Optimization of a Fragment-Based Screening Hit toward Potent DOT1L Inhibitors Interacting in an Induced Binding Pocket

Article abstract of DOI:10.1021/acsmedchemlett.6b00168

Mixed lineage leukemia (MLL) gene rearrangement induces leukemic transformation by ectopic recruitment of disruptor of telomeric silencing 1-like protein (DOT1L), a lysine histone methyltransferase, leading to local hypermethylation of H3K79 and misexpression of genes (including HoxA), which drive the leukemic phenotype. A weak fragment-based screening hit identified by SPR was cocrystallized with DOT1L and optimized using structure-based ligand optimization to yield compound 8 (IC₅₀ = 14 nM). This series of inhibitors is structurally not related to cofactor SAM and is not interacting within the SAM binding pocket but induces a pocket adjacent to the SAM binding site.

Full text of DOI:10.1021/acsmedchemlett.6b00168

Letter
pubs.acs.org/acsmedchemlett
Optimization of a Fragment-Based Screening Hit toward Potent
DOT1L Inhibitors Interacting in an Induced Binding Pocket
Clemens Scheufler, Henrik Mo
Kim S. Beyer, Ralph Tiedt, and Fred
̈
bitz, Christoph Gaul, Christian Ragot, Cel
́ ́ ́
ine Be, Cesar Fernandez,
́
eric Stauffer*
́
Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland
*
S Supporting Information
ABSTRACT: Mixed lineage leukemia (MLL) gene rearrange-
ment induces leukemic transformation by ectopic recruitment
of disruptor of telomeric silencing 1-like protein (DOT1L), a
lysine histone methyltransferase, leading to local hyper-
methylation of H3K79 and misexpression of genes (including
HoxA), which drive the leukemic phenotype. A weak
fragment-based screening hit identified by SPR was cocrystal-
lized with DOT1L and optimized using structure-based ligand
optimization to yield compound 8 (IC = 14 nM). This series
5
0
of inhibitors is structurally not related to cofactor SAM and is not interacting within the SAM binding pocket but induces a
pocket adjacent to the SAM binding site.
KEYWORDS: Dot1L, lysine histone methyltransferase, inhibitor, fragment-based screen, structure-based design
he importance of the epigenetic control of gene
expression and its deregulation in cancer has been
researchers at Epizyme led to the discovery of the clinical
candidate EPZ-5676 by modifying in several iterations the
T
10,11
under growing focus over the recent years. Histone
methyltransferases (HMTs) are a class of enzymes that
introduce methyl marks on lysines and arginines of histone
proteins. The lysine methyltransferases (KMTs) are responsible
for mono-, di-, and trimethylation of the ε-amino group of
histone lysines using the cofactor S-adenosyl methionine
methionine moiety attached to adenosine.
Other research-
ers, at Baylor College and at the Structural Genomics
Consortium, have disclosed similar DOT1L inhibitors with
12,13
some modifications of the adenosyl moiety.
All of these
inhibitors were shown to interact with DOT1L at the SAM
binding pocket and display a SAM-competitive behavior. In this
report, we are disclosing the discovery of new DOT1L
inhibitors, which do not interact within the SAM binding
pocket but induce a pocket adjacent to the SAM binding site.
The superimposition of the cocrystal structures of SAM and
fragment hit 1 bound to DOT1L shows no spatial overlap of
the two ligands (Figure 1). However, in a biochemical assay, 1
and its analogues are SAM-competitive because, upon binding,
they are engaging the lid loop of the SAM binding pocket and
form a conformation incompatible with efficient SAM binding.
