Discovery of Potent, Selective, and Structurally Novel Dot1L Inhibitors by a Fragment Linking Approach

Article abstract of DOI:10.1021/acsmedchemlett.6b00519

Misdirected catalytic activity of histone methyltransferase Dot1L is believed to be causative for a subset of highly aggressive acute leukemias. Targeting the catalytic domain of Dot1L represents a potential therapeutic approach for these leukemias. In the context of a comprehensive Dot1L hit finding strategy, a knowledge-based virtual screen of the Dot1L SAM binding pocket led to the discovery of 2, a non-nucleoside fragment mimicking key interactions of SAM bound to Dot1L. Fragment linking of 2 and 3, an induced back pocket binder identified in earlier studies, followed by careful ligand optimization led to the identification of 7, a highly potent, selective and structurally novel Dot1L inhibitor.

Full text of DOI:10.1021/acsmedchemlett.6b00519

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Letter
The Discovery of Potent, Selective and Structurally
Novel Dot1L Inhibitors by a Fragment Linking Approach
Henrik Moebitz, Rainer Machauer, Philipp Holzer, Andrea Vaupel, Frederic Stauffer,
Christian Ragot, Giorgio Caravatti, Clemens Scheufler, César Fernández, Ulrich
Hommel, Ralph Tiedt, Kim S Beyer, Chao Chen, Hugh Zhu, and Christoph Gaul
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00519 • Publication Date (Web): 14 Feb 2017
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The Discovery of Potent, Selective and Structurally Novel
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Henrik Möbitz,‡ Rainer Machauer,‡ Philipp Holzer,‡ Andrea Vaupel,‡ Frédéric Stauffer,‡ Christian
Ragot,‡ Giorgio Caravatti,‡ Clemens Scheufler,‡ Cesar Fernandez,‡ Ulrich Hommel,‡ Ralph Tiedt,‡ Kim
S. Beyer,‡ Chao Chen,† Hugh Zhu,† and Christoph Gaul*‡
‡Novartis Institutes for Biomedical Research, Basel, Switzerland
†Novartis Institutes for Biomedical Research, Shanghai, China
Dot1L, protein lysine methyltransferase, inhibitor, mixed lineage leukemia, fragment linking
ABSTRACT: Misdirected catalytic activity of histone methyltransferase Dot1L is believed to be causative for a subset of highly
aggressive acute leukemias. Targeting the catalytic domain of Dot1L represents a potential therapeutic approach for these leukemi-
as. In the context of a comprehensive Dot1L hit finding strategy, a knowledge-based virtual screen of the Dot1L SAM binding
pocket led to the discovery of 2, a non-nucleoside fragment mimicking key interactions of SAM bound to Dot1L. Fragment linking
of 2 and 3, an induced back pocket binder identified in earlier studies, followed by careful ligand optimization led to the identifica-
tion of 7, a highly potent, selective and structurally novel Dot1L inhibitor.
Acute myeloid leukemia (AML) is a genetically heteroge-
neous disease associated with a wide range of chromosomal
aberrations with strong prognostic power. Translocations af-
fecting the chromatin modifying protein MLL (mixed lineage
leukemia) are found in 5-10% of patients with AML (many of
whom have contracted AML as a late effect after prior chemo-
therapy) and in 70% of infants with acute lymphoblastic leu-
kemia (ALL). There is strong genetic evidence that Dot1L, a
histone methyltransferase targeting H3K79, is required for
leukemogenesis driven by the most common MLL transloca-
tions (e.g. MLL-AF4, MLL-AF9, MLL-AF10, MLL-AF6).1-7
Beyond MLL-translocated leukemia, recent literature reports
describe Dot1L dependence in AML with MLL-PTD fusions,8
NUP98-NSD1 translocations,9 IDH mutations,10 and HoxA
overexpression.9,11
ing.20 In reality, though, this potential is rarely fulfilled due to
the stringent requirements on binding geometry and mutually
compatible binding conformations. There are few cases that
approach additive binding potency, e.g. thrombin (pIC50: 3.5 +
4.9 ⇒ 8.9)21 and BCL2 (pIC50: 3.5 + 2.2 ⇒ 5.9).22 Superaddi-
tive linking is mostly restricted to fragmentation of highly
potent ligands (for e.g. MMPs, avidin),20 and a case study
showed that a favorable entropic component is indeed respon-
sible for the high linking efficiency.23 Some of the most suc-
cessful examples that used linking prospectively involve the
dimerization of ligands binding to dimeric enzymes with prox-
imal binding sites, e.g. FBPase (pIC50: 2*3.2 ⇒ 7.8)24 and
XIAP.25
As described in earlier reports,18,19 our Dot1L hit finding
campaigns by fragment-based screening and high-throughput
screening led to the discovery of chemical matter binding to an
induced pocket adjacent to the binding site of co-factor S-
adenosylmethionine (SAM). Some of the identified ligands
extended into the methionine pocket of the SAM binding site.
