Based on the ongoing demand for preclinical lead molecules, BellBrook Labs developed universal high throughput screening assays for drug discovery using direct detection of nucleotides and homogenous fluorescent readouts. 

Their proprietary Transcreener^® and AptaFluor Assay Kits are robust, repeatable, easy-to-use enzyme activity assays for drug discovery. By directly detecting the common product of an enzymatic reaction, e.g. ADP for kinases, Transcreener provides a universal method that can be used across an entire target family. Other universal assay methods introduce multiple coupling steps, creating more interference that provide false positives and negatives. Direct detection and far-red fluorescent readouts (by fluorescence polarization, fluorescence intensity or TR-FRET) limit interference, producing accurate results that accelerate your drug discovery.

One of their focus is also on new targets, such as cGAMP/cGAS.  Cyclic GAMP synthase (cGAS) is a recently discovered enzyme that is rapidly emerging as a master regulator of the immune system and thus as a compelling therapeutic target for debilitating autoimmune diseases, including lupus, as well as for cancer immunotherapy. However, the methods for screening for cGAS inhibitors were cumbersome and expensive enough to keep most companies from pursuing it. BellBrookLabs developed the first simple HTS assay for cGAS, which has been used by NIH to identify novel inhibitors.

AdipoGen Life Sciences provides the complete BellBrook Labs product range in Switzerland and Italy. For an overview please see below or contact us if you request further information.

For Product Inquiries: info@adipogen.com

Transcreener^® and AptaFluor Assay Kits can be used for:

• Screen for small molecule inhibitors
• Determine enzymatic activity
• Profile inhibitor potency / determine IC50 values
• Measure residence time / off-rate / dissociation of lead molecules with target
• Inhibitor selectivity profiling
• Mechanism of action studies

Important Adavantages to Competitive Assays are:

• Universal – Use with virtually any kinase
• Direct detection of nucleotide enzyme products eliminates complex coupling steps that add to assay interference
• The sensitivity of Transcreener enables accurate data, generating Z’ values of greater than 0.7 at substrate conversion levels less than 10%
• Saves you time and development costs by using a single set of reagents for the entire kinase enzyme family
• Your choice of FP, FI, and TR-FRET readouts with certified performance on major multimode readers
• Overnight reagent and signal stability means you get reliable, robust screening data in large automated screens
• Safe, non-radioactive assay method
• Measure enzyme activity in real-time allowing for residence time determination in an HTS format.

Transcreener^® Assay Technology?

Transcreener^® is a universal assay method that can be used across entire families of nucleotide-dependent enzymes. Rather than using separate assays for a multitude of specific reaction products, such as phosphorylated proteins or lipids, a single nucleotide detection assay can be used for all of the enzymes that generate a common nucleotide product. For example, ADP detection can be used as a universal kinase assay method for any protein, lipid or carbohydrate kinase.

Transcreener^® assays rely on direct, highly specific detection of nucleotides using antibodies that are able to differentiate between nucleotides on the basis of a single phosphate group. Selectivities for the product nucleotides vs. the substrates (e.g., ADP vs. ATP for a kinase assay) range from 150-fold to over 1000-fold. This exquisite selectivity allows detection of nucleotide enzyme products in the presence of an excess of the substrate nucleotide (e.g, ADP detection in the presence of excess ATP for kinase assays), a requirement for measuring enzyme initial velocity.

To generate a signal, the Transcreener^® Assays use a homogenous, competitive immunoassay format in which the antibodies are paired with high-affinity fluorescent tracers. Displacement of the tracer by the nucleotide being detected causes a change in its fluorescence properties. The FP assay is the simplest; it requires just the tracer and the unmodified antibody; the change in polarization is caused by the increased rotational mobility of the tracer when it is displaced. For the TR-FRET and FI formats, the antibody is conjugated with a molecule that changes the magnitude of the tracer fluorescence when it is bound, and this effect is lost when the tracer is displaced.

Transcreener^® assays have fewer reagents and a less complex mechanism than any other nucleotide detection assay, which generally require coupling enzymes to convert a nucleotide to a product that can generate a signal with a reporter enzyme. Each coupling and reporter enzyme is a potential target for the compounds being screened, which increases the risk of false positives or of missing a hit, and requires additional wells for counter-screening. (Some ADP detection assays even use kinases as coupling enzymes, further complicating their use for kinase inhibitor screening.)

