IFEMS, an Interactive Finite Element Modeling System Using a CAD/CAM System

A method of coupling a CAD/CAM system with a general purpose finite element mesh generator is described. The three computer programs which make up the interactive finite element graphics system are discussed

High-throughput screening identifies a bisphenol inhibitor of SV40 large T antigen ATPase activity

The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ∼0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose-response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC50 of 41 μM in the primary assay and 6.2 μM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo

Discovery of a Novel Compound with Anti-Venezuelan Equine Encephalitis Virus Activity That Targets the Nonstructural Protein 2

Abstract Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection. Author Summary Alphaviruses occur worldwide, causing significant diseases such as encephalitis or arthritis in humans and animals. In addition, some alphaviruses, such as VEEV, pose a biothreat due to their high infectivity and lack of available treatments. To discover small molecule inhibitors with lead development potential, we used a cell-based assay to screen 348,140 compounds for inhibition of a VEEV-induced cytopathic effect. The screen revealed a scaffold with high inhibitory VEEV cellular potency and low cytotoxicity liability. While most previously reported anti-alphavirus compounds inhibit host proteins, evidence supported that this scaffold targeted the VEEV nsP2 protein, and that inhibition was associated with viral replication. Interestingly, compound resistance studies with VEEV mapped activity to the N-terminal domain of nsP2, to which no known function has been attributed. Ultimately, this discovery has delivered a small molecule-derived class of potent VEEV inhibitors whose activity is coupled to the nsP2 viral protein, a novel target with a previously unestablished biological role that is now implicated in viral replication.This research was supported by the following funding sources: NIH R03MH087448-01A1, University of Louisville Internal Research Initiate grant to DHC, USAMRAA W81XWH-10-2-0064 and W81XWH-08-2-0024 to CBJ. Screening was provided by the Southern Research Specialized Screening Center (U54HG005034-0) and chemistry through the University of Kansas Specialized Chemistry Center (U54HG005031). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Novel approach to identify inhibitors of iron acquisition systems of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that has been declared by the World Health Organization as a “priority 1 critical pathogen” needing immediate new strategies for chemotherapy. During infection, P. aeruginosa uses redundant mechanisms to acquire ferric, heme (Hm), or ferrous iron from the host to survive and colonize. Significant efforts have been undertaken to develop siderophore blockers to inhibit ferric iron acquisition by P. aeruginosa, but there is a lack of inhibitors that can block Hm or ferrous iron acquisition by P. aeruginosa. We developed and employed a targeted high-throughput screen (HTS) and identified a molecule(s) that can specifically inhibit the Hm and ferrous iron acquisition systems of P. aeruginosa. Our targeted approach relies on screening a small-molecule library against P. aeruginosa under three growth conditions, where the only variable was the iron source (ferric, Hm, or ferrous iron). Each condition served as a counterscreen for the other, and we identified molecules that inhibit the growth of P. aeruginosa in the presence of only Hm or ferrous iron. Our data indicate that econazole, bithionate, and raloxifene inhibit the growth of P. aeruginosa in the presence of Hm and that oxyquinoline inhibits the growth of P. aeruginosa in the presence of ferrous iron. These iron-specific inhibitors do not interfere with the activity of meropenem, a commercial antipseudomonal, and can also increase meropenem activity. In conclusion, we present a proof of concept of a successful targeted conditional screening method by which we can identify specific iron acquisition inhibitors. This approach is highly adaptable and can easily be extended to any other pathogen. IMPORTANCE Since acquiring iron is paramount to P. aeruginosa’s survival and colonization in the human host, developing novel strategies to block the access of P. aeruginosa to host iron will allow us to starve it of an essential nutrient. P. aeruginosa uses siderophore, heme, or ferrous iron uptake systems to acquire iron in the human host. We have developed a novel approach through which we can directly identify molecules that can prevent P. aeruginosa from utilizing heme or ferrous iron. This approach overcomes the need for the in silico design of molecules and identifies structurally diverse biologically active inhibitor molecules. This screening approach is adaptable and can be extended to any pathogen. Since Gram-negative pathogens share many similarities in iron acquisition at both the mechanistic and molecular levels, our screening approach presents a significant opportunity to develop novel broad-spectrum iron acquisition inhibitors of Gram-negative pathogens.Microbiology and Molecular Genetic

Spectrum of antiviral activity of CID15997213.

<p>IC₅₀ measured in a cell-based CPE assay (µM) with triplicate data points for VEEV 3526, TrD, CHIKV and RSV. IC₅₀ v*alue presented here for VEEV TC-83 is the mean from 17 independent experiments.</p>†<p>Log difference in progeny virus titers between in the absence/presence of the compound at 5 µM was >6. 0.05 MOI of VEEV TC-83 was used for infection.</p>‡<p>IC₅₀ measured in Neuro 2A cell line.</p

HTS of 348K compounds and identification of the hit compound.

<p>A flow diagram of various assays used in the screen. The number of hits remaining after each run is indicated in bold.</p

CID15997213 targets viral nsP2.

<p>(<b>A</b>) Time of addition study. Test compound, CID15997213, was added to the designated wells by replenishing the culture media with fresh culture media containing 5 µM of the compound at the time points denoted on the x axis. The graph denotes the virus titers at 16 hours post-infection from various time of addition points. Each data point is the mean from two independent replicates with duplication in titration. (<b>B</b>) Location of the mutations in the CID15997213 resistant viruses. The mutations mapped within the N-terminus of nsP2 protein (pink). There were no missense mutations in either nsP1, nsP3 or nsP4 genes. * Methyl-transferase like domain. (<b>C</b>) Sequence alignment of nsP2 alphaviruses. Amino acid sequences of nsP2 of following alphaviruses were aligned with Clustal W (<a href="http://www.clustal.org" target="_blank">www.clustal.org</a>): VEEV (L01442.2, GenBank Access No. hereafter), EEEV (NC_003899), WEEV (NC_003908), Fort Morgan virus (FMV, NC_013528), Ross River virus (RRV, NC_001544), Semliki Forest virus (SFV, NC_003215), O'nyong-nyong virus (ONYV, NC_001512.1), CHIKV (L37661.3), Sindbis virus (SINV, NP_740671.1). Y102 position is highlighted in red.</p