Fragment-based screening using surface plasmon resonance
(
SAM) as methyl source and are modulating directly and
indirectly, via “readers”, the chromatin structure, the gene
expression, and the transcription regulation. An etiological role
of KMTs in different types of cancers has been proposed, and
pharmacological tool compounds targeting KMTs have been
identified to address the relationship between alteration of
1
−3
histone methylation and malignant transformation. In MLL-
rearranged leukemia, DOT1L is aberrantly recruited by MLL
fusion proteins and facilitates the transcription of genes critical
for leukemia such as homobox (HOX) genes and myeloid
1
4
4
(
(
SPR) was applied to an immobilized DOT1L construct
ecotropic viral integration site 1 homologue (MEIS1). DOT1L
amino acids 1−416) encompassing the N-terminal catalytic
domain. We identified compound 1 as a weak binder with an
was described as the only known KMT responsible for
5
methylations of lysine K79 on histone H3 until a recent
estimated equilibrium binding constant of K ≈ 50 μM. The
report suggested that the SET domain containing interleukin-5
response element II binding protein (RE-IIBP) also acts as a
D
biochemical activity of this hit was measured by a scintillation
proximity assay (SPA), using tritium labeled cofactor SAM at
concentration equal to KM and excess of biotinylated
nucleosome as substrate (Supporting Information), and
resulted in a moderate inhibition (IC50 = 320 μM) (Table
1). Further, the compound was confirmed to bind DOT1L by
6
HMT for H3K79. No antagonizing demethylase for this site
was identified so far. Structurally, DOT1L distinguishes itself
from the other KMTs by the absence of the common SET
domain and is more closely related to the arginine
7
methyltransferase (RMT) class.
Reported DOT1L inhibitors to date are mostly related to
cofactor SAM and its byproduct S-adenosyl homocysteine
Received: April 21, 2016
Accepted: June 1, 2016
8
(
SAH) with the exception of a recent report of a micromolar
9
inhibitor discovered by virtual screening. Pioneering work by
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00168
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ACS Medicinal Chemistry Letters
Letter
Table 1. Biochemical Activity of Compounds 1−10 with
General Formula
Figure 1. Superimposition of SAM bound DOT1L cocrystal structure
(
(
(
PDB 1nw3) and compound 1 bound DOT1L cocrystal structure
PDB 5dtm). The ligand and lid loop of the SAM binding pocket
residues 126−140, Pro130 and Phe131 shown as sticks) are colored
in blue for Dot1L·SAM and magenta for Dot1L·1. The flexible loop
2
for structural visualization and figure preparation.
96−309 at the crystal interface is omitted for clarity. PyMol was used
20
1
3 ε
NMR protein observation experiments on C -methionine
1
5
labeled samples and subsequently was cocrystallized with
DOT1L. Protein crystal structure analysis revealed the details
of the binding mode (Figure 2A). The 2,6-dichlorophenyl
moiety of compound 1 acts as a hydrophobic anchor occupying
a hydrophobic cavity formed by side chain movements of
Met147, Leu143, Phe239, and Tyr312. The 3-(2-N-
methylaminocarbonyl)pyrrolyl moiety is sandwiched between
Phe243, Pro130, and Phe131 engaging the flexible lid loop of
the SAM pocket in a novel conformation by π−π stacking. Such
reorganization of the lid loop was also induced by the
compounds described by Chen et al. in the Companion
1
6
Paper. Compound 1 also makes several weak, often water-
mediated hydrogen bonds: the carbonyl group with Asn241,
the N−H of the pyrrole with the backbone carbonyl of Ser311,
and the N-methyl amide with Asp241 and Ser311 via two water
molecules (Figure 2A).
a
b
SPA assay; geometric means of duplicates. Ligand efficiency (LE)
calculated from IC50 as surrogate for Kd.
Compound 1 was considered a suboptimal fragment hit.
First, 1 is a large fragment with 19 heavy atoms and a molecular
17
weight of 300 Da. Its interactions with the protein are
dominated by hydrophobic and stacking interactions. The
hydrogen bond interactions with the protein are suboptimal in
distance and orientation or are water-mediated. As a
consequence, the ligand efficiency of 1 (LE = 0.25) is at the
low end of what one would expect for a good fragment hit
Asn241. An acetylamino substituent (compound 2, IC50 = 4.3
μM), where the carbonyl can approach Asn241 at H-bonding
distance, was found to be optimal. Another way to extend the
stacking capability of the initial hit (compound 1) was realized
by linking the 2,6-dichlorophenylcarbonyl moiety to a bicyclic
1
8
(Table 1). In addition, compound 1 is inducing its pocket,
heteroaryl system such as 6-quinolinyl (compound 3, IC50
139 μM).
=
potentially limiting the impact of structure-based ligand
optimization due to the expected plasticity of the induced
pharmacophore. However, with a good understanding of the
limitations of our chemical starting point and extensive X-ray
crystallographic support available, we initiated hit optimization
by fragment growing.