However, to our surprise, chemical matter occupying the
adenosine pocket of the SAM binding site was not discovered
by the abovementioned techniques. We decided to develop
alternative hit finding approaches to close this potential oppor-
tunity gap.
Targeting Dot1L, either by inhibiting the catalytic domain
or the interaction with its fusion protein complex partner, is a
potential therapeutic approach for the abovementioned leuke-
mias. Epizyme’s pioneering work in discovering catalytic
Dot1L inhibitors resulted in the identification of the clinical
candidate EPZ-5676.12 We and other research groups have
discovered alternative means to inhibit Dot1L.13-19
Herein, we describe the discovery of potent, selective and
structurally novel Dot1L inhibitors via fragment linking.
Fragment linking is an attractive strategy due to the promise of
additive binding potency. In theory, the entropic benefit from
the restriction of translational freedom and the displacement of
waters by the linker might even result in superadditive link-
During our mode of action studies with the earlier identified
induced pocket binders, it was realized that all of these ligands
function through a SAM-competitive mode of action. Interest-
ingly, though, adenosine can bind to Dot1L in the presence of
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ACS Medicinal Chemistry Letters
the induced pocket binders. This observation triggered the
generation of a ternary X-ray crystal structure of adenosine
(IC50 = 100 ꢀM) and induced pocket binder 1, a potent Dot1L
inhibitor (IC50 = 190 nM),19 complexed to Dot1L (Figure 1
and 2).
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Figure 3. The virtual screen for SAM site binders was based on
the SAM adenosine site pharmacophore (pdb code 5mvs; blue:
acceptor; red: donor; orange: aromatic; grey: excluded volume),
resulting in substructure and pharmacophore searches in which R2
was required to contain a suitable hydrogen bond donor.
Figure 1. Chemical structure and biochemical potency of
adenosine and 1.
As observed for the Dot1L-compound 1 cocrystal structure,
the flexible loop of Dot1L comprised of amino acids 126-140,
which forms the “lid” of the SAM binding pocket in the SAM-
bound state, is folded over and collapsed onto ligand 1 (Figure
2). The interactions of 1 with Dot1L are identical in the binary
versus ternary complex. Adenosine does not interact with the
flexible loop in the ternary complex, but otherwise forms the
same contacts with Dot1L as in the binary complex.
All hits discovered from both approaches were submitted to
cocrystallization experiments with Dot1L in the absence and
presence of 1. Gratifyingly, the ternary X-ray structure for one
of the virtual screen hits, pyrrolopyrimidine 2, a weak, frag-
ment-like Dot1L inhibitor (IC50 = 240 ꢀM, MW = 217 Da,
clogP = 0.73, HBD = 2, HBA = 2) could be obtained (Figure 4
and 5).
Figure 4. Chemical structure and biochemical potency of 2 and 3.
The overall protein conformation and the binding mode of
ligand 1 are identical to the ternary complex discussed in Fig-
ure 2. As envisioned, 2 occupies the adenosine pocket of
Dot1L. The NH of the pyrrolopyrimidine core serves as a hy-
drogen bond donor, interacting with the side chain of Asp222,
while the adjacent nitrogen acts as a hydrogen bond acceptor
for the backbone NH of Phe223 (Figure 5). Further, the prima-
ry amine of 2 is forming a salt bridge with Glu186 and coordi-
nates a structural water, mimicking the bidentate coordination
of Glu186 by the ribose moiety of SAM in the Dot1L-SAM
complex. Importantly, the pyrrolopyrimidine core is nicely
sandwiched between the side chains of Lys187 and Phe223
(Figure 5). Overall, the interactions of 2 with Dot1L are com-
parable to the purine-protein contacts in the SAM-bound
Dot1L structure, validating the pharmacophore model used in
the virtual screen (Figure 5 insert).
