Development and validation of a generic methyltransferase enzymatic assay based on an SAH riboswitch.

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) - the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z' > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1.

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z' value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2'3'cGAMP compared with 2'3'c-diGMP and showed no activity with 3'3'c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors.

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 µM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay.

ATP depletion and ADP formation are generic detection methods used for the identification of kinase and other ATP-utilizing enzyme inhibitors in high-throughput screening campaigns. However, the most widely used nucleotide detection approaches require high ATP consumption rates or involve the use of coupling enzymes, which can complicate the selection of lead compounds. As an alternative, we have developed the Transcreener (BellBrook Labs, Madison, WI) platform, which relies on the direct immunodetection of nucleotides. Here we describe the development of antibodies with >100-fold selectivity for ADP versus ATP, which enable robust detection of initial velocity rates (Z' > 0.7 at 10% substrate consumption) at ATP concentrations ranging from 0.1 microM to 1,000 microM in a competitive fluorescence polarization (FP) immunoassay. Competitive binding experiments indicate similar affinities for other nucleotide diphosphates, including 2' -deoxy ADP, GDP, and UDP. The antibody-tracer complex and the red-shifted, ratiometric FP signal are stable for at least 24 h at room temperature, providing suitable conditions for high-throughput screening. A method for calculating a kinase ATP Km with this FP immunoassay is also presented. The Transcreener ADP assay provides a simple, generic assay platform for inhibitor screening and selectivity profiling that can be used for any ADP-generating enzyme.

Evaluating PI3 kinase isoforms using Transcreener ADP assays.

Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The Transcreener ADP Assay relies on homogeneous immunodetection of adenosine diphosphate (ADP), using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP--the invariant product of all kinase reactions--provides complete flexibility for varying lipid substrate parameters. The authors used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The nonphysiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP- and TR-FRET-based assays, and there was excellent concordance (r(2)=0.93) in the IC(50) values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. Adenosine triphosphate (ATP) hydrolysis in the absence of substrate was detected with the PI3Kalpha isoform, and inhibitors affected PI3Kalpha intrinsic ATP hydrolysis activity similarly to lipid phosphorylation.

High-Throughput Assay for RhoGEFs Based on the Transcreener® GDP Assay.

We describe a high-throughput screening (HTS)-compatible method for detecting GTPase exchange factor (GEF) activity based on stimulation of GDP formation by Rho GTPases. The method is based on the fact that GDP dissociation is the rate-limiting step in the Rho GTPase catalytic cycle, so by accelerating its release a GEF causes an increase in the steady-state rate of GDP formation. The Transcreener® GDP GTPase Assay, a fluorescence polarization immunoassay (FPIA), is used to detect GDP formation in a homogeneous format.

A High-Throughput Method for Measuring Drug Residence Time Using the Transcreener ADP Assay.

Analysis of drug-target residence times during drug development can result in improved efficacy, increased therapeutic window, and reduced side effects. Residence time can be estimated as the reciprocal of the dissociation rate ( koff) of an inhibitor from its target. The traditional methods for measuring koff require synthesis of labeled ligands or low-throughput label-free methods. To provide an alternative that is better suited to an automated high-throughput screening (HTS) environment, we adapted a classic "jump dilution" catalytic assay method for determination of koff values for kinase inhibitor drugs. We used the Transcreener ADP2 Kinase assay as a universal, homogenous method to monitor the recovery of kinase activity as the drugs dissociated from preformed inhibitor-kinase complexes. We measured residence times for several drugs that bind the epidermal growth factor receptor (EGFR), ABL1, and Aurora kinases and found that the rank ordering of inhibitor koff values correlated with literature values determined using ligand binding assays. Moreover, very similar results were obtained using the Transcreener assay with fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Förster resonance energy transfer (TR-FRET) detection modes. This HTS-compatible, generic assay method should facilitate the use of residence time as a parameter for compound prioritization and optimization early in kinase drug discovery programs.

Analysis of ligand-dependent recruitment of coactivator peptides to estrogen receptor using fluorescence polarization.

Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ERalpha or ERbeta in an in vitro high-throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ERalpha and ERbeta with affinity ranging from 20-500 nm. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17beta-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, whereas antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC50) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17beta-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen, resulting in distinct EC50 values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ERbeta was observed in the presence of the ERbeta-selective agonist diarylpropionitrile, but not the ERalpha-selective ligand propyl pyrazole triol. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.

Development and Validation of a Universal High-Throughput UDP-Glycosyltransferase Assay with a Time-Resolved FRET Signal.

Glycosyltransferase enzymes play diverse metabolic and regulatory roles by catalyzing the transfer of sugar molecules to protein, lipid, and carbohydrate acceptors, and they are increasingly of interest as therapeutic targets in a number of diseases, including metabolic disorders, cancer, and infectious diseases. The glycosyltransferases are a challenging target class from an assay development perspective because of the diversity of both donor and acceptor substrates and the lack of suitable glycan detection methods. However, many glycosyltransferases use uridine 5'-diphosphate (UDP) sugars as donor substrates, and detection of the free UDP reaction product provides a generic approach for measuring the activity of those enzymes. To exploit this approach for a broadly applicable high-throughput screening (HTS) assay for discovery of glycosyltransferase inhibitors, we developed a Transcreener(®) assay for immunodetection of UDP with a time-resolved Förster resonance energy transfer (TR-FRET) signal. We optimized the assay for detection of glycosyltransferase activity with nucleotide diphosphate (NDP) sugars at concentrations from 10 µM to 1 mM, achieving Z' values of 0.6 or higher. The assay was validated by orthogonal pooled screening with 8,000 compounds using polypeptide N-acetylgalactosaminyltransferase T3 as the target, and the hits were confirmed using an orthogonal readout. The reagents and signal were both stable for more than 8 h at room temperature, insuring robust performance in automated HTS environments. The TR-FRET-based UDP detection assay provides a broadly applicable approach for screening glycosyltransferases that use a UDP-sugar donor.

Biochemical Assay Development for Histone Methyltransferases Using a Transcreener-Based Assay for S-Adenosylhomocysteine.

Epigenetic regulation has been implicated in diverse diseases including cancer, diabetes, and inflammation, and high-throughput screening for histone methyltransferase (HMT) inhibitors is an area of intense drug discovery effort. HMTs catalyze the transfer of methyl group from S-adenosylmethionine (SAM) to lysine or arginine on histone tails forming the methylated products and S-adenosylhomocysteine (SAH). HMTs are challenging to incorporate into biochemical assays for a number of reasons. They have slow turnovers and low Km values for SAM, which leads to low levels of product formation, and thus requires very sensitive detection methods and/or high levels of enzyme. They also have diverse acceptor substrate requirements, ranging from peptides to intact nucleosomes. Additionally, some HMTs function as complexes of three or more proteins. Developing assays for individual HMTs, including sourcing and acquiring high quality enzymes and acceptor substrates, therefore can be laborious and expensive. We recently developed the Transcreener(®) EPIGEN Methyltransferase assay, a sensitive SAH detection method with a fluorescence polarization readout, to enable universal HMT detection independent of acceptor substrate. To facilitate screening and profiling of HMTs, we describe the development of turnkey assay systems for thirteen HMTs including identification of optimal acceptor substrates and their concentrations, optimization of detection reagents, determination of initial velocity enzyme concentrations, and measurement of inhibitor potencies.

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

RGS21, a regulator of taste and mucociliary clearance?

