High-affinity small-molecule inhibitors of the menin-mixed lineage leukemia (MLL) interaction closely mimic a natural protein-protein interaction

Article abstract of DOI:10.1021/jm401868d

The protein-protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin-MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ～288000 small molecules. We determined menin-inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin-MLL interaction with IC₅₀ = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.

Full text of DOI:10.1021/jm401868d

Article
pubs.acs.org/jmc
Open Access on 01/28/2015
High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage
Leukemia (MLL) Interaction Closely Mimic a Natural Protein−Protein
Interaction
Shihan He,†,# Timothy J. Senter,‡,§,∥,⊥,# Jonathan Pollock,† Changho Han,‡,§,∥ Sunil Kumar Upadhyay,†
Trupta Purohit,† Rocco D. Gogliotti,‡,§,∥ Craig W. Lindsley,‡,§,∥,⊥ Tomasz Cierpicki,†
Shaun R. Stauffer,‡,§,∥,⊥ and Jolanta Grembecka*,†
†Department of Pathology, University of Michigan, Ann Arbor, 1150 West Medical Center Drive, MSRBI, Room 4510D, Michigan,
48109, United States
‡Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
§Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, Tennessee 37232, United States
∥Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United
States
⊥Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
S
* Supporting Information
ABSTRACT: The protein−protein interaction (PPI) be-
tween menin and mixed lineage leukemia (MLL) plays a
critical role in acute leukemias, and inhibition of this
interaction represents a new potential therapeutic strategy
for MLL leukemias. We report development of a novel class of
small-molecule inhibitors of the menin−MLL interaction, the
hydroxy- and aminomethylpiperidine compounds, which
originated from HTS of ∼288000 small molecules. We
determined menin−inhibitor co-crystal structures and found
that these compounds closely mimic all key interactions of
MLL with menin. Extensive crystallography studies combined
with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin−
MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells,
validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach
for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.
important proof-of-principle that small-molecule inhibitors of
PPIs may serve as novel therapeutic agents and justifies the
efforts in developing novel PPI inhibitors.
INTRODUCTION
■
Protein−protein interactions (PPIs) play a critical role in many
biological processes and in a broad spectrum of human diseases,
including cancer,1 and small-molecule modulators of PPIs are
highly desired to serve as chemical tools and potential
therapeutics. Despite these needs, identification of small-
molecule inhibitors of PPIs is considered challenging due to
multiple reasons, including large interacting areas, lack of well-
defined binding pockets, and flexibility of residues on PPI
interfaces,2,3 significantly limiting successful development of
PPI modulators. Discovery of cell-permeable small-molecule
inhibitors of PPIs provides an additional challenge due to
increased molecular weight of PPI inhibitors often required to
achieve high potency.4 On the other hand, recent successes
with a number of PPI inhibitors3,5−8 demonstrate that some
PPIs are amenable to inhibition by small molecules. More
importantly, advancing small-molecule inhibitors of PPIs into
clinical trials, such as the Bcl-2 protein family inhibitor ABT-
2639 and the MDM2 inhibitor RG7112,10 provides an
The protein−protein interaction between menin and mixed
lineage leukemia (MLL) plays a critical role in acute leukemias
with translocations of the MLL gene.11 Fusion of MLL with one
out of over 60 partner genes results in expression of chimeric
MLL fusion proteins, which enhance proliferation of
hematopoietic cells and block hematopoietic differentiation,
ultimately leading to acute leukemias.12 The MLL leukemias
represent a heterogeneous group of acute myeloid leukemias
(AML) and acute lymphoblastic leukemias (ALL), accounting
for about 5−10% of acute leukemias in adults13 and ∼70% of
acute leukemias in infants.14 Patients with MLL leukemias have
very poor prognosis and respond poorly to currently available
treatments,12,15 with only about 35% overall five-year survival
Received: December 5, 2013
Published: January 28, 2014
© 2014 American Chemical Society
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rate,16 emphasizing the urgent need for development of novel
therapies.
CHEMISTRY
■
Synthesis of Hydroxy- and Aminomethylpiperidine
The MLL fusion proteins preserve the N-terminal MLL
fragment of approximately 1400 amino acids fused with the
fusion partner.15,17−19 Importantly, the N-terminal fragment of
MLL, retained in all MLL fusion proteins, is involved in the
interactions with menin,11,20,21 and this interaction plays a
critical role in the MLL−fusion protein mediated leukemogenic
transformations.11,22 Menin is a highly specific binding partner
of MLL and MLL fusion proteins required for regulation of
target genes expression, including HOXA9 and MEIS1 genes,
both of which are essential for leukemogenic activity of MLL
fusions.11 Therefore, menin represents a critical oncogenic
cofactor of MLL fusion proteins in acute leukemias, and
disruption of the protein−protein interaction between menin
and MLL with small molecules represents a very attractive
therapeutic strategy to develop new targeted drugs for the MLL
leukemia patients.
Inhibitors of Menin−MLL Interaction. The HTS hit MIV-1
(1), 4-(3-(4-benzhydrylpiperidin-1-yl)propoxy)benzonitrile
(compound 3) and 1-(3-phenoxypropyl)piperidin-4-yl)-
diphenylmethanol, (compound 4, MIV-2), Table 1, were
Table 1. Structures and IC50 Values for
Hydroxymethylpiperidine Inhibitors of the Menin−MLL
Interaction
Menin interacts with two MLL fragments located within the
N-terminal region, with MBM1 (menin-binding motif 1
corresponding to MLL4−15) representing the high affinity
menin binding motif.20 We previously reported a high
resolution crystal structure of the menin−MBM1 complex,
which demonstrated that MLL binds to a very large central
cavity on menin.23,24 Furthermore, we developed the
thienopyrimidine class of the menin−MLL inhibitors, which
represents the first class of small molecules targeting this
protein−protein interaction reported to date.23,25 The
thienopyrimidine compounds bind to the MLL binding site
on menin and mimic a subset of the critical MLL interactions
with menin but are incapable of interacting with the P10
pocket,23 which appears to limit their further chemical
optimization into more potent, drug-like molecules. Further-
more, the MLL derived peptidomimetics were recently
reported as potent in vitro inhibitors of the menin−MLL
interaction;26 however, cellular activity of these compounds was
not provided, suggesting that optimization of their properties to
identify therapeutically useful compounds is required. These
limitations, together with the pressing need to develop menin−
MLL inhibitors suitable for in vivo studies in animal models of
MLL leukemia, emphasize a clear demand for identification of
novel menin−MLL inhibitors with distinct chemical scaffolds
suitable for optimization of potency and physicochemical
properties.
Here, we report development of a novel class of hydroxy- and
aminomethylpiperidine inhibitors of the menin−MLL inter-
action, which we initially discovered by HTS of ∼288000 small
molecules. These compounds directly bind to menin at the
MLL binding site and specifically block the menin−MLL
protein−protein interaction. Crystal structures of menin−
inhibitor complexes guided medicinal chemistry optimization,
resulting in MIV-6R (IC50 = 56 nM and Kd = 85nM), which
demonstrated strong and selective activity in MLL leukemia
cells. Overall, this work provides a novel and attractive
molecular scaffold together with extensive structural data for
menin−ligand complexes, thus paving the way toward further
development of these compounds into chemical probes or new
potential therapeutics. Importantly, our studies demonstrate the
success of HTS in identifying PPI inhibitors and how an
effective structure-guided optimization enabled development of
nanomolar inhibitors, providing yet another example of PPI
interface amenable to inhibition by small molecules.
readily prepared starting from either diphenyl(piperidin-4-
yl)methanol (compounds 1 and 4) or 4-benzhydrylpiperidine
(compound 3) using a one-pot two-step procedure.27 Full
experimental details can be found within Supporting
Information. Synthesis of the hydroxymethyl piperidine aryl
and diaryl analogues 2, 5−10, 12, 18, and 21 (Table 1, Figures
4−5), cyclopentyl analogues 11 and 13−17 (Figure 4), and
head group and tail group piperidine analogues 19−20 (Figure
5) were prepared according to Schemes 1−4. Our initial
strategy, involving preinstallation of the nitrile tail group prior
to Grignard addition, led to final products contaminated with
trace amounts of an inseparable side-product presumed to
result from Grignard addition to the nitrile (R3 = CN). To
circumvent this issue, we performed a selective alkylation of
piperidines 22a−b using 1-bromo-3-chloropropane to afford
chloride intermediates 23a−b in good yield (Scheme 1).
