A Cell-Based β-Lactamase Reporter Gene Assay for the CREB Signaling Pathway

The Cyclic-AMP Response Element Binding (CREB) proteins comprise a family of transcription factors that stimulate or repress the expression of a wide variety of genes by binding to nucleotide sequences known as cAMP Response Elements. CREB-mediated transcription has been implicated in a wide variety of important physiological processes, including long-term memory, and enhancement of CREB signaling has been suggested as an attractive therapeutic strategy for human memory disorders. To identify small molecule compounds that enhance CREB pathway signaling, we have optimized and validated a cell-based β-lactamase reporter gene CREB pathway assay in 1536-well plate format. The LOPAC library of 1280 compounds was screened in triplicate in this assay on a quantitative high throughput screening (qHTS) platform. A variety of compounds which affect known members of the CREB pathway were identified as active, including twelve known phosphodiesterase (PDE) inhibitors, and forskolin, a known activator of adenylate cyclase, thus validating the assay’s performance. This qHTS platform assay will facilitate identification of novel small molecule CREB signaling enhancers, which will be useful for chemical genetic dissection of the CREB pathway and as starting points for potentially memory-enhancing therapeutics

Firefly luciferase in chemical biology: A compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter

Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds, found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes including FLuc-catalyzed formation of multisubstrate-adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays compounds acting as FLuc inhibitors yield paradoxical luminescence increases, data on compounds acquired from FLuc-dependent assays requires careful analysis as described in this report

Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP3) assay measures 3H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M1 acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers

Feasibility of [F-18]fluoropivalate hybrid PET/MRI for imaging lower and higher grade glioma: a prospective first-in-patient pilot study

Purpose: MRI and PET are used in neuro-oncology for the detection and characterisation of lesions for malignancy to target surgical biopsy and to plan surgical resections or stereotactic radiosurgery. The critical role of short-chain fatty acids (SCFAs) in brain tumour biology has come to the forefront. The non-metabolised SCFA radiotracer, [18F]fluoropivalate (FPIA), shows low background signal in most tissues except eliminating organs and has appropriate human dosimetry. Tumour uptake of the radiotracer is, however, unknown. We investigated the uptake characteristics of FPIA in this pilot PET/MRI study. Methods: Ten adult glioma subjects were identified based on radiological features using standard-of-care MRI prior to any surgical intervention, with subsequent histopathological confirmation of glioma subtype and grade (lower-grade – LGG – and higher-grade – HGG – patients). FPIA was injected as an intravenous bolus injection (range 342–368 MBq), and dynamic PET and MRI data were acquired simultaneously over 66 min. Results: All patients tolerated the PET/MRI protocol. Three patients were reclassified following resection and histology. Tumour maximum standardised uptake value (SUVmax,60) increased in the order LGG (WHO grade 2) < HGG (WHO grade 3) < HGG (WHO grade 4). The net irreversible solute transfer, Ki, and influx rate constant, K1, were significantly higher in HGG (p < 0.05). Of the MRI variables studied, DCE-MRI-derived extravascular-and-extracellular volume fraction (ve) was high in tumours of WHO grade 4 compared with other grades (p < 0.05). SLC25A20 protein expression was higher in HGG compared with LGG. Conclusion: Tumoural FPIA PET uptake is higher in HGG compared to LGG. This study supports further investigation of FPIA PET/MRI for brain tumour imaging in a larger patient population. Clinical trial registration: Clinicaltrials.gov, NCT04097535

Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors

The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography

Identification of small molecule compounds that inhibit the HIF-1 signaling pathway

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Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex

Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade

Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 µL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (∼25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel therapeutics for other neglected tropical diseases