Synthesis and structure-activity relationship investigation of adenosine-containing inhibitors of histone methyltransferase DOT1L

Article abstract of DOI:10.1021/jm300917h

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 K_i values as low as 0.5 nM. These compounds also show high selectivity (>4500-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 that 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.

Full text of DOI:10.1021/jm300917h

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationship Investigation of
Adenosine-Containing Inhibitors of Histone Methyltransferase
DOT1L
Justin L. Anglin,† Lisheng Deng,† Yuan Yao,† Guobin Cai,† Zhen Liu,† Hong Jiang,† Gang Cheng,†
Pinhong Chen,† Shuo Dong,§ and Yongcheng Song*,†
§
†Department of Pharmacology and Department of Medicine, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030,
United States
S
* Supporting Information
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 (>4500-fold) over three
other histone methyltransferases. Structure−activity relation-
ships (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 that 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.
INTRODUCTION
■
Post-translational modifications of a lysine residue of histone
play important roles in gene regulation.1 These chemical
modifications include acetylation and methylation, which
change the electrostatic and/or steric properties of histone
and thereby control the accessibility of genes wrapped around
the histone core. Histone methyltransferase (HMT) consists of
a family of >50 enzymes that methylate the side chain amino
(or guanidine) group of a lysine (or arginine) residue of histone
or other proteins.2,3 As schematically illustrated in Figure 1, all
HMTs use S-adenosyl-L-methionine (SAM) as the methyl
donor (enzyme cofactor), producing methylated substrates as
well as S-adenosyl-L-homocysteine (SAH). Aberrant histone
methylations have been linked to many types of cancer,4 and
there is therefore great interest in developing inhibitors of these
enzymes.5 However, only a limited number of compounds have
been reported to be HMT inhibitors.5−8
DOT1L is a histone lysine methyltransferase that specifically
methylates the residue Lys79 of histone 3 (H3K79).9,10 The
catalytic domain of human DOT1L is highly conserved from
yeasts to mammals. There are two features that make DOT1L a
unique HMT. First, H3K79 is located in the ordered histone
octamer core structure, while the methylation sites of all other
HMTs are in the unordered tail of a histone protein. Second,
DOT1L is the only histone lysine methyltransferase that
belongs to class I methyltransferases. All other histone lysine
methyltransferases are class V methyltransferases with a
conserved SET domain. Of particular interest is that DOT1L
Figure 1. DOT1L-catalyzed reaction and inhibitors.
has been proposed to be a target for acute leukemia with MLL
(mixed lineage leukemia) gene translocations.11−13 As
Received: June 25, 2012
Published: August 27, 2012
© 2012 American Chemical Society
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compared to other leukemias, this subtype of leukemia has a
poor prognosis with a 5 year survival of <40%.14 Studies
showed that several predominant fusion partners of onco-MLL
genes, including AF4, AF9, AF10, and ENL, can recruit
DOT1L, which methylates H3K79, and causes overexpression
of leukemia relevant genes (such as Hoxa9 and Meis1) that
eventually leads to leukemia initiation. In addition, H3K79
hypermethylation was also found to be a hallmark of MLL-
rearranged leukemia. A potent inhibitor EPZ004777 (1, Figure
1) was recently reported, which can selectively inhibit the
proliferation of MLL-rearranged leukemia cell lines and prolong
the life spans of experimental animals in a mouse model of
human MLL leukemia.7a This further pharmacologically
validated DOT1L as an antileukemia target. However, because
of its very short half-life in plasma, 1 has a low plasma
concentration of ∼0.5 μM even when supplied continuous with
an implanted pump. Clearly, more stable inhibitors are needed.
Our early work also disclosed a structure-based approach to
the identification of several potent DOT1L inhibitors, such as
2−4 (Figure 1), showing that introduction of an N6-substituent
on the adenine ring can provide high selectivity for DOT1L.7b
Likely due to containing a reactive N-2-iodoethyl group (a
precursor of aziridinium), these DOT1L selective inhibitors do
not have specific activity in killing MLL-rearranged leukemia
cells. In a parallel effort to find competitive DOT1L inhibitors,
we also identified urea-containing compounds that possess
strong inhibitory activity against DOT1L. Here, we report the
synthesis, structure−activity relationships (SAR), and biological
activities of 55 compounds targeting DOT1L. In addition,
isothermal titration calorimetry (ITC) was used to investigate
the binding affinities of selected inhibitors to DOT1L.
