Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells.

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.

NMR Reporter Assays for the Quantification of Weak-Affinity Receptor-Ligand Interactions.

Biophysical methods are widely employed in academia and the pharmaceutical industry to detect and quantify weak molecular interactions. Such methods find broad application in fragment-based drug discovery (FBDD). In an FBDD campaign, a suitable affinity determination method is key to advancing a project beyond the initial screening phase. Protein-observed (PO) nuclear magnetic resonance (NMR) finds widespread use due to its ability to sensitively detect very weak interactions at residue-level resolution. When there are issues precluding the use of PO-NMR, ligand-observed (LO) NMR reporter assays can be a useful alternative. Such assays can measure affinities in a similar range to PO-NMR while offering some distinct advantages, especially with regard to protein consumption and compound throughput. In this paper, we take a closer look at setting up such assays for routine use, with the aim of getting high-quality, accurate data and good throughput. We assess some of the key characteristics of these assays in the mathematical framework established for fluorescence polarization assays with which the readers may be more familiar. We also provide guidance on setting up such assays and compare their performance with other affinity determination methods that are commonly used in drug discovery.

An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen.

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.

A Fluorescence Anisotropy-Based Comprehensive Method for the In Vitro Screening of COI1-JAZs Agonists and Antagonists.

The phytohormone (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) causes protein-protein interactions (PPI) between F-box Protein CORONATINE INSENSITIVE 1 (COI1) and JASMONATE ZIM DOMAIN (JAZ) transcriptional repressor. A total of 13 JAZ subtypes are encoded in the genome of Arabidopsis thaliana; however, their genetic redundancy obfuscates the individual function of each JAZ. One approach to decipher this redundant signaling network is chemical genetics, using small molecules specific to individual JAZ subtype, for which a reliable and high-throughput screening system of the ligands for all combinations of COI1-JAZs would be indispensable. In this chapter, we describe a fluorescence anisotropy-based quantitative screening system for the ligands of COI1-JAZ co-receptors. Our method is applicable to agonists and antagonists of the COI1-JAZs.

Reviewing Hit Discovery Literature for Difficult Targets: Glutathione Transferase Omega-1 as an Example.

Early stage drug discovery reporting on relatively new or difficult targets is often associated with insufficient hit triage. Literature reviews of such targets seldom delve into the detail required to critically analyze the associated screening hits reported. Here we take the enzyme glutathione transferase omega-1 (GSTO1-1) as an example of a relatively difficult target and review the associated literature involving small-molecule inhibitors. As part of this process we deliberately pay closer-than-usual attention to assay interference and hit quality aspects. We believe this Perspective will be a useful guide for future development of GSTO1-1 inhibitors, as well serving as a template for future review formats of new or difficult targets.

A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase.

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining ß-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.

Small molecule aptamer assays based on fluorescence anisotropy signal-enhancer oligonucleotides.

Herein, we design novel fluorescence anisotropy (FA) aptamer sensing platforms dedicated to small molecule detection. The assay strategy relied on enhanced fluctuations of segmental motion dynamics of the aptamer tracer mediated by an unlabelled, partially complementary oligonucleotide. The signal-enhancer oligonucleotide (SEO) essentially served as a free probe fraction revealer. By targeting specific regions of the signalling functional nucleic acid, the SEO binding to the unbound aptamer triggered perturbations of both the internal DNA flexibility and the localized dye environment upon the free probe to duplex structure transition. This potentiating effect determined increased FA variations between the duplex and target bound states of the aptameric probe. FA assay responses were obtained with both pre-structured (adenosine) and unstructured (tyrosinamide) aptamers and with dyes of different photochemical properties (fluorescein and texas red). The multiplexed analysis ability was further demonstrated through the simultaneous multicolour detection of the two small targets. The FA method appears to be especially simple, sensitive and widely applicable.

Fluorescence anisotropy (polarization): from drug screening to precision medicine.

