Tomatidine reduces Chikungunya virus progeny release by controlling viral protein expression

Tomatidine, a natural steroidal alkaloid from unripe green tomatoes has been shown to exhibit many health benefits. We recently provided in vitro evidence that tomatidine reduces the infectivity of Dengue virus (DENV) and Chikungunya virus (CHIKV), two medically important arthropod-borne human infections for which no treatment options are available. We observed a potent antiviral effect with EC50 values of 0.82 μM for DENV-2 and 1.3 μM for CHIKV-LR. In this study, we investigated how tomatidine controls CHIKV infectivity. Using mass spectrometry, we identified that tomatidine induces the expression of p62, CD98, metallothionein and thioredoxin-related transmembrane protein 2 in Huh7 cells. The hits p62 and CD98 were validated, yet subsequent analysis revealed that they are not responsible for the observed antiviral effect. In parallel, we sought to identify at which step of the virus replication cycle tomatidine controls virus infectivity. A strong antiviral effect was seen when in vitro transcribed CHIKV RNA was transfected into Huh7 cells treated with tomatidine, thereby excluding a role for tomatidine during CHIKV cell entry. Subsequent determination of the number of intracellular viral RNA copies and viral protein expression levels during natural infection revealed that tomatidine reduces the RNA copy number and viral protein expression levels in infected cells. Once cells are infected, tomatidine is not able to interfere with active RNA replication yet it can reduce viral protein expression. Collectively, the results delineate that tomatidine controls viral protein expression to exert its antiviral activity. Lastly, sequential passaging of CHIKV in presence of tomatidine did not lead to viral resistance. Collectively, these results further emphasize the potential of tomatidine as an antiviral treatment towards CHIKV infection.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

Adenovirus vectored IFN-α protects mice from lethal challenge of Chikungunya virus infection

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that is responsible for numerous large and geographical epidemics, causing millions of cases. However, there is no vaccine or therapeutics against CHIKV infection available. Interferon-alpha (IFN-α) has been shown to produce potent antiviral responses during viral infection. Herein we demonstrated the use of an adenovirus-vectored expressed mouse IFN-α (mDEF201) as a prophylactic and therapeutic treatment against CHIKV in vivo. 6-day-old BALB/c mice were pre- or post-treated intranasally with single dose of mDEF201 at 5 x 10⁶ PFU per mouse and challenged with lethal dose of CHIKV. Complete survival protection was observed in mice upon a single dose of mDEF201 administration 1 days prior to virus challenge. Viral load in the serum and multiple organs were significantly reduced upon mDEF201 administration in a dose dependent manner as compare with adenovirus 5 vector placebo set. Histological analysis of the mice tissue revealed that mDEF201 could significantly reduce the tissue morphological abnormities, mainly infiltration of immune cells and muscle fibre necrosis caused by CHIKV infection. In addition, administration of mDEF201 at 6 hours post CHIKV challenge also showed promising inhibitory effect against viral replication and dissemination. In conclusion, single-dose of intranasal administration with mDEF201 as a prophylactic or therapeutic agent within 6 hours post CHIKV infection is highly protective against a lethal challenge of CHIKV in the murine model.     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication

Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV) replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2) that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R₇) sequence to favor their intracellular translocation. Thus, when fused to R₇, interference peptides (100 μM) were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target.     

Discovery of orally active chalcones as histone lysine specific demethylase 1 inhibitors for the treatment of leukaemia

Histone lysine specific demethylase 1 (LSD1) has emerged as an attractive molecule target for the discovery of potently anticancer drugs to treat leukaemia. In this study, a series of novel chalcone derivatives were designed, synthesised and evaluated for their inhibitory activities against LSD1 in vitro. Among all these compounds, D6 displayed the best LSD1 inhibitory activity with an IC₅₀ value of 0.14 μM. In the cellular level, compound D6 can induce the accumulation of H3K9me1/2 and inhibit cell proliferation by inactivating LSD1. It exhibited the potent antiproliferative activity with IC₅₀ values of 1.10 μM, 3.64 μM, 3.85 μM, 1.87 μM, 0.87 μM and 2.73 μM against HAL-01, KE-37, P30-OHK, SUP-B15, MOLT-4 and LC4-1 cells, respectively. Importantly, compound D6 significantly suppressed MOLT-4 xenograft tumour growth in vivo, indicating its great potential as an orally bioavailable candidate for leukaemia therapy.     