OBJECTIVES/HYPOTHESIS: Motile cilia of airway epithelial cells help to expel harmful inhaled material. Activation of bitterant-responsive G protein-coupled receptors (GPCRs) is believed to potentiate cilia beat frequency and mucociliary clearance. In this study, we investigated whether regulator of G protein signaling-21 (RGS21) has the potential to modulate signaling pathways connected to airway mucociliary clearance, given that RGS proteins modulate GPCR signaling by acting as GTPase-accelerating proteins (GAPs) for the Gα subunits of heterotrimeric G proteins. STUDY DESIGN: This is a pilot investigation to determine if RGS21, a potential tastant specific RGS gene, is expressed in sinonasal mucosa, and to determine its specific Gα substrate using in vitro biochemical assays with purified proteins. METHODS: Rgs21 expression in sinonasal mucosa was determined using quantitative, real-time PCR and a transgenic mouse expressing RFP from the Rgs21 promoter. Rgs21 was cloned, over-expressed, and purified using multistep protein chromatography. Biochemical and biophysical assays were used to determine if RGS21 could bind and accelerate the hydrolysis of GTP on heterotrimeric Gα subunits. RESULTS: Rgs21 was expressed in sinonasal mucosa and lingual epithelium. Purified recombinant protein directly bound and accelerated GTP hydrolysis on Gα subunits. CONCLUSIONS: Rgs21 is expressed in sinonasal mucosa, is amenable to purification as a recombinant protein, and can bind to Gα(i/o/q) subunits. Furthermore, RGS21 can accelerate the hydrolysis rate of GTP on Gαi subunits. This provides evidence that RGS21 may be a negative regulator of bitterant responses. Future studies will be needed to determine the physiological role of this protein in mucociliary clearance.

Evaluating modulators of "Regulator of G-protein Signaling" (RGS) proteins.

"Regulator of G-protein Signaling" (RGS) proteins constitute a class of intracellular signaling regulators that accelerate GTP hydrolysis by heterotrimeric Gα subunits. In recent years, RGS proteins have emerged as potential drug targets for modulation by small molecules. Described in this unit are high-throughput screening procedures for identifying modulators of RGS protein-mediated GTPase acceleration (GAP activity), for assessment of RGS domain/Gα interactions (most avid in vitro when Gα is bound by aluminum tetrafluoride), and for validation of candidate GAP-modulatory molecules with the single-turnover GTP hydrolysis assay.

Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay.

Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids, and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation; however, most HMT assay methods are either not amenable to a high-throughput screening (HTS) environment or are applicable to a limited number of enzymes. The authors developed a generic methyltransferase assay method using fluorescent immunodetection of adenosine monophosphate (AMP), which is formed from the MT reaction product S-adenosylhomocysteine in a dual-enzyme coupling step. The detection range of the assay; its suitability for HTS, including stability of reagents following dispensing and after addition to reactions; and the potential for interference from drug-like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well-characterized Transcreener AMP/GMP assay, the authors have developed a robust HTS assay for HMTs that should be broadly applicable to other types of methyltransferases as well.

Development and validation of a transcreener assay for detection of AMP- and GMP-producing enzymes.

Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3',5'-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC(50) ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z' values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP- and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin- activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal:background inherent in substrate depletion assays, and the potential for interference with coupling enzymes.

Two Galpha(i1) rate-modifying mutations act in concert to allow receptor-independent, steady-state measurements of RGS protein activity.

RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate Galpha subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from Galpha and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a Galpha(i1) mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. Galpha(i1)(R178M/A326S) GTPase activity was stimulated 6- to 12-fold by RGS proteins known to act on Galpha(i) subunits and not affected by those unable to act on Galpha(i), demonstrating that the Galpha/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of Galpha( i1)(R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on Galpha(i1)(R178M/A326S), the authors developed a Transcreener fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Galpha(i1)(R178M/A326S) with a homogeneous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling.

Transcreener: screening enzymes involved in covalent regulation.

Enzymes that catalyse group transfer reactions comprise a significant fraction of the human proteome and are a rich source of drug targets because of their role in covalent regulatory cycles. Phosphorylation, glycosylation, sulfonation, methylation and acetylation represent some of the key types of group transfer reactions that modulate the function of diverse biomolecules through covalent modification. Development of high-throughput screening methods for these enzymes has been problematic because of the diversity of acceptor substrates. Recently, the authors developed a novel assay platform called Transcreener that relies upon fluorescence detection of the invariant reaction product of a group transfer reaction, usually a nucleotide. This platform enables screening of any isoform in a family of group transfer enzymes, with any acceptor substrate, using the same assay reagents.