The N-methylamide of compound 1 was modified to a 3-
pyridyl in order to extend the stacking moiety and to maintain
the hydrogen bond acceptor capability of the amide via the
pyridyl nitrogen. In addition, a substituent was introduced at
position 5 of the pyridyl to replace the water-mediated
hydrogen bond of the amide NH by a direct interaction with
Analysis of the cocrystal structure of compound 3 with
DOT1L (5dtq) indicates that the 2,6-dichlorophenyl is filling
optimally the hydrophobic backpocket limiting the opportunity
for gaining affinity in this part of the molecule. Notably, the
carbonyl oxygen of compound 3 is not engaging Asn241 in a
hydrogen bond interaction (Figure 2B). In order to open a new
growth vector at this position, an isosteric replacement of the
ketone by a methylamino group was shown to be suitable
(compound 4, IC50 = 98 μM). Formally, compounds 3 and 4
could be still considered fragments with a molecular weight of
300 Da, equal to our initial hit (compound 1). The ligand
B
DOI: 10.1021/acsmedchemlett.6b00168
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ACS Medicinal Chemistry Letters
Letter
acceptor for a direct hydrogen bond with Asn241 triggered an
unexpected binding mode shift as shown by cocrystallization of
DOT1L with compound 5 (Figure 2C, 5dtr). Although the 2,6-
dichlorophenyl hydrophobic anchor is still interacting in the
same pocket as observed for compounds 1 and 3, Phe131 of the
flexible SAM pocket lid loop is swinging around the quinoline
bicycle and now is forming an edge-to-face interaction with the
quinoline, which is flanked on the other face by Leu143 and
Val310. The methoxy group is not acting as a hydrogen bond
acceptor for Asn241 as originally designed but rather as a donor
of three pseudo hydrogen bonds from the polarized C−H of
the methyl group to a water molecule, Ser140 and Pro130
(
Figure 2C).
The growth vector emerging from the amino group at
position 6 of the quinoline is a quite narrow channel formed by
Phe131, Ser140, Val169, Phe239, and Asn241 (Figure S1). We
further optimized compound 5 by modifying the quinolinyl
stacking moiety to the equipotent but more electron deficient
quinazolinyl, and by introducing an amino group at position 4
of the quinazoline in order to form a hydrogen bond interaction
with the hydroxyl group of Ser140. We then extended the N-
Me to an N-propargyl substituent (compound 6, IC50 = 0.92
μM) to optimally pass through the narrow hydrophobic
channel (Figure S1). Compound 6, containing 23 heavy
atoms, is the most efficient ligand (LE = 0.36) reported herein,
and the jump in efficiency compared to compound 4 is mostly
driven by the introduction of the 4-amino hydrogen bond
donor that is optimally interacting with Ser140.
The cocrystal structure of compound 5 indicates that the
narrow hydrophobic channel opens toward a solvent accessible
pocket, with Asn241 and Ser140 providing the first polar
interaction opportunities. We could demonstrate that growing
the terminal position of 6 with a 5-methylpyridin-2-yl was
favorable, positioning a hydrogen bond acceptor for Ser140
(
compound 7, IC50 = 0.047 μM). Docking of compound 7
revealed that Ser140 is sandwiched between the hydrogen bond
donor amino group at position 4 of the quinazoline and the
pyridyl hydrogen bond acceptor (Figure 3). The cocrystal
structure of a closely related inhibitor to compound 7 with
DOT1L confirms a similar binding mode as for compound 5
and the predicted bidentate interaction with the Ser140
Figure 2. X-ray cocrystal structures of Dot1L (gray) with 1 (A), 3 (B),
and 5 (C). Amino acids engaged in key interactions with the ligand
(
blue) are illustrated as sticks. (Water mediated) Hydrogen bonds
with Dot1L are shown as dotted red lines. The binding mode flip from
(gray) to 5 (blue) is shown as insert in (C).
3
efficiencies of compounds 3 and 4 (LE = 0.26 and 0.28,
respectively) slightly exceed that of compound 1 despite the
fact that we decreased the hydrogen bond capabilities of those
new ligands while improving their stacking/hydrophobic
interactions. In contrast to the cocrystal structure of DOT1L
with EPZ-5676 (4hra) where the lid loop is partially disordered,
1
and 3 are engaging the lid loop and interact efficiently with
Phe131 (Figures 1 and S2).
Figure 3. Docking of compound 7, highlighting chelation of Ser140, in
the cocrystal structure of DOT1L·5. View of the ligand going through
the narrow hydrophobic channel to reach Ser140 is shown as insert.