Addition of Grignard reagents to 23a−b provided the crude
carbinol chlorides in high conversion. Subsequent alkylation
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Scheme 1. Synthesis of Aryl Hydroxymethylpiperidines 2, 5−10, 12, 18, and 21
Scheme 2. Synthesis of Cyclopentyl Hydroxymethylpiperidines 11 and 13−17
Scheme 3. Synthesis of Aminomethylpiperidine 19
Scheme 4. Synthesis of Sulfonamide Containing Hydroxymethylpiperidine 20
using the appropriate phenol reagent led to final target
compounds (Scheme 1).
Preparation of tertiary carbinamine, 4-(3-(4-(cyclopentyl-
(amino)(phenyl)methyl)piperidin-1-yl)propoxy)benzonitrile
(compound 19, MIV-6), was accomplished via solvolysis of
carbinol 11 in the presence of sodium azide in chloroform and
sulfuric acid (Scheme 3). The crude azide was reduced using
palladium on carbon with atmospheric hydrogen in EtOH to
afford amine 19. Despite efforts to screen for alternative
conditions to improve solvolysis and formation of the azide
intermediate, facile formation of a major elimination side
product leading to olefin remained problematic. Resolution of
racemic 19 was accomplished using chiral supercritical fluid
chromatography (SFC) to afford single enantiomers 19S and
19R for which the stereochemical configuration was sub-
sequently inferred on the basis of the absolute configuration
observed in the electron density map of the X-ray structure of
the respective menin complex with 19.
Synthesis of cyclopentyl hydroxymethyl piperidines 11
(MIV-3) and 13−17 is outlined in Scheme 2. To circumvent
low yields due to competing reduction of the sterically hindered
ketones by alkyl Grignard reagents,28 we employed a strategy
involving introduction of the saturated cycloalkyl followed by a
second aryl or alkyl group (Scheme 2). Thus, initial
introduction of the cycloalkyl was accomplished using a
known two-step procedure starting from N-benzylpiperidine-
4-carbonitrile to give key starting aminoketone 24 (Scheme
2).29 Subsequent one-pot double alkylation using 24 and 1-
bromo-3-chloropropane afforded piperidine 25 in 78% yield.
Grignard or stabilized aryl lithium additions afforded final target
compounds 11 and 13−17 in moderate to good yield.
Overaddition of these aryl Grignard and lithium reagents to
the nitrile was not evident for substrate 25, and all final
compounds displayed excellent purity (>98%).
Preparation of 20 began with 2-pyridyl lithium addition to
benzyl protected ketone 26 to provide 27 in excellent yield
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(Scheme 4). Hydrogenolysis of the N-benzyl of 27 using
Pearlman’s catalyst overnight in ethanol with heating, followed
by alkylation of the crude deprotected piperidine using chloride
28, afforded target sulfonamide 20.
an essential pharmacophore within MIV-1 required for efficient
inhibition of the menin−MLL interaction (synthetic proce-
dures used for preparation of hydroxy- and aminomethylpiper-
idine compounds are provided in Schemes 1−4). We found
that removal of the hydroxyl group from MIV-1 in the linker
region, resulting in 4-(3-(4-(hydroxydiphenylmethyl)piperidin-
1-yl)propoxy)benzonitrile (compound 2, MIV-2, Figure 1c),
slightly increased the activity (IC50 = 10.8 μM, Table 1). Thus,
to facilitate synthesis and reduce the number of resulting
stereoisomers, we eliminated the central hydroxyl group from
subsequent analogues. In contrast, removal of the hydroxyl
group from the quaternary carbon in the head group region
resulted in compound 3 (Table 1) with decreased inhibitory
activity by more than 20-fold, emphasizing the importance of
this group in binding to menin. Similarly, the binding affinity
was strongly decreased by removing the nitrile within the tail
group region, which resulted in compound 4 with IC50 > 250
μM (Table 1). On the basis of these data, we concluded that
MIV-2 (Figure 1c) represents an essential pharmacophore for
the hydroxymethylpiperidine class of menin−MLL inhibitors.
These findings remain in a very good agreement with the
structural data on the menin−inhibitor complexes (see below),
demonstrating that all structural features present in MIV-2 are
required for effective interactions with the corresponding
binding pockets on menin.
Probing the Hydrophobic Head Group Region to
Improve Potency of Menin−MLL Inhibitors. We used
MIV-2 as a lead compound for medicinal chemistry
optimization to improve potency of hydroxymethylpiperidine
class. First, we explored replacement of one of the phenyl rings
at the headgroup region of MIV-2 (Figure 1c) with different
hydrophobic groups (R2 substituents, Table 1). Interestingly,
we found a very pronounced structure−activity relationship
(SAR) for modifications at this site (Table 1). The inhibitory
activity of 4-(3-(4-(hydroxy(phenyl)methyl)piperidin-1-yl)-
propoxy)benzonitrile (compound 5, MIV-nc), with hydrogen
replacing the phenyl group, was very weak (IC50 = 234 μM)
and did not improve upon addition of a methyl group at this
position (6, Table 1). Introducing more bulky hydrophobic
substituents resulted in increased inhibition of the menin−MLL
interaction. For example, n-butyl (7) or cyclopropyl (8)
substituents yielded about 20-fold increase in activity versus
MIV-nc (Table 1). Furthermore, analogues with iso-propyl (9)
and cyclobutyl (10) groups had further improved IC50 values
(IC50 = 4.1 and 4.0 μM, respectively, Table 1). Finally,
cyclopentyl was identified as the most optimal saturated
carbocycle at this position, resulting in 4-(3-(4-(cyclopentyl-
(hydroxy)(phenyl)methyl)piperidin-1-yl)propoxy)benzonitrile
(compound 11, MIV-3),27 with an IC50 of 390 nM measured
for the racemic mixture. Further increase in ring size decreased
activity, as cyclohexyl analogue 12 was about 4-fold less active
than MIV-3 (Table 1). On the basis of these data, we
concluded that cyclopentyl was the preferred hydrophobic
group at R2 position.
