Synthesis and Structure–Activity Relationship Investigation of Adenosine-Containing Inhibitors of Histone Methyltransferase DOT1L

Abstract

Histone3-lysine79 (H3K79) methyltransferase DOT1L has been found to be a drug target for acute leukemia with MLL (mixed lineage leukemia) gene translocations. A total of 55 adenosine-containing compounds were designed and synthesized, among which several potent DOT1L inhibitors were identified with Ki values as low as 0.5 nM. These compounds also show high selectivity (>4,500-fold) over three other histone methyltransferases. Structure activity relationships (SAR) of these compounds for their inhibitory activities against DOT1L are discussed. Potent DOT1L inhibitors exhibit selective activity against the proliferation of MLL-translocated leukemia cell lines MV4;11 and THP1 with EC50 values of 4–11 μM. Isothermal titration calorimetry studies showed two representative inhibitors bind with a high affinity to the DOT1L:nucleosome complex, and only compete with the enzyme cofactor SAM (S-adenosyl-L-methionine), but not the substrate nucleosome.

DOT1L as a therapeutic target for the treatment of DNMT3A-mutant acute myeloid leukemia

“…[21][22][23][24][25] Here, we report a possible role for DOT1L in leukemias with mutations of DNMT3A. Given the high prevalence of DNMT3A mutations across a variety of hematologic malignancies and that in most clinical studies DNMT3A mutations are associated with a particularly poor prognosis, 1,3,4,[31][32][33] identifying a novel therapeutic target is of substantial clinical impact.…”

“…Eluted DNA was used to prepare a library (Illumina ChIP-seq kit) and then sequenced on an Illumina HiSequation (100-base paired-end). Raw reads were quality trimmed (Trimgalore) and mapped (mm9) (Bowtie 2.0.6 21 or EPZ004777 23 or dimethyl sulfoxide (DMSO) control. For time-dependent assays, cells were incubated in 3 mM EPZ004777 23 or DMSO control.…”

“…To explore this hypothesis, we examined the relative methylation of H3K79 of the DNMT3A-mutant human cell line OCI AML3, which harbors the most common and wellcharacterized type of DNMT3A mutation, the dominant-negative acting R882 mutation 5,6 compared with the MLLr cell line, THP1, and KG-1 cells that have WT DNMT3A and MLL. Mass spectrometry demonstrated that OCI AML3 DNMT3A-mutant cells had decreased unmethylated H3K79 and increased H3K79me2 in the compared with DNMT3A WT cells ( To explore H3K79 methylation as a potential therapeutic target in DNMT3A-mutant AML, we tested the efficacy of pharmacologic DOT1L inhibition in vitro using 2 specific DOT1L inhibitors with comparable potency and specificity: SYC-522 21 and EPZ004777. 23 We treated the only known human cell lines with DNMT3A mutations, R882 mutant OCI AML3, and OCI AML2 cells which have a functionally uncharacterized non-R882 mutation (and a possible cryptic MLL rearrangement 28 ), with SYC-522 or EPZ004777.…”

“…[17][18][19][20] Pharmacologic inhibition of DOT1L has shown promising preclinical activity in MLL-rearranged leukemia and is now being tested in adult and pediatric clinical trials. [21][22][23][24][25] MLL rearrangements rarely cooccur with DNMT3A mutations in AML. 3,4,7 The essential mutual exclusivity 7 of these lesions and the overexpression of Dot1l in our murine model led us to hypothesize that MLL rearrangements and DNMT3A mutations are distinct epigenetic aberrations that converge on a common mechanism, resulting in dysregulated gene expression mediated by H3K79 methylation.…”

Chromatin proteins and modifications as drug targets

“…In a preclinical setting, such studies are readily conducted but can pose significant challenges in clinical studies involving cancer patients. In an attempt to address these shortcomings, further modifications of the pyrrolopyrimidine core of the EPZ004777 have been investigated 22 as an approach to designing second--generation DOT1L…”