James Inglese

Protein kinases that phosphorylate activated G protein-coupled receptors.

G protein‐coupled receptor kinases (GRKs) are a family of serine/threonine protein kinases that specifically recognize agonist‐occupied, activated G protein‐coupled receptor proteins as substrates. Phosphorylation of an activated receptor by a GRK terminates signaling by that receptor, by initiating the uncoupling of the receptor from heterotrimeric G proteins. Six distinct mammalian GRKs are known, which differ in tissue distribution and in regulatory properties. The intracellular localization of GRKs to membrane‐bound receptor substrates is the most important known regulatory feature of these enzymes. Rhodopsin kinase (GRK1) requires a post‐translationally added farnesyl isoprenoid to bind to light‐activated rhodopsin. The β‐adrenergic receptor kinases (GRK2 and GRK3) associate with heterotrimeric G protein βγ‐subunits, released upon receptor activation of G proteins, for membrane anchorage. The recently‐described GRKs 4, 5, and 6 comprise a distinct subgroup of GRKs. These kinases utilize distinct mechanisms for membrane localization, which are just beginning to be defined. All GRKs appear to play the same general cellular role of desensitizing activated G protein‐coupled receptors, but utilize distinctly individual means to the same end.—Premont, R. T., Inglese, J., Lefkowitz, R. J. Protein kinases that phosphorylate activated G protein‐coupled receptors. FASEB J. 9, 175–182 (1995)

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.

Apparent activity in high-throughput screening: origins of compound-dependent assay interference.

Expansive compound collections made up of structurally heterogeneous chemicals, the activities of which are largely undefined, present challenging problems for high-throughput screening (HTS). Foremost is differentiating whether the activity for a given compound in an assay is directed against the targeted biology, or is the result of surreptitious compound activity involving the assay detection system. Such compound interference can be especially difficult to identify if it is reproducible and concentration-dependent - characteristics generally attributed to compounds with genuine activity. While reactive chemical groups on compounds were once thought to be the primary source of compound interference in assays used in HTS, recent work suggests that other factors, such as compound aggregation, may play a more significant role in many assay formats. Considerable progress has been made to profile representative compound libraries in an effort to identify chemical classes susceptible to producing compound interference, such as compounds commonly found to inhibit the reporter enzyme firefly luciferase. Such work has also led to the development of practices that have the potential to significantly reduce compound interference, for example, through the addition of non-ionic detergent to assay buffer to reduce aggregation-based inhibition.

Identification of oxadiazoles as new drug leads for the control of schistosomiasis

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends almost exclusively on praziquantel. Millions of people are treated annually with praziquantel, and drug-resistant parasites thus are likely to evolve. Phosphinic amides and oxadiazole 2-oxides, identified from a quantitative high-throughput screen, were shown to inhibit a parasite enzyme, thioredoxin glutathione reductase (TGR), with activities in the low micromolar to low nanomolar range. Incubation of parasites with these compounds led to rapid inhibition of TGR activity and parasite death. The activity of the oxadiazole 2-oxides was associated with a donation of nitric oxide. Treatment of schistosome-infected mice with 4-phenyl-1,2,5-oxadiazole-3-carbonitrile-2-oxide led to marked reductions in worm burdens from treatments against multiple parasite stages and egg-associated pathologies. The compound was active against the three major schistosome species infecting humans. These protective effects exceed benchmark activity criteria set by the World Health Organization for lead compound development for schistosomiasis.

Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening

Background The propensity of compounds to produce adverse health effects in humans is generally evaluated using animal-based test methods. Such methods can be relatively expensive, low-throughput, and associated with pain suffered by the treated animals. In addition, differences in species biology may confound extrapolation to human health effects. Objective The National Toxicology Program and the National Institutes of Health Chemical Genomics Center are collaborating to identify a battery of cell-based screens to prioritize compounds for further toxicologic evaluation. Methods A collection of 1,408 compounds previously tested in one or more traditional toxicologic assays were profiled for cytotoxicity using quantitative high-throughput screening (qHTS) in 13 human and rodent cell types derived from six common targets of xenobiotic toxicity (liver, blood, kidney, nerve, lung, skin). Selected cytotoxicants were further tested to define response kinetics. Results qHTS of these compounds produced robust and reproducible results, which allowed cross-compound, cross-cell type, and cross-species comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited species- or cell type–specific cytotoxicity. Closely related cell types and analogous cell types in human and rodent frequently showed different patterns of cytotoxicity. Some compounds inducing similar levels of cytotoxicity showed distinct time dependence in kinetic studies, consistent with known mechanisms of toxicity. Conclusions The generation of high-quality cytotoxicity data on this large library of known compounds using qHTS demonstrates the potential of this methodology to profile a much broader array of assays and compounds, which, in aggregate, may be valuable for prioritizing compounds for further toxicologic evaluation, identifying compounds with particular mechanisms of action, and potentially predicting in vivo biological response.

Fluorescence Spectroscopic Profiling of Compound Libraries

Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions commonly utilized in HTS. The quantitative HTS paradigm was utilized to test each sample at seven or more concentrations over a 4-log range in 1,536-well format. Raw fluorescence was compared with fluorophore standards to compute a normalized response as a function of concentration and spectral region. More than 5% of library members were brighter than the equivalent of 10 nM 4-methyl umbelliferone, a common UV-active probe. Red-shifting the spectral window by as little as 100 nm was accompanied by a dramatic decrease in autofluorescence. Native compound fluorescence, fluorescent impurities, novel fluorescent compounds, and the utilization of fluorescence profiling data are discussed.

Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression

High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87–91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC50 = 7 ± 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.

Gβγ interactions with PH domains and Ras-MAPK signaling pathways

Abstract G-protein-coupled receptor signaling and receptor tyrosine kinase (RTK) signaling are two mechanisms of transmembrane communication used by numerous extracellular agents and stimuli. The βγ-subunit complex of G proteins mediates many of the functions associated with G-protein-coupled receptor signaling and may even provide a means to link G proteins to RTK-initiated cascades. This connection may be mediated by the pleckstrin homology domain, a modular domain found in many signaling proteins that interact with G βγ .

Illuminating Insights into Firefly Luciferase and Other Bioluminescent Reporters Used in Chemical Biology

Understanding luciferase enzymology and the structure of compounds that modulate luciferase activity can be used to improve the design of luminescence-based assays. This review provides an overview of these popular reporters with an emphasis on the commonly used firefly luciferase from Photinus pyralis (FLuc). Large-scale chemical profile studies have identified a variety of scaffolds that inhibit FLuc. In some cell-based assays, these inhibitors can act in a counterintuitive way, leading to a gain in luminescent signal. Although formerly attributed to transcriptional activation, intracellular stabilization of FLuc is the primary mechanism underlying this observation. FLuc inhibition and stabilization can be complex, as illustrated by the compound PTC124, which is converted by FLuc in the presence of ATP to a high affinity multisubstrate adduct inhibitor, PTC124-AMP. The potential influence these findings can have on drug discovery efforts is provided here.

Quantitative Analyses of Aggregation, Autofluorescence, and Reactivity Artifacts in a Screen for Inhibitors of a Thiol Protease

The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of nonspecific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds, and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.5% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.