Synthesis, optimization, and evaluation of novel small molecules as antagonists of WDR5-MLL interaction

Article abstract of DOI:10.1021/ml300467n

The WD40-repeat protein WDR5 plays a critical role in maintaining the integrity of MLL complexes and fully activating their methyltransferase function. MLL complexes, the trithorax-like family of SET1 methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they have been widely implicated in various cancers. Antagonism of WDR5 and MLL subunit interaction by small molecules has recently been presented as a practical way to inhibit activity of the MLL1 complex, and N-(2-(4-methylpiperazin-1-yl)-5-substituted- phenyl) benzamides were reported as potent and selective antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compound 2 (K_dis = 7 μM), leading to identification of more potent antagonist 47 (K_dis = 0.3 μM).

Full text of DOI:10.1021/ml300467n

Letter
pubs.acs.org/acsmedchemlett
Synthesis, Optimization, and Evaluation of Novel Small Molecules as
Antagonists of WDR5‑MLL Interaction
Yuri Bolshan,†,§ Matthaus Getlik,‡ Ekaterina Kuznetsova,† Gregory A. Wasney,† Taraneh Hajian,†
̈
Gennadiy Poda,‡ Kong T. Nguyen,† Hong Wu,† Ludmila Dombrovski,† Aiping Dong,†
Guillermo Senisterra,† Matthieu Schapira,† Cheryl H. Arrowsmith,† Peter J. Brown,† Rima Al-awar,‡
Masoud Vedadi,*,† and David Smil*,†,§
†Structural Genomics Consortium, University of Toronto, 101 College Street, MaRS Centre, South Tower, Toronto, Ontario M5G
1L7, Canada
‡Medicinal Chemistry Platform, Ontario Institute for Cancer Research, 101 College Street, MaRS Centre, South Tower, Toronto,
Ontario M5G 0A3, Canada
S
* Supporting Information
ABSTRACT: The WD40-repeat protein WDR5 plays a critical role in
maintaining the integrity of MLL complexes and fully activating their
methyltransferase function. MLL complexes, the trithorax-like family of SET1
methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they
have been widely implicated in various cancers. Antagonism of WDR5 and MLL
subunit interaction by small molecules has recently been presented as a practical
way to inhibit activity of the MLL1 complex, and N-(2-(4-methylpiperazin-1-yl)-
5-substituted-phenyl) benzamides were reported as potent and selective
antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compound
2 (Kdis = 7 μM), leading to identification of more potent antagonist 47 (Kdis = 0.3 μM).
KEYWORDS: WDR5, MLL, SET1 methyltransferase complex, peptide binding site, small molecule antagonists
he SET1-family of human histone methyltransferases
MLLs have been reported to be essential for embryonic
T(SET1A, SET1B, MLL1, MLL2, MLL3, and MLL4) development and biological processes14−17 and have been
widely implicated in a variety of cancers.18−22 As almost all
contribute to active chromatin via mono-, di-, and trimethyla-
tion of lysine 4 on histone H3.1−3 MLLs are S-adenosyl
methionine (SAM) dependent lysine methyltransferases that
MLLs show minimal or no activity in the absence of the
complex components (WDR5 in particular),13 identifying
WDR5−MLL interaction antagonists has been proposed as a
potentially selective alternative to active site directed inhibitors
of MLL methyltransferase activity. Such antagonists could serve
as a starting point for the development of potential therapeutics
to treat MLL-rearranged leukemias or other related cancers.23
Recent reports detailing the use of short arginine containing
peptides to bind WDR5 and disrupt its interaction with MLL,24
along with the use of the WIN motif containing peptides to
reduce the levels of H3K4 dimethylation by the MLL core
complex in vitro,9 provide additional rationale for targeting
WDR5 as a means to antagonize the MLL and SET1 family of
HMTs. However, to the best of our knowledge, the only
reported nonpeptide, small molecule antagonist of the WD40-
repeat domain is a racemic 1,1′-binaphthyl-2,2′-dicarboxylic
acid, SCF-I2 (1, Figure 1), an inhibitor of yeast Cdc4.25
As part of our ongoing research involving the discovery and
development of potent, selective, and cell-permeable small
molecule inhibitors (chemical probes) for epigenetic proteins,26
work optimally when in complex with other components;
WDR5 (WD40 repeat protein 5), RbBP5 (retinoblastoma-
binding protein-5), ASH2L (absent small homeotic disks-2-
like), and DPY-30 (WRAD).4 WDR5 has been shown to be
essential for complex integrity and for fully activating MLL
methyltransferase function.5−7
In the peptide-bound structure, WDR5 adopts a donut-
shaped WD40-repeat fold with a deep central cavity that
normally accommodates an arginine side chain of interacting
peptides.8 WDR5 binds to the “WDR5 INteracting” (WIN)
motif of MLL1 through the peptidyl arginine-binding cleft.8−12
A crystal structure of WDR5 in complex with MLL1 WIN
peptide revealed the critical role of arginine 3765 of MLL1 in
complex formation.9 A similar interaction has been reported for
WDR5 and the WIN region of all SET1 family lysine
methyltransferases, with reported KD values of 1.13, 0.16,
2.03, 0.03, 0.26, and 0.10 μM for MLL1-4, SET1A, and SET1B
WIN peptides, respectively.13 Although there is significant
divergence in the amino acid sequences of WIN motifs close to
the critical arginine residue, the plasticity of the WDR5 arginine
binding pocket allows such an interaction with the WIN motifs
of all MLLs.13
Received: December 20, 2012
Accepted: February 4, 2013
Published: February 4, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Compounds 2 and 4−18
Figure 1. Structures of reported WD40-repeat domain antagonists.
we recently disclosed several novel compounds that antagonize
WDR5−MLL interaction, including WDR5-0102 (2) (Figure
1).23 This proof of principle study prompted us to undertake
the SAR-driven optimization of prototypic antagonist WDR5-
0102 (2) described in this Letter, leading eventually to
preparation of the more potent antagonist 47.
Significantly, a high resolution X-ray crystal structure we
obtained for WDR5 in complex with 2 (PDB code 3SMR)
showed the compound to be residing within the binding pocket
(Figure 2), which normally accommodates the arginine side
a
Reagents and conditions: (a) 2-chlorobenzoyl chloride, pyridine,
CH2Cl2, 4 h, rt. (b) Amine, N,N-diisopropylethylamine, DMF, 2 h, 80
°C. (c) Trifluoroacetic acid, CH2Cl2, 1 h, rt.
benzamides 2 and 5−18 in high overall yields. In several cases
(13, 15, 17), an additional step involving Boc deprotection was
required.
These compounds (Table 1) were then evaluated using a
fluorescence polarization (FP) assay, detecting the displace-
Table 1. Modifications of the N-Methylpiperazine Moiety at
C-2
no.
R1 (2° amine)
1-methylpiperazine
Kdis (μM)
2
4
6.9 1.5
F