Nearly one-third of the encoded proteome is comprised of secretory proteins that enable communication between cells and organ systems, playing a ubiquitous role in human health and disease. High-throughput detection of secreted proteins would enhance efforts to identify therapies for secretion-related diseases. Using the Z mutant of alpha-1 antitrypsin as a human secretory model, we have developed 1536-well high-throughput screening assays that utilize acoustic droplet ejection to transfer nanoliter volumes of sample for protein quantification. Among them, the acoustic reverse phase protein array (acoustic RPPA) is a multiplexable, low-cost immunodetection technology for native, endogenously secreted proteins from physiologically relevant model systems like stem cells that is compatible with plate-based instrumentation. Parallel assay profiling with the LOPAC1280 chemical library validated performance and orthogonality between a secreted bioluminescent reporter and acoustic RPPA method by consistently identifying secretory modulators with comparable concentration response relationships. Here, we introduce a robust, multiplexed drug discovery platform coupling extracellular protein quantification by acoustic RPPA with intracellular and cytotoxicity analyses from single wells, demonstrating proof-of-principle applications for human induced pluripotent stem cell-derived hepatocytes.

CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.

Measurement of gene expression for high-throughput screening is an increasingly used technique that has been developed for not only gene dosage disorders resulting from disease-associated copy number variations, but also for induction/repression of genes modulating the severity of a disease phenotype. Traditional methods have employed transient or stable transfection of reporter constructs in which a single reporter is driven by selected regulatory elements from the candidate gene. However, individual regulatory elements are inherently unable to capture the integrated regulation of multiple enhancers at the endogenous locus, and random reporter insertion can result in neighborhood effects that impact the physiological responsiveness of the reporter. Therefore, we outline a general method of employing genome editing to insert reporters into the 3' UTR of a candidate gene, which has been used successfully in our studies of the Pmp22 gene associated with Charcot-Marie-Tooth disease. The method employs genome editing to insert two nonhomologous reporters that maximize the efficiency of identification of biologically active molecules through concordant responses in small molecule screening. We include a number of aspects of the design and construction of these reporter assays that will be applicable to creation of similar assays in a variety of cell types.

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >10 12 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.

Reporter-biased artifacts-i.e., compounds that interact directly with the reporter enzyme used in a high-throughput screening (HTS) assay and not the biological process or pharmacology being interrogated-are now widely recognized to reduce the efficiency and quality of HTS used for chemical probe and therapeutic development. Furthermore, narrow or single-concentration HTS perpetuates false negatives during primary screening campaigns. Titration-based HTS, or quantitative HTS (qHTS), and coincidence reporter technology can be employed to reduce false negatives and false positives, respectively, thereby increasing the quality and efficiency of primary screening efforts, where the number of compounds investigated can range from tens of thousands to millions. The three protocols described here allow for generation of a coincidence reporter (CR) biocircuit to interrogate a biological or pharmacological question of interest, generation of a stable cell line expressing the CR biocircuit, and qHTS using the CR biocircuit to efficiently identify high-quality biologically active small molecules. © 2017 by John Wiley & Sons, Inc.

Parkin, an E3 ubiquitin ligase, is a central mediator of mitochondrial quality control and is linked to familial forms of Parkinson's disease (PD). Removal of dysfunctional mitochondria from the cell by Parkin is thought to be neuroprotective, and pharmacologically increasing Parkin levels may be a novel therapeutic approach. We used genome-editing to integrate a coincidence reporter into the PARK2 gene locus of a neuroblastoma-derived cell line and developed a quantitative high-throughput screening (qHTS) assay capable of accurately detecting subtle compound-mediated increases in endogenous PARK2 expression. Interrogation of a chemogenomic library revealed diverse chemical classes that up-regulate the PARK2 transcript, including epigenetic agents, drugs controlling cholesterol biosynthesis, and JNK inhibitors. Use of the coincidence reporter eliminated wasted time pursuing reporter-biased false positives accounting for ∼2/3 of the actives and, coupled with titration-based screening, greatly improves the efficiency of compound selection. This approach represents a strategy to revitalize reporter-gene assays for drug discovery.