Direct detection also means that the protocol is the simplest and most flexible available: run your enzyme reaction, add Transcreener^® reagents with stop mix, and read plates. Or the Transcreener^® detection reagents can be added at the start of the enzyme reaction to continuously monitor enzyme activity; e.g., for measuring inhibitor residence times with “jump dilution” kinase assays. Some coupled methods require extra liquid addition and incubation steps, which complicates assay automation and precludes running them in a continuous mode.

Thousands of cellular reactions use nucleotides, including most of the enzymes that catalyze posttranslational modifications of proteins such as phosphorylation and methylation. Because of their central role in signal transduction, many PTM enzymes have become drug targets. Though protein kinases get the most attention, acetyltransferases, methyltransferases, and glycosyltransferases are also clinically validated targets, and many more PTM-related drugs are in the pipeline. Transcreener^® assays were developed to enable HTS efforts targeting PTM enzymes, as well as other types of nucleotide-dependent enzymes, including ATPases, GTPases, and phosphodiesterases.

For Detailed information please see the Transcreener^® HTS Assay Overview Page and download BellBrook Labs Guide to "Measuring Drug-Target Residence Times with Biochemical Assay"

For Application Notes

For Instrument Compatibility Table

For FAQs

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AptaFluor – Leveraging the Power of Aptamers for Drug Discovery

Epigenetic regulation has been shown to be a contributing factor in a variety of diseases. The discovery of methyltransferase inhibitors is currently an area of intense activity. Methyltransferase enzymes methylate a variety of substrates using S-adenosylmethionine (SAM) as the methyl donor leaving the common product S-adenosylhomocysteine (SAH).

The AptaFluor SAH Methyltransferase Assay uses a natural occurring aptamer, or riboswitch, that selectively binds with SAH, the invariant product of methyltransferase reactions. The exquisite affinity and selectivity of the riboswitch combined with a positive TR-FRET signal enable screening and profiling of methyltransferases with unparalleled sensitivity.

By directly detecting SAH in a homogenous format, it is possible to perform screening and enzymatic studies with virtually any methyltransferase. The assay can be used as a tool to aid researchers in hit identification and characterization in a quest to develop new methyltransferase inhibitors as treatments for disease.

Key Attributes/Advantages:

• Direct Detection of SAH with a TR-FRET Readout
• Ultra-Sensitive
• Use with Physiological SAM Concentrations Lower than 100nM
• Outstanding Reagent and Signal Stability
• Excellent Z’ under a variety of assay conditions
• Simple Mix-and-Read
• Universal Assay – Use with Virtually Any Methyltransferase

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Transcreener and Aptafluor Assays Overview

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NEW & UNIQUE Transcreener^® cGAMP cGAS Assay

The Transcreener^® cGAMP cGAS FP Assay (Prod. No. BBL-3024) is a far-red, competitive fluorescence polarization (FP) assay. Because the antibody is highly selective for cGAMP, the assay can be used to measure activity of the cyclic GMP-AMP synthase (cGAS) enzyme which converts ATP and GTP, to cGAMP. cGAS is a recently discovered enzyme that acts as a foreign DNA sensor that induces an immune response via activation of the stimulator of interferon genes (STING) receptor. By directly measuring cGAMP with a highly selective antibody, it is possible to assay the activity of cGAS while screening large compound libraries for inhibitors. This assay can be used to i) measure enzymatic activity of cGAS, ii) screen compound libraries for cGAS inhibitors, iii) quantify inhibitor potency or iv) determine inhibitor-cGAS residence time. 

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Transcreener^® and AptaFluor Methyltransferase Assays

Epigenetic regulation has been shown to be a contributing factor in a variety of diseases. The discovery of methyltransferase inhibitors is currently an area of intense activity. Methyltransferase enzymes methylate a variety of substrates using S-adenosylmethionine (SAM) as the methyl donor leaving the common product S-adenosylhomocysteine (SAH).

