Selective inhibitors of histone methyltransferase DOT1L: Design, synthesis, and crystallographic studies

Article abstract of DOI:10.1021/ja206312b

Histone H3-lysine79 (H3K79) methyltransferase DOT1L plays critical roles in normal cell differentiation as well as initiation of acute leukemia. We used structure- and mechanism-based design to discover several potent inhibitors of DOT1L with IC₅₀ values as low as 38 nM. These inhibitors exhibit only weak or no activities against four other representative histone lysine and arginine methyltransferases, G9a, SUV39H1, PRMT1 and CARM1. The X-ray crystal structure of a DOT1L-inhibitor complex reveals that the N6-methyl group of the inhibitor, located favorably in a predominantly hydrophobic cavity of DOT1L, provides the observed high selectivity. Structural analysis shows that it will disrupt at least one H-bond and/or have steric repulsion for other histone methyltransferases. These compounds represent novel chemical probes for biological function studies of DOT1L in health and disease.

Full text of DOI:10.1021/ja206312b

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Selective Inhibitors of Histone Methyltransferase DOT1L: Design,
Synthesis, and Crystallographic Studies
Yuan Yao,†,§ Pinhong Chen,†,§ Jiasheng Diao,† Gang Cheng,† Lisheng Deng,† Justin L. Anglin,†
B. V. Venkataram Prasad,‡ and Yongcheng Song*,†
†Department of Pharmacology and ‡Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, United States
S Supporting Information
b
ABSTRACT: Histone H3-lysine79 (H3K79) methyltrans-
ferase DOT1L plays critical roles in normal cell differentia-
tion aswell as initiation of acute leukemia. We used structure-
and mechanism-based design to discover several potent
inhibitors of DOT1L with IC50 values as low as 38 nM.
These inhibitors exhibit only weak or no activities against
four other representative histone lysine and arginine methyl-
transferases, G9a, SUV39H1, PRMT1 and CARM1. The X-
ray crystal structure of a DOT1Lꢀinhibitor complex reveals
Figure 1. Mechanism of catalysis of DOT1L.
that the N6-methyl group of the inhibitor, located favorably
in a predominantly hydrophobic cavity of DOT1L, provides
HKMTs containing a SET domain (which are class-V methyl-
the observed high selectivity. Structural analysis shows that
transferases), it belongs to the class-I methyltransferase family. In
it will disrupt at least one H-bond and/or have steric repul-
addition, DOT1L is the only known enzyme that specifically
catalyzes methylation of the histone H3-lysine79 (H3K79)
sion for other histone methyltransferases. These compounds
represent novel chemical probes for biological function
residue located in the nucleosome core structure, while other
studies of DOT1L in health and disease.
methylation sites are in the unordered N-terminal tail of the
histone. Moreover, the clinical importance of DOT1L as well as
the H3K79 methylation is that DOT1L has been found to be
necessary and sufficient for the initiation and maintenance of
leukemia with mixed-lineage leukemia (MLL) gene transloca-
tions.12ꢀ14 This type of leukemia accounts for ∼75% of infant
and ∼10% of adult acute leukemia and has a particularly poor
prognosis.15 DOT1L therefore represents a novel target for inter-
vention. It is of interest that while this manuscript was being
revised for publication, a DOT1L inhibitor that possesses
selective activity against MLL leukemia was disclosed.16
DOT1L catalyzes an SN2 reaction of the H3K79 ε-NH2 of the
substrate nucleosome with the methyl group of S-(50-adenosyl)-
L-methionine (SAM), which is the cofactor of the enzyme, as
schematically illustrated in Figure 1. One of the reaction pro-
ducts, S-(50-adenosyl)-L-homocysteine (SAH), has been known
to be a nonselective inhibitor of many methyltransferases, inclu-
ding DOT1L.17 We also found that it inhibits recombinant
human DOT1L (catalytic domain 1ꢀ472)10 with a Ki value of
160 nM (Table 1). However, SAH cannot be used as a probe in
cell biology or in vivo, since it is quickly degraded to become
adenosine and homocysteine by SAH hydrolase,18 keeping a
cellular SAM/SAH molar ratio of ∼40:1.19 In addition, selec-
tivity is of importance for a DOT1L inhibitor to be a useful probe,
since other histone lysine and arginine methyltransferases also
use SAM and histone/nucleosome as their cofactor and sub-
strate, respectively.5,6
he human genome is packed into chromatins, which are
composed of millions of repetitive units known as nucleo-
T
somes. A single nucleosome includes a fragment of DNA (∼147