Introduction of a methoxy group at position 4 of the
quinoline (compound 5, IC = 39 μM) designed to provide an
5
0
C
DOI: 10.1021/acsmedchemlett.6b00168
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ACS Medicinal Chemistry Letters
Letter
a
hydroxyl group (structure not shown). Finally, the most
optimal group that we have identified to go through the narrow
channel and interact in the pocket at the exit of the channel was
the N-(4-hydroxy-4-(1-(2-hydroxyethyl)-1H-imidazol-2-yl)but-
Scheme 2. Synthesis of Compounds 4−10
2
-yn-1-yl) substituent (compound 8, IC50 = 0.014 μM).
We hypothesized that the shift of binding mode observed
between compounds 3 and 5 was not the result of changing the
carbonyl to the N-Me group but rather the consequence of the
methoxy group serving as a hydrogen bond donor for Ser140.
We went back to investigate the initial binding mode using 3-
methyl-4-oxo-3,4-dihydroquinazolinyl as stacking moiety with a
hydrogen bond acceptor only at position 4 and extending
through the narrow channel with a 3-pyridylpropargyl
substituent in order to position adequately the nitrogen
acceptor to interact with Ser140 hydroxyl (compound 9, IC50
=
0.84 μM), similarly to what was achieved with compound 7.
Further optimization was accomplished with the introduction
of a second symmetrical nitrogen and a methyl at position 2 of
the pyrimidine (compound 10, IC50 = 0.089 μM). The
cocrystal structure of compound 10 with DOT1L is a
confirmation of the predicted binding mode comparable to 3.
The quinazolinone stacking moiety of 10 is sandwiched
between Phe243 and Phe131 and is engaged in a hydrogen
bond interaction with Asn241 (structure not shown).
Compound 2 was obtained by Friedel−Crafts acylation with
,6-dichlorobenzoyl chloride of the corresponding heteroaryl
2
intermediate. Compound 3 was obtained by manganese dioxide
oxidation of the alcohol resulting from the attack on 2,6-
dichlorobenzaldehyde with the Grignard originating from 6-
bromoquinoline (Scheme 1). Syntheses of compounds 4−10
a
Scheme 1. Synthesis of Compounds 2 and 3
a
Reagents and conditions: (a) 2,6-dichloroaniline, Cs CO , Xantphos,
2
3
Pd (dba) ·CHCl , dioxane, 100 °C, 17−19 h, yields 69−81%; (b) (i)
2
3
3
5
7
5% NaH in oil, DMF, rt, 30 min; (ii) CH I, rt, 2−15 h, yields 73−
3
5%; (c) (i) 55% NaH in oil, DMF, rt, 30 min; (ii) 4-methoxybenzyl
chloride, rt, 145 min, yield 95%; (d) (i) TFA/water, 90 °C, 48 h, yield
8
8%; (ii) POCl , DMF, 100 °C, 3 h, yield 99%; (e) NH , dioxane, 170
3
3
°
C, 16 h, yield 97%; (f) (i) 55−60% NaH in oil, DMF, rt, 20−30 min;
a
(ii) propargyl bromide, rt, 105−120 min, yields 64−92%; (g) 2-
bromo-5-methylpyridine, CuI, Pd(PPh ) Cl , NEt , NMP, rt, 17 h,
Reagents and conditions: (a) (i) 1-Boc-pyrrole-2-boronic acid pinacol
3
2
2
3
ester, Pd(dbpf)Cl , Cs CO , THF/water, 50 °C, 13 h; (ii) TFA, rt, 1
2
2
3
yield 63%; (h) (i) EtMgBr, THF, rt, 1 h; (ii) 1-(2-((tert-
butyldimethylsilyl)oxy)-1H-imidazole-2-carbaldehyde, rt, 20 min,
h, yield 98%; (b) 2,6-dichlorobenzoyl chloride, AlCl , CH Cl , rt, 25.5
19
3
2
2
h, yield 8%; (c) (i) methylmorpholine, n-BuLi, THF/hexane, −78 °C,
yield 65%; (j) HF·pyridine, THF, rt, 1.5 h, yield 22%; (k) 3-
1
h; (ii) MgBr ·Et O, −78 °C, 90 min; (iii) 2,6-dichlorobenzaldehyde,
2
2
t
iodopyridine, CuI, Pd(PhCN) Cl , Bu PHBF , iPr NH, dioxane, rt,
2
2
3
4
2
THF, −78 °C, 3.5 h, yield 24%; (d) MnO , CHCl , rt, 94 h, yield 36%.