Figure 2. Active site of the DOT1L:2 structure (PDB code: 3SR4),
with compound 2 shown as a ball and stick model and hydrogen
bonds as dotted lines.
Table 1. Structures of Inhibitors and Their Ki Values against
DOT1L
compd
R−
Ki (μM)
SAH
2
−H
0.16
0.29
2.0
2.0
3.8
2.8
7.8
6.5
8.9
1.1
−CH3
−allyl
−c-Pr
−i-Pr
−n-Bu
5
6
7
RESULTS AND DISCUSSION
8
■
Inhibitor Design and SARs for DOT1L Inhibition. The
poor pharmacokinetics of 1 is likely due to its 7-deaza-
adenosine moiety, which can be recognized by many human
enzymes, such as adenosine deaminase, hydrolase, or
nucleosidase, leading to a rapid degradation. Other compounds
containing adenosine or an adenosine-like group suffer from a
similar metabolic instability.15,16 In our early work,7b we found
that DOT1L, but not other HMTs, can well tolerate an
additional substituent as big as a benzyl group at the N6
position of the adenine ring, and the crystal structure of the
DOT1L:2 complex shows that the N6-methyl group is located
in a mainly hydrophobic pocket, surrounded by Phe223,
Leu224, Val249, Lys187, and Pro133 shown in Figure 2.
Therefore, we first explore the SAR at the N6 position of
adenine, in an effort to find potent DOT1L inhibitors having a
substituted adenosine moiety that could possess improved
metabolic stability.
9
−C(CH3)3
10
11
12
−CH2CH2CH(CH3)2
−CH2C(CH3)3
−CH2Ph
active (Ki = 6.5−8.9 μM). In addition, compound 12 having an
N6-benzyl group has the second best inhibitory activity in this
series with a Ki of 1.1 μM. These results show that the small
methyl group in 2 provides the best activity as well as
selectivity.
Next, we wanted to replace the highly polar, charged amino
acid moiety (at physiological pH) of compound 2 and SAH,
since it renders these compounds limited bioavailability.
Although the amino acid group forms five H-bonds with
DOT1L, the pocket also contains several hydrophobic residues
including Phe239, Val169, Thr139, and Tyr 136 (Figure 2),
suggesting that a more lipophilic side chain might also be
favorable. To this end, 15 compounds with a general structure
in Chart 1 were synthesized and tested for their inhibition
against DOT1L, and the results are also shown in Chart 1. In
general, these compounds are inactive or only weakly active.
Compound 13 (Ki = ∼100 μM) is decarboxy-SAH, but it is
>600× less active than SAH. Compound 14 with a shorter
linker has a Ki value of 25 μM, being slightly more active than
13. Changing −S− to −NH− resulted in an inactive compound
15. Compounds 16−18 having a carboxyl, hydroxyl, or
propargyl group possess no activity against DOT1L. Com-
pounds 19−25 containing a phenyl or hetercyclic aromatic ring
are also inactive. However, compounds 26 and 27 with a
carbamate group, which are actually the precursors for making
First, we synthesized a series of SAH derivatives 2 and 5−12
containing nine N6-substituents. The structures and inhibitory
activities of these compounds are shown in Table 1, together
with those of SAH, the common product of HMT-catalyzed
reactions and a nonselective HMT inhibitor. Compound 2 with
an N6-methyl is still a potent DOT1L inhibitor with a Ki value
of 290 nM. Although it is slightly less active than SAH (Ki =
160 nM), its methyl group provides excellent selectivity for
DOT1L, with only weak or no activity against other HMTs.7b
Compounds 5−8 with an allyl, cyclopropyl, isopropyl, and n-
butyl group, respectively, have considerably reduced activity
with Ki values of 2.0−3.8 μM, as compared to 2. Compounds
9−11 bearing a branched butyl or pentyl chain are even less
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Chart 1. Structures and Activities of Compounds 13−27
compounds 13 and 15, were found to be weak inhibitors of
DOT1L with Ki values of 50 and 46 μM. These two
compounds are of interest, since they possess greatly improved
lipophilicity as well as unexpected, higher activity than 13 and
15.