base pairs) wound around a disklike histone octamer consisting
of two histone H2A, H2B, H3, and H4 proteins. Post-transla-
tional epigenetic modifications on several lysine and arginine
residues of histones, such as methylation and acetylation, control
the accessibility of the DNA, thereby regulating the expression or
silencing of a gene.1 It has been widely recognized that in
addition to gene mutations, aberrant epigenetic modifications
play an important role in the initiation of many diseases, such as
cancer.2ꢀ4 Great interest has therefore been generated in study-
ing histone-modifying enzymes (e.g., histone methyltransferases)
as well as their functions in pathogenesis. Histone methyltrans-
ferases include a large family of dozens of histone lysine methyl-
transferases (HKMTs) and histone/protein arginine methyltran-
sferases (PRMTs),5,6 many of which have recently been found
to play critical roles in cell differentiation, gene regulation, DNA
recombination, and damage repair.7 Therefore, small-molecule
inhibitors of histone methyltransferases represent useful chemi-
cal probes for these biological studies as well as potential thera-
peutics.8 However, very few inhibitors of HKMTs and PRMTs
have been discovered and developed.8,9
We are particularly interested in human histone lysine methyl-
transferase DOT1L,10,11 which is highly conserved from yeasts
to mammals. DOT1L is a unique HKMT in that unlike all other
Received: July 11, 2011
Published: September 21, 2011
r
2011 American Chemical Society
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We analyzed the crystal structure of the DOT1LꢀSAM
complex11 as well as those of all other histone methyltransferases
available in the Protein Data Bank and found one structural
feature that is unique to the binding of SAM to DOT1L, which
can be exploited to design selective DOT1L inhibitors. As shown
in Figure S1A in the Supporting Information (SI), the 6-NH2
group of SAM forms only one H-bond with DOT1L with a large
hydrophobic cavity nearby. However, the 6-NH2 group of bound
SAM or SAH has two H-bonds with PRMTs (which also are class-
I methyltransferases), such as CARM1 (also known as PRMT4),
as shown in Figure S1B. All other HKMTs, such as G9a, are SET-
domain methyltransferases having a completely different struc-
ture. The binding conformation of SAM/SAH to these enzymes is
distinctfrom thatof DOT1L, with the 6-NH2 group facing toward
the protein and forming two H-bonds (Figure S1C). We thus
hypothesized that N6-substituted SAH analogues such as 1 and 2
(Chart 1)would be potent and selective DOT1L inhibitors. This
turned out to be the case. Compounds 1 and 2 were synthesized
from N6-substituted adenosine (see the Experimental Section in
the SI). Compound 1, having only one extra ꢀCH3 group in com-
parisonwith SAH, was still found to be a potentDOT1L inhibitor,
with a Ki value of 290 nM (Table 1 and Figure S2), butit possesses
only weak or no inhibitory activities against two PRMTs
(CARM1 and PRMT1) and two HKMTs (G9a and SUV39H1),
with Ki values of 22.7 to >100 μM (Table 1). In contrast, SAH
remains an inhibitor of all these enzymes, with Ki values of 0.4ꢀ
4.9 μM. Similarly, compound 2, N6-benzyl-SAH, has good
activity toward DOT1L (Ki = 1.1 μM) but is very weak against
CARM1 and PRMT1 (Ki = 18 and 21.2 μM, respectively) and
inactive toward G9a and SUV39H1 (Table 1).
Next, X-ray crystallography was used to investigate how com-
pound 1 binds to DOT1L, with a particular interest in the bind-
ing site of the N6-methyl group that provides excellent selec-
tivity. We determined the crystal structure of the DOT1Lꢀ1
complex at 2.5 Å. Details of the data processing and refinement
are shown in Table S1 in the SI, and the overall structure and
proteinꢀligand interactions in the DOT1Lꢀ1 complex are illus-
trated in Figure S3. As shown in Figure 2A, the protein and the
SAH moiety of the inhibitor are superimposed with those of
the previously reported DOT1LꢀSAM structure,11 with a root-
mean-square deviation (rmsd) of 0.2 Å. As a result, all of the 10
H-bonds as well as the other interactions between the ligand and
the protein remain essentially intact (Figure 2B), in agreement
with the potent inhibitory activity of 1. The N6-methyl group is
nicely inserted into a hydrophobic cavity, surrounded by Phe223,
Leu224, Val249, Lys187, and Pro133 (Figure 2B,C). In addition,
its orientation allows the 6-NH group to form a H-bond with
Asp222 that is important to the binding of the adenine ring.