RESULTS AND DISCUSSION
■
Identification of Novel Inhibitors of Protein−Protein
Interaction between Menin and MLL. To identify new
inhibitors of the menin−MLL interaction, we performed a high
throughput screening of ∼288000 small molecules at the NIH
Molecular Libraries Probe Production Centers Network
(MLPCN, https://mli.nih.gov/mli) using a fluorescence polar-
ization (FP) assay with a fluorescein-labeled MLL-derived
peptide MBM1.20 A stepwise procedure, including FP assay for
primary screening followed by homogenous time resolved
fluorescence (HTRF) assay for secondary screening and NMR
saturation transfer difference (STD) experiments to validate
direct binding of compounds to menin, was applied to identify
menin−MLL inhibitors (detailed description of the HTS screen
is provided at PubChem Bioassay, AID 1766: http://pubchem.
ncbi.nlm.nih.gov/assay/assay.cgi?aid=1766). The most potent
compound identified in the screen was MLS001171971 (4-(2-
hydroxy-3-(4-(hydroxydiphenylmethyl)piperidin-1-yl)-
propoxy)benzonitrile, compound 1, MIV-1), belonging to the
hydroxymethylpiperidine class, which exhibited a half maximal
inhibitory concentration (IC50) value of 12.8 μM for inhibition
of the menin−MLL interaction (Figure 1a). We also identified
Figure 1. Discovery of hydroxymethylpiperidine class of menin−MLL
inhibitors by HTS. (a) Structure and activity of the most potent hit
from HTS, MIV-1. (b) NMR saturation transfer difference (STD)
experiments demonstrating binding of MIV-1 to menin and
competition with MLL (competition STD). (c) Structure and activity
for MIV-2 representing the essential pharmacophore for the
hydroxymethylpiperidine class of menin−MLL inhibitors.
a compound with a similar structure, MLS001175799
(Supporting Information Figure 1), but with a 6-fold weaker
activity (IC50 = 77 μM) than MIV-1. We validated direct
binding of MIV-1 to menin using saturation transfer difference
(STD) NMR experiments, resulting in a strong STD effect
(Figure 1b). Importantly, addition of the MLL peptide strongly
decreased the STD effect observed for MIV-1 (Figure 1b),
demonstrating that MIV-1 and MLL compete for binding to
menin. These results confirm reversible and specific binding of
MIV-1 hydroxymethylpiperidine compound to menin.
MIV-3 represents a racemic mixture, and to assess the
activity of individual enantiomers, we separated the racemic
mixture by chiral SFC. Interestingly, the R enantiomer, MIV-3R
(11b, stereochemistry assigned based on the crystal structures,
see below), was only about 2-fold more potent than the S
enantiomer (11a, MIV-3S) as assessed by FP assay (IC50 = 270
nM and 529 nM for MIV-3R and S, respectively, Figure 2a).
We subsequently measured the dissociation constants (Kd) for
binding of both enantiomers of MIV-3 to menin using
Determination of the Essential Pharmacophore with-
in Hydroxymethylpiperidine Class of Menin−MLL
inhibitors. First, we performed synthetic efforts to identify
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Figure 2. MIV-3 binds to menin with nanomolar affinity. (a) Structure and activity for MIV-3 and two individual enantiomers of this compound.
(b,c) Isothermal titration calorimetry experiments demonstrating direct and specific binding of both enantiomers of MIV-3 to menin. N corresponds
to the stoichiometry of ligand binding to menin.
isothermal titration calorimetry (ITC) and found that they both
bind to menin with nanomolar affinities (Kd = 285 and 952 nM
for R and S isomers, respectively, Figure 2b,c), consistent with
the IC50 values, Figure 2a. Overall, development of MIV-3R
represents over 40-fold improvement in the activity as
compared to the parent compound MIV-1. Interestingly, we
found a relatively small difference in inhibition of the menin−
MLL interaction by both enantiomers of MIV-3, Figure 2.
Hydroxymethylpiperidine Inhibitors Mimic the Most
Critical Interactions of MLL with Menin. To establish the
molecular determinants of MIV-3 binding to menin and
understand relatively small differences in the binding affinity
between the two enantiomers, we determined the crystal
structures of menin in complex with both enantiomers of MIV-
3 (Figure 3a,b). We found that they both bind in extended
conformations to the MLL binding site on menin and occupy
three hydrophobic pockets: F9, P10, and P13 (Figure 3). There
is a very close overlap of the binding modes of both
enantiomers of MIV-3, with the only difference being an
alternate positioning of the head group substituents between
the F9 and P10 hydrophobic pockets on menin (Figure 3c).
The menin−MIV-3 interactions are mostly mediated by the
hydrophobic contacts, with only one direct hydrogen bond
formed between the nitrile group of both enantiomers of MIV-
3 and the indole nitrogen of Trp341 on menin (Figure 3c).
Additional water mediated hydrogen bond involves hydroxyl at
the head group region of MIV-3R and S and the Asp180 side
chain on menin (Figure 3c). The importance of the hydrogen
bonds involving head group hydroxyl and tail group nitrile is
reflected by more than 20-fold decrease in the activity of the
analogues deficient in the corresponding functional groups
(compounds 3 and 4, Table 1). Of note, binding of both
enantiomers of MIV-3 to menin does not induce conforma-
tional changes of the protein.
(Leu177, Phe238, Ser155, Met278, Figure 3c) and backbone of
Asp180 and His181, while the cyclopentyl ring binds to the P10
pocket and interacts with the side chains of Ala242, Cys241,
Tyr276, Phe238, and Ser155, Figure 3c. In the case of MIV-3S
enantiomer, the positions of cyclopentyl and phenyl rings are
swapped between these two pockets on menin (Figure 3c). The
piperidine ring and tail groups in both enantiomers of MIV-3
bind to menin in the same manner, extending toward the P13
pocket and Trp341. The alkoxy portion of the linker fits into
the P13 pocket formed by Tyr319 and Tyr323 (Figure 3c),
while the benzonitrile moiety extends beyond the P13 pocket,
where it approaches Met322 and Glu363 side chains to form a
hydrogen bond with the side chain of Trp341 (Figure 3c). The
interaction of the benzonitrile moiety is of particular interest as
this region of the binding site is not occupied by the MLL
peptide or our previously identified thienopyrimidine class of
menin−MLL inhibitors (Figure 3c,d). Thus this unique and
important interaction identified for the new hydoxymethylpi-
peridine class offers the possibility to introduce additional
contacts with menin to further improve affinity through
rationally designed modifications. Overall, the binding mode
of MIV-3 explains the SAR data (Table 1) and is consistent
with the essential pharmacophore identified for this class of
menin−MLL inhibitors (Figure 1c).
In our previous studies, we found that three hydrophobic
residues in the MLL derived peptide MBM1, namely Phe9,
Pro10, and Pro13, have the most critical contributions for
binding to menin and their mutations to alanine residues
reduce binding affinity by 30−1000-fold.20,23 Superposition of
MLL MBM1 peptide and both enantiomers of MIV-3 bound to
menin demonstrate a close overlap of these small-molecule
inhibitors with the critical residues of MLL required for potent
binding to menin (Figure 3d). Specifically, the phenyl ring in
the head group of the more potent enantiomer MIV-3R
overlaps with the side chain of Phe9 in MLL, while the
cyclopentyl ring in MIV-3R adopts a similar position and
conformation as the Pro10 side chain in MLL (Figure 3d), thus
In the co-crystal structure with more potent enantiomer,
MIV-3R, the phenyl ring fits very well into the F9 pocket on
menin formed by the side chains of hydrophobic residues
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4a, Table 1). As demonstrated above, the phenyl ring in MIV-
3R closely mimics the interactions of MLL Phe9 with menin,
and therefore it was used to design further modifications. In the
crystal structure of menin in complex with the MI-2−2
thienopyrimidine inhibitor, we found a very favorable C−F---
CO dipolar interaction between a fluorine atom in MI-2−2
and backbone of His181.23 On the basis of the menin−MIV-3R
co-crystal structure, we anticipated that introducing fluorine
into position 3 of the phenyl ring should result in similar
contacts and in improved activity. Indeed, introduction of a 3-
fluoro to give 4-(3-(4-(cyclopentyl(3-fluorophenyl)(hydroxy)-
methyl)piperidin-1-yl)propoxy)benzonitrile (compound 13,
MIV-4) resulted in a 2-fold improvement in potency versus
MIV-3 (Figure 4b). We validated the binding mode of MIV-4
to menin by solving the crystal structure of the complex (Figure
4c, Supporting Information Table 1), which confirmed the
presence of dipolar interaction between the fluorine atom in
MIV-4 (in R enantiomer) and the backbone amide of His181
on menin. Introducing a chloro substituent at the same position
(15) or incorporation of additional fluorine to the phenyl ring
(14) resulted in a weaker inhibitory activity (Figure 4b).
