Assay Development for Histone Methyltransferases

A major focus of our pediatric cancer research is the discovery of chemical probes to further our understanding of the biology of leukemia harboring fusion proteins arising from chromosomal rearrangements, and to develop novel specifically targeted therapies. The NUP98-NSD1 fusion protein occurs in a highly aggressive subtype of acute myeloid leukemia after rearrangement of the genes NUP98 and NSD1. The methyltransferase activity of NSD1 is retained in the fusion, and it gives rise to abnormally high levels of methylation at lysine 36 on histone 3, enforcing oncogene activation. Therefore, inhibition of the methyltransferase activity of NUP98-NSD1 may be considered a viable therapeutic strategy. Here, we report the development and validation of a highly sensitive and robust luminescence-based assay for NSD1 and other methyltransferases that use S-adenosylmethionine (SAM) as a methyl donor. The assay quantifies S-adenosylhomocysteine (SAH), which is produced during methyl transfer from SAM. SAH is converted enzymatically to adenosine monophosphate (AMP); in the process, adenosine triphosphate (ATP) is consumed and the amount of ATP remaining is measured using a luminescent assay kit. The assay was validated by pilot high-throughput screening (HTS), dose-response confirmation of hits, and elimination of artifacts through counterscreening against SAH detection in the absence of NSD1. The known methyltransferase inhibitor suramin was identified, and profiled for selectivity against the histone methyltransferases EZH2, SETD7, and PRMT1. HTS using the luminescent NSD1 assay described here has the potential to deliver selective NSD1 inhibitors that may serve as leads in the development of targeted therapies for NUP98-NSD1-driven leukemias.

Section: Evaluation Of Nsd1 Inhibitor Selectivitymentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Evaluation Of Nsd1 Inhibitor Selectivitymentioning

confidence: 99%

See 1 more Smart Citation

A Sensitive Luminescent Assay for the Histone Methyltransferase NSD1 and Other SAM-Dependent Enzymes

Drake¹,

Watson²,

Kisielewski³

et al. 2014

“…Conditions for other enzymes in the 15 or 20 HMT profiling panels were as previously described. 51 For certain experiments, the methyltransferase activity was determined as TCA-precipitable counts from 25 or 50 mL reactions, as single determinations, in a scintillation/filter plate assay. Assay conditions were the same as described above, but in addition to chicken core histones, HeLa mono/di-nucleosomes were also used (0.05 mg/mL as DNA; *0.8 mM histone H3).…”

Section: Methyltransferase Assaysmentioning

confidence: 99%

Development and Use of Assay Conditions Suited to Screening for and Profiling of SET-Domain-Targeted Inhibitors of the MLL/SET1 Family of Lysine Methyltransferases

Ferry

Smith

Denney

et al. 2015

Self Cite

Methylation of histone H3 lysine-4 (H3K4) is an important, regulatory, epigenetic post-translational modification associated with actively transcribed genes. In humans, the principal mediators of this modification are part of the MLL/SET1 family of methyltransferases, which comprises six members, MLLs1-4 and SET1A/SET1B. Aberrations in the structure, expression, and regulation of these enzymes are implicated in various disease states, making them important potential targets for drug discovery, particularly for oncology indications. The MLL/SET1 family members are most enzymatically active when part of a ''core complex,'' the catalytic SET-domain-containing subunits bound to a subcomplex consisting of the proteins WDR5, RbBP5, Ash2L and a homodimer of DPY-30 (WRAD 2 ). The necessity of MLL/SET1 members to bind WRAD 2 for full activity is the basis of a particular drug development strategy, which seeks to disrupt the interaction between the MLL/SET1 subunits and WDR5. This strategy is not without its theoretical and practical drawbacks, some of which relate to the ease with which complexes of Escherichia coli-expressed MLL/SET1 and WRAD 2 fall apart. As an alternative strategy, we explore ways to stabilize the complex, focusing on the use of an excess of WRAD 2 to drive the binding equilibria toward complex formation while maintaining low concentrations of the catalytic subunits. The purpose of this approach is to seek inhibitors that bind the SET domain, an approach proven successful with the related, but inherently more stable, enhancer of zeste homolog 2 (EZH2) complex.

“…10 Multiple biochemical assays have been reported in the drug discovery effort for small-molecule inhibitors against different epigenetic enzymes. [11][12][13][14][15] However, cell-based assays are more biologically relevant and are critical in elucidating the mechanism of action for these inhibitors. Recently, two groups published two cell-based assays detecting H3K27me3 levels; the first was a dissociationenhanced lanthanide fluorescence immunoassay (DELFIA Ò ) 16 and the second was a LanthaScreen Ò assay.…”

Section: Introductionmentioning

confidence: 99%

Development of Multiple Cell-Based Assays for the Detection of Histone H3 Lys27 Trimethylation (H3K27me3)

Qian

Lü

et al. 2013

Self Cite

Posttranslational modification of histone proteins in eukaryotes plays an important role in gene transcription and chromatin structure. Dysregulation of the enzymes involved in histone modification has been linked to many cancer forms, making this target class a potential new area for therapeutics. A reliable assay to monitor small-molecule inhibition of various epigenetic enzymes should play a critical role in drug discovery to fight cancer. However, it has been challenging to develop cell-based assays for high-throughput screening (HTS) and compound profiling. Recently, two homogeneous cell-based assay kits using the AlphaLISA Ò and LanthaScreen Ò technologies to detect trimethyl histone H3 Lysine 27 have become commercially available, and a heterogeneous cell assay with modified dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA Ò ) format has been reported. To compare their pros and cons, we evaluated, optimized, and validated these three assay formats in three different cell lines and compared their activities with traditional Western blot detection of histone methylation inhibition by using commercial and in-house small-molecule inhibitors. Our data indicate that, although all four formats produced acceptable results, the homogeneous AlphaLISA assay was best suited for HTS and compound profiling due to its wider window and ease of automation. The DELFIA and Western blot assays were useful as validation tools to confirm the cell activities and eliminate potential false-positive compounds.