implemented and potential antiviral lead compound(s) has been identified [34]. The development and validation of a cytopathic effect (CPE)-based assay led to the screening of the NIH Molecular Libraries Small Molecule Repository (MLSMR), with 194,950 small molecule compounds then. Further studies, using various primary, secondary and confirmatory assays, confirmed 185 structures that were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library [34]. Based on the results from the previous studies [34], we selected and evaluated the virostatic efficacy of a cluster of active compounds, in particular, the aminothiophenecarboxylic acid derivatives. Furthermore, aiming to understand their mechanism of action, various studies were carried out to determine which viral life stage(s) these compounds acted on, focusing on how these virostatic compounds protect cells from BTV-induced apoptosis. Results Thiophene Derivatives as Lead Virostatic Agents against BTV Based on the previously identified six compound clusters [34], we selected 2–4 representative compounds from each cluster and assessed their virostatic efficacies against BTV, including the 50% effective concentration (EC50) and the 90% effective concentration (EC90). The EC50 of these lead compounds were determined between 0.7 mM and 20 mM, respectively (results not shown). Interestingly, the four thiophene derivative com- pounds from cluster I, i.e. compound 001 through 004 (C001, C002, C003 and C004), showed potent virostatic efficacy against BTV, especially the aminothiophenecarboxylic acid derivatives C001 and C003. Using the ten-concentration dose response assay, the EC50s of C001 and C003 were determined at 0.6960.15 mM and 1.7660.73 mM, respectively (Fig. 1A and B). The EC90s of C001 and C003 were at 6.2061.39 mM and 15.8666.56 mM, respectively (Fig. 1A and B). The above results indicated that both C001 and C003 could be potential novel virostatic agents against BTV. Additionally, the 50% cytotox- icity concentration (CC50) of both C001 and C003 were over 100 mM (Fig. 1A and B). Accordingly, the selective indexes (SI50), which equals to EC50/CC50, were above 145 and 57 for C001 and C003, respectively, indicating that both compounds were highly selective against BTV. Considering the compound solubility and availability, we selected C003 as the lead virostatic agent for further evaluation, including structure- activity relationship (SAR) analysis, chemical modification and mechanism of action studies. Improving C003 Virostatic Efficacy via Chemical Modification C003, or 2-{[3-(2-furyl)acryloyl]amino}-4-(4-methylphenyl)-3- thiophenecarboxylic acid, is an aminothiophenecarboxylic acid derivative with a structure showed in Figure 2. To improve the potential of C003 as an anti-BTV lead structural framework, it was divided into 5 zones for synthetic target goals. Figure 3 illustrates our results from a variation in zone 5 by replacing the furan of C003 with a thiophene (IC2a or C052) and pyridine (IC3a or C055). Our approach to C052 (IC2a) and C055 (IC3a) involved a convergent pathway whereby (1) the Gewald reaction was employed for the thiophene unit (IC4 of zone 1) and (2) a Horner- Wadsworth-Emmons reaction of 2-thiophenecarboxaldehyde (IC5) and 2-pyridinecarboxaldehyde (IC6) with malonic acid followed by acid chloride formation provided a means to IC7 and Virostatic Agents against Bluetongue Virus Figure 1. The virostatic efficacy and cytotoxicity were showed in (A) C003 and (B) C001. Cells were infected with BTV at MOI of 0.01 with or without C003 or C001 at various concentrations. At 72 h.p.i, cell viability (triangles) or cytotoxicity (circles) was measured using the CellTiter-Glo reagent. Each data point represents means and standard deviation (SD) from five replicates. doi:10.1371/journal.pone.0043341.g001 Figure 2. The compound structure of C003 was showed. The compound was divided into five zones as indicated. doi:10.1371/journal.pone.0043341.g002 IC8, the zone 5 modifiers. Bringing these units (IC4 and IC7/ IC8) together produced IC9 and IC 10. Saponification of IC9 and IC10 gave the sodium salts IC2b and IC3b, which, upon acidification, yielded the desired final products C052 (IC2a) and C055 (IC3a) (Fig. 3). Because of their potential as prodrugs of C052 (IC2a) and C055 (IC3a), esters IC9 and IC10, were also evaluated for their virostatic efficacies. Interestingly, while other analogs including C055 did not show any improvement of its virostatic efficacy, the de novo synthesized analogs, C052, showed an improved virostatic efficacy with EC50 at 0.2760.12 mM and EC90 of 2.561.04 mM, respectively (Fig. 4). Similarly, C052 showed very little toxicity with CC50 at 82.69 mM. Consequently, the SI50 of C052, were at 306. Comparing with the EC50 and SI50 of C003, the EC50 of C052 were 6.5 times lower, and the SI50 was 5.4 times higher, indicating that the virostatic efficacy of C003 could be further improved via SAR and chemical modifications, leading to a more potent and selective virostatic agent against BTV. PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43341