In this study, we describe a robust automated assay for the identification of MV inhibitors. The protocol implemented here does not necessitate modification of the pathogen itself and is suitable for the screen of non-attenuated wild type viral isolates. This is a major advantage over a previously reported screening strategy (White et al. 2007
), which, although suitable for hit identification, relied on an attenuated MV recombinant expressing eGFP as an additional transcription unit. The current protocol thus maximizes the likelihood that identified inhibitors show activity against primary virus strains currently endemic in the field. While optimized here for the identification of MV blockers, the protocol should be readily adaptable to other viral targets, provided infection coincides with a strong cytopathic effect and triggers breakdown of the host cell monolayer.
Fixation and permanent stain of assay plates with crystal violet is cost-effective and eliminates tight time constraints for obtaining automated readouts. It furthermore allows re-assessment of plates visually and microscopically to ensure complete absence of infectious centers in individual candidate wells. This double assessment has greatly reduced the amount of primary hit candidates (from 2070 to 60), while minimizing labor-intensive manual counterscreening efforts. At the same time, its accuracy is reflected in the high 59% hit confirmation rate obtained in secondary assays.
Importantly, in-silico data base mining followed by biotesting confirmed that our hit identification regimen is not over-stringent, which can lead to extensive discarding of potentially promising compounds. Our assessment of the frequency of compounds falsely tested inactive in the primary screen was based on the assumption that structural analogs of confirmed hits should have a higher likelihood for antiviral activity than randomly selected library entries. The highest density of false-negatives should thus be found among those analogs, biasing the assessment heavily towards a higher false-negative rate. However, dose-response curves generated for eight independent sets of analogs revealed that most analogs designated inactive by the primary assay indeed had no active in counterscreening exercises. In this biased subgroup, the false-negative rate was 12%, generating confidence that a majority of active structural compound classes present in the library may have been discovered.
Highly effective antiretroviral therapy has confirmed that combined administration of antivirals with distinct targets is desirable. Both overall boosting of inhibitory activity and a reduced rate of viral escape through the development of resistance have been observed (Bartlett et al. 2001
; Bartlett et al. 2006
; Murphy et al. 2001
). By analogy, similar beneficial effects will most likely apply to inhibitors of other viral pathogens. Our protocol has therefore been designed with a maximum potential for the identification of a mechanistically diverse array of paramyxovirus blockers in a single screen. Since virus-induced cytopathicity is monitored after completion of multiple infectious cycles, inhibitors of viral entry (through blockage of receptor binding or membrane fusion), the viral RdRp complex, or of particle assembly are likely to be identified.
Basic classification of confirmed hits in blockers of the viral entry machinery and inhibitors acting post-entry suggests that the screen has indeed returned a mechanistically diverse group of compounds that interfere with different steps of the viral life cycle. At present, we cannot completely exclude that general suppression of host cell protein biosynthesis by entry blocker candidates biases the plasmid-based cell-to-cell fusion assay. Naturally, this would coincide with high cytotoxicity, however. Thus, it appears unlikely based on our cytotoxicity assessment. Importantly, none of the hits shows cross-resistance with the MV RdRp activity inhibitor AS-136A. This may reflect that either none of the new hits blocks RdRp activity or that those hits blocking RdRp share no overlapping target sites with AS-136A. In either case, these findings open future avenues for enhancing antiviral activity through combination of functionally distinct hit classes with each other and/or with AS-136A.
The potential for the identification of assembly inhibitors in particular stands in contrast to the previously reported rMV-eGFP based screening protocol, which made the identification of compounds acting downstream of RdRp activity and thus eGFP expression unlikely. Interestingly, activity of confirmed hits discovered in the previous screen is restricted to the morbillivirus genus (MV and to a lesser degree the closely related CDV) and does not extend to more distantly related members of the paramyxovirinae (White et al. 2007
). In this context it is intriguing to speculate that newly identified hits with a broader target range may interfere with late stages in the viral life cycle, possibly by targeting cellular components that are uniformly exploited by different members of the family for particle assembly.
Such specific targeting of host cell components stands in contrast to the mechanism of activity of promiscuous compounds that are frequently found in HTS exercises (McGovern et al. 2002
; McGovern and Shoichet 2003
). The latter are thought to function through non-specific recruitment of effector molecules examined in the assay to larger compound aggregates (McGovern et al. 2002
; McGovern and Shoichet 2003
). Reflecting activity through aggregate formation, promiscuous compounds are characterized by active concentrations in the micromolar range and essentially flat structure-activity profiles. In the case of the hit compounds identified in our screen, future in-depth mechanistic characterization will be required to uncover the molecular nature of the targeted pathogen or host components. Nonetheless, high target specificity of the MV- and morbillivirus-specific compounds with active concentrations in the nanomolar range of the most potent representatives of the group with broader target range (i.e. compound 22407448) supports inhibition through specific docking and argues against a promiscuous, aggregation-based mechanism of action.
In conclusion, we have developed a robust, cost-effective protocol for automated screening of MV inhibitors that should be readily transferable to other members of the paramyxovirus family. Implementation of the assay has confirmed high accuracy of primary hit identification and has yielded a diverse set of confirmed hits that target distinct steps of the viral life cycle. Efficacy testing and future hit-to-lead chemistry will explore the developmental potential of selected compound classes.