High-throughput screening (HTS) has historically been used by the pharmaceutical industry to rapidly test hundreds of thousands of compounds to identify potential drug candidates. More recently, academic groups have used HTS to identify new chemical probes or small interfering RNA (siRNA) that can serve as experimental tools to examine the biology or physiology of novel proteins, processes, or interactions. HTS presents a significant challenge with the vast and complex nature of data generated. This report describes MScreen, a web-based, open-source cheminformatics application for chemical library and siRNA plate management, primary HTS and dose-response data handling, structure search, and administrative functions. Each project in MScreen can be secured with passwords or shared in an open information environment which enables collaborators to easily compare data from many screens, providing a useful means to identify compounds with desired selectivity. Unique features include compound, substance, mixture, and siRNA plate creation and formatting; automated dose-response fitting and quality control (QC); and user, target, and assay method administration. MScreen provides an effective means to facilitate HTS information handling and analysis in the academic setting so that users can efficiently view their screening data and evaluate results for follow-up.

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate-detection reagents, such as quinaldine red (QR). While such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70’s weak enzymatic activity have combined to create significant challenges in high throughput screening. To overcome these difficulties, we have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal. Biochem. 2005, 342:254–259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, we tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~ 0.6, CV ~8%) and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70-family members.

Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses cause serious and potentially fatal central nervous system infections in humans and are high-priority potential bioterrorism agents. There are currently no widely available vaccines or licensed therapies for these virulent pathogens. To identify potential novel antiviral drugs, we developed a cell-based assay with a western equine encephalitis virus replicon that expresses a luciferase reporter gene and screened a small molecule diversity library of 51,028 compounds. We identified and validated a thieno[3,2-b]pyrrole compound with a half maximal inhibitory concentration of <10 µmol/L, a selectivity index >20, and potent activity against live virus in cultured neuronal cells. Furthermore, a structure-activity relationship analysis with 20 related compounds identified several with enhanced activity profiles, including 6 with submicromolar half maximal inhibitory concentrations. In conclusion, we have identified a novel class of promising inhibitors with potent activity against virulent neurotropic alphaviruses.

Physical differences in small molecule binding between enzymes and non-enzymes were found through mining the protein-ligand database, Binding MOAD (Mother of All Databases). The data suggest that divergent approaches may be more productive for improving the affinity of ligands for the two classes of proteins. High-affinity ligands of enzymes are much larger than those with low affinity, indicating that the addition of complementary functional groups is likely to improve the affinity of an enzyme inhibitor. However, this process may not be as fruitful for ligands of non-enzymes. High- and low-affinity ligands of non-enzymes are nearly the same size, so modest modifications and isosteric replacement might be most productive. The inherent differences between enzymes and non-enzymes have significant ramifications for scoring functions and structure-based drug design. In particular, non-enzymes were found to have greater ligand efficiencies than enzymes. Ligand efficiencies are often used to indicate druggability of a target, and this finding supports the feasibility of non-enzymes as drug targets. The differences in ligand efficiencies do not appear to come from the ligands; instead, the pockets yield different amino acid compositions, despite very similar distributions of amino acids in the overall protein sequences.