It is therefore clear that introducing a substituent at N6 does
not significantly affect the binding of SAH to DOT1L. However,
our experiments show the N6-substituted SAH analogues 1 and 2
cannot bind to other HKMTs and PRMTs strongly (Table 1),
suggesting that any substitution at this position disrupts at least
Table 1. Ki or IC50 values (μM) of Methyltransferase
Inhibitors
DOT1L
CARM1
PRMT1
G9a
SUV39H1
SAHa
1a
2a
3b
4b
5b
6b
0.16
0.29
1.1
0.40
>100
18
0.86
22.7
21.2
22.0
2.7
0.57
4.9
>100
>100
>100
1.8
>100
>100
>100
>100
>100
>100
15.7
0.038
0.12
0.11
46.4
1.1
>100
>100
>100
>100
>100
>100
a Ki values. b IC50 values.
Chart 1. Structures of DOT1L Inhibitors
Figure 2. X-ray crystal structure of the human DOT1Lꢀ1 complex. (A) Superposition of the structures of DOT1Lꢀ1 (with C atoms in green) and
DOT1LꢀSAM (in purple), with an rmsd of 0.2 Å. For clarity, only protein backbones are shown. (B) Close-up view of the active site of the DOT1Lꢀ1
structure, with 10 H-bonds shown as dotted lines. (C) Electrostatic potential surface (with 25% transparency) of the DOT1Lꢀ1 complex, showing that
the N6-methyl group of 1 is located in a hydrophobic cavity. 1 is shown as a space-filling model.
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Scheme 1. General Synthesis of Compounds 4ꢀ6a
Compound 4 was found to be an extremely potent inhibitor of
DOT1L (IC50 = 38 nM; Table 1), almost quantitatively inacti-
vating DOT1L. Interestingly, it possesses relatively weak or no
inhibitory activity toward other methyltransferases, with IC50
values of 1.1 to >100 μM, respectively, showing a high selectivity
(>29-fold). It is remarkable that because of the complicated
enzyme kinetics of histone methyltransferases involving covalent
binding of inhibitor 4 (or 3) to the substrate, we measured IC50
values for each enzyme using a minimal enzyme concentration
(50ꢀ100 nM), Km of SAM, and saturated concentration of the
substrate. Under these assay conditions, the IC50 values may be
used to compare the relative inhibitory abilities of each com-
pound across these enzymes. Although 4 does not have an
N6-substituent, the locally more hydrophobic environment at
the binding site of the putative aziridinium intermediate of 4 in
DOT1L might account for the selectivity, since it could protect
the highly reactive aziridinium cation from nonspecific hydro-
lysis. The corresponding sites in other histone methyltransferases
are either exposed to the solvent (for SET-domain HKMTs) or
polar (for PRMTs). We synthesized compounds 5 and 6, which
are N6-substituted analogues of 4, using the general approach in
Scheme 1. These two compounds also exhibited potent activity
against DOT1L, with IC50 values of 120 and 110 nM, respec-
tively (Table 1). As expected, their N6-methyl and benzyl groups
provide excellent selectivity: 5 and 6 are essentially inactive
against other methyltransferases, showing that these compounds
could have wide applications in probing the biological functions
of DOT1L.
a Reagents and conditions: (i) acetone, SOCl2; (ii) phthalimide, PPh3,
diisopropyl azodicarboxylate; (iii) NH2NH2, 80 °C; (iv) ethyl bromoa-
cetate, NEt3; (v) LiAlH4; (vi) BOC2O; (vii) ClCOOMe, DMAP, NEt3;
(viii) BOC2O, DMAP; (ix) DIBAL, ꢀ78 °C; (x) NaCNBH3, HCl,
MeOH; (xi) PPh3, I2, imidazole, 0 °C; (xii) HCl-dioxane.
one H-bond and/or changes the binding conformation of the
adenine ring, thereby causing a considerable affinity loss. In addi-
tion, for SET-domain HKMTs, any N6-substituent leads to into-
lerable steric repulsion with the protein, preventing these com-
pounds from binding. These results show that N6-substituted
SAH analogues are selective inhibitors of DOT1L and provide a
structural basis for further inhibitor design and development.