The AptaFluor SAH Methyltransferase TR-FRET Assay (Prod. No. BBL-3023) is a universal assay for enzymes that convert S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), with a far-red, time-resolved Försterresonance-energy-transfer (TR-FRET) readout. The assay is designed specifically for high-throughput screening (HTS), with a simple mix-and-read format and 2 liquid addition steps. It is run in an endpoint mode: methyltransferase (MT) enzyme reactions are quenched with a universal stop reagent (provided) and then the detection reagents are added. The assay offers reagent stability and compatibility with commonly used multimode plate readers. The exquisite affinity and selectivity of the riboswitch combined with a positive TR-FRET signal enable screening and profiling of methyltransferases with unparalleled sensitivity. Far-red tracer further minimizes interference from fluorescent compounds and light scattering.

The Transcreener^® EPIGEN Methyltransferase FP Assay (Prod. No. BBL-3017) is a competitive far-red fluorescence polarization (FP) assay. It combines the extensively validated Transcreener^® AMP2/GMP2 Assay, which relies on fluorescent immunodetection of AMP, with coupling enzymes that convert SAH to AMP.

Both assays can be used with any enzymes that convert S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). Examples include enzymes within the histone methyltransferase (HMT) and DNA methyltransferase (DNMT) families.

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STANDARD Transcreener^® ADP2 Assay

Transcreener ADP2 Kinase Assay Kits rely on direct detection of ADP using a proprietary antibody that binds with exquisite specificity and affinity to ADP, with negligible cross-reactivity to ATP. This exquisite selectivity allows direct detection of ADP in the presence of an excess of ATP, a requirement for kinase activity assays run under initial velocity conditions (e.g., ≤10% ATP consumption). The Transcreener ADP2 assay is available in three fluorescent readouts, which provide a safe HTS-compatible alternative to cumbersome radioassay methods and are more sensitive and less subject to interference than other detection methods. All readouts use far-red tracers to minimize compound interference and lower false-positive rates.

i) The Transcreener® ADP2 TR-FRET Assay (Prod. No. BBL-3011) is a is competitive immunoassay with a far-red time-resolved Förster resonance-energy-transfer readout.

ii) The Transcreener® ADP2 FP Assay (Prod. No. BBL-3010) is a competitive far-red fluorescence polarization (FP) assay.

iii) The Transcreener® ADP2 FI Assay (Prod. No. BBL-3013) is a competitive red fluorescence intensity (FI) assay.

Because they are highly selective for ADP, these assays can be used with any enzyme that converts ATP to ADP, regardless of what other substrates are used. Examples of enzymes include protein, lipid, and carbohydrate kinases, ATPases, DNA helicases, carboxylases, and glutamine synthetase.

 

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Transcreener^® AMP2/GMP2 Assay

Transcreener^® AMP2/GMP2 Assay Kits detect any AMP/GMP producing enzyme (e.g., ligases, synthetases, phosphodiesterases) using any precursor substrate, including cAMP, cGMP, ATP, or NAD. The assay relies on a highly specific monoclonal antibody that recognizes AMP or GMP with more than 1,000-fold selectivity over substrate nucleotides, including ATP, cAMP, or cGMP. It is the only activity assay method for direct detection of unlabeled AMP or GMP; i.e. without using coupling enzymes. The Transcreener^® AMP2/GMP2 Assay is available in two fluorescent readouts, which provide a safe HTS-compatible alternative to cumbersome radioassay methods and are more sensitive and less subject to interference than other detection methods. All readouts use far-red tracers to minimize compound interference and lower false-positive rates.

i) The Transcreener^® AMP2/GMP2 FP Assay (Prod. No. BBL-3015) is a competitive far-red fluorescence polarization (FP) assay.

ii) The Transcreener^® AMP2/GMP2 TR-FRET Assay (Prod. No. BBL-3020) is competitive immunoassay with a far-red time-resolved Förster resonance-energy-transfer readout.

The assays can be used with any enzyme that produces AMP or GMP, regardless of the substrate. Examples of enzymes include ubiquitin, small ubiquitin-related modifiers (SUMO), nucleic acid and protein ligases, phosphodiesterases (PDEs) and synthetases.

 

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Transcreener^® GDP GTPase Assay

The Transcreener^® GDP GTPase assay is a homogenous, fluorescent immunoassay that monitors the activity of GTPases along with GEF and GAP partners. The assay directly measures the amount of GDP produced in an in vitro GTPase reaction. A highly selective, high-affinity antibody enables nanomolar GDP detection in the presence of excess GTP.

