Demethylase Assay Services

Reaction Biology offers assays for both Jumonji C-domain containing (JmjCs) and lysine-specific demethylases (LSD) histone demethylase subfamilies.

LSDs are flavin-dependent monoamine oxidases that catalyze the demethylation of Kme2 or Kme1, producing peroxide (H2O2) and formaldehyde (H2CO) in the process. JmjCs are Fe(II)/2-oxoglutarate-dependent dioxygenases that use a reactive Fe(IV)-oxoferryl species to catalyze hydroxylation reactions.

  • Three assay formats are available to measure the activity of demethylases
  • Low scale, large scale screening and high-throughput options as well as hit to lead screening options are available
  • Please ask us for assay development and research collaboration options
  • All demethylase enzymes are produced in house and available for purchase.

Histone Demethylase Enzyme Family

Histone demethylases (HDMs) aid in the removal of methyl groups (—CH3) from specific sites on histones. The phylogenetic tree shows the relationships between genes predicted to encode HDMs.

 

phylogenetic tree of human histone demethylase targets for drug discovery and compound screening

Assay Details

Assay format: HTRF

HTRF (Homogeneous Time-Resolved Fluorescence) is an assay technology that combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR) for measuring molecular interactions in a homogeneous format.

HTRF makes use of rare earth complexes such as Europium or Terbium cryptates. These chelates are very stable under a wide range of chemical conditions, are not subject to photobleaching, and show high kinetic stability. HTRF allows for enhanced assay performance compared to conventional fluorescent probes in terms of sensitivity, assay window, and robustness.

HTRF assay principle for compound screening of demethylase proteins performed with biotinylated histones

Example of KDM4A assay procedure with HTRF. Enzymatic demethylation of the biotinylated histone H3 lysine 36 residue is detected by a Europium cryptate-labeled antibody specific to the histone methylation status (M2). The histone substrate peptide is biotinylated and binds to streptavidin carrying the acceptor conjugate XL665.

 

demethylase screening example on KDM4A for IC50 value determination

KDM4A was inhibited by reference compounds 2,4-PDCA and IOX1. 2,4 PDCA (IC50 = 138 nM) does inhibit the enzyme 15 x more than IOX1 (IC50 = 2014 nM). Shown is a concentration-response curve with 10 inhibitor concentrations performed in duplicates.

Assay format: Amplex Red

The Amplex Red reagent is a fluorogenic substrate for horseradish peroxidase for quantification of the activity of enzymes that produce peroxide, such as demethylase LSD1. The Amplex Red reagent reacts with peroxide in a 1:1 stoichiometry to produce the red-fluorescent oxidation product, resorufin, which can be quantified by a luminometer.

Assay format: AlphaLISA

AlphaLISA assays stand for amplified luminescent proximity homogeneous assay.

alphaLISA assay principle for compound screening of demethylases

Demethylase assay procedure with AlphaLISA for example KDM4D. Enzymatic demethylation of the biotinylated histone H3 lysine 9 residue is detected by antibodies specific to the histone methylation status (M2) which are coupled to AlphaLISA acceptor beads. Streptavidin covered donor beads interact with the histone substrate peptide via the biotin tag. The proximity of donor and acceptor beads enables singlet oxygens, a radical oxygen form, to transfer energy to the acceptor beads inducing fluorescence.

 

demethylase compound screening example KDM4_D with IC50 value determination

Example data of KDM4D inhibited by reference compounds 2,4-PDCA (IC50 = 331 nM). Shown is a concentration-response curve with 10 inhibitor concentrations performed in duplicates.

Assay setup

Setups: Single-dose screening in duplicates or IC50 value determination with 5 or 10 concentrations. Other screening formats are available upon request.

Controls: No inhibitor (DMSO vehicle) control and for every assay, one target-specific control compound is tested in 10-dose IC50 format. 

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies.

Report: The raw data, % enzyme activity and control compound IC50 values will be reported in Excel format for single-dose assays. For IC50 orders, raw data, IC50 values, and curve fitting will be delivered in Excel format. Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.

Screening facility: This assay is performed at our screening facility in Malvern, PA, US.

Compound requirements: In brief, for a standard project, 20ul of a 10mM DMSO stock or solid material is needed. Less material is needed for large scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.