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16 | SEPTEMBER 1, 2016 | GENengnews.com | Genetic Engineering & Biotechnology News Mary Anne Jelinek, Ph.D. Biochemical assays are a common approach used to identify inhibitors of epigenetic-mod- ifying enzymes. Challenges that arise when designing these assays are centered on choos- ing histone substrates that are affordable, perform robustly, and most closely mimic the cellular environment in which the small molecule will ultimately function. This tuto- rial discusses substrate options for epigenetic enzyme activity assays and demonstrates the importance of substrate selection in the de- velopment of successful biochemical assays. In the past decade there has been tremen- dous progress in our understanding of the mechanisms that lead to changes in cellular epigenetic landscapes and how these changes influence cell development, differentiation, and disease. Post-translational modification (PTM) of the unstructured histone tails is one of the principle epigenetic mechanisms that regulate chromatin structure and gene ex- pression through a highly regulated process that requires the participation of enzymes that deposit and remove these modifications, as well as the effector proteins that bind the PTMs (also known as the "writers", "eras- ers", and "readers", respectively). Together, these formulate the histone pat- terns that dictate alterations in chromatin structure and, consequently, the interactions with underlying DNA that ultimately affect gene transcription. Although there are over 80 different known histone modifications, the most widely studied modifications are acetylation, which is associated with active chromatin and positively correlated with gene expression, and methylation, which, depending on the amino acid residue being modified and the degree of methylation, can be associated with either active or repressed chromatin. In addition to influencing gene expres- sion, it has more recently been observed that alterations in histone modifications are correlated with disease. There is growing evidence in the literature demonstrating a link between aberrant histone modification patterns and the onset and progression of several human pathologies, including auto- immune, neurological, inflammatory, and neoplastic disorders. In cancer, mutations have been identi- fied not only in histones, but in all classes of epigenetic-modifying enzymes, including histone methyltransferases (HMTs), histone demethylases (HDMs), histone acetyltrans- ferases (HATs), and histone deacetylases (HDACs). The presence of these mutations and associated aberrant epigenetic land- scapes in cancer has gained the attention of the pharmaceutical industry, resulting in significant investment in scientific resources to develop small molecule therapeutics that target the epigenetic machinery in order to inhibit or reverse cancer progression. Efforts to develop epigenetic drug thera- pies have proven highly successful. Along with the discovery of inhibitors within all epigenetic enzyme classes, a novel class of inhibitors have also been identified that block nonenzymatic effector proteins, such as BRD4, from interacting with their modi- fied lysine targets. There are currently 5 FDA approved drugs on the market that have epi- genetic modes of action and greater than 15 more in single or combination clinical trials. The effort to develop new and improved epigenetic inhibitors continues, and one of the common approaches utilized to identify inhibitors are homogeneous biochemical as- says that use recombinant enzymes and sub- strates to screen for changes in enzymatic activity. Assays are typically configured to either detect an altered substrate, such as the gain or a loss of a histone modification, or to detect a cofactor by-product of the enzy- matic reaction. Both assay readout types present unique challenges. The former requires specific re- agents, typically antibodies, capable of dis- criminating between subtle changes in the modification status of the substrate (e.g., the gain or loss of a CH3- methyl group). There- fore, the choice of substrate may affect assay performance, as antibody directed detection of a modification might differ between pep- tide, protein, or multi-protein substrates. By contrast, assays that measure the con- version of a cofactor will not have detection variations due to substrate selection but may have more complex readouts that require coupling to additional enzymatic reactions. Monitoring B Cell Killing in Real-Time LABEL-FREE KILLING ASSAY REAL-TIME MONITORING KILLING KINETICS INCREASED SENSITIVITY - LOWER EFFECTOR TO TARGET RATIO SIMPLE WORKFLOW - WITHOUT EXTRA SAMPLE HANDLING AUTOMATIC DATA PLOTTING - PRECLUDING SUBJECTIVE DATA VETTING For more informaঞon, please visit: www.aceabio.com/b-cell-killing-assay xCELLigence ® Immunotherapy Kit B Cell Killing Assay Choosing the Right Substrate Is Critical Biochemical Assays for Epigenetic Enzymes DRUG DISCOVERY Assay Tutorial Figure 2. (A) PRC2 favors mononucleosomes over other substrate alternatives. Activity of PRC2, a complex of EZH2, EED, SUZ12, and RbAp46/48, toward a variety of substrates was measured using an HTRF assay detecting conversion of SAM to SAH. This assay format enables direct comparison of the various substrates but requires more enzyme. (B) SETD2 has an absolute requirement for nucleosome substrates. Activity of SETD2 toward a variety of substrates was measured using an HTRF assay. Figure 1. Substrate choices for epigenetic enzymes.