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

Abstract

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.

Discovery of Potent 2-Aryl-6,7-dihydro-5H-pyrrolo[1,2-a]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design

“…HMT activity studies were performed at Reaction Biology Corp. using purified recombinant human MLL1 complex (MLL1 aa 3745–3969 plus WRAD), purified HeLa oligonucleosomes as substrate, and S -adenosyl- L -[methyl-3H]methionine (SAM) as the methylation cofactor. 31 MLL1 HMT inhibition potency rank order tracked with affinity rank order ( 6e > 6a > 6b >> 6c-6d ); even though weak potency was observed with IC 50 values in the micromolar range. A large functional to biochemical shift of ~2800-fold (HMT IC 50 / TR-FRET K i ) based on the TR-FRET competition assay was observed.…”

“…To this end, potent compounds from series 6 were profiled for their functional inhibition of WRAD-mediated MLL1 H3K4 histone-methyl-transferase (HMT) activity. HMT-activity studies were performed at Reaction Biology Corporation using purified recombinant human MLL1 complex (MLL1 aa 3745–3969 plus WRAD), purified HeLa oligonucleosomes as substrate, and S -adenosyl- l -[methyl-3 H ]­methionine (SAM) as the methylation cofactor . The MLL1-HMT-inhibition-potency rank order was tracked with the affinity rank order ( 6e > 6a > 6b ≫ 6c – 6d ); weak potency was observed with IC 50 values in the micromolar range.…”

“…Purified oligonucleosomes were obtained from HeLa cells (primarily oligomers of 3–6 units, 600–1200 bp DNA), and S-adenosyl-L-[methyl- 3 H]methionine (SAM) was used as the methylation cofactor. 31 S -Adenosylhomocysteine (SAH) was used as a positive control. Reaction conditions consisted of 7–10 nM of MLL1 complex, 0.05 mg/mL HeLa oligonucleosomes, and 1 μM of SAM substrate.…”

“…The reaction-buffer conditions were 50 mM Tris-HCl (pH 8.5), 5 mM MgCl 2 , 50 mM NaCl, 0.01% Brij35, 1 mM DTT, and 1% DMSO. Purified oligonucleosomes were obtained from HeLa cells (primarily oligomers of 3–6 units, 600–1200 bp of DNA), and S -adenosyl- l -[methyl- 3 H]­methionine (SAM) was used as the methylation cofactor …”

Targeting human SET1/MLL family of proteins

“…While this approach is invaluable for the initial discovery and characterization of methyltransferases, its low‐throughput methodology and limited dynamic range renders it unsuitable for compound screening and discovery of chemical probes (potent and selective inhibitors or antagonists). To address this issue, we and others have developed a series of assays that accommodate the requirements of medium‐ or high‐throughput screening . In addition to facilitating the discovery of chemical probes targeting a number of methyltransferases, the development of these assays has also provided a means to more thoroughly characterize the biochemical activity of many HMTs, including the SET1 family of methyltransferases.…”