developed a novel dual pseudovirion-based HTS assay system with lentiviral pseudotyped particles. This system focuses on finding inhibitors of the step of viral entry, and minimizes the risks associated with hazardous viruses, allowing handling at BSL-2, when specialized BSL-3 facilities would otherwise be necessary. The DEP assay also identifies grossly toxic compounds and reduces the number of false positive hits introduced by compounds able to non-specifically inhibit shared cellular functions of many viruses, for example endocytosis. We demonstrate that this system reduces the number of false positive hits significantly, allowing earlier identification of promising inhibitors. Indeed, if envelopes are chosen with care, this assay allows simultaneous screening of two unrelated envelopes of interest that can also act as internal controls for each other.
In earlier screens we found that many promising inhibitors of entry mediated by pH-dependent viruses such as SARS-CoV, were able to inhibit other viruses, likely due to toxic or other more subtle effects on cellular function. These hits were often not cytotoxic in common laboratory cell lines, but showed sufficient signs of toxicity in primary cell types such as hepatocytes not to be considered likely drug candidates (data not shown). Thus, although pan-viral family antivirals are an attractive goal, we have generally avoided such leads as a somewhat unrealistic objective, and the dual envelope assay allows the rapid and early identification and elimination of such compounds. Ironically, notable exceptions to this rule are the protease inhibitors described in this study. These compounds act through a very defined mechanism, blocking envelope processing and/or viral capsid uncoating and can clearly be demonstrated to inhibit filoviruses, coronaviruses, henipaviruses and reoviruses (Chandran et al., 2005
; Ebert et al., 2002
; Pager et al., 2006
; Pager and Dutch, 2005
; Schornberg et al., 2006
; Simmons et al., 2005
). Thus, care must be taken when choosing partner virus envelopes, not to use envelopes with overlapping functions, receptors or specific targets. For example, the use of Ebola GP with SARS-CoV S would have eliminated the detection of the WRR series of compounds, as entry mediated by both viral envelopes is blocked by CTSL inhibitors. Indeed, the assay could be reversed if a common entry mechanism were suspected, in order to deliberately search for specific inhibitors of this mechanism by only selecting hits that reduced both viruses.
Based on this assay system, we successfully initiated screens of two libraries for compounds that would target the viral entry process of SARS-CoV. In particular, inhibitors of the essential step of SARS-CoV spike proteolysis, which, in cell lines at least, occurs in the endosomes of target cells (Huang et al., 2006
; Simmons et al., 2005
) were identified. It is hoped that compounds identified in these assays to be specifically able to inhibit the endsomal proteolytic processing of SARS-CoV Spike will be useful in animal studies for determining whether the CTSL/endosomal route of entry or surface protease-mediated fusion route is most important in vivo. Many of these hits were dipeptide epoxyketones, and had previously been reported to inhibit one of the coronavirus proteases (Goetz et al., 2007
). Two of these epoxyketones, WRR 182 and WRR 183, are isomers of each other. This affects their biological properties, with WRR 183 being 10-fold more potent against the SARS-CoV chymotrypsin-like protease. Nevertheless, both compounds appear to inhibit SARS-CoV replication in vitro with apparently similar abilities (Goetz et al., 2007
). We demonstrate here that WRR 182, but not 183, is highly active against SARS-CoV spike-mediated entry. Thus, the antiviral properties of these two isomers are explained. The R isomer likely acts through the inhibition of CTSL activity. In contrast, the S isomer inhibits replication due to the fact that it binds irreversibly to the 3Clpro protease. Compounds capable of inhibiting both CTSL and 3Clpro would target the virus at multiple steps of the life cycle, thus increasing their efficiency. This would also likely lessen the chances of drug resistance being established. With the WRR 495 compound we demonstrate that this is indeed possible. Unfortunately, this compound, unlike the parental compounds, is highly toxic. It is possible that this is due to WRR 495 becoming broadly active against many cellular proteases, although we have not demonstrated this. Nevertheless, WRR 495 represents the potential that other, less toxic, dually active third generation compounds can be developed.
In conclusion, this highly sensitive, rapid, and reproducible assay system has been successfully applied for the screening of entry inhibitors of SARS-CoV. Moreover, it can be easily adapted to screen for entry inhibitors of other viruses with careful selection of matching partner virus envelopes of no interference. In particular, human coronaviruses together with other respiratory viruses, such as metapneumovirus and parainfluenzavirus, can cause significant morbidity and mortality, particularly among immunocompromised patients. HTS assays for entry inhibitors, such as the DEP assay described here, should provide good lead candidates for anti-viral drug development against SARS-CoV and other viruses.