INTRODUCTION: Fluorescence anisotropy (FA) is one of the major established methods accepted by industry and regulatory agencies for understanding the mechanisms of drug action and selecting drug candidates utilizing a high-throughput format. AREAS COVERED: This review covers the basics of FA and complementary methods, such as fluorescence lifetime anisotropy and their roles in the drug discovery process. The authors highlight the factors affecting FA readouts, fluorophore selection and instrumentation. Furthermore, the authors describe the recent development of a successful, commercially valuable FA assay for long QT syndrome drug toxicity to illustrate the role that FA can play in the early stages of drug discovery. EXPERT OPINION: Despite the success in drug discovery, the FA-based technique experiences competitive pressure from other homogeneous assays. That being said, FA is an established yet rapidly developing technique, recognized by academic institutions, the pharmaceutical industry and regulatory agencies across the globe. The technical problems encountered in working with small molecules in homogeneous assays are largely solved, and new challenges come from more complex biological molecules and nanoparticles. With that, FA will remain one of the major work-horse techniques leading to precision (personalized) medicine.

Selective inhibitor of platelet-activating factor acetylhydrolases 1b2 and 1b3 that impairs cancer cell survival.

Platelet-activating factor acetylhydrolases (PAFAHs) 1b2 and 1b3 are poorly characterized serine hydrolases that form a complex with a noncatalytic protein (1b1) to regulate brain development, spermatogenesis, and cancer pathogenesis. Determining physiological substrates and biochemical functions for the PAFAH1b complex would benefit from selective chemical probes that can perturb its activity in living systems. Here, we report a class of tetrahydropyridine reversible inhibitors of PAFAH1b2/3 discovered using a fluorescence polarization-activity-based protein profiling (fluopol-ABPP) screen of the NIH 300,000+ compound library. The most potent of these agents, P11, exhibited IC50 values of ∼40 and 900 nM for PAFAH1b2 and 1b3, respectively. We confirm selective inhibition of PAFAH1b2/3 in cancer cells by P11 using an ABPP protocol adapted for in situ analysis of reversible inhibitors and show that this compound impairs tumor cell survival, supporting a role for PAFAH1b2/3 in cancer.

A 1536-well fluorescence polarization assay to screen for modulators of the MUSASHI family of RNA-binding proteins.

RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10 µM. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs.

Carbon nanotube signal amplification for ultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition.

A versatile sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) is developed for the simple and ultrasensitive monitoring of DNA methyltransferase (MTase) activity and inhibition in homogeneous solution. This method uses a dye-labeled DNA probe that possess a doubled-stranded DNA (dsDNA) part for Mtase and its corresponding restriction endonuclease recognition, and a single-stranded DNA part for binding MWCNTs. In the absence of MTase, the dye-labeled DNA is cleaved by restriction endonuclease, and releases very short DNA carrying the dye that cannot bind to MWCNTs, which has relatively small FP value. However, in the presence of MTase, the specific recognition sequence in the dye-labeled DNA probe is methylated and not cleaved by restriction endonuclease. Thus, the dye-labeled methylated DNA product is adsorbed onto MWCNTs via strong π-π stacking interactions, which leads to a significant increase in the FP value due to the enlargement of the molecular volume of the dye-labeled methylated DNA/MWCNTs complex. This provides the basic of a quantitative measurement of MTase activity. By using the MWCNT signal amplification approach, the detection sensitivity can be significantly improved by two orders of magnitude over the previously reported methods. Moreover, this method also has high specificity and a wide dynamic range of over five orders of magnitude. Additionally, the suitability of this sensing platform for MTase inhibitor screening has also been demonstrated. This approach may serve as a general detection platform for sensitive assay of a variety of DNA MTases and screening potential drugs.

Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.

A high-throughput-compatible fluorescence anisotropy-based assay for competitive inhibitors of Escherichia coli UDP-N-acetylglucosamine acyltransferase (LpxA).

LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses.

Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in α-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with α-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus.

Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples.

Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 µM and 0.8 µM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.

Improvement of inhibitor identification for heat shock protein 90α by utilizing a red-shifted fluorescence polarization probe.

