Discovery of a Selective Inhibitor for the YEATS Domains of ENL/AF9

Abstract

Eleven-nineteen leukemia (ENL) contains an epigenetic reader domain (YEATS domain) that recognizes lysine acylation on histone 3 and facilitates transcription initiation and elongation through its interactions with the super elongation complex (SEC) and the histone methyl transferase DOT1L. Although it has been known for its role as a fusion protein in mixed lineage leukemia (MLL), overexpression of native ENL, and thus dysregulation of downstream genes in acute myeloid leukemia (AML), has recently been implicated as a driver of disease that is reliant on the epigenetic reader activity of the YEATS domain. We developed a peptide displacement assay (histone 3 tail with acylated lysine) and screened a small-molecule library totaling more than 24,000 compounds for their propensity to disrupt the YEATS domain–histone peptide binding. Among these, we identified a first-in-class dual inhibitor of ENL ( Kd = 745 ± 45 nM) and its paralog AF9 ( Kd = 523 ± 53 nM) and performed “SAR by catalog” with the aim of starting the development of a chemical probe for ENL.

AFF4 regulates osteogenic differentiation of human dental follicle cells

“…In addition, the YEATS region of ENL and AF9, a novel acetyl-binding module that is directly associated with histone acetylation and H3K79 methylation, is an indispensable structure involved in enhancing transcriptional stimulation. 35,36 Formation of an SEC with AFF1 or AFF4 is a primary control factor in cancer pathogenesis and development. SEC-like 2 (SEC-L2) or SEC-like 3 (SEC-L3), containing factor AFF2 or AFF3, respectively, was also discovered through biochemical isolation.…”

Structural insights into interaction mechanisms of alternative piperazine-urea YEATS domain binders in MLLT1

“…An early attempt exploiting a peptide-based strategy with 2-furancarbonyl lysine led to a competitive nanomolar binder 24 , demonstrating potential inhibitions of the YEATS protein module. Our recent screening efforts alternatively revealed a number of small molecule binders for MLLT1/3 21,25 . One chemotype identified was benzimidazole-amide, and extensive modification focused on this scaffold led to the development of the potent and selective MLLT1/3 inhibitor (SGC-iMLLT) with IC50 of ~0.26 μM and KD of 113 nM 26 .…”

“…We therefore aimed to search for other scaffolds distinct from benzimidazole-amide, which is the only characterized binder to date. Analyses of the results from our previous screening campaign revealed a number of piperazine-1-carboxamide hits 25 , suggesting that this piperazine-urea scaffold could serve an alternative acetyl-lysine mimetic moiety for MLLT1/3. Interestingly, this chemical class has been extensively used previously in development of inhibitors with good pharmacokinetic properties for diverse targets, such as fatty acid amide hydrolase (FAAH) 27 , melanocortin subtype-4 receptor (MC4R) 28 and the receptor for the chemokine CCL2 (CCR2 or known also as MCP-1) 29 .…”

“…Based on our previous work 25,30 , we selected three piperazine-1-carboxamide hits (1, 2 and 3) and for comparison three benzimidazole-amide derivatives of similar sizes (4, 5 and 6), and first determined the crystal structures of their complexes with MLLT1 ( Figure 1). We observed surprisingly different binding modes and interactions between these two chemotypes to the protein.…”

A structure-based approach towards identification of inhibitory fragments for eleven-nineteen-leukemia protein (ENL) YEATS domain

“…Both co-crystal structures of ENL with 12 and 19 provided valuable structural insights into the binding mode highlighting for example the importance of direct attachments of the aromatic moieties to the R 1 and R 2 position of the amide core, which may provide fundamental keys not only for a decrease of flexibility of both ligands and Y78 but for enhancing optimal conditions for strong π-π contacts. In parallel, development of a high-throughput assay and screening identified similar chemotypes, in essence a benzimidazole-amide hit with low micromolar affinity 30 .…”