Methods to identify the bioactive diversity within natural product extracts (NPEs) continue to evolve. NPEs constitute complex mixtures of chemical substances varying in structure, composition, and abundance. NPEs can therefore be challenging to evaluate efficiently with high-throughput screening approaches designed to test pure substances. Here we facilitate the rapid identification and prioritization of antimalarial NPEs using a pharmacologically driven, quantitative high-throughput-screening (qHTS) paradigm. In qHTS each NPE is tested across a concentration range from which sigmoidal response, efficacy, and apparent EC(50)s can be used to rank order NPEs for subsequent organism reculture, extraction, and fractionation. Using an NPE library derived from diverse marine microorganisms we observed potent antimalarial activity from two Streptomyces sp. extracts identified from thousands tested using qHTS. Seven compounds were isolated from two phylogenetically related Streptomyces species: Streptomyces ballenaensis collected from Costa Rica and Streptomyces bangulaensis collected from Papua New Guinea. Among them we identified actinoramides A and B, belonging to the unusually elaborated nonproteinogenic amino-acid-containing tetrapeptide series of natural products. In addition, we characterized a series of new compounds, including an artifact, 25-epi-actinoramide A, and actinoramides D, E, and F, which are closely related biosynthetic congeners of the previously reported metabolites.

Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot–Marie–Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin.

A library of 367 protein kinase inhibitors, the GSK Published Kinase Inhibitor Set (PKIS), which has been annotated for protein kinase family activity and is available for public screening efforts, was assayed against the commonly used luciferase reporter enzymes from the firefly, Photinus pyralis (FLuc) and marine sea pansy, Renilla reniformis (RLuc). A total of 22 compounds (∼6% of the library) were found to inhibit FLuc with 10 compounds showing potencies ≤1 µM. Only two compounds were found to inhibit RLuc, and these showed relatively weak potency values (∼10 µM). An inhibitor series of the VEGFR2/TIE2 protein kinase family containing either an aryl oxazole or benzimidazole-urea core illustrate the different structure activity relationship profiles FLuc inhibitors can display for kinase inhibitor chemotypes. Several FLuc inhibitors were broadly active toward the tyrosine kinase and CDK families. These data should aid in interpreting the results derived from screens employing the GSK PKIS in cell-based assays using the FLuc reporter. The study also underscores the general need for strategies such as the use of orthogonal reporters to identify kinase or non-kinase mediated cellular responses.

The structural integrity of myelin formed by Schwann cells in the peripheral nervous system (PNS) is required for proper nerve conduction and is dependent on adequate expression of myelin genes including peripheral myelin protein 22 (PMP22). Consequently, excess PMP22 resulting from its genetic duplication and overexpression has been directly associated with the peripheral neuropathy called Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent type of CMT. Here, in an attempt to identify transcriptional inhibitors with therapeutic value toward CMT1A, we developed a cross-validating pair of orthogonal reporter assays, firefly luciferase (FLuc) and β-lactamase (βLac), capable of recapitulating PMP22 expression, utilizing the intronic regulatory element of the human PMP22 gene. Each compound from a collection of approximately 3,000 approved drugs was tested at multiple titration points to achieve a pharmacological end point in a 1536-well plate quantitative high-throughput screen (qHTS) format. In conjunction with an independent counter-screen for cytotoxicity, the design of our orthogonal screen platform effectively contributed to selection and prioritization of active compounds, among which three drugs (fenretinide, olvanil, and bortezomib) exhibited marked reduction of endogenous Pmp22 mRNA and protein. Overall, the findings of this study provide a strategic approach to assay development for gene-dosage diseases such as CMT1A.

The chemical diversity of nature has tremendous potential for the discovery of molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, and macro- and microorganisms has curtailed their use in lead discovery. Here, we describe a process for leveraging the concentration-response curves 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 improves the probability that labor-intensive subsequent steps of reculturing, 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.

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug-drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z'-factors of >or=0.5. Seven- to 15-point concentration-response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format.

The authors describe how room temperature storage of a 1120-member compound library prepared in either DMSO or in a hydrated-DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z' factors of 0.71 and 0.62, with 17% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes, respectively. The authors tested the library using quantitative high-throughput screening to generate potency values for every library member, which was measured at 7 time intervals spanning 37 weeks. They calculated the minimum significant ratio (MSR) from these potency values at each time interval and found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed from approximately 2.0 to 5.0. The hydrated conditions showed similar performance in both MSR progression and analytical quality control results. Based on this study, the authors recommend that DMSO samples be stored in 1536-well plates for <4 months at room temperature. Furthermore, the study illustrates the degree and time scale of apparent compound potency changes due to sample storage.