Therefore, structure-based designed 3-fluoro substituted phenyl
represents the most preferred substituent identified thus far to
interact with the F9 pocket on menin (Figure 4b,c and Table
1).
In the next step, we performed optimization of the R2
substituent that interacts with the P10 pocket. In the complex
of menin with more potent MIV-3R enantiomer, this pocket is
occupied by the cyclopentyl ring, which is not optimal for
further derivatization and contributes significantly to the
lipophilic character of the molecule (cLogP = 5.6 for MIV-3;
cLogP calculations performed using Molinspiration software,
www.molinspiration.com). Because the crystal structure of
MIV-3S reveals that the P10 pocket can also accommodate
phenyl ring (Figure 3b,c) with only 2-fold decreased IC50 value
(Figure 2a), we selected phenyl for further optimization of the
R2 substituent as an approach to streamline derivatization and
SAR. The analysis of the apo structure of menin and the
complex with MIV-3S revealed the presence of a water
molecule, which forms a hydrogen bond with Tyr 276, located
in a proximity to the ortho position of the phenyl ring in MIV-
3S (not shown). Therefore, we introduced a nitrogen atom via
replacement of phenyl with a pyridine or thiazole rings to
engage in favorable interactions with this structural water
molecule. These efforts resulted in 4-(3-(4-(cyclopentyl-
(hydroxy)(pyridin-2-yl)methyl)piperidin-1-yl)propoxy)-
benzonitrile (compound 16, MIV-5) and 4-(3-(4-(cyclopentyl-
(hydroxy)(thiazol-2-yl)methyl)piperidin-1-yl)propoxy)-
benzonitrile (compound 17), both of which showed improved
potency and increased polarity versus MIV-3, Figure 4d. We
confirmed the existence of a water-mediated hydrogen bond
between MIV-5 and Tyr276 by solving the crystal structure of
the complex (Figure 4e). As predicted, despite using the
racemic mixture of MIV-5 for co-crystallization with menin,
only the S enantiomer was observed in the structure (Figure
4e). This was further confirmed by testing separated
enantiomers of MIV-5, showing a ∼4-fold greater inhibitory
activity for the S enantiomer (IC50 = 195 nM). Then we
combined optimal substituents identified to interact with F9
and P10 pockets and synthesized the hybrid 3-fluorophenyl-2-
Figure 3. MIV-3 binds to the MLL binding site on menin. (a,b)
Crystal structures of menin in complex with MIV-3R (a, PDB code
4GO3) and MIV-3S (b, PDB code 4GO4) with 2Fo − Fc electron
density maps for ligands contoured at the 1σ level. Menin is shown in
surface representation and location of F9, P10, and P13 pockets is
labeled. (c) Superposition of the menin−MIV-3R (carbon atoms in
green) and menin−MIV-3S (carbon atoms in magenta) crystal
structures showing binding mode of ligands and key protein residues
in the binding site. The F9, P10, and P13 pockets are labeled. Dashed
lines represent hydrogen bonds between the ligands and protein; w
represents a water molecule. (d) Superposition of the crystal structures
of menin−MIV-3R (green carbons) and the menin−MLL (blue
carbons, PDB code 4GQ6) complexes. Menin is shown in surface
representation with atoms colored according to the atom type. The F9,
P10, and P13 pockets are labeled.
mimicking very closely the critical MLL interactions with
menin by structurally and conformationally similar moieties.
Furthermore, the alkoxy portion of the linker in MIV-3R
mimics the interactions of MLL Pro13 with menin, while the
benzonitrile moiety extends beyond the P13 pocket toward
previously unexplored region of the binding site. Overall, the
potent activity of MIV-3R results from mimicking key
interactions of MLL with menin in F9, P10, and P13 pockets
as well as from additional contacts beyond the P13 pocket,
which provides further opportunity for optimization of these
compounds.
Structure-Based Design of Nanomolar Inhibitors of
the Menin−MLL Interaction. We then used the crystal
structure of menin in complex with both enantiomers of MIV-3
for rational design of new analogues to further improve potency
and physicochemical properties of this class of compounds.
Four regions in MIV-3 were explored for modifications
(Figures 4 and 5) to rationally design new compounds based
upon structural data. First, we explored additional optimization
of R1 substituents that bind to the F9 pocket on menin (Figure
pyridyl congener 18, with ∼3-fold improved IC50 value (IC50
=
90 nM for R enantiomer of 18, Figure 4d), which represents
the most potent hydroxmethylpiperidine compound reported
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Figure 4. Structure-based optimization of hydroxymethylpiperidine compounds in the head group region. (a) General structure of the
hydroxymethylpiperidine class showing positions of R1 and R2 substituents, which bind to the F9 and P10 pockets, respectively. (b) Structures and
inhibitory activities of MIV-3 analogues with modifications introduced at R1. IC50 values are provided for the racemic mixtures. (c) Crystal structure
of menin in complex with MIV-4 (green carbon atoms, PDB code 4GO6) showing the interaction of fluorine atom (light blue) in MIV-4 and menin
backbone (gray carbon atoms). Close distances involving fluorine from MIV-4 are shown as dashed lines. (d) Structures and activities for analogues
with modifications introduced simultaneously at R1 and R2. IC50 values are provided for the racemic mixtures, unless indicated. (e) Crystal structure
of menin in complex with MIV-5 (green carbon atoms, PDB code 4GO5) showing water mediated hydrogen bond with Tyr276.
here. Importantly, introduction of heterocyclic versus phenyl
ring to occupy the P10 pocket substantially increased polarity
(cLogP value reduced by 1 order of magnitude) and may also
reduce potential for oxidative metabolism of these compounds.
The crystal structure of MIV-3 enantiomers with menin
revealed that the solvent exposed hydroxyl group in the head
group region is involved in a water mediated hydrogen bond
with Asp180 (Figure 5b). On the basis of structural data, we
anticipated that introducing a positively charged group, such as
an amino moiety, should result in additional favorable
electrostatic interactions with adjacent Asp180 and other acidic
residues located in this region of the binding site (e.g., Asp153,
Glu 359, Figure 5b). Indeed, MIV-6 (19, Figure 5a,b), which
represents the aminomethylpiperidine class, showed almost 6-
menin with Kd = 85 nM (Figure 5a and Supporting Information
Figure 2). These results demonstrate the importance of
considering long-range electrostatic interactions in designing
protein ligands to achieve efficient binding.