Table 2. Structures of Compounds 26−58 and Their Ki
Values against DOT1L
To find compounds with improved potency, three related
series of compounds based on the structures of 26 and 27 were
synthesized, including carbamates, amides, and ureas, and the
results are summarized in Table 2. Compound 28, a thioether
analogue of 27, possesses essentially the same activity (Ki = 47
μM), while changing the benzyl group to an ethyl for
compound 29 makes it inactive, showing that an aromatic
ring is favored for the R2 group (in the general structure shown
in Table 2). Introducing an additional isopropyl substituent
into the 5′-N atom for compound 30 (Ki = 22 μM) resulted in
a ∼2-fold activity increase, as compared to its parent compound
27. We next sought whether other functional groups can
provide improved inhibitory potency. Analogous amide
compounds 31−33 were found to be inactive. A reversed
amide compound 34 (with the same skeleton length) as well as
a reversed carbamate compound 35, however, exhibit ∼2×
improved inhibition against DOT1L with Ki values of 22 and
16 μM, as compared to 26−28. Urea compound 36 was found
to have a greatly enhanced activity with a Ki value of 1.9 μM.
Compounds 37−40 containing a different R2 group were
therefore synthesized to optimize this position. Although 37−
39 having 2-furanyl-methyl, propyl, and cyclohexyl group are
less active than 36, compound 40 with a Ki of 0.55 μM exhibits
∼3× more activity, showing that a phenyl group here is more
favorable than an alkyl or aryl-substituted alkyl group. In
addition, these results demonstrate that both −NH− moieties
of the urea group are very important, with each one offering
∼25 to >50-fold activity improvements, as compared to less
favored −O− and −CH2− groups (e.g., compound 36 vs 28, as
well as compound 40 vs 34/35). These two −NH− could serve
as H-bond donors to increase the binding affinity to DOT1L.
Next, compounds 41 and 42 having 2- and 4-methylene linkers,
respectively, were prepared and found to possess less activity
(Ki = 18 and 1.1 μM) than compound 40 with a 3-methylene
linker. This shows that a three −CH2− linker between the urea
and the 5′-position represents the optimal length for the
inhibition.
Compounds 43−54 that are derivatives of 40 were
synthesized in an effort to find an appropriate substituent on
the phenyl ring. As can be seen in Table 2, all of the 4-
substituents in 43−49 (Ki = 58−350 nM), including isopropyl,
tert-butyl, −Cl, −Br, −I, −CF3, and −OCF3, provide improved
activity, as compared to the parent compound 40 (Ki = 550
nM). Among these, bulky tert-butyl (in 44) and electron-
withdrawing −CF3 group (in 48) render >8-fold potency
enhancement. For halogen substituents (in compounds 45−
47), a trend of −Cl > −Br > −I was observed. Compound 50
with a 2-ethyl substituent possesses a slightly decreased activity
(Ki = 690 nM), while 51 having a 3-ethyl exhibits a 4× stronger
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inhibition (Ki = 130 nM). 2,4-Difluoro-substituted compound
52 is less active than 40, while another disubstituted compound
53 with 3,5-di-CF3 represents one of the best inhibitors with a
Ki value of 80 nM. Compound 54 with a 1-naphthyl R2 group
(i.e., 2,3-disubstituted phenyl) is considerably less active (Ki =
2700 nM) than compound 40.
We next synthesized compound 55 by replacing the −S− of
44 with a −N(i-Pr)− linkage, which in compound 30 shows
improved activity. Compound 55 was found to be an extremely
potent DOT1L inhibitor with a Ki value of 0.46 nM. The large
activity boost (as compared to that of 44 with a Ki of 70 nM)
might be due to coordinated protein conformational changes
induced by cobinding of the N-(4-tert-butylphenyl)urea side
chain and the −N(i-Pr)− group of 55. Introducing a N6-methyl
group led to compound 56, which possesses a similar inhibitory
activity against DOT1L (Ki = 0.76 nM), while compounds 57
and 58 having N6-allyl and benzyl groups were found to be less
active with Ki values of 12 and 22 nM, respectively.