Small molecule modulators are critical for dissecting and understanding signaling pathways at the molecular level. Interleukin 6 (IL-6) is a cytokine that signals via the JAK-STAT pathway and is implicated in cancer and inflammation. To identify modulators of this pathway, we screened a chemical collection against an IL-6 responsive cell line stably expressing a beta-lactamase reporter gene fused to a sis-inducible element (SIE-bla cells). This assay was optimized for a 1536-well microplate format and screened against 11 693 small molecules using quantitative high-throughput screening (qHTS), a method that assays a chemical library at multiple concentrations to generate titration-response profiles for each compound. The qHTS recovered 564 actives with well-fit curves that clustered into 32 distinct chemical series of 13 activators and 19 inhibitors. A retrospective analysis of the qHTS data indicated that single concentration data at 1.5 and 7.7 microM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and non-selective series, we found four activator and one inhibitor series that modulated SIE-bla cells but did not show similar activity in reporter gene assays induced by EGF and hypoxia. Small molecules within these series will make useful tool compounds to investigate IL-6 signaling mediated by JAK-STAT activation.

Assays for ATPases have been enabled for high-throughput screening (HTS) by employing firefly luciferase to detect the remaining ATP in the assay. However, for any enzyme assay, measurement of product formation is a more sensitive assay design. Recently, technologies that allow detection of the ADP product from ATPase reactions have been described using fluorescent methods of detection. We describe here the characterization of a bioluminescent assay that employs firefly luciferase in a coupled-enzyme assay format to enable detection of ADP levels from ATPase assays (ADP-Glo, Promega Corp.). We determined the performance of the ADP-Glo assay in 1,536-well microtiter plates using the protein kinase Clk4 and a 1,352 member kinase focused combinatorial library. The ADP-Glo assay was compared to the Clk4 assay performed using a bioluminescence ATP-depletion format (Kinase-Glo, Promega Corp). We performed this analysis using quantitative HTS (qHTS) where we determined potency values for all library members and identified approximately 300 compounds with potencies ranging from as low as 50 nM to >10 microM, yielding a robust dataset for the comparison. Both assay formats showed high performance (Z'-factors approximately 0.9) and showed a similar potency distribution for the actives. We conclude that the bioluminescence ADP detection assay system is a viable generic alternative to the widely used ATP-depletion assay for ATPases and discuss the advantages and disadvantages of both approaches.

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.

A computational method is described that simulates the EPR microwave power saturation characteristics of a protein sample containing an EPR signal from a buried free radical in a frozen solution containing paramagnetic spin-lattice relaxation-enhancement agents such as [Dy(III)HEDTA], where HEDTA is N-hydroxyethylenediaminetriacetate. The specific experiment modeled in this work is the EPR progressive power saturation experiment, where a series of spectra are recorded with increasing microwave power incident on the sample. A fit of the data series yields a characteristic power at half saturation, or P-1/2, that is proportional to the spin-lattice relaxation rate of the signal observed in the sample. The addition of DyHEDTA to the protein solution increases P-1/2 through dipole-dipole coupling to the buried free radical by an amount strongly dependent on the distance r from the fast-relaxing DyHEDTA to the slow-relaxing radical. The measured value of P-1/2 reflects an ensemble of protein molecules in which each buried spin interacts with a specific distribution of DyHEDTA molecules. To simulate the effect of DyHEDTA on the P-1/2 of the buried radical spin, we have created a Monte Carlo-type method that models the power-saturation characteristics of the bulk sample by creating saturation profiles of individual, randomly oriented spin ensembles. These individual spin ensembles incorporate arbitrary structural data for the protein as a region where the individual molecules of DyHEDTA are spatially excluded from the calculation, as opposed to previous methods where simple geometric models were employed. The calculation method is validated mathematically and is used to simulate experimental data from the heme-nitric oxide-containing protein systems myoglobin and horseradish peroxidase to demonstrate the effectiveness of the new simulation method.

Spin-labeled Cys89 of the soluble methane monooxygenase regulatory protein (MMOB) from Methylococcus capsulatus (Bath) binds within 15 +/- 4 A of the hydroxylase (MMOH) diiron center, placing the MMOB docking site in the MMOH "canyon" region on iron-coordinating helices E and F of the alpha-subunit.