Figure 2. X-ray crystal structure of Dot1L with 1 and adenosine
(pdb code 5mvs). Cartoon and surface representation of Dot1L
(grey), flexible loop of Dot1L (126-140) (lightblue), stick model
of ligands 1 (green) and adenosine (blue). Asp161 is shown as red
surface for orientation. The flexible loop has been omitted from
the surface representation for clarity.
With this information in hand, we chose to pursue two ave-
nues to discover adenosine pocket binders. First, a fragment-
based second-site screen by NMR was run with the first site
(“the induced pocket”) being blocked with 1. Second, a
knowledge-based virtual screen of the Novartis corporate ar-
chive for fragments resembling kinase hinge binders that satis-
fy an adenosine-based pharmacophore was conducted (Figure
3). This screen made use of our in house kinase inhibitor col-
lection (mainly in the form of intermediates) and roughly 200
compounds were selected for a high concentration biochemi-
cal screen.
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Figure 5. Detailed interactions of 2 with Dot1L from a ternary X-
ray crystal structure of Dot1L (grey) with 2 (blue) and 1 (not dis-
played) (pdb code 5mw3). Amino acid side chains engaging in
key interactions with the ligand illustrated as sticks, polar contacts
highlighted as dotted red lines (protein hydrogen bonds in grey).
Insert: Overlay of adenosine (grey) and 2 (blue) based on the ter-
nary structures. Only subtle conformational changes were ob-
served on the protein side between the two ternary complexes.
Figure 6. Model of ternary complex of 2 and 3 bound to Dot1L
based on the individual X-ray cocrystal structures of 2 and 3.
Surface representation of Dot1L (grey), stick model of ligands 2
(blue) and 3 (grey). Green arrow represents linking vector be-
tween 2 and 3. Insert: Overlay of 2 and 3 with a model of 6 (grey).
Both analogs, with the three-carbon and the four-carbon
linker, were synthesized. To our delight, compounds 4 and 5
turned out to be highly potent Dot1L inhibitors (IC50 = 4 nM
for both compounds) (Figure 7). Of note, and assuming that
IC50 = 2*Ki for both compounds, the binding energy (ꢁG = -
RTlnKi) of 4 and 5 is approximately the sum of the binding
energy of fragments 2 and 3 (additive linking with linking
coefficient E = 3.7 M-1).23,26
Two approaches to capitalize on the discovery of the adeno-
sine replacement 2 were considered. Compound 2 could either
be optimized by stepwise growing or by linking to fragment-
like binders of the “induced pocket”. The latter approach was
prioritized, however, ligand 1 was deemed to be not well suit-
ed for linking to 2 due to an unfavorable vector geometry /
attachment point situation. In contrast, a different “induced
pocket” binder, the previously discovered indole urea 3 (IC50
=
9.0 ꢀM, MW = 314 Da) (Figure 4),18 seemed to be an attrac-
tive option for attempting fragment linking to 2.
A ternary model of 2 and 3 complexed to Dot1L was built
based on the individual X-ray cocrystal structures of 2 and 3
(Figure 6). As indicated by the green arrow, it became appar-
ent that the two ligands are perfectly lined up for linking via
the primary amine of 2 and the amide NH2 of 3 by either a
three- or four-carbon aliphatic chain.
Figure 7. N-Linking of 2 and 3 by a C3 or C4 linker to give 4 and
5.
Linked compound 4 was amenable for further potency op-
timization. Methylation of the secondary amine of 4 led to the
dramatically more potent analog 6 (IC50 = 0.15 nM) (Figure
8). We speculate that the additional methyl group stabilizes the
almost eclipsed syn conformation of 4 (central torsion 20°) to
a regular gauche conformation (central torsion 58°),27 although
shielding of the hydrogen bond may further contribute to the
more than 20-fold reduced IC50. Of note, the N-methyl deriva-
tive of fragment 2 is roughly equipotent to 2 (IC50 = 141 ꢀM),
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IC50 (MV4-11, prolif.)*
5
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formally resulting in superadditive linking with fragment 3 to
compound 6 (E = 0.14 M-1). The high linking efficiency is
remarkable, considering that the binding mode of the initial
fragment 2 (and its N-Me analog) is likely to be slightly al-
tered (Figure 6 insert).