A mechanism-based inhibitor design was exploited to find
selective DOT1L inhibitors with improved potency. Compound
3 (Chart 1) was initially synthesized. The rationale is that it can
undergo intramolecular cyclization at neutral pH to form a reac-
tive aziridinium intermediate20,21 that may be covalently bound
to the ε-NH2 group of H3K79 (Figure S4). Compound 3 was
found to exhibit only weak enzyme inhibition against DOT1L,
with an IC50 value of 15.7 μM. We reasoned that compound 4
with one more ꢀCH2ꢀ group could be a better inhibitor, since
the two CꢀN bonds (∼1.47 Å each) in 3 are considerably shorter
than the CꢀS bonds (∼1.82 Å) in SAM/SAH. The crystal struc-
tures of DOT1L show that SAM as well as 1 bind to the protein in
a fully extended conformation, suggesting that the amino acid
moiety of 3 might not be able to reach its optimal binding site in
DOT1L. Compound 4 has not been made before, and our syn-
thetic route isshown in Scheme 1. The 20,30-dihydroxylsof adeno-
sine were selectively protected with an acetonide, after which the
50-hydroxyl was converted to an ꢀNH2 group, using a Mitsunobu
reaction followed by treatment with hydrazine. The product was
alkylated with ethyl bromoacetate and reduced with LiAlH4 to
afford compound 7. The tert-butyl ester of L-glutamic acid was
first protected with one tert-butoxycarbonyl (BOC) group and
its δ-carboxyl converted to a methyl ester. It was necessary to
protect the amino group with a second BOC before reduction to
give aldehyde 8. Compounds 7 and 8 were subjected to a reductive
amination to produce compound 9, whose free hydroxyl group
was converted to an iodide with PPh3/I2, affording compound 4
after acidic deprotection.
In summary, this work is of interest for a number of reasons.
First, DOT1L, a specific histone H3K79 methyltransferase, plays
a critical role in normal cell differentiation as well as the initiation
and maintenance of acute leukemia with MLL gene transloca-
tions. DOT1L inhibitors therefore represent novel chemical
probes for functional studies of DOT1L as well as potential thera-
peutics for leukemia. Second, we used structure- and mechanism-
based design to synthesize and identify several potent DOT1L
inhibitors with IC50 values as low as 38 nM. These compounds
exhibit only weak or no inhibitory activities on four other repre-
sentative histone lysine and arginine methyltransferases. Third,
we determined the crystal structure of the DOT1Lꢀ1 complex,
which revealed the structural basis for the excellent selectivity.
The methyl group of the inhibitor is located favorably in a hydro-
phobic cavity of DOT1L, while it disrupts at least one H-bond
and/or has steric repulsions for all other histone methyltrans-
ferases. Thisfinding should haveimplications for the future design
and development of DOT1L inhibitors.
’ ASSOCIATED CONTENT
S
Supporting Information. Figures S1ꢀS4, Table S1, ex-
b
perimental section, and complete ref 16. This material is available
free of charge via the Internet at http://pubs.acs.org. Coordi-
nates and structure factors of the DOT1Lꢀ1 complex have been
deposited in the Protein Data Bank as entry 3SR4.
’ AUTHOR INFORMATION
Corresponding Author
ysong@bcm.edu
Author Contributions
§These authors contributed equally.
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’ ACKNOWLEDGMENT
This work was supported by a grant (RP110050) from the
Cancer Prevention and Research Institute of Texas (CPRIT) to
Y.S. and a grant (Q1279) from the Robert A. Welch Foundation
to B.V.V.P. The recombinant human DOT1L (1-472) expression
plasmid was kindly provided by Dr. Yi Zhang (University of
North Carolina). We thank the staff of the X-ray Crystallography
Facility at Baylor College of Medicine for assistance with data
collection. We also thank Dr. Timothy Palzkill (Baylor College of
Medicine) for useful discussion on enzyme kinetics.
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