Synthesis and activity testing of compounds 2 and 5−58
targeting DOT1L provide several novel observations for SAR.
First, a series of urea-containing adenosine derivatives were
found to be potent DOT1L inhibitors with Ki values as low as
0.46 nM, with each of the two −NH− moieties of the urea
offering 25- to >50-fold activity enhancements. Second, 3- or 4-
substituted phenyl is a preferred R2 group. Third, introducing a
third substituent at the 5′-position, such as an isopropyl, can
further improve the inhibitory activity. Fourth, a 3-methylene
linker between the urea and the 5′-position is the optimal
length for DOT1L inhibition. Fifth, an N6-substituent on the
adenine ring in general decreases the activity, while a small
substituent at this position, such as methyl (as found in
compound 56 with a Ki of 0.76 nM), could help provide
desired selectivity as well as stability, without much activity loss.
Biological Activity of Selected DOT1L Inhibitors. From
the above SAR studies, we have obtained several highly potent
DOT1L inhibitors having Ki values as low as 0.46 nM. We next
investigated whether these compounds have enzyme activity
against other HMTs. Selectivity is of importance for developing
clinically useful DOT1L inhibitors that target the SAM binding
site, since all HMTs use a similar mechanism of catalysis. Our
previous structure-guided design showed a N6 substituent, such
as the methyl in compound 2, provides excellent selectivity for
DOT1L (Table 3).7b Potent DOT1L inhibitors 55−58 were
inhibitor SAH, compounds 55−58 exhibit essentially no activity
against all of these three HMTs, showing excellent selectivity
(>4500-fold).
Next, we tested the activities of compounds 55−58 against
the proliferation of three human leukemia cell lines, MV4;11,
THP1, and NB4. MV4;11 and THP1 are MLL gene
translocated leukemia cells, harboring MLL-AF4 and MLL-
AF9 oncogenes, respectively, while NB4 possesses a wild-type
MLL. Figure 3 shows time-dependent activities of two
representative compounds 55 and 56 on MV4;11 cells at
four different concentrations (i.e., 1, 3, 10, and 30 μM). These
two potent DOT1L inhibitors work very slowly in blocking the
proliferation of the MLL-translocated cells. For example, at 10
μM, they had negligible activity against the cell growth even on
day 10. However, 55 and 56 (at 10 μM) were able to exhibit
61.7 and 43.4% cell growth inhibition on day 15 as well as more
pronounced activities (91.7 and 77.6% inhibition) on day 20.
Other doses of these two compounds showed a similar
tendency of activity. In addition, as shown in Table 4,
compounds 55 and 56 exhibit good cell growth inhibition
against MLL-translocated cells MV4;11 and THP1 with EC50
values of 4.4−11.0 μM but are almost devoid of activity (EC50
> 77 μM) in a 20 day treatment against NB4 leukemia cells
bearing a wild-type MLL gene. This indicates these two
DOT1L inhibitors act in a mechanism drastically different from
that of traditional chemotherapeutic agents, such as cisplatin,
which is able to kill all of these leukemia cells within a short
period of time (standard 2 day incubation) with EC50 values of
0.59−3.0 μM (Table 4). Previous biological studies7a
demonstrate that the slow action of DOT1L inhibitors is likely
due to a relatively long time required for cellular events that
lead to cell growth inhibition, including blocked H3K79
methylation, followed by decreased levels of mRNA expression
for methyl-H3K79 targeted genes, as well as ultimately the
depletion of the gene products (proteins) key to the cell
proliferation. As can be seen in Table 4, two other potent
DOT1L inhibitors 57 and 58 also exhibit good activity against
MV4;11 and THP1 with EC50 values of 5.9 − 11.2 μM,
although they (especially for 58) also have activity on NB4,
showing a decreased selectivity.
Molecular Modeling and ITC Studies. Our early X-ray
crystallographic study revealed that the binding site of the N6-
subtituent is actually a mainly hydrophobic pocket that directly
exposes to the solvent (Figure 2).7b This could explain that
although this group provides high selectivity for DOT1L, it
does not increase the binding affinity (Table 1). However, the
big, hydrophobic, urea-containing side chains render both
greatly enhanced activity and selectivity. The next question of
interest is where these urea side chains bind in DOT1L. X-ray
crystallography is the method of choice to determine this.