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+
*[nM], [h], all data are the results of at least two assay runs with
the mean value reported. The coefficient of variation was less than
60% in all cases. Biochemical IC50 values were determined at KM
for SAM. Ki values were determined by applying the Morrison
tight binding model (Supporting Information).
Furthermore, a series of bicyclic 5,6-, 6,5-, and 6-6-systems
was explored as indole replacements. It turned out that ben-
zothiophene is one of the most suitable cores, as exemplified
by compound 7, a highly active Dot1L inhibitor (IC50 < 0.1
nM) (Figure 8).
The X-ray cocrystal structure of 7 bound to Dot1L con-
firmed the expected binding mode (Figure 9). The urea glycine
amide core overlaid perfectly with 3 in the ternary complex,
the only marked difference being a flip of the 0-Cl aryl due to
an interaction of the pyridine nitrogen with Ser140. The amino
piperidine was slightly pushed down relative to 2 in the ter-
nary complex, so that the charged amine replaces the crystal
water from the ternary complex and assumes a gauche con-
formation.
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Figure 8. Optimization of indole 4 to benzothiophene 7.
Analog 7 is characterized by a Ki in the very low pM range
and a very long on-target residence time (τ >5 h, the detection
limit of our internal SPR assay) as assessed by surface plas-
mon resonance experiments (Table 1). Importantly, 7 performs
equal or better than EPZ-5676 in a head-to-head comparison
in our cellular assays. It potently suppresses H3K79 dimethyl-
ation (IC50 = 3 nM), the direct product of the Dot1L-catalyzed
Figure 9. X-ray cocrystal structure of Dot1L with 7 (pdb code
5mw4). Surface representation of Dot1L (grey), stick model of
ligand 7 (blue). Amino acid side chains engaging in key interac-
tions with the ligand illustrated as sticks, polar contacts highlight-
ed as dotted red lines. Insert: Overlay of the purine region of 7
(blue) and 3 (grey, crystal water in red) from the ternary structure.
reaction, as well as the activity of the HoxA9 promoter (IC50
=
17 nM) in HeLa and Molm-13 cells, respectively (Table 1).
Furthermore, 7 very effectively inhibits proliferation of the
human MLL-rearranged leukemia cell line MV4-11 carrying
the oncogenic MLL-AF4 fusion (IC50 = 5 nM) (Table 1).
Noteworthy, 7 displays a favorable selectivity profile against a
panel of 22 PKMTs and PRMTs with no inhibitory activity up
to 50 ꢀM (Supporting Information).
The synthetic route to linked analogs is modular and allows
for parallel SAR interrogation of the adenosine pocket binder,
the induced pocket binder and the linker. As an example, the
synthesis of 7 is shown in Scheme 1. Adenosine pocket binder
2 is readily prepared in two steps from commercially available
(R)-tert-butyl piperidin-3-ylcarbamate 8. The linker building
block 11, accessed from Cbz-protected glycine 10 by amide
bond formation with 3,3-diethoxypropan-1-amine, is fused to
2 by reductive amination. Methylation of 12, followed by Cbz-
deprotection and CDI-mediated urea formation with amino-
benzothiophene 15 completes the synthesis of 7 in a conver-
gent manner. Please note that 15 is prepared by Suzuki cou-
pling from 14, the synthesis for which has been described ear-
lier.18
Table 1. Biochemical, biophysical and cellular Characteri-
zation of lead compound 7
EPZ-
5676
7
IC50 (Dot1L SPA)*
<0.1
0.002
>5
<0.1
0.012
>5
Ki (Dot1L SPA)*
τ (Dot1L SPR)+
Scheme 1. Synthetic route to compound 7.a
IC50 (HeLa, H3K79me2 ELISA)*
IC50 (Molm-13, HoxA9 RGA)*
3
7
17
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We are grateful to our medicinal chemistry colleagues Christophe
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Mura, Rainer Tschan, Meral Ilhan, Michael Hediger, and Stephan
Kläusler for their scientific contributions. We thank Paul J. West-
wood, Kerstin Pollehn, Dirk Erdmann, Claudio Thoma and An-
dreas Theuer for developing and supporting project-related bio-
physical, biochemical and cellular assays. The authors would like
to acknowledge Elke Koch, Céline Be, Aurelie Winterhalter, Julia
Klopp, Aude Izaac and Patrick Graff for their contributions to the
protein structural work. The crystallographic experiments were
performed on the X10SA beamline at the Swiss Light Source,
Paul Scherrer Institut, Villigen, Switzerland supported by the
team of Expose GmbH.
