BellBrook Labs will exhibit and present posters at the upcoming Discovery on Target conference in Boston, MA. At the event BellBrook will demonstrate applications for its suite of high throughput screening tools, including residence time determination, methyltransferase activity measurement, studying ectonucleotidase enzymes, screening for kinase inhibitors, and more!
Discovery on Target 2018
Sheraton Boston Hotel
Boston, Massachusetts USA
Visit Booth #405 to Get Your Hard Copy of Our New Guide:
Learn How to Determine Drug-Target Residence Times with Biochemical Assays in this Free Guide Drug-target residence time has become a critical component in the discovery of new therapeutics. BellBrook Labs has recently published a guide to help describe the use of a proven “jump-dilution” method along with BellBrook’s Transcreener Assay platform to help streamline efforts.
There has been an increasing focus in understanding cancer and tumor growth by studying the role of tumor immunosurveillance. Cancer cells employ several elegant ways to avoid the antitumor immune response. One well-studied mechanism is the generation of adenosine, an important signaling molecule involved in antitumor T cell response suppression. Adenosine is generated by the hydrolysis of extracellular ATP released by dying tumor cells. The conversion of ATP into adenosine is mediated by the family of ectonucleotidases. These membrane-bound enzymes hydrolyze nucleotides to nucleosides and are crucial for maintaining immune homeostasis. The subfamily includes ectonucleoside triphosphate diphosphohydrolase-1, also known as CD39, ENTPD1, or NTPDase1 that hydrolyzes ATP and ADP to AMP and CD73 or 5` Nucleotidase, that hydrolyze AMP to adenosine. These enzymes are emerging as extremely prominent immune-oncology targets for drug discovery. As the only HTS method capable of direct detection of nucleotides, the Transcreener platform is uniquely suited for measuring ectonucleotidase activity with the high sensitivity and low levels of interference required for a successful HTS campaign. The homogenous assays use a far-red fluorescence polarization (FP) or TR-FRET readout and they can be broadly applicable to ectonucleotidases. We developed a simple biochemical assay for measuring CD39 activity using the Transcreener AMP2 Assay. The assay provides robust detection of AMP production (Z’ > 0.6) with sub-nanomolar amounts of CD39. Initial pilot screens have demonstrated robust assay performance (Z’ = 0.6 – 0.7) and IC50s determined for tool compounds of CD39 were consistent with published values. The Transcreener GDP2 Assay was coupled with an Adenosine Kinase enzyme to detect the production of adenosine using CD73. Adenosine Kinase converts the adenosine to GDP which can be detected using the GDP2 antibody. The availability of HTS-compatible enzyme assay methods will accelerate the discovery of inhibitors for CD39 and CD73 that play a role in tumor immunity and other diseases impacted by adenosine signaling.
Kinases are one of the most highly validated target families, yet only a small fraction of the kinome has been exploited therapeutically. ADP detection enables a universal assay method that has been broadly adopted for HTS efforts targeting kinases. The Transcreener ADP2 Kinase assay uses homogenous detection of ADP with a choice of FP, FI, or TR-FRET readouts. Transcreener is the only assay that directly detects ADP without any secondary signaling components, making it the simple yet flexible. Other ADP detection methods rely on multiple coupled enzyme steps that are inherently prone to interference and require time-consuming counter-screens to triage false positives. Here we provide data demonstrating advantages of the Transcreener ADP2 Kinase Assay for HTS and examples of its use in important hit-to-lead applications, including dose-response curves (IC50) and inhibitor dissociation rate (koff) measurements. The Transcreener ADP2 assay provides sensitive detection of initial velocity using ATP concentrations from 1 µM to 1 mM, which encompasses the full range of kinase Km values. The assay is more sensitive than other ADP detection methods, especially at lower ATP concentrations, which reduces consumption of costly enzymes. Compatibility with 96, 384 and 1536 well formats and extensive validation on all major multimode readers provide flexibility with respect to equipment and throughput. Overnight reagent and signal stability at room temperature allows maximal flexibility for liquid handling and assay protocols – an important consideration in large volume screens – without compromising data quality. The assay also has distinct advantages in SAR and MOA studies. The high sensitivity allows accurate dose-response measurements for potent inhibitors; typically kinase concentrations less than 5 nM can be used, ensuring that inhibitor depletion does not impact IC50 values. Transcreener can also be used in kinetic mode, which simplifies assay development and provides flexibility for MOA studies; e.g., determining drug-target residence times.
Histone methyltransferases (HMTs) produce many different methylated products, and assay methods that detect S-adenosylhomocysteine (SAH) therefore offer some advantages over methods that detect specific methylation events. However, direct detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, HMTs are slow enzymes, and current non-radioactive SAH detection methods are not sufficiently sensitive to allow detection of many HMTs using physiological concentrations of SAM. To overcome this technical gap, we leveraged a naturally occurring SAH-sensing RNA aptamer, or “riboswitch”, that binds SAH with nanomolar affinity and exquisite selectivity. We engineered split-aptamer sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time-resolved Förster resonance energy transfer (TR-FRET) signals. The AptaFluor Methyltransferase Assay, allows robust detection of SAH (Z’ > 0.7) at concentrations below 10 nM, with signal stability of at least six hours in the presence of typical HMT assay components. Here we compare the AptaFluor™ Methyltransferase Assay to two other HTS-compatible HMT assay methods based on a) immunodetection with an HTRF® readout and b) a coupled enzyme assay with a luminescent readout. We used PRMT4 to compare the assays for detection of HMT activity, as this enzyme as this enzyme is representative of typical low Km HMTs (Km = 140 nM) and it has attracted interest as a therapeutic target. All three assay methods allow robust detection of PRMT4 activity using 200 nM SAM in 2-hour reactions. The greater sensitivity of the AptaFluor assay allowed practical detection using significantly less PRMT4 (EC50 = 2.8 nM) which can be advantageous for high volume screening and for dose-response measurement with high-affinity inhibitors. Interestingly, the difference in sensitivity between AptaFluor and the other two assay methods was greater when saturating SAM (2 µM) was used. By enabling direct SAH detection with the sensitivity required for physiological HMT reaction conditions, the AptaFluor Methyltransferase Assay should provide a valuable tool for epigenetic drug discovery.