However, we have not obtained quality single crystals of
DOT1L in complex with any one of these urea-containing
inhibitors. We explored molecular modeling (docking) to
predict the binding site of these groups, using the crystal
structure of the DOT1L:SAM complex10 as the docking
template. As representatively shown in Figure S1 in the
Supporting Information for compound 36, all of these urea-
containing groups are invariably predicted by the docking
Table 3. Ki Values (μM) of DOT1L Inhibitors on Other
HMTs
compd
DOT1L
PRMT1
CARM1
SUV39H1
SAH
2
0.16
0.40
22.7
0.86
>100
>100
>100
>100
>100
4.9
>100
>100
>100
>100
>100
0.29
55
56
57
58
0.00046
0.00076
0.012
0.022
>100
>100
>100
>100
chosen to be tested against three representative HMTs, that is,
PRMT1, CARM1 (or called PRMT4), and SUV39H1. PRMT1
and CARM1 are histone/protein arginine methyltransferases,
which also belong to class I methyltransferases and share
certain similar structural features as DOT1L. SUV39H1 is a
typical SET domain histone lysine methyltransferase, belonging
to the class V methyltransferases having a distinct structure. As
can be seen in Table 3, drastically different from nonselective
program Glide17 in Schrodinger18 (version: 2010) to extend
̈
into the nucleosome (substrate) binding pocket of DOT1L
through the “lysine binding channel”, which holds the tightly
clustered −CH2CH2CH2− linkers of 10 docking structures of
36 (Figure S1a in the Supporting Information). The bulky and
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Figure 3. Time-dependent activity against MLL leukemia cell line MV4;11.
Table 4. EC50 Values (μM) for Leukemia Cells
Table 5. Kd Values (μM) for Ligand Binding to DOT1L
compd
MV4;11
THP1
NB4
SAH
55
56
nucleosome
0.014
a
55
4.4
5.6
8.1
11.0
10.0
11.1
2.5
101
77.0
31.3
9.5
DOT1L
0.36
0.15
0.17
0.15
a
a
a
a
56
57
58
DOT1L: nucleosome
0.066
0.073
0.13
0.086
NT
a
a
a
11.2
5.9
1 μM SAH NT
NT
NT
a
a
a
2.5 μM
NT
NT
NT
b
SAH
cisplatin
0.59
b
3.0
a
a
a
a
DOT1L: with
nucleosome
5 μM SAH NT
0.20
0.24
NT
NT
a
With a 20 day treatment. With a 2 day treatment.
a
a
10 μM
SAH
NT
NT
NT
a
a
a
20 μM
SAH
NT
0.55
NT
NT
lipophilic aromatic group is predicted to be located in a mainly
hydrophobic pocket on the nucleosome binding surface and the
urea moiety to form a H-bond with the protein (Figure S1b in
the Supporting Information). These results seem to be
reasonable, since the amino acid binding pocket in the
DOT1L:SAM structures has no room to hold such long and
bulky side chains.
a
NT, not tested.
The docking studies, if real, indicate that these urea-
containing compounds are bisubstrate type inhibitors, which
occupy the binding sites of SAM and nucleosome. These
inhibitors should therefore compete with both SAM and the
substrate nucleosome. Structurally similar inhibitor 1 was
reported to be competitive with SAM.7a A kinetic study from
the same group also showed the Km value for nucleosome is 8.6
nM19 but did not report whether 1 competes with nucleosome
on binding to DOT1L. It is known that the binding of
nucleosome to DOT1L involves very large surface areas
including those from histone proteins (H3, H4, H2A, and
H2B) and the DNA around the histone core.10 However, the
Kd (dissociation constant) value of nucleosome has not been
measured.
We used ITC to determine the binding affinities of inhibitors
55, 56, SAH, and the substrate nucleosome. First, each of these
compounds was titrated individually into DOT1L in a 50 mM
HEPES buffer (pH 7.0) with 150 mM NaCl. As summarized in
Table 5 and representatively shown in Figure 4, the Kd values
for SAH, 55, 56, and nucleosome were determined to be 360,
170, 150, and 14 nM, respectively. The high-affinity binding of
nucleosome to DOT1L (Kd = 14 nM) is consistent with their
large surface interactions as well as the reported Km value. The
binding affinity of SAH is also comparable to its Ki value (160
nM, Table 1) as determined by the enzyme inhibition assay.