Finally, we used the crystal structure of menin with MIV-5 to
guide replacement of the nitrile, with the overall goal to further
improve polarity while retaining the hydrogen bond with
Trp341 (Figure 5d). On the basis of the menin−MIV-5
structure, we designed sulfonamide group as the optimal
replacement of nitrile. Indeed, the sulfonamide analogue 20
(MIV-7, IC50 = 114 nM) showed ∼3-fold improved inhibitory
activity versus the corresponding nitrile analogue MIV-5
(Figure 5c), while 21 demonstrated a modest ∼1.5-fold
improvement in IC50 versus the corresponding nitrile derivative
18 (Figures 4d, 5c). The binding mode of MIV-7 to menin was
validated by crystallographic studies, confirming the existence
of the hydrogen bonds with the side chain of Trp341 and also
the presence of an additional water-mediated hydrogen bond
with Glu363 (Figure 5d), resulting in increased potency. These
data clearly validate the sulfonamide as a viable replacement for
the nitrile in this class of menin−MLL inhibitors, which allows
fold increase in inhibiting the menin−MLL interaction (IC50
=
67nM for racemic mixture of MIV-6) versus MIV-3. The
binding mode of MIV-6 was not affected by incorporating the
amino group, as validated by the crystal structure of menin in
complex with MIV-6R enantiomer (Figure 5b). The MIV-6R
represents the most potent inhibitor of the menin−MLL
interaction reported here (IC50 = 56 nM), which binds to
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Figure 5. Structure-based optimization of polar interactions with menin. (a) Structures and activities of analogues with substitution of a hydroxyl
(marked as -X) at the head group region. IC50 values are provided for the racemic mixtures, unless indicated. (b) Comparison of the crystal
structures of menin in complex with MIV-6R (bottom, PDB code 4GO8) and MIV-3R (top) showing polar interactions between the head group
region and menin. Inhibitors are shown in stick representation (green carbon atoms); menin is presented as a gray ribbon. Selected side chains on
menin involved in contacts with compounds are shown in stick representation; w represents a water molecule. Hydrogen bonds are shown as dashed
lines with distances in angstroms. E359 is present in two alternative conformations. (c) Structures and activities of analogues with substitution of
nitrile at the tail group region. IC50 values are provided for the racemic mixtures. (d) Comparison of the crystal structures of menin in complex with
MIV-7 (bottom, PDB code 4GO7) and MIV-5 (top) showing the interactions of inhibitor tail groups with menin. Structural representation of the
protein and ligand and labeling the same as in (b).
for significant increase in polarity relative to the nitrile
analogues (cLogP is reduced by an order of magnitude,
cLogP = 3.4 for MIV-7 versus 4.4 for MIV-5).
immunoprecipitation (co-IP) experiment in HEK-293 cells
transfected with Flag−MLL-AF9 to evaluate whether the
compounds can effectively disrupt the menin−MLL fusion
protein interaction in mammalian cells (Figure 6a). Treatment
with low micromolar concentrations of both MIV-6 and MIV-
3R very effectively disrupts the menin−MLL-AF9 interaction in
cells, with more pronounced effect observed for MIV-6 (Figure
6a). Importantly, the expression levels of menin and MLL-AF9
were not affected upon treatment (Figure 6a). These data
clearly demonstrate that both MIV-6 and MIV-3R can reach
the target protein and effectively disrupt the menin−MLL
fusion protein interaction in human cells.
MIV-6 and MIV-3R Disrupt Menin−MLL Fusion Protein
Interaction in Cells and Affect Expression of MLL Fusion
Protein Downstream Targets. To assess the mechanism of
action of this new class of menin−MLL inhibitors, we evaluated
their effect on blocking the activity of MLL fusion proteins in
leukemia cells. For this purpose, we selected the most potent
inhibitor we developed harboring the amino group, MIV-6
(IC50 = 67 nM), and corresponding hydroxyl analogue, MIV-
3R (IC50 = 270 nM). First, we performed the co-
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Figure 6. Biological activity of hydroxy- and aminomethylpiperidine menin−MLL inhibitors. (a) Co-immunoprecipitation experiment in HEK293
cells transfected with MLL-AF9. (b) Quantitative real-time PCR performed in MLL-AF9 transformed murine bone marrow cells after 6 days of
treatment with compounds. Expression of Hoxa9 and Meis1 has been normalized to β-actin. Data represent mean values for duplicates SD. The
experiment was performed two times. (c) Titration curves from MTT cell viability assay performed for MIV-6 and MIV-3R after 7 days of treatment
of MLL-AF9 and Hoxa9/Meis1 (HM-2) transformed murine bone marrow cells. Cell growth inhibition (GI50) values are provided for treatment of
MLL-AF9 cells. Data represent mean values of quadruplicates SD. The experiment was performed three times. (d) Titration curves from MTT cell
viability assay performed for MIV-6 and MIV-3R after 7 days of treatment of human MLL leukemia cell lines (MV4;11 and MOLM-13) and control
cell line HL-60 (non-MLL leukemia cell line). Cell growth inhibition (GI50) values are provided for MV4;11 and MOLM-13 cell lines. Data
represent mean values of quadruplicates SD. The experiment was performed three times. (e) Quantification of CD11b expression in MLL-AF9
transformed murine bone marrow cells treated for 6 days with MIV-6 and MIV-3R as detected by flow cytometry. Data represent the mean values
for duplicates SD. (f) Wright−Giemsa-stained cytospins for MLL-AF9 transformed murine bone marrow cells after 7 days of treatment. Black line
represents the scale bar (50 μm).
The menin−MLL fusion protein interaction is required for
the maintenance of high expression level of HOXA9 and MEIS1
in MLL leukemia cells and for leukemic transformation by MLL
fusions.11 To assess whether MIV-6 and MIV-3R affect the
expression level of HOXA9 and MEIS1, we performed real-time
quantitative PCR (qRT-PCR) experiments in MLL-AF9
transformed murine bone marrow cells (BMC). Indeed,
treatment with both MIV-6 and MIV-3R resulted in a strong
and dose dependent reduction in the expression level of Hoxa9
and Meis1 as compared to the DMSO control (Figure 6b), with
about 50% decrease in Hoxa9 level upon treatment with 3 μM
of MIV-6. The effects on expression level of target genes
correlates well with the in vitro inhibition observed for these
compounds as MIV-6 showed about 4-fold more pronounced
effect on Hoxa9 expression. Importantly, the negative control
compound, MIV-nc, which is a very weak menin−MLL
inhibitor (IC50 = 234 μM, Table 1), did not show any effect
on Hoxa9 and Meis1 expression (Figure 6b). These results
demonstrate that MIV-6 and MIV-3R inhibit the menin−MLL
fusion protein interaction in cells and block the MLL−fusion
protein dependent gene expression, emphasizing on-target
effects for these compounds and validating their specific
mechanism of action.
MIV-6 and MIV-3R Selectively Block Proliferation and
Induce Differentiation in MLL Leukemia Cells. Disruption
of the menin−MLL fusion protein interaction is expected to
result in growth arrest and differentiation of MLL leukemia
cells.11,25 Therefore, we tested activity of MIV-6 and MIV-3R
in murine bone marrow cells transformed with either MLL-AF9
or Hoxa9/Meis1 (HM-2), the later served as a negative control
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cell line. As expected, strong and dose-dependent inhibition of
cell proliferation was observed for both compounds in MLL-
AF9 transformed BMCs (Figure 6c), with GI50 = 1.1 μM for
MIV-6, which showed ∼4-fold more pronounced effect than
MIV-3R (GI50 = 4.5 μM), consistent with its higher in vitro
activity. In contrast, these two compounds have a very limited
effect on proliferation of Hoxa9/Meis1 transformed BMCs
(Figure 6c), demonstrating selectivity toward MLL fusion
transformed cells. Similar effects were observed in human MLL
leukemia cell lines. Treatment of MV4;11 and MOLM-13 cells
harboring MLL-AF4, MLL-AF9 fusion proteins, respectively,
with MIV-6 and MIV-3R resulted in a strong and dose
dependent inhibition of cell proliferation, with a more
pronounced effect observed for MIV-6 (Figure 6d). In contrast,
marginal effect on cell proliferation was seen upon treatment of
HL-60 promyelocytic leukemia cells, which served as a negative
control cell line (Figure 6d).