2
3
1
7.75 h, yield 20%; (l) 5-bromo-2-methylpyrimidine, CuI, Pd-
(
PPh ) Cl , NEt , NMP, rt, 19.5 h, yield 22%.
3
2
2
3
involve an initial Buchwald−Hartwig coupling of 2,6-dichlo-
roaniline on the suitable bromo-bicyclic heteroaryl partner
followed by alkylation of the aniline nitrogen. Extension of the
terminal position of the propargyl group was achieved by
Sonogashira coupling with the suitable halogenoheteroaryl or
by deprotonation and nucleophilic attack on a known aldehyde.
For some of the derivatives, further functional group
manipulations were applied as shown in Scheme 2.
In conclusion, we discovered a new class of DOT1L
inhibitors by optimizing a weak fragment-based screening hit
displaying suboptimal interactions in an induced binding
pocket. By fine-tuning the stacking interactions, replacing
water mediated hydrogen bond interactions by direct hydrogen
bonds, and opening a new vector for fragment growing, we
were able to increase affinity by more than 4 orders of
magnitude. We have witnessed a binding mode shift triggered
by very subtle modifications of the ligand. Both binding modes
were exploited, based on our structural understanding, for
further specific optimization. Finally, we demonstrated for the
first time the possibility to potently inhibit DOT1L catalytic
activity without interacting in the SAM binding site.
D
DOI: 10.1021/acsmedchemlett.6b00168
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ACS Medicinal Chemistry Letters
Letter
M.; Brown, P. J.; Schapira, M. Bromo-deaza-SAH: A potent and
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00168.
■
selective DOT1L inhibitor. Bioorg. Med. Chem. 2013, 21, 1787−1794.
*
S
(13) Deng, L.; Zhang, L.; Yao, Y.; Wang, C.; Redell, M. S.; Dong, S.;
Song, Y. Synthesis, activity and metabolic stability of non-ribose
containing inhibitors of histone methyltransferase DOT1L. MedChem-
Comm 2013, 4, 822−826.
(14) Retra, K.; Irth, H.; van Muijlwijk-Koezen, J. E. Surface Plasmon
Figures S1 and S2, synthetic procedures, compound
characterizations, and assay protocols (PDF)
Resonance biosensor analysis as a useful tool in FBDD. Drug Discovery
Today: Technol. 2010, 7, e181−e187.
(15) Gelis, I.; Bonvin, A. M. J. J.; Keramisanou, D.; Koukaki, M.;
AUTHOR INFORMATION
Corresponding Author
E-mail: frederic.stauffer@novartis.com.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
*
Gouridis, G.; Karamanou, S.; Economou, A.; Kalodimos, C. G.
Structural Basis for Signal-Sequence Recognition by the Translocase
Motor SecA as Determined by NMR. Cell 2007, 131, 756−769.
(16) Chen, C.; Zhu, H.; Stauffer, F.; Caravatti, G.; Vollmer, S.;
Machauer, R.; Holzer, P.; Mo
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bitz, H.; Scheufler, C.; Klumpp, M.;
Tiedt, R.; Beyer, K. S.; Calkins, K.; Guthy, D.; Kiffe, M.; Zhang, J.;
Gaul, C. Discovery of Novel Dot1L Inhibitors through a Structure-
Based Fragmentation Approach. ACS Med. Chem. Lett. 2016, 10.1021/
acsmedchemlett.6b00167. Companion paper.
Notes
(17) Jhoti, H.; Williams, G.; Rees, D. C.; Murray, C. W. The ‘rule of
The authors declare the following competing financial
interest(s): Authors are shareholders of Novartis and/or
employees of Novartis.
three’ for fragment-based drug discovery: where are we now? Nat. Rev.
Drug Discovery 2013, 12, 5−7.
(18) Bembenek, S. D.; Tounge, B. A.; Reynolds, C. H. Ligand
efficiency and fragment-based drug discovery. Drug Discovery Today
ACKNOWLEDGMENTS
■
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The authors thank Aurelie Winterhalter, Julia Klopp, Patrick
Graff, Elke Koch, Paul James Westwood, Christophe Mura, and
Aurore Roustan for excellent technical assistance.
(19) Berdini, V.; Saxty, G.; Angibaud, P. R.; Querolle, O. A. G.;
Poncelet, V. S.; Roux, B.; Meerpoel, L. Anticancer pyridopyrazines via
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