However, the measured Kd values of 55 and 56 are >200×
larger than their Ki values of 0.46 and 0.76 nM. Next, we
investigated the binding affinities of these ligands to the
DOT1L:nucleosome (1:1 molar ratio, 5 μM) complex, which
better mimics the enzyme inhibition assay given the tight
binding of nucleosome to DOT1L. Compounds SAH, 55, and
Figure 4. Representative ITC results and fitting curves for inhibitor
binding to DOT1L.
56 were found to have considerably higher affinities to the
DOT1L:nucleosome complex with Kd values of 150, 66, and 86
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a
Scheme 1
a
Reagents and conditions: (i) Ac2O, CH3CN, Et3N, reflux. (ii) SOCl2, DMF, CHCl3, reflux. (iii) NH3, MeOH. (iv) R1NH2, EtOH, 80 °C, 2 h. (v) 2
equiv of SOCl2, acetone, pyridine, overnight, then aqueous NH3. (vi) RH, reflux. (vii) Acetone, SOCl2, CH(OMe)3. (viii) Thioacetic acid, PPh3,
diisopropyl azodicarboxylate, THF. (ix) NaOMe, then RBr, MeOH. (x) HCl, dioxane/H2O. (xi) Phthalimide, PPh3, diisopropyl azodicarboxylate,
THF. (xii) NH2NH2, EtOH, 80 °C. (xiii) RBr or RI, diisopropylethylamine, DMF. (ixv) Acetone, AcOH, NaCNBH3, MeOH. (xv) Methyl acrylate,
MeOH. (xvi) LiAlH4, THF. (xvii) RNCO, CH2Cl2.
nM, respectively (Table 5). The enhanced affinity for SAH can
be conceivable since SAH and nucleosome have different
binding sites in DOT1L. However, for compounds 55 and 56,
these experimental results are inconsistent with our docking
studies, which suggest that these two molecules are competitive
with nucleosome and (if real) should exhibit greatly reduced
binding affinities to DOT1L in the presence of 5 μM
nucleosome (≫Kd of 14 nM). The ITC experiments therefore
showed that these two inhibitors do not compete with
nucleosome. One possibility is due to the flexibility of
DOT1L, which could render appropriate conformational
changes at the active site of DOT1L upon the binding of
inhibitor 55/56. Such an induced fit could allow cobinding of
55 (or 56) and nucleosome. In addition, we used ITC to
determine whether 55 is competitive with SAH. Compound 55
was titrated into the DOT1L:nucleosome complex in the
presence of increasing concentrations of SAH. As shown in
Table 5 and Figure S2 in the Supporting Information, the Kd
values of 55 measured in the presence of 1, 2.5, 5, 10, and 20
μM SAH increase in a linear fashion (r2 = 0.97), showing a
competitive mode of action.
The large difference between the Kd and Ki values of 55 and
56 measured by ITC and enzyme inhibition assay, respectively,
could also be due to the protein conformational changes
induced by these ligands. The rapidly formed initial EI
(enzyme:inhibitor) complex could undergo a time-dependent
conversion to a more tightly bound complex EI*.20 For the
enzyme inhibition assays, there was a preincubation of an
inhibitor with DOT1L:nucleosome, which allows the formation
of EI*. However, there was no preincubation for the ITC
experiments.
Chemistry. The general methods for synthesizing com-
pounds 2 and 5−58 are shown in Scheme 1. N6-substituted
adenosine 59 was prepared from inosine by triacetate
protection, conversion to 6-chloro by treatment with SOCl2,
deprotection, and heating with a primary amine. Compounds 2
and 5−12 were synthesized from 59 according to our previous
method.7b Compounds 13−25 were made by heating a thiol or
amine with 5′-adenosyl chloride 60 readily prepared from
adenosine. Other 5′-S-containing compounds 26, 28, 29, and
31−54 were prepared from 2′,3′-acetonide-protected adeno-
sine by a Mitsunobu reaction with thioacetic acid, followed by
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paper (Whatman) that binds histone H3 protein, washed three times
with 50 mM NaHCO3, dried, and transferred into a scintillation vial
containing 2 mL of scintillation cocktail. The radioactivity on the filter
paper was measured with a Beckman LS-6500 scintillation counter.