We then assessed the effect of hydroxy- and amino-
methylpiperidine compounds on differentiation of MLL fusion
protein dependent cells. Indeed, the MLL-AF9 BMCs undergo
differentiation upon treatment with MIV-6 and MIV-3R, as
assessed by flow cytometry analysis of expression level of
CD11b, which serves as a differentiation marker of myeloid
cells. Treatment with more potent MIV-6 induces more
pronounced increase in CD11b expression (Figure 6e). In
addition, Wright−Giemsa staining of MLL-AF9 BMCs treated
with low micromolar concentrations of MIV-6 and MIV-3R
revealed significant changes in morphology of these cells, such
as decreased nuclei to cytoplasm ratio, multilobed nuclei, and
highly vacuolated cytoplasm, which clearly demonstrate
myeloid differentiation upon treatment with both inhibitors
(Figure 6f). Treatment with higher concentration of MIV-6 (6
μM) resulted in terminal differentiation (Figure 6f). All these
results further confirm on-target effects and specific mechanism
of action for the hydroxy- and aminomethylpiperidine
inhibitors.
interactions with Asp180 on menin (Figure 5b). The resulting
compound MIV-6R (IC50 = 56 nM, Kd = 85 nM) provides
∼230-fold improvement in the activity versus the initial HTS
hit MIV-1. Importantly, despite a relatively low molecular
weight (Mw = 416 Da), MIV-6 inhibits the menin−MLL
interaction with an affinity similar to the 4-fold larger 12-amino
acid MLL-derived peptide. Consequently, the ligand efficiency
(LE) index31 for this compound is relatively high (LE = 0.31 for
MIV-6R) as compared to 0.24 average value reported for PPI
inhibitors.5 Furthermore, MIV-6 has significantly improved
polarity (cLogP = 2.9) versus MIV-3 (cLogP = 5.6).
The co-crystal structure of menin in complex with
compounds reported here revealed that they mimic the key
interactions of MLL with menin by occupying three pockets on
menin (F9, P10, and P13), which are the most critical for MLL
binding. Importantly, these compounds also extend beyond the
P13 pocket toward an additional region on menin, which does
not participate in MLL binding, providing a further interaction
site for potency optimization and modulation of physicochem-
ical properties of menin−MLL inhibitors. We utilized this site
for improvement in compound binding affinity by introducing
the benzonitrile group to form a hydrogen bond with Trp341
and for increase in compound polarity by introducing
sulfonamide as an efficient replacement of nitrile (Figure 5d).
The later example clearly demonstrates the potential for further
optimization of physicochemical properties for this class of
menin−MLL inhibitors.
The newly developed hydroxy- and aminomethylpiperidine
class of compounds have very effective and selective activities in
cell-based experiments. The most potent compound, MIV-6,
efficiently inhibited proliferation and induced hematopoietic
differentiation in MLL leukemia cells and strongly reduced the
expression level of key MLL−fusion protein target genes.
Overall, these data demonstrate that the novel class of menin−
MLL inhibitors reported here has very promising on-target
biological activity. On the basis of structural studies, these
compounds represent an attractive chemical scaffolds for
further optimization as they closely mimic all critical
interactions of MLL with menin, including the interactions
with the P10 pocket, which remains unoccupied by the
previously reported thienopyrimidine class of menin−MLL
inhibitors.23 Our studies will pave the way toward further
optimization of these compounds into chemical probes for in
vivo studies in MLL leukemia models and for potential
therapeutic applications. This work provides another example
of PPI interface amenable to potent inhibition by small
molecules, strongly supporting the efforts in developing
inhibitors for novel PPIs as chemical probes and/or potential
therapeutic agents.
CONCLUSIONS
■
Menin is a critical oncogenic cofactor of MLL fusion proteins,
and the protein−protein interaction between menin and MLL
fusion proteins represents a validated and attractive therapeutic
target in acute leukemias with translocations of MLL
gene.11,21,30 Specific inhibition of this protein−protein
interaction with small molecules could lead to development
of novel targeted therapy for acute leukemias with MLL
translocations.25 The difficulty in developing potent small-
molecule inhibitors of the menin−MLL interaction partly arises
from a very large size of the binding site on menin (over 5000
Å3),23 as small-molecule inhibitors can occupy only a small
portion of this site.
EXPERIMENTAL SECTION
In the present study, we report development of a novel class
of the menin−MLL inhibitors, the hydroxy- and amino-
methylpiperidine compounds, which we identified by a high
throughput screening of over ∼288000 small molecules at the
NIH MLPCN. Medicinal chemistry optimization of the HTS
hit MIV-1 (IC50 = 10.8 μM) led to a very significant
improvement of inhibitory activity, resulting in development
of MIV-3 (IC50 = 270 and 530 nM for R and S enantiomers,
respectively, Figure 2a). The co-crystal structures of both
enantiomers of MIV-3 with menin were used to design new
analogues with improved binding affinities. The most
pronounced activity increase was obtained after replacing the
hydroxyl group with the amino group, leading to electrostatic
■
General Chemistry Information. The structures of the presented
compounds were characterized by 1H, 13C NMR, and mass
spectrometry (HRMS). Purity (>95%) was assessed by RP-HPLC.
All NMR spectra were recorded on a Bruker 400, 500, or 600 MHz
1
instruments. H chemical shifts are reported in δ values relative to
residual solvent signals in ppm. Data are reported as follows: chemical
shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, br = broad resonance, m = multiplet), coupling constant (Hz).
Low resolution mass spectra were obtained on an Agilent 1200 series
6130 mass spectrometer. High resolution mass spectra were recorded
on a Waters Q-TOF API-US plus Acuity system with ES as the ion
source. Analytical thin layer chromatography was performed on
Sorbent Technologies 250 μm silica plates. Visualization was
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accomplished via UV light, and/or the use of potassium permanganate
solution followed by application of heat. Analytical HPLC was
performed on an HP1100 with UV detection at 214 and 254 nm along
with ELSD detection, LC/MS (J-Sphere80-C18, 3.0 mm × 50 mm, 4.1
min gradient, 7%[0.1%TFA/H2O]:93%[CH3CN]). Preparative RP-
HPLC purification was performed on a custom HP1100 automated
purification system with collection triggered by mass detection or
using a Gilson Inc. preparative UV-based system using a Phenomenex
Luna C18 column (50 × 30 mm ID, 5 μm) with an acetonitrile
(unmodified)−water (0.5 mL/L NH4OH) custom gradient. Normal-
phase silica gel preparative purification was performed using an
automated Combi-flash Companion from ISCO. Semipreparative
purifications were carried out via stacked injections on a Waters
Investigator SFC using a 10 mm × 250 mm Chiral Technologies
Chiralpak ID column heated to 40 °C. Analytical separations were
carried out on an Agilent 1260 Infinity SFC using a 4.6 mm × 250 mm
Chiral Technologies Chiralpak ID column heated to 40 °C. Solvents
for extraction, washing, and chromatography were HPLC grade. All
reagents were purchased from Aldrich Chemical Co. and were used
without purification. All polymer-supported reagents were purchased
from Argonaut Technologies and Biotage.
concentration in vacuo afforded a crude oil which was purified by
flash column chromatography (9:1 CH2Cl2/MeOH) to afford a
colorless oil comprising an inseperable mixture of the desired azide
and an elimination byproduct in 30 mg that was carried on to the next
step. 4-(3-(4-(Azido(cyclopentyl)(phenyl)methyl)piperidin-1-yl)-
propoxy)benzonitrile (30 mg, 0.07 mmol) was dissolved in degassed
EtOH (0.5 mL) and Pd/C (2.7 mg) added in one portion. Reaction
was placed under a balloon of H2 gas and allowed to stir for 4 h at
ambient temperature. The reaction was filtered over Celite and rinsed
with MeOH. The filtrate was concentrated to afford an oil. RP-HPLC
preparative purification afforded the desired product as a TFA salt.