Heat shock protein-90 (HSP90) is an ATP-dependent molecular chaperone with intrinsic ATPase activity. HSP90 is required for the stability and function of client proteins, many of which are involved in oncogenesis. Thus, identification of HSP90 inhibitors would potentially lead to the discovery of cancer therapeutics. Here, we present a high-throughput screening campaign utilizing two geldanamycin (GM)-labeled probes in a fluorescence polarization (FP) assay. For the primary screen, a previously reported green BODIPY-labeled GM (GM-BODIPY) was used to evaluate a library collection of about 400,000 compounds. From this screen, 3058 compounds showed >30% inhibition. To distinguish true positives from compound interference, a confirmatory screen was deemed necessary. Accordingly, a red-shifted FP binding assay was developed using GM labeled with red BODIPY. This tool enabled reliable identification of promising HSP90α inhibitors.

High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1.

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

Development of a novel fluorescence polarization-based assay for studying the ß-catenin/Tcf4 interaction.

Aberrant activation of the Wnt/ß-catenin signaling pathway is associated with a wide range of human cancers. The interaction of ß-catenin with T cell factor (Tcf) is a key step in activation of proliferative genes in this pathway. Interruption of this interaction would be a valuable strategy as a tumor therapy. In this study, we developed a novel fluorescein isothiocyanate (FITC)-labeled Tcf4-derived probe for identification of inhibitors of the ß-catenin/Tcf4 interaction using a fluorescence polarization assay. This assay shows high potential for use in high-throughput screening for the discovery of inhibitors of the ß-catenin/Tcf4 interaction.

Optimization of the structure-switching aptamer-based fluorescence polarization assay for the sensitive tyrosinamide sensing.

In this paper, a structure-switching aptamer assay based on a fluorescence polarization (FP) signal transduction approach and dedicated to the L-tyrosinamide sensing was described and optimized. A fluorescently labelled complementary strand (CS) of the aptamer central region was used as a probe. The effects of critical parameters such as buffer composition and pH, temperature, aptamer:CS stoichiometry, nature of the dye (Fluorescein (F) or Texas Red (TR)) and length of the CS (15-, 12-, 9- and 6-mer) on the assay analytical performances were evaluated. Under optimized experimental conditions (10 mM Tris-HCl, 5 mM MgCl(2) and 25 mM NaCl, pH 7.5 temperature of 22°C and stoichiometry 1:1), the results showed that, for a 12-mer CS, the F dye moderately increased the method sensitivity in comparison to the TR label. The F labelled 9-mer CS, however, did not allow the hybrid formation with the functional nucleic acid, thus emphasizing the importance of the nature of the fluorophore. In contrast, the same 9-mer CS labelled with the TR dye was able to effectively associate with the aptamer and was easily displaced upon target binding as demonstrated by a significant improvement of the sensitivity and a detection limit of 250 nM, comparable to those reported with direct aptasensing methods. The present study demonstrates that not only the CS length but also the nature of the dye played a preponderant role in the performance of the structure-switching aptamer assay, highlighting the importance of interdependently controlling these two factors for an optimal FP-based sensing platform.

Development of filtration-based time-resolved fluorescence assay for the high-throughput screening of urotensin II receptor antagonist.

The time-resolved fluorescence (TRF) receptor binding assay has many advantages over the traditional radioligand binding assay in terms of sensitivity and reproducibility for the screening of receptor ligands. The TRF-based urotensin receptor (UT) binding assay with an automatic vacuum filtration system was developed and evaluated for the high-throughput screening of UT receptor antagonists. For this assay development, the human recombinant urotensin II (UII) was modified by labeling europium at its N-terminal position (Eu-UII) and used as a fluorescent tracer. The microsomal membrane fraction of UT receptor was prepared from HEK293 cells stably expressing the human UT receptor. The 50% inhibitory concentration (IC(50)) values of UII from competition binding assays with Eu-UII were 2.76 nM, which is very similar to that of fluorescence polarization (FP)-based UT receptor binding experiment (2.18 nM). Comparing with the FP-based receptor binding assay for UII (Z' factor, 0.36), the current TRF assay presented improved Z' factor (0.76) with a relatively higher signal-to-background ratio (1.5 and 2.1, respectively). The known high-affinity UT receptor antagonists, palosuran and SB657510, exhibited IC(50) values of 23.6 and 73.4 nM, respectively, which were consistent with the IC(50) values from FP-based receptor binding assay (30.6 and 78.7 nM, respectively). These results suggest that our filtration-based TRF UT receptor binding assay can achieve the desired sensitivity with higher reproducibility to adapt for the high-throughput screening of compound libraries.