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, studying CD73 and CD39, screening for kinase inhibitors, using Transcreener for a cGAS drug discovery program, and more!
Discovery on Target 2019
Westin Copley Place
Boston, Massachusetts USA
Visit Booth #403 – Let’s Talk Drug Discovery!
Ectonucleotidases are plasma membrane-bound enzymes with externally-oriented active sites that metabolize nucleotides to nucleosides and are crucial for maintaining immune homeostasis. CD73, also known as ecto-5’ nucleotidase, is a GPI anchored cell surface protein that plays a critical role in adenosinergic signaling. CD73 has both enzymatic and non-enzymatic functions in cells. As an enzyme, CD73 catalyzes the hydrolysis of AMP into adenosine and phosphate. Recent studies have shown a key role for adenosine in immunosuppression in the tumor microenvironment with ectonucleotidases emerging as promising immuno-oncology targets, with a number of groups working on small molecule and antibody-based inhibitors. We developed a simple biochemical assay for measuring CD73 activity based on the Transcreener ADP2 Assay. The assay uses Adenosine Kinase as a coupling enzyme to convert adenosine into AMP and ADP in the presence of ATP. The assay requires only sub picomolar amounts of CD73 with robust detection of Adenosine production (Z’ > 0.7). Initial pilot screens have demonstrated potential hits while IC50 values determined for a test compound PSB 12379, was consistent with published values. The homogenous assay uses a far-red fluorescence polarization (FP) or TR-FRET readout. Targeting the CD73 pathway, in combination with other potentially complementary immune pathways, is a potential strategy to help activate an anti-tumor immune response. The availability of HTS-compatible Transcreener assay methods from BellBrook Labs will accelerate the discovery of inhibitors for these ectonucleotidases, such as CD73, that play a role in tumor immunity and other diseases impacted by adenosine signaling.
Ectonucleotidases are plasma membrane-bound enzymes with externally-oriented active sites that metabolize nucleotides to nucleosides and are crucial for maintaining immune homeostasis. The ectonucleoside triphosphate diphosphohydrolase-1, also known as CD39, ENTPD1, or NTPDase1 hydrolyzes ATP and ADP to AMP. AMP can further be processed to adenosine leading to a significant impact on many disease states. Recent studies have shown a key role for adenosine in immunosuppression in the tumor microenvironment, and ectonucleotidases are emerging as promising immuno-oncology targets. 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 availability of HTS-compatible enzyme assay methods will accelerate the discovery of inhibitors for CD39 and related ectonucleotidases that play a role in tumor immunity and other diseases impacted by adenosine signaling.
When interrogating kinases for therapeutic purposes, developing an enzymatic activity assay can be a vital component of any successful lead development program. Off-the-shelf assay kits can provide a versatile solution, and some can be used for HTS as well as hit-to-lead applications. Detection of enzymatic products like ADP enables a universal method for measuring the activity of virtually any kinase. This flexibility helps eliminate the need for laborious internal assay development and provides the user with a robust solution. The Transcreener® ADP2 Kinase Assay uses homogenous detection of ADP with a choice of FP, FI, or TR-FRET readouts. Although other methods of ADP detection are available, Transcreener is the only with direct detection of ADP. Alternative assays use complex coupling mechanisms, making them prone to assay interference that generate false positives and thus requires time-consuming counter screens to triage. The assay provides sensitive detection of kinase initial velocity over a broad range of ATP concentrations, critical for screening such a diverse class of targets. Compatibility with ultra-miniaturized formats, along with outstanding overnight and reagent signal stability provide flexibility in liquid handling while producing data that yield dependable results. And while Transcreener’s versatility and simplicity make it an excellent fit for HTS, the assay also has distinct advantages in SAR and MOA studies. Sensitivity affords the use of the assay at low enzyme concentrations and helps determine accurate IC50 measurements for potent inhibitors. Transcreener can also be used in kinetic mode, which simplifies early stage assay development and enables high throughput assessment of drug-target residence time. Here we demonstrate the Transcreener ADP2 Kinase Assay as a powerful HTS approach for discovering, evaluating, selecting, and improving small molecule kinase inhibitors.
Detection of foreign nucleic acids is an important first line of defense in the immune response to microbial pathogens. However, aberrant induction of type I interferons (IFN) by self-nucleic acids causes debilitating autoimmune diseases such as Aicardi–Goutieres Syndrome (AGS), systemic lupus erythematosus (SLE) and Sjogren’s syndrome. A number of recent studies have clearly established that a key molecular trigger for nucleic acid-driven type I IFN induction is the production of the unique cyclic dinucleotide, cyclic GAMP (cGAMP), by the cytosolic DNA sensor, cyclic GAMP synthase (cGAS). The cGAS apoenzyme is enzymatically inactive; binding of non-specific dsDNA induces a transition to an active conformation that catalyzes the formation of cGAMP from ATP and GTP. cGAMP binds to the STING (stimulator of interferon genes) receptor to initiate the signaling for induction of type I IFNs. Thus the cGAS enzyme senses the primary signal for a type I IFN response and amplifies it in the form of a second messenger. Knockout studies in animal models have clearly indicated that inhibiting cGAS is a promising approach for therapeutic intervention in monogenic type I interferonopathies such as AGS and, by extension, complex diseases such as SLE.
We developed a cGAS enzymatic assay with fluorescence polarization (FP) and time-resolved Forster resonance energy transfer (TR-FRET) readouts based on our Transcreener® HTS platform. We used the FP assay to screen 100,000 compounds with full-length human cGAS, resulting in the identification of four novel chemotypes. Following confirmation of hits and removal of compounds with visually evident reactivity or metabolic liabilities, we thoroughly triaged non-stoichiometric inhibitors, aggregators, DNA intercalators and redox-active compounds using a battery of established assays. Initial studies revealed two promising chemotypes (BBL40783 and BBL50101) with favorable structural, physicochemical and ADME/PK properties that function via distinct mechanisms. BBL40783 exhibited good concordance between biochemical IC50 and Kd determined by surface plasmon resonance (1.26 µM, 2.4 µM, respectively). BBL50101 and related analogs did not bind appreciably in SPR but were found to stabilize cGAS in thermal shift assays (TSA) in the presence of ATP, GTP, and dsDNA, suggesting that this chemotype may bind specifically to dimerized cGAS. We used SAR-driven medicinal chemistry with both chemotypes to increase the potency into the nanomolar range. We obtained a high-resolution crystal structure of a BBL40783 analog in complex with cGAS and demonstrated cGAS-specific cellular activity with the same compound. These efforts establish a strong foundation for development of first-in-class lead molecules targeting cGAS for autoimmune disease.