IC50 values were obtained by using a standard sigmoidal dose response
curve fitting in Prism (version 5.0, GraphPad Software, Inc., La Jolla,
CA). The reported IC50 values were the mean values from at least
three experiments. Ki values for less potent inhibitors (IC50 > 0.8 μM)
were calculated using the Cheng−Prusoff equation Ki = IC50/(1 +
[SAM]/Km). For more potent inhibitors (IC50 ≤ 0.8 μM), Ki values
were calculated using the Morrison tight binding model fitting in
Prism 5.0. Enzyme inhibition assays for PRMT1, CARM1, and
SUV39H1 were performed as described previously.7b
Proliferation Inhibition Assays. MV4;11, THP1, and NB4 cell
lines were obtained from ATCC (American Type Culture Collection,
Manassas, VA). We used an XTT cell viability assay kit available from
Roche to assess the viability of suspension leukemia cells. In brief, 5 ×
105 cells/well in 96-well plates were treated with an increasing
concentration (0.1−100 μM) of a compound in RPMI1640 medium
supplemented with 10% FBS (fetal bovine serum), penicillin (100 U/
mL), and streptomycin (100 μg/mL). After a given period of time, cell
viability was evaluated with the XTT assay and monitored at 490 nm
with a Beckman DTX-880 microplate reader. All of the experiments
were done in triplicate. The EC50 values were calculated by using a
standard sigmoidal dose response curve fitting in Prism 5.0.
in situ hydrolysis of the thioester product with NaOMe and a
one-pot alkylation with a bromide in MeOH. A Mitsunobu
reaction of 2′,3′-acetonide-protected adenosine with phthali-
mide, followed by NH2NH2 treatment, gives rise to adenosine
derivative 61 having a 5′-NH2, which was alkylated with Z-
protected 3-iodopropylamine to give compound 27. To make
N-isopropyl-containing compounds 55−58, compound 61 was
subjected successively to a reductive amination with acetone
and NaCNBH3 to add an N-isopropyl group, a Michael
addition with methyl acrylate, and a reduction with LiAlH4 to
give compound 62 with a terminal −OH. The −OH was then
converted, by a Mitsunobu reaction followed by NH2NH2
treatment, to an −NH2, which was further reacted with an
isocyanate, affording, after depretection of 2′,3′-acetonide,
compounds 55−58.
CONCLUSION
■
This work provides the synthesis, SAR, and ITC studies of a
series of inhibitors of human HMT DOT1L, a novel target for
acute leukemia with MLL gene translocations. First, a total of
55 adenosine-containing compounds were designed and
synthesized, among which several highly potent DOT1L
inhibitors were identified with Ki values as low as 0.5 nM.
Second, SAR analysis of these compounds shows that (1)
replacing the amino acid moiety of SAH with an N-phenyl-
substituted urea functional group leads to a series of potent and
selective DOT1L inhibitors; (2) replacing the −S− as found in
SAH to an −N(isopropyl)− group offers additional activity
enhancement; (3) the optimal linker between the urea and the
5′-groups is −CH2CH2CH2−; and (4) a small substituent (e.g.,
methyl) at the N6-position of adenine ring renders high
selectivity without much activity loss. Third, several represen-
tative DOT1L inhibitors demonstrate selective activity against
the proliferation of MLL-rearranged leukemia cells with the
EC50 values of 4−11 μM. However, it takes a relatively long
time (>10 days) for these compounds to exert growth arrest,
showing a different mechanism of action from traditional
chemotherapeutic drugs. Finally, ITC experiments showed
urea-containing inhibitors 55 and 56 are able to bind with a
high affinity (Kd: 66 and 86 nM) to the DOT1L:nucleosome
complex, and only compete with SAM/SAH, but not the
substrate nucleosome, on binding to DOT1L.