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REFERENCES
(1) Nguyen, A. T.; Taranova, O.; He, J.; Zhang, Y. Dot1L, the
H3K79 Methyltransferase, Is Required for MLL-AF9-Mediated Leu-
kemogenesis. Blood 2011, 117, 6912–6922.
(2) Jo, S. Y.; Granowicz, E. M.; Maillard, I.; Thomas, D.; Hess, J.
L. Requirement for Dot1L in Murine Postnatal Hematopoiesis and
Leukemogenesis by MLL Translocation. Blood 2011, 117, 4759–
4768.
(3) Chang, M.-J.; Wu, H.; Achille, N. J.; Reisenauer, M. R.; Chou,
C.-W.; Zeleznik-Le, N. J.; Hemenway, C. S.; Zhang, W. Histone H3
Lysine 79 Methyltransferase Dot1 Is Required for Immortalization by
MLL Oncogenes. Cancer Res. 2010, 70, 10234–10242.
(4) Bernt, K. M.; Zhu, N.; Sinha, A. U.; Vempati, S.; Faber, J.;
Krivtsov, A. V.; Feng, Z.; Punt, N.; Daigle, A.; Bullinger, L.; Pollock,
R. M.; Richon, V. M.; Kung, A. L.; Armstrong, S. A. MLL-
Rearranged Leukemia Is Dependent on Aberrant H3K79 Methylation
by Dot1L. Cancer Cell 2011, 20, 66–78.
(5) Deshpande, A. J.; Chen, L.; Fazio, M.; Sinha, A. U.; Bernt, K.
M.; Banka, D.; Dias, S.; Chang, J.; Olhava, E. J.; Daigle, S. R.;
Richon, V. M.; Pollock, R. M.; Armstrong, S. A. Leukemic Trans-
formation by the MLL-AF6 Fusion Oncogene Requires the H3K79
Methyltransferase Dot1L. Blood 2013, 121, 2533–2541.
(6) Chen, L.; Deshpande, A. J.; Banka, D.; Bernt, K. M.; Dias, S.;
Buske, C.; Olhava, E. J.; Daigle, S. R.; Richon, V. M.; Pollock, R. M.;
Armstrong, S. A. Abrogation of MLL-AF10 and CALM-AF10-
Mediated Transformation through Genetic Inactivation or Pharmaco-
logical Inhibition of the H3K79 Methyltransferase Dot1L. Leukemia
2013, 27, 813–822.
(7) Krivtsov, A. V; Feng, Z.; Lemieux, M. E.; Faber, J.; Vempati,
S.; Sinha, A. U.; Xia, X.; Jesneck, J.; Bracken, A. P.; Silverman, L.
B.; Kutok, J. L.; Kung, A. L.; Armstrong, S. A. H3K79 Methylation
Profiles Define Murine and Human MLL-AF4 Leukemias. Cancer
Cell 2008, 14, 355–368.
(8) Kühn, M. W. M.; Hadler, M. J.; Daigle, S. R.; Koche, R. P.;
Krivtsov, A. V.; Olhava, E. J.; Caligiuri, M. A.; Huang, G.; Bradner,
J. E.; Pollock, R. M.; Armstrong, S. A. MLL Partial Tandem Duplica-
tion Leukemia Cells Are Sensitive to Small Molecule Dot1L Inhibi-
tion. Haematologica 2015, 100, e190-e193.
(9) Deshpande, A. J.; Deshpande, A.; Sinha, A. U.; Chen, L.;
Chang, J.; Cihan, A.; Fazio, M.; Chen, C.; Zhu, N.; Koche, R.; Dzhe-
kieva, L.; Ibáñez, G.; Dias, S.; Banka, D.; Krivtsov, A.; Luo, M.;
Roeder, R. G.; Bradner, J. E.; Bernt, K. M.; Armstrong, S. A. AF10
Regulates Progressive H3K79 Methylation and HOX Gene Expres-
sion in Diverse AML Subtypes. Cancer Cell 2014, 26, 896–908.
