Characterization of polyoxometalate I as an inhibitor of RNA-dependent RNA polymerase of Foot and Mouth Disease virus [abstract]

Abstract only availableFoot and Mouth Disease (FMD) is a highly contagious disease that affects a variety of domesticated cloven-hoofed animals including cattle, swine, sheep and goats, as well as several wild animal species. FMD outbreaks are currently controlled with mass-extermination of livestock. The financial cost of potential outbreaks would be immense. This disease is caused by foot-and-mouth disease virus (FMDV), a non-enveloped, single-stranded, positive-sense RNA virus. The purpose of our investigation is to identify chemicals that interfere with the replication of FMDV. As part of this effort we have identified a polyoxometalate inhibitor (polyoxometalate I). We have cloned, expressed and purified FMDV RdRp. We use steady-state kinetic experiments and polymerization assays to characterize the inhibitory activity of the polyoxometalate I, determining the precise inhibitory potential and the mechanism of inhibition. Preliminary results show that polyoxometalate I inhibits the FMDV RdRp surprisingly efficiently with an IC50 of 0.5uM. Current experiments are focusing on a detailed kinetic characterization of the mechanism of action for this inhibitor. This research may provide insights that lead to new treatment options to prevent the further spread of FMD to unaffected animals.USD

Novel inhibitors of Foot and Mouth Disease Virus (FMDV) Targeting the RNA-Dependent RNA Polymerase activity of 3Dpol [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionFoot-and-Mouth Disease Virus (FMDV) is a positive stranded picornavirus which infects cloven-hoofed animals, such as cattle, pigs and sheep, and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination could be used but it would require at least 7 days to trigger an effective immune response. On the contrary, the use of antiviral drugs is expected to have prophylactic and/or therapeutic effects almost immediately. However, there are currently no approved FMDV inhibitors. Here we have applied a combination of screening, biochemical, virological, and molecular modeling tools to discover, validate, and characterize novel inhibitors of FMDV replication. Using a luciferase-based assay we have screened a chemical library of compounds and have identified two compounds, 5-chloro-3-(thiophen-2-yl-sulfanylmethyl)-1-benzothiophene 1,1-dioxide (or C7F8) and N'1-thieno[2,3-d]pyrimidin-4-yl-4-chloro-1-benzenesulfonohydrazide (or C5D9) that inhibited the RNA-dependent RNA polymerase activity of FMDV replicase (3Dpol) with IC50 values of 2.5 μM and 15 μM respectively. These compounds were shown to be specific inhibitors of FMDV 3Dpol and not nucleic acid chelators, as they did not affect activity of other viral polymerases using the same nucleic acid substrate. Molecular modeling docking experiments suggest that both inhibitors bind at a pocket proximal to, but distinct from, the NTP binding site of 3Dpol, thereby affecting indirectly RNA synthesis. C7F8 and C5d9 were not cytotoxic at concentrations up to at least 100 ||M. Importantly, C5D9 exhibited antiviral activity and suppressed virus production in FMDV-infected cells with 50% and 90% effective concentrations (EC50 and EC90) of 10 ||M and 20 ||M, respectively. The results indicate that 3Dpol inhibitors can be promising anti-FMDV agents for use as alternative or supplementary options to contain future outbreaks of FMD

Characterization of a novel MR-detectable nanoantioxidant that mitigates the recall immune response

In many human diseases, the presence of inflammation is associated with an increase in the level of reactive oxygen species (ROS). The resulting state of oxidative stress is highly detrimental and can initiate a cascade of events that ultimately lead to cell death. Thus, many therapeutic attempts have been focused on either modulating the immune system to lower inflammation or reducing the damaging caused by ROS. Berlin et al. reported the development of a novel nanoantioxidant known as poly(ethylene glycol)-functionalized-hydrophilic carbon clusters (PEG-HCCs). They showed that PEG-HCCs could be targeted to cancer cells, utilized as a drug delivery vector, and can even be visualized ex vivo. Our work here furthers this work and characterizes Gd-DTPA conjugated PEG-HCCs and explores the potential for in vivo tracking of T cells in live mice. We utilized a mouse model of delayed-type hypersensitivity (DTH) to assess the immunomodulatory effects of PEG-HCCs. The T1-agent Gd-DTPA was then conjugated to the PEG-HCCs and T1 measurements, and T1-weighted MRI of the modified PEG-HCCs was done to assess their relaxivity. We then assessed if PEG-HCCs could be visualized both ex vivo and in vivo within the mouse lymph node and spleen. Mice treated with PEG-HCCs showed significant improvements in the DTH assay as compared to the vehicle (saline)-treated control. Flow cytometry demonstrated that splenic T cells are capable of internalizing PEG-HCCs whereas fluorescent immunohistochemistry showed that PEG-HCCs are detectable within the cortex of lymph nodes. Finally, our nanoantioxidants can be visualized in vivo within the lymph nodes and spleen of a mouse after addition of the Gd-DTPA. PEG-HCCs are internalized by T cells in the spleen and can reduce inflammation by suppression of a recall immune response. PEG-HCCs can be modified to allow for both in vitro and in vivo visualization using MRI