Antiviral activity of an N-allyl acridone against dengue virus

Background

Dengue virus (DENV), a member of the family Flaviviridae, is at present the most widespread causative agent of a human viral disease transmitted by mosquitoes. Despite the increasing incidence of this pathogen, there are no antiviral drugs or vaccines currently available for treatment or prevention. In a previous screening assay, we identified a group of N-allyl acridones as effective virus inhibitors. Here, the antiviral activity and mode of action targeted to viral RNA replication of one of the most active DENV-2 inhibitors was further characterized.

Results

The compound 10-allyl-7-chloro-9(10H)-acridone, designated 3b, was active to inhibit the in vitro infection of Vero cells with the four DENV serotypes, with effective concentration 50% (EC₅₀) values in the range 12.5-27.1 μM, as determined by virus yield inhibition assays. The compound was also effective in human HeLa cells. No cytotoxicity was detected at 3b concentrations up to 1000 μM. Mechanistic studies demonstrated that virus entry into the host cell was not affected, whereas viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR. The addition of exogenous guanosine together with 3b rescued only partially the infectivity of DENV-2.

Conclusions

The acridone derivative 3b selectively inhibits the infection of Vero cells with the four DENV serotypes without a direct interaction with the host cell or the virion but interfering specifically with the intracellular virus multiplication. The mode of antiviral action for this acridone apparently involves the cellular enzyme inosine-monophospahe dehydrogenase together with another still unidentified target related to DENV RNA synthesis.     

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC₅₀ values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.     

Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication

Background

Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection.

Methods

Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection.

Results

Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1.

Conclusion

Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.     

Fusion of Protegrin-1 and Plectasin to MAP30 Shows Significant Inhibition Activity against Dengue Virus Replication

Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC₅₀) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.     

Identification,Design and Bio-Evaluation of Novel Hsp90 Inhibitors by Ligand-Based Virtual Screening

Heat shock protein 90 (Hsp90), whose inhibitors have shown promising activity in clinical trials, is an attractive anticancer target. In this work, we first explored the significant pharmacophore features needed for Hsp90 inhibitors by generating a 3D-QSAR pharmacophore model. It was then used to virtually screen the SPECS databases, identifying 17 hits. Compound S1 and S13 exhibited the most potent inhibitory activity against Hsp90, with IC₅₀ value 1.61±0.28 μM and 2.83±0.67 μM, respectively. Binding patterns analysis of the two compounds with Hsp90 revealed reasonable interaction modes. Further evaluation showed that the compounds exhibited good anti-proliferative effects against a series of cancer cell lines with high expression level of Hsp90. Meanwhile, S13 induced cell apoptosis in a dose-dependent manner in different cell lines. Based on the consideration of binding affinities, physicochemical properties and toxicities, 24 derivatives of S13 were designed, leading to the more promising compound S40, which deserves further optimization.     

Curcumin as a Potent and Selective Inhibitor of 11β-Hydroxysteroid Dehydrogenase 1: Improving Lipid Profiles in High-Fat-Diet-Treated Rats

Background

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activates glucocorticoid locally in liver and fat tissues to aggravate metabolic syndrome. 11β-HSD1 selective inhibitor can be used to treat metabolic syndrome. Curcumin and its derivatives as selective inhibitors of 11β-HSD1 have not been reported.

Methodology

Curcumin and its 12 derivatives were tested for their potencies of inhibitory effects on human and rat 11β-HSD1 with selectivity against 11β-HSD2. 200 mg/kg curcumin was gavaged to adult male Sprague-Dawley rats with high-fat-diet-induced metabolic syndrome for 2 months.

Results and Conclusions

Curcumin exhibited inhibitory potency against human and rat 11β-HSD1 in intact cells with IC₅₀ values of 2.29 and 5.79 µM, respectively, with selectivity against 11β-HSD2 (IC₅₀, 14.56 and 11.92 µM). Curcumin was a competitive inhibitor of human and rat 11β-HSD1. Curcumin reduced serum glucose, cholesterol, triglyceride, low density lipoprotein levels in high-fat-diet-induced obese rats. Four curcumin derivatives had much higher potencies for Inhibition of 11β-HSD1. One of them is (1E,4E)-1,5-bis(thiophen-2-yl) penta-1,4-dien-3-one (compound 6), which had IC₅₀ values of 93 and 184 nM for human and rat 11β-HSD1, respectively. Compound 6 did not inhibit human and rat kidney 11β-HSD2 at 100 µM. In conclusion, curcumin is effective for the treatment of metabolic syndrome and four novel curcumin derivatives had high potencies for inhibition of human 11β-HSD1 with selectivity against 11β-HSD2.     