The mixture was treated with a StratoSpheres SPE MP-carbonate resin
1
cartridge to give title compound as a free base in 20 mg (2%). H
NMR (600 MHz, CDCl3) δ 7.54 (2H, d, J = 6.0 Hz), 7.43 (2H, d, J =
7.2 Hz), 7.31 (2H, t, J = 7.2 Hz), 7.22 (1H, t, J = 7.2 Hz), 6.91 (2H, d,
J = 6.0 Hz), 4.02 (2H, t, J = 8.4 Hz), 3.05−2.96 (2H, m), 2.62 (1H,
m), 2.47 (2H, br s), 1.96−1.89 (4H, m), 1.73 (1H, m), 1.62−1.40
(9H, m), 1.29−1.24 (2H, m), 1.13−1.05 (2H, m). 13C NMR (150.9
MHz, CDCl3) δ (ppm): 162.4, 144.5, 134.1, 127.6, 127.5, 126.1,
119.42, 115.3, 103.9, 66.9, 61.3, 55.2, 54.7, 54.6, 46.4, 45.6, 27.6, 27.1,
26.7, 26.6, 26.5, 25.9, 25.6. HRMS (ES+, M + H) calcd for
C28H30N2O2, 418.858; found, 418.2857. Chiral separation: Semi-
preparative purifications were carried out via stacked injections on a
Waters Investigator SFC using a 10 mm × 250 mm Chiral
Technologies Chiralpak ID column heated to 40 °C. The eluent was
50% MeOH (0.1% DEA) in CO2 at a flow rate of 15 mL/min.
Backpressure was maintained at 100 bar. The first eluting peak (19S),
retention time = 3.97 min, was inferred as the S stereoisomer based
upon the absolute configuration observed in the electron density map
of the X-ray structure of the MIV-6R−menin complex. The second
eluting peak (19R), retention time = 5.38 min, was inferred as the R
stereoisomer based upon the absolute configuration observed in the
electron density map of the X-ray structure of the MIV-6R−menin
complex.
Expression and Purification of Human Menin. The expression
and purification of menin have been described previously.20,23
High Throughput Screening. The fluorescence polarization
assay20,23 was used for HTS of 288000 compounds at NIH MLPCN.
Specifically, menin at 200 nM and the 12 amino acid MLL-derived
peptide (MLL4−15) labeled with fluorescein or Texas Red, used at 25
and 50 nM, respectively, were utilized in the primary screening with
the dual fluorescence polarization read-out at 535 and 632 nm. The
primary screening was performed in 1536-well format using 4 μL final
volume and four concentrations of each compound (61, 11.7, 2.25, 0.5
μM). Compounds, which showed dose-dependent inhibition in
primary screen or at least 20% inhibition at the highest concentration
in both FP read-outs were selected for confirmation screen, resulting
in 240 compounds representing primary screening hits. The
homogenous time resolved FRET (HTRF) assay, with His−menin
to bind to anti-His Eu3+ cryptate donor beads and biotinylated
MLL4−15 peptide to bind to streptavidin-XL-665 acceptor beads, was
applied for confirmation screening performed at the concentration
range 383 μM to 0.01 nM using 2-fold dilutions of compounds
identified as hits in the primary screen. Donor and acceptor beads for
HTRF assay were purchased from Cisbio International. Total 62
compounds were validated to inhibit the menin−MLL interaction in
the confirmation HTRF assay and were subsequently tested in NMR
STD and competition NMR STD experiments (see below) for direct
and specific binding to menin. MLS001171971 (4-(2-hydroxy-3-(4-
(hydroxydiphenylmethyl)piperidin-1-yl)propoxy)benzonitrile, com-
pound 1, MIV-1) was the most potent compound identified by
HTS to inhibit the menin−MLL interaction and validated to bind to
menin by NMR STD. Detailed description of the HTS screen is
provided at PubChem Bioassay, AID 1766: http://pubchem.ncbi.nlm.
nih.gov/assay/assay.cgi?aid=1766.
Procedures for Synthesis of Compounds. Synthesis of
presented compounds was performed according to Schemes1−4.
Full experimental procedures, analytical data, NMR spectra, and chiral
separation of compounds reported here can be found within
Supporting Information. Synthesis and analytical data for representa-
tive compounds from hydroxy- (MIV-3) and aminomethylpiperidine
(MIV-6) classes are provided below.
Synthesis of 4-(3-(4-(Cyclopentyl(hydroxy)(phenyl)methyl)-
piperidin-1-yl)propoxy)benzonitrile, 11 (MIV-3). 4-(3-(4-
(Cyclopentanecarbonyl)piperidin-1-yl)propoxy)benzonitrile (30 mg,
0.09 mmol) was dissolved in THF (0.7 mL). Phenylmagnesium
bromide (1.0 M in THF) was added dropwise to the solution with
stirring. Reaction was then warmed to 50 °C, and stirring was
continued for 2 h. Reaction was quenched with satd aqueous NH4Cl
and extracted with EtOAc. The combined organic fractions were
washed with saturated NaCl and dried over Na2SO4. Concentration in
vacuo provided the crude product, which was purified by flash column
chromatography (9:1 CH2Cl2/MeOH) to provide 46 mg of the
desired products (74% yield). 1H NMR (400 MHz, CDCl3) δ (ppm):
7.54 (2H, d, J = 9 Hz), 7.37 (2H, d, J = 8 Hz), 7.30 (2H, t, J = 8 Hz),
7.22 (1H, t, J = 7 Hz), 6.88 (2H, d, J = 9 Hz), 4.07 (2H, t, J = 6 Hz),
3.21 (2H, m), 2.67 (3H, m), 2.19 (2H, m), 2.05 (2H, m), 1.95 (1H,
m), 1.82 (1H, m), 1.72 (1H, m), 1.64 (1H, m), 1.59−1.40 (7H, m),
1.25 (1H, m), 1.07 (1H, m). 13C NMR (100.6 MHz, CDCl3) δ (ppm):
162.4, 143.9, 134.0, 127.7, 126.5, 126.4, 119.4, 115.3, 103.8, 79.6, 68.9,
55.2, 54.5, 54.4, 45.8, 45.3, 27.5, 27.3, 26.7, 26.4, 26.1, 26.0, 25.6.
HRMS (ES+, M + H) calcd for C27H35N2O2, 419.2699; found,
419.2698. Chiral separation: Semipreparative purifications were carried
out via stacked injections on a Waters Investigator SFC using a 10 mm
× 250 mm Chiral Technologies Chiralpak IA column heated to 40 °C.
The eluent was 55% EtOH (0.1% DEA) in CO2 at a flow rate of 15
mL/min. Backpressure was maintained at 100 bar. The first eluting
peak (11S), retention time = 0.95 min, was inferred as the S
stereoisomer based upon the absolute configuration observed in the
electron density map of the X-ray structure of the MIV-3S−menin
complex. The second eluting peak (11R), retention time = 2.3 min, was
inferred as the R stereoisomer based upon the absolute configuration
observed in the electron density map of the X-ray structure of the
MIV-3R−menin complex.
Synthesis of 4-(3-(4-(Cyclopentyl(amino)(phenyl)methyl)-
piperidin-1-yl)propoxy)benzonitrile, 19 (MIV-6). A CHCl3 (4.78
mL, 0.25 M) solution of 4-(3-(4-(cyclopentyl(hydroxy)(phenyl)-
methyl)piperidin-1-yl)propoxy)benzonitrile (1.0 g, 2.39 mmol) and
sodium azide (1.16 g, 17.9 mmol) was cooled to 0 °C. To the solution
H2SO4 was added dropwise (0.28 mL, 9.3 mmol). The mixture was
allowed to warm to rt over 4 h with stirring, then cooled to 0 °C and
treated with NH4OH until pH was basic. The biphasic solution was
extracted with CH2Cl2 (3×) and the organic layers combined and
dried over MgSO4. Concentration under reduced pressure and
Biochemical Characterization of Menin−MLL Inhibitors.
