Design of a fluorescent ligand targeting the S-adenosylmethionine binding site of the histone methyltransferase MLL1

Abstract

The histone methyltransferase MLL1 has been linked to translocation-associated gene fusion in childhood leukemias and is an attractive drug target. High-throughput biochemical analysis of MLL1 methyltransferase activity requires the production of at least a trimeric complex of MLL1, RbBP5 and WDR5 to elicit robust activity. Production of trimeric and higher order MLL1 complexes in the quantities and reproducibility required for high-throughput screening presents a significant impediment to MLL1 drug discovery efforts. We present here a small molecule fluorescent ligand (FL-NAH, 6) that is able to bind to the S-adenosylmethionine (SAM) binding site of MLL1 in a manner independent of the associated complex members. We have used FL-NAH to develop a fluorescence polarization-based SAM displacement assay in a 384-well format targeting the MLL1 SET domain in the absence of associated complex members. FL-NAH competes with SAM and is displaced from the MLL1 SET domain by other SAM-binding site ligands with Kdisp values similar to the higher-order complexes, but is unaffected by the H3 peptide substrate. This assay enables screening for SAM-competitive MLL1 inhibitors without requiring the use of trimeric or higher order MLL1 complexes, significantly reducing screening time and cost.

Mechanisms and Inhibitors of Histone Arginine Methylation

“…[97] Our efforts were also applied to create fluorophore-labeled SAM analogs to set up fluorescent-based binding assays to study cofactor competitive inhibitors. [98] For unknown reasons, we did not detect any salient binding of the fluorescent SAM analogs with PRMTs (unpublished data). It may be that the PRMTs cannot accommodate the fluorescein group, which is supported by previous observations that certain SAM analogs of lesser bulk have also been poor cofactors in the absence of active site engineering.…”

Targeting human SET1/MLL family of proteins

“…This assay was optimized for screening in 384‐well format with a Z′‐factor of 0.6 . Very recently, we also developed a SAM displacement assay for MLL1 . In this assay, a small molecule fluorescent ligand (FL‐NAH) that is able to bind to the SAM binding site of MLL1 in a manner independent of the associated complex members was used to develop a fluorescence polarization‐based SAM displacement assay in 384‐well format.…”

“…FL‐NAH binds to MLL1 SET domain in the absence of associated complex members and competes with SAM, SAH, and the fungal metabolite sinefungin, but not with a peptide corresponding to residues 1–25 of histone H3. This assay enables screening for SAM‐competitive MLL1 inhibitors without requiring the use of trimeric or higher order MLL1 complexes, significantly reducing screening time and cost …”

Hyperhomocysteinemia in ApoE-/- Mice Leads to Overexpression of Enhancer of Zeste Homolog 2 via miR-92a Regulation

“…The modification of histones by methylation of the lysine residues has been shown to regulate gene expression by affecting chromatin structure. Histone methyltransferase (HMTs) may catalyze the transfer of a methyl group from SAM to a lysine residue on H3 [23]. For instance, methylation state of H3K9 is a strong characteristic of transcriptional repression [24], but trimethylation state of H3K4 is correlated with gene activation [25].…”