High throughput screening (HTS) is an integral part of a highly collaborative approach to drug discovery at the University of Michigan. The HTS lab is one of four core centers that provide services to identify, produce, screen and follow-up on biomedical targets for faculty. Key features of this system are: protein cloning and purification, protein crystallography, small molecule and siRNA HTS, medicinal chemistry and pharmacokinetics. Therapeutic areas that have been targeted include anti-bacterial, metabolic, neurodegenerative, cardiovascular, anti-cancer and anti-viral. The centers work in a coordinated, interactive environment to affordably provide academic investigators with the technology, informatics and expertise necessary for successful drug discovery. This review provides an overview of these centers at the University of Michigan, along with case examples of successful collaborations with faculty.
Emerging evidence suggests that inflammation provides a link between obesity and insulin resistance. The noncanonical IκB kinases IKKε and TANK-binding kinase 1 (TBK1) are induced in liver and fat after high fat diet by NF-κB activation, and in turn initiate a program of counter-inflammation that preserves energy storage. Here, we report the discovery of a small molecule inhibitor of these kinases called amlexanox. Treatment of obese mice with amlexanox elevates energy expenditure through increased thermogenesis, producing weight loss, improved insulin sensitivity and decreased steatosis in obese mice. Because of its record of safety in patients, amlexanox may be an interesting candidate for clinical evaluation in the treatment of obesity and related disorders.
ΔFosB protein accumulates in the striatum in response
chronic administration of drugs of abuse, L-DOPA, or stress, triggering
long lasting neural and behavioral changes that underlie aspects of
drug addiction, abnormal involuntary movements (dyskinesia), and depression.
ΔFosB binds AP-1 DNA consensus sequences found in promoters
of many genes and can both repress or activate gene transcription.
In the striatum, ΔFosB is thought to dimerize with JunD to form
a functional transcription factor, though strikingly JunD does not
accumulate in parallel. One explanation is that ΔFosB can recruit
different partners, including itself, depending on the neuron type
in which it is induced and the chronic stimulus, generating protein
complexes with different effects on gene transcription. To develop
chemical probes to study ΔFosB, a high-throughput screen was
carried out to identify small molecules that modulate ΔFosB
function. Two compounds with low micromolar activity, termed C2 and
C6, disrupt the binding of ΔFosB to DNA via different mechanisms,
and in in vitro assays stimulate ΔFosB-mediated transcription.
In cocaine-treated mice, C2 significantly elevates mRNA levels of
the AMPA glutamate receptor GluR2 subunit with specificity, a known
target gene of ΔFosB that plays a role in drug addiction and
endogenous resilience mechanisms. C2 and C6 show different activities
against ΔFosB homodimers compared to ΔFosB/JunD heterodimers,
suggesting that these compounds can be used as probes to study the
contribution of different ΔFosB-containing complexes on the
regulation of gene transcription in biological systems and to assess
the utility of ΔFosB as a therapeutic target.
ΔFosB; high throughput screening; transcription
factor; drug addiction; dyskinesia; depression
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.
chemoinformatics; data analysis software; open source; high-throughput screening
► Nematode neuropeptide G protein-coupled receptors are good targets for anthelmintic discovery. ► Neuropeptides encoded on the Caenorhabditis elegans flp-2 gene are bioactive in parasite assays. ► The receptor for flp-2 peptides has been functionally expressed in mammalian cells and yeast. ► The recombinant yeast expressing this receptor is the focus of an ongoing drug discovery effort.
Profound neuropeptide diversity characterizes the nematode nervous system, but it has proven challenging to match neuropeptide G protein-coupled receptors (GPCR) with their cognate ligands in heterologous systems. We have expressed the Caenorhabditis elegans GPCR encoded in the locus T19F4.1, previously matched with FMRFamide-like peptides encoded on the flp-2 precursor gene, in mammalian cells and in the yeast Saccharomyces cerevisiae. Pharmacological characterization revealed that the receptor is potently activated by flp-2 peptides in CHO cells (∼10 nM EC50) and in yeast (∼100 nM EC50), signaling through a Gqα pathway in each system. The yeast GPCR expression system provides a robust assay for screening for agonists of the flp-2 receptor and is the target of an ongoing high-throughput screening exercise.
flp, FMRFamide-like protein gene; FLP, FMRFamide-like peptide; FLP2-R, receptor for peptides encoded on flp-2; FLIPR, fluorescence imaging plate reader; GPCR, G protein-coupled receptor; nlp, neuropeptide-like protein gene; FLP; Caenorhabditis elegans; flp2; Neuropeptide
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.
unfolded protein response; endoplasmic reticulum stress; cell-based assay; luciferase reporter; natural products
Homocitrate synthase (HCS) catalyzes the first step of L-lysine biosynthesis in fungi by condensing acetyl-Coenzyme A and 2-oxoglutarate to form 3R-homocitrate and Coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust, fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of ~41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized over 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases and enzymes involved in lipid metabolism.
homocitrate synthase; antifungal; high-throughput screen; fluorescent assay; MMBC; ThioGlo 1; acetyltransferase
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.
phosphate; malachite green; ATPase; molecular chaperone; fluorescence assay
Shigella flexneri is a human enteropathogen that infects ca. 165 million people and claims more than 1 million lives per year worldwide. Although shigellosis has been considered a disease of the “Third World,” like many other contagious diseases, it does occur in developed countries. The emergence of drug and multi-drug-resistant strains of Shigella emphasize the need for novel antibiotic development. VirF, an AraC-type transcriptional regulator, is responsible for the expression of all downstream virulence factors that control intracellular invasion and cell-to-cell spread of Shigella. Gene knockout studies have validated that inhibition of VirF expression is sufficient to block the normal life cycle of Shigella in the host and thereby increase susceptibility to the host immune system. The authors have developed a high-throughput, cell-based assay to monitor inhibition of VirF using β-galactosidase as a reporter protein. Using an avirulent strain of Shigella, they have screened libraries containing ~42,000 small molecules. Following confirmation and dose-response analysis, they have identified 25 compounds that demonstrate VirF inhibition in vivo ≥55% in comparison to the controls and little general antibacterial activity (measured by cell growth, OD600). The authors are in the process of confirming these “hits” in several secondary assays to assess the mechanism of action.
VirF; Shigella flexneri; AraC family; HTS; transcriptional activators
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.
Guanine nucleotide-exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytokskeletal changes, motility, growth, survival, and gene transcription. The RhoGEF Leukemia-Associated RhoGEF (LARG) is a member of the Regulators of G-protein Signaling Homology Domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide binding assay utilizing BODIPY-Texas Red-GTPγS (BODIPY-TR-GTPγS), we performed a ten-thousand compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a non-fluorescent radioactive guanine nucleotide binding assay measuring LARG-stimulated [35S] GTPγS binding to RhoA, thus ruling out non-specific fluorescent effects. All five compounds selectively inhibited LARG-stimulated RhoA [35S] GTPγS binding, but had little to no effect upon RhoA or Gαo [35S] GTPγS binding. Therefore, these five compounds should serve as promising starting points for the development of small molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.
high-throughput screening; fluorescence polarization; RhoGEF; RhoA; LARG; Drug Discovery
Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.
Furin; BACE; TGN; Prohormone Convertase; Alzheimers; SEAP; Alkaline Phosphatase; NSAIDs