Molecular Modeling. Docking studies were performed using the
program Glide17 as previously reported.21 The crystal structures of the
DOT1L:SAM complex10 were prepared using the module “protein
preparation wizard” in Maestro22 in Schrodinger18 with default protein
̈
parameters: water molecules were removed, hydrogens were added,
and inhibitors were extracted. H-bonds were then optimized, and the
protein was energy-minimized using OPLS_2005 force field. A
receptor grid, which is large enough to contain the whole active site,
was generated using Glide without constraints. Compounds were built,
minimized using OPLS_2005 force field in Maestro, and then docked
into the prepared protein structure using Glide (docking parameters:
standard-precision and dock flexibly).
ITC. Using a previously reported method,23 ITC experiments were
performed with a Nano ITC LV (190 μL) instrument from TA
Instruments (New Castle, DE) at 279 K, at which DOT1L is more
stable during the experiments (lasting for ∼2 h). A 190 μL solution
containing DOT1L (5 μM) (or a 1:1 DOT1L:nucleosome complex (5
μM) containing an increasing concentration of SAH from 0 to 20 μM)
in 50 mM HEPES buffer (pH 7.0) with 150 mM NaCl was added into
the sample cell. A ligand (40−60 μM) solution in the same buffer (50
μL) was transferred into the syringe of the instrument. Upon reaching
temperature equilibrium, 25 injections (2 μL each) of the ligand were
used for the titration. ITC data were imported into NanoAnalyze
software (TA Instruments, New Castle, DE). The titration baseline
was corrected, and Kd values for the binding of the ligand were
obtained by fitting into the independent model in the software. All
experiments were repeated for at least one time to ensure the reported
data are reliable.
EXPERIMENTAL SECTION
■
All reagents were purchased from Alfa Aesar (Ward Hill, MA) or
Aldrich (Milwaukee, WI). Compounds were characterized by 1H
NMR on a Varian (Palo Alto, CA) 400 MR spectrometer. The purities
of all compounds were determined by a Shimadzu Prominence HPLC
with a Zorbax C18 or C8 column (4.6 mm × 250 mm) monitored by
1
UV absorbance at 254 nm or H (at 400 MHz) absolute spin-count
ASSOCIATED CONTENT
■
quantitative NMR analysis using imidazole as an internal standard. The
purities of all compounds were found to be >95%. The synthesis and
characterization of compounds 5−58 can be found in Experimental
Section in the Supporting Information.
S
* Supporting Information
Figures S1 and S2 and Experimental Section showing docking
results, linear correlation of Kd values of 55 with the competing
SAH concentrations, and detailed compound synthesis and
characterization. This material is available free of charge via the
Internet at http://pubs.acs.org.
Enzyme Inhibition. Expression, purification, and inhibition of
recombinant human DOT1L (catalytic domain 1−472) were
performed according to our previous published methods.7b In brief,
compounds with concentrations ranging from 1 nM to 100 μM were
incubated with DOT1L (100 nM) and 1.5 μM oligo-nucleosome in 20
μL of 20 mM Tris buffer (containing 1 mM EDTA, 0.5 mM DTT, and
50 μg/mL BSA, pH 8.0) for 10 min. A 0.76 μM (equaling to the Km)
AUTHOR INFORMATION
■
Corresponding Author
3
concentration of H-SAM (10 Ci/mM; Perkin-Elmer) was added to
*Tel: 713-798-7415. E-mail: ysong@bcm.edu.
initiate the reaction. After 30 min at 30 °C, the reaction was stopped
by adding SAH to a final concentration of 100 μM. Fifteen microliters
of reaction mixture was then transferred to a small piece of P81 filter
Notes
The authors declare no competing financial interest.
8072
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Journal of Medicinal Chemistry
Article
Arrowsmith, C. H.; Brown, P. J.; Simeonov, A.; Vedadi, M.; Jin, J.
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ACKNOWLEDGMENTS
■
This work was supported by a grant (RP110050) from the
Cancer Prevention and Research Institute of Texas (CPRIT) to
Y.S. J.L.A. was supported by a training fellowship from the Keck
Center of the Gulf Coast Consortia, on the Pharmacological
Sciences Training Program, National Institute of General
Medical Sciences (NIGMS), T32GM089657.
ABBREVIATIONS USED
■
HMT, histone methyltransfers; H3K79, histone3-lysine79;
ITC, isothermal titration calorimetry; MLL, mixed lineage
leukemia; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-
homocysteine; SAR, structure−activity relationship
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