(10) Sarkaria, S. M.; Christopher, M. J.; Klco, J. M.; Ley, T. J.
Primary Acute Myeloid Leukemia Cells with IDH1 or IDH2 Muta-
tions Respond to a Dot1L Inhibitor in Vitro. Leukemia 2014, 28,
2403–2406.
aReagents and conditions: (a) 4-chloro-7H-pyrrolo[2,3-
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d]pyrimidine, Hünig’s base, BuOH, 52 h, 80 °C, quant.; (b)
HCl(aq), dioxane, 4 h, rt, then H+ exchange resin, MeOH and elu-
tion with NH3, MeOH, 95%; (c) TPTU, Hünig’s base, DMA, 7
min, 0 °C, then 3,3-diethoxypropan-1-amine, 45 min, rt, 67%; (d)
2, NaOAc, MeOH/CH2Cl2, 30 min, rt, then NaBH3CN, 16 h, rt,
58%; (e) 37% formaldehyde(aq), NaBH3CN, AcOH, MeOH, 45
min, rt, 80%; (f) 10% Pd-C, H2, MeOH/THF, 7.5 h, rt, 84%; (g) 2-
methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine,
PdCl2(Amphos), Na2CO3(aq), EtOH/toluene, 20 min, 60 °C, 36%;
(h) CDI, MeCN/DMF, 1h, rt, then 15, NEt3, 1 h, 45 °C, 33%.
In summary, we have discovered by customized screening
an attractive fragment 2 mimicking the adenosine pocket in-
teractions of SAM bound to Dot1L. Fragment linking of 2 and
3, an induced pocket binder identified in earlier studies, deliv-
ered a highly potent, selective and structurally novel Dot1L
inhibitor 7, lacking any nucleoside-like features. Future re-
search is directed at the improvement of ADME properties,
with a focus on the reduction of hydrogen bond donors and
number of rotatable bonds.
ASSOCIATED CONTENT
PyMol was used for structural visualization and figure prepara-
tion.28 Synthetic procedures, compound characterization and assay
protocols. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: christoph.gaul@novartis.com
(11) Methods for the Detection and Treatment of Leukemias that
are Responsive to Dot1L Inhibition. WO2015/017863.
Author Contributions
(12) Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.;
Basavapathruni, A.; Jin, L.; Boriack-Sjodin, P. A.; Allain, C. J.;
Klaus, C. R.; Raimondi, A.; Scott, M. P.; Waters, N. J.; Chesworth,
R.; Moyer, M. P.; Copeland, R. A.; Richon, V. M.; Pollock, R. M.
Potent Inhibition of Dot1L as Treatment of MLL-Fusion Leukemia.
Blood 2013, 122, 1017–1025.
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script.
ACKNOWLEDGMENT
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(13) Shen, C.; Jo, S. Y.; Liao, C.; Hess, J. L.; Nikolovska-Coleska,
2,6-Dichlorophenyl Hydrophobic Anchor and Interacting in an In-
Z. Targeting Recruitment of Disruptor of Telomeric Silencing 1-like
(Dot1L): Characterizing the Interactions between Dot1L and Mixed
Lineage Leukemia (MLL) Fusion Proteins. J. Biol. Chem. 2013, 288,
30585–30596.
(14) Yu, W.; Chory, E. J.; Wernimont, A. K.; Tempel, W.; Scop-
ton, A.; Federation, A.; Marineau, J. J.; Qi, J.; Barsyte-Lovejoy, D.;
Yi, J.; Marcellus, R.; Iacob, R. E.; Engen, J. R.; Griffin, C.; Aman, A.;
Wienholds, E.; Li, F.; Pineda, J.; Estiu, G.; Shatseva, T.; Hajian, T.;
Al-Awar, R.; Dick, J. E.; Vedadi, M.; Brown, P. J.; Arrowsmith, C.
H.; Bradner, J. E.; Schapira, M. Catalytic Site Remodelling of the
Dot1L Methyltransferase by Selective Inhibitors. Nat. Commun. 2012,
3, 1288-1298.
(15) Yao, Y.; Chen, P.; Diao, J.; Cheng, G.; Deng, L.; Anglin, J.