The Transcreener^® GDP assay is available in three fluorescent readouts, which provide a safe HTS-compatible alternative to cumbersome radioassay methods and are more sensitive and less subject to interference than other detection methods. All readouts use far-red tracers to minimize compound interference and lower false-positive rates.

i) The Transcreener^® GDP TR-FRET Assay (Prod. No. BBL-3021) is competitive immunoassay with a far-red time-resolved Förster resonance-energy-transfer readout.

ii) The Transcreener^® GDP FP Assay (Prod. No. BBL-3009) is a competitive far-red fluorescence polarization (FP) assay.

iii) The Transcreener^® GDP FI Assay (Prod. No. BBL-3014) is a competitive red fluorescent intensity (FI) assay.

This mix-and-read fluorescent GTPase assay can be used to follow the progress of any enzyme that produces GDP, including monomeric small G proteins, such as CDC42 and Gα subunits of heterotrimeric G proteins, such as Gαi1. It can be used for screening GTPases, fucosyltransferase or mannosyltransferase and covers a broad range of GTP concentrations.

 

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Transcreener^® UDP2 Assay

The Transcreener^® UDP2 Assay Kits rely on direct detection of UDP and is compatible with any enzyme class that produces UDP. The Transcreener^® UDP2 Assay is available in two fluorescent readouts, which provide a safe HTS-compatible alternative to cumbersome radioassay methods and are more sensitive and less subject to interference than other detection methods. All readouts use far-red tracers to minimize compound interference and lower false-positive rates.

i) The Transcreener^® UDP2 FP Assay (Prod. No. BBL-3018) is a far-red, competitive fluorescence polarization (FP) assay.

ii) The Transcreener^® UDP2 TR-FRET Assay (Prod. No. BBL-3022) is competitive immunoassay with a far-red time-resolved Förster resonance-energy-transfer readout.

Because it is highly selective for UDP, the assay can be used with any enzyme that produces UDP, regardless of what other substrates are used. Examples include glycosyltransferases, galactosyltransferases, glucuronyltransferases, N-acetylglucosamyltransferases, N-acetylgalactosyltransferases, xylosyltransferases, and glycogen, cellulose, lactose and hyaluronan synthases.

 

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NEW  Transcreener^® CD73 Assay

The Transcreener® CD73 FP Assay is a far-red, competitive fluorescence polarization (FP) assay. The assay is designed to be used with enzymes such as ecto-5’-nucleotidase (also known as 5’-nucleotidase, NT5E, Cluster of Diferentiation 73, or CD73), that produce the product adenosine (ADO). The Transcreener CD73 FP Assay is simple biochemical assay for measuring CD73 activity based on the Transcreener ADP2 Assay. The assay uses a coupling enzyme to convert adenosine into AMP and ADP in the presence of ATP. The assay provides a powerful tool to screen entire compound libraries for CD73 modulators to help find new therapeutics for diseases such as cancer.

The Transcreener assay is designed specifically for high throughput screening (HTS), with a single addition, mix-and-read format. It offers reagent stability and compatibility with commonly used multimode plate readers.

The Transcreener CD73 FP Assay provides the following benefits:

• A simple single addition CD73 activity assay capable of HTS.
• Excellent data quality (Z’ ≥ 0.7) and signal (≥85 mP polarization shift) at ADO ranges between 0.3 μM and 3 μM.
• Far-red tracer further minimizes interference from fluorescent compounds and light scattering.

 

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NEW  Corning Plates for Transcreener Assays - Choose the Right Plate for your Assay  (Out of Stock due to COVID-19)

Transcreener® HTS Assays rely on direct immunodetection of nucleotides with a far-red readout in a simple mix-and-read format. The assays are available in three different detection modes, fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Forster resonance energy transfer (TR-FRET). A good microplate reader and the right choice of plates to differentiate specific signal versus background signal or “noise” is crucial to the success of these assays.

To achieve the best results be sure to:

- Use polystyrene, non-binding plates
- Make sure the plate has the correct working volume
- Use black plates for FP & FI
- Use white plates for TR-FRET

 

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