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

Background

In the present study, we examined the inhibitory effects of a methanolic extract, dichloromethane fraction, water layer, and polyhydroxylated sterols (1–4) isolated from the Vietnamese starfish Protoreaster nodosus on pro-inflammatory cytokine (IL-12 p40, IL-6, and TNF-α) production in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) using enzyme-linked immunosorbent assays (ELISA).

Results

The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC₅₀ values ranging from 0.60 ± 0.01 to 26.19 ± 0.64 μg/mL. Four highly pure steroid derivatives (1–4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6 (IC_50s = 3.11 ± 0.08 and 1.35 ± 0.03 μM), and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40 (IC_50s = 0.01 ± 0.00 and 1.02 ± 0.01 μM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production.

Conclusion

This is the first report of the anti-inflammatory activity from the starfish P. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.     

Pedilanthus tithymaloides Inhibits HSV Infection by Modulating NF-κB Signaling

Pedilanthus tithymaloides (PT), a widely used ethnomedicinal plant, has been employed to treat a number of skin conditions. To extend its utility and to fully exploit its medicinal potential, we have evaluated the in vitro antiviral activity of a methanolic extract of PT leaves and its isolated compounds against Herpes Simplex Virus type 2 (HSV-2). Bioactivity-guided studies revealed that the extract and one of its constituents, luteolin, had potent antiviral activity against wild-type and clinical isolates of HSV-2 (EC₅₀ 48.5–52.6 and 22.4–27.5 μg/ml, respectively), with nearly complete inhibition at 86.5–101.8 and 40.2–49.6 μg/ml, respectively. The inhibitory effect was significant (p<0.001) when the drug was added 2 h prior to infection, and was effective up to 4 h post-infection. As viral replication requires NF-κB activation, we examined whether the observed extract-induced inhibition of HSV-2 was related to NF-κB inhibition. Interestingly, we observed that treatment of HSV-2-infected cells with extract or luteolin suppressed NF-κB activation. Although NF-κB, JNK and MAPK activation was compromised during HSV replication, neither the extract nor luteolin affected HSV-2-induced JNK1/2 and MAPK activation. Moreover, the PT leaf extract and luteolin potently down-regulated the expression of tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6, NO and iNOS and the production of gamma interferon (IFN-γ), which are directly involved in controlling the NF-κB signaling pathway. Thus, our results indicate that both PT leaf extract and luteolin modulate the NF-κB signaling pathway, resulting in the inhibition of HSV-2 replication.     

Design and green synthesis of novel quinolinone derivatives of potential anti-breast cancer activity against MCF-7 cell line targeting multi-receptor tyrosine kinases

A new set of 4,6,7,8-tetrahydroquinolin-5(1H)-ones were designed as cytotoxic agents against breast cancer cell line (MCF-7) and synthesised under ultrasonic irradiation using chitosan decorated copper nanoparticles (CS/CuNPs) catalyst. The new compounds 4b, 4j, 4k, and 4e exhibited the most potent cytotoxic activity of IC₅₀ values (0.002 − 0.004 µM) comparing to Staurosporine of IC₅₀; 0.005 μM. The latter derivatives exhibited a promising safety profile against the normal human WI38 cells of IC₅₀ range 0.0149 − 0.048 µM. Furthermore, the most promising cytotoxic compounds 4b, 4j were evaluated as multi-targeting agents against the RTK protein kinases; EGFR, HER-2, PDGFR-β, and VEGFR-2. Compound 4j showed promising inhibitory activity against HER-2 and PDGFR-β of IC₅₀ values 0.17 × 10⁻³, 0.07 × 10⁻³ µM in comparison with the reference drug sorafenib of IC₅₀; 0.28 × 10⁻³, 0.13 × 10⁻³ µM, respectively. In addition, 4j induced apoptotic effect and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells.     

Thiazolides,a New Class of Antiviral Agents Effective against Rotavirus Infection,Target Viral Morphogenesis,Inhibiting Viroplasm Formation

Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC₅₀s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC₅₀s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.