Inhibition of the menin−MLL interaction by small molecules was
assessed by fluorescence polarization (FP) assay using the protocol
described previously.20,25
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NMR Spectroscopy. NMR samples for the saturation transfer
difference (STD) experiments contained 2.5 μM menin solution in 50
mM phosphate buffer, 50 mM NaCl, 1 mM DTT, pH = 7.5, with 5%
of D2O. The MIV-1 compound was added as stock solutions in
DMSO to final 100 μM concentration and 5% DMSO. All NMR
experiments were recorded at 25 °C using 600 MHz Bruker Avance III
spectrometer. For the STD experiments, we used 2 s irradiation using
a train of 50 ms Gaussian pulses centered at 0 ppm with published
pulse sequence.32 Samples for the competition STD experiments
contained additional MLL1−46 peptide at final concentration of 10 μM,
and the measurements were carried out in the same way as described
above for STD experiments.
Isothermal Titration Calorimetry. Menin was extensively
dialyzed at 4 °C against ITC buffer (50 mM phosphate, pH 7.5, 50
mM NaCl, 1 mM β-mercaptoethanol) and degassed prior to
measurement. Compounds were dissolved in DMSO and diluted
with the ITC buffer to final concentrations (50−100 μM, 5% DMSO).
Protein solution was adjusted to contain 5% DMSO final
concentration. The titrations were performed using a VP-ITC titration
calorimetric system (MicroCal) at 25 °C. The calorimetric cell,
containing menin (concentrations in the range 5−10 μM), was titrated
with the compounds (50−100 μM) injected in 10 μL aliquots. Data
was analyzed using Origin 7.0 (OriginLab) to obtain Kd and
stoichiometry.
Real-Time PCR. Effect of menin−MLL inhibitors on expression
level of Hoxa9 and Meis1 was assessed by real-time quantitative PCR
(qRT-PCR) using the protocol described previously.23
Expression of CD11b. MLL-AF9 transformed bone marrow cells
were plated in 12-well plates at an initial concentration of 3 × 105
cells/mL and treated with compounds or 0.25% DMSO. Media were
changed at days three and five with viable cell concentration restored
to 3 × 105 cells/mL and compounds resupplied. Seven days after the
experiment was setup, the 1.5 × 105 cells were harvested and washed
with FACS buffer (PBS, 1% FBS, 0.1% NaN3). Cells were resuspended
in 100 μL of FACS buffer and incubated with 1 μL of Pacific Blue rat
antimouse CD11b antibody (BD BioLegend) at 4 °C for 30 min. Cells
were then washed, resuspended in 100 μL of Annexin V binding
buffer, and incubated with 4 μL of Annexin V-FITC (BD Biosciences)
and 6 μL of propidium iodide (1 mg/mL, Sigma-Aldrich) at room
temperature for 10 min before being analyzed by flow cytometry.
Cytospin/Wright−Giemsa Staining. Mouse bone marrow cells
transduced with MLL-AF9 were plated in 12-well plates (1 mL/well)
at an initial concentration of 3 × 105/mL cells, treated with
compounds or 0.25% DMSO and incubated at 37 °C in a 5% CO2
incubator. Cytospins were prepared as described previously25 at
designated time points.
ASSOCIATED CONTENT
■
Crystallization of Menin Complexes with Small-Molecule
Inhibitors. For co-crystallization experiments, 2.5 mg/mL menin was
incubated with small-molecule inhibitors (MIV-3R, MIV-3S, MIV-4,
MIV-5, MIV-6R, MIV-7) at 1:3 molar ratio. Crystals were obtained
using the sitting drop technique at 10 °C by applying the procedure
described previously.23 Prior to data collection, crystals were
transferred into a cryosolution containing 20% PEG550 MME and
flash-frozen in liquid nitrogen.
S
* Supporting Information
Crystallography data, experimental data, characterization of
compounds by HRMS (ES+, M + H), 1H NMR and 13C NMR,
and chiral SFC traces. This material is available free of charge
via the Internet at http://pubs.acs.org.
Accession Codes
The atomic coordinate files have been deposited in the Protein
Data Bank with the PDB codes 4GO3, 4GO4, 4GO5, 4GO6,
4GO7, and 4GO8.
Crystallographic Data Collection and Structure Determi-
nation. Diffraction data for menin and menin complexes were
collected at the 21-ID-D and 21-ID-F beamlines at the Life Sciences
Collaborative Access Team at the Advanced Photon Source. Data were
processed with HKL-2000.33 Structures of the complexes were
determined by molecular replacement using MOLREP with the apo-
structure of human menin (PDB code: 4GPQ) as a search model in
molecular replacement. The model was refined using REFMAC,34
COOT,35 and the CCP4 package.36 In the final stages, refinement was
performed with addition of the TLS groups defined by the TLSMD
server.37 Validation of the structures was performed using MOLPRO-
BITY38 and ADIT.39 Details of data processing and refinement are
summarized in Supporting Information Table 1. Coordinates and
structure factors have been deposited in the Protein Data Bank.
Co-immunoprecipitation Experiments. HEK293 cells were
transfected with Flag−MLL-AF9 plasmid using Fugene 6 (Roche).
Forty-eight hours after transfection, cells were treated with compounds
(0.25% final DMSO concentration) or DMSO for 12 h. Whole cell
lysates were immunoprecipitated with ANTI-FLAG M-2 magnetic
beads (Sigma-Aldrich) and analyzed by SDS-PAGE and Western
blotting. For more details, see Grembecka et al.25
Viability Assays. MLL-AF9 transformed mouse bone marrow cells
were prepared as described previously.40 MV4;11, KOPN-8, ML-2,
and MOLM-13 cells were cultured in RPMI-1640 medium with 10%
FBS, 1% penicillin/streptomycin, and NEAA. For viability assay,
MOLM-13 (1 × 105/mL), MV4;11 (1 × 105/mL), HL-60 (2 × 105/
mL), and MLL-AF9 mouse bone marrow cells (2.5 × 104/mL) were
plated (1 mL/well), treated with compounds or 0.25% DMSO, and
cultured at 37 °C for 7 days. Media were changed at day four with
viable cell number restored to the original concentration, and
compounds were resupplied. Then 100 μL of cell suspension were
transferred to 96-well plates for each sample in quadruplicates. A
Vybrant MTT cell proliferation assay kit (Molecular Probes) was
employed. Plates were read for absorbance at 570 nm using a
PHERAstar BMG microplate reader. The experiments were performed
three times in quadruplicated with calculation of mean and standard
deviation for each condition.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 734-615-9319. Fax: 734-615-0688. E-mail: jolantag@
umich.edu.
Author Contributions
#S.H. and T.J.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by the National Institute of Health
grants: R03 (1R03MH084875) to J.G. and T.C., R01
(1R01CA160467) to J.G., MLPCN grants (1U54
MH084659) a Leukemia and Lymphoma Society (LLS) TRP
grant (6116-12) to J.G., LLS Scholar (1215-14) to J.G.,
American Cancer Society grants (RSG-11-082-01-DMC to
T.C. and RSG-13-130-01-CDD to J.G.), ACS MEDI
Predoctoral Fellowship (T.J.S), and the Department of
Pathology, University of Michigan. Use of the Advance Photon
Source was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences under
contract number DE-AC02-06CH11357. Use of the LS-CAT
Sector 21 was supported by the Michigan Economic Develop-
ment Corporation and the Michigan Technology Tri-Corridor
for the support of this research program (grant 085P1000817).
We thank Mathew Mulder for HRMS determination and
Nathan Kett for preparative chiral SFC. We thank Jay L. Hess
and Andrew G. Muntean for cell lines.
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Journal of Medicinal Chemistry
Article
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ABBREVIATIONS USED
■
FP, fluorescence polarization; HTRF, homogenous time
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MLPCN, Molecular Libraries Probe Production Centers
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