L.; Prasad, B. V. V.; Song, Y. Selective Inhibitors of Histone Methyl-
transferase Dot1L: Design, Synthesis, and Crystallographic Studies. J.
Am. Chem. Soc. 2011, 133, 16746–16749.
(16) Anglin, J. L.; Deng, L.; Yao, Y.; Cai, G.; Liu, Z.; Jiang, H.;
Cheng, G.; Chen, P.; Dong, S.; Song, Y. Synthesis and Structure-
Activity Relationship Investigation of Adenosine-Containing Inhibi-
tors of Histone Methyltransferase Dot1L. J. Med. Chem. 2012, 55,
8066–8074.
duced Binding Pocket. ACS Med. Chem. Lett. 2016, 7, 730-734.
(20) Ichihara, O.; Barker, J.; Law, R. J.; Whittaker, M. Compound
Design by Fragment-Linking. Mol. Inf. 2011, 30, 298–306.
(21) Howard, N.; Abell, C.; Blakemore, W.; Chessari, G.; Con-
greve, M.; Howard, S.; Jhoti, H.; Murray, C. W.; Seavers, L. C. A.;
van Montfort, R. L. M. Application of Fragment Screening and Frag-
ment Linking to the Discovery of Novel Thrombin Inhibitors. J. Med.
Chem. 2006, 49, 1346 – 1355.
(22) Petros, A. M.; Dinges, J.; Augeri, D. J.; Baumeister, S. A.; Be-
tebenner, D. A.; Bures, M. G.; Elmore, S. W.; Hajduk, P. J.; Joseph,
M. K.; Landis, S. K.; Nettesheim, D. G.; Rosenberg, S. H.; Shen, W.;
Thomas, S.; Wang, X.; Zanze, I.; Zhang, H.; Fesik, S. W. Discovery
of a Potent Inhibitor of the Antiapoptotic Protein Bcl-XL from NMR
and Parallel Synthesis. J. Med. Chem. 2006, 49, 656–663.
(23) Borsi, V.; Calderone, V.; Fragai, M.; Luchinat, C.; Sarti, N.
Entropic Contribution to the Linking Coefficient in Fragment Based
Drug Design: A Case Study. J. Med. Chem. 2010, 53, 4285–4289.
(24) Hebeisen, P.; Kuhn, B.; Kohler, P.; Gubler, M.; Huber, W.;
Kitas, E.; Schott, B.; Benz, J.; Joseph, C.; Ruf, A. Allosteric FBPase
Inhibitors Gain 10(5) Times in Potency When Simultaneously Bind-
ing Two Neighboring AMP Sites. Bioorg. Med. Chem. Lett. 2008, 18,
4708–4712.
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2
3
4
5
6
7
8
9
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13
14
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54
55
56
57
58
59
60
(17) Anglin, J. L.; Song, Y. A. Medicinal Chemistry Perspective
for Targeting Histone H3 Lysine-79 Methyltransferase Dot1L. J.
Med. Chem. 2013, 56, 8972–8983.
(25) Dimeric IAP Inhibitors. WO2012/080271.
(26) Shuker, S.B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Dis-
covering High-Affinity Ligands for Proteins: SAR by NMR. Science
1996, 274, 1531-1534.
(27) Observation from unpublished X-ray cocrystal structures of 4
and 6 bound to Dot1L.
(18) Chen, C.; Zhu, H.; Stauffer, F.; Caravatti, G.; Vollmer, S.;
Machauer, R.; Holzer, P.; Möbitz, H.; Scheufler, C.; Klumpp, M.;
Tiedt, R.; Beyer, K. S.; Calkins, K.; Guthy, D.; Kiffe, M.; Zhang, J.;
Gaul, C. The Discovery of Novel Dot1L Inhibitors through a Struc-
ture-Based Fragmentation Approach. ACS Med. Chem. Lett. 2016, 7,
735-740.(19) Scheufler, C.; Be, C.; Fernandez, C.; Moebitz, H.;
Ragot, C.; Beyer, K.; Gaul, C. Stauffer, F. Optimization Towards
Potent Dot1L Inhibitors of a Fragment-Based Screen Hit Containing a
(28) The PyMOL Molecular Graphics System, Version 1.5.0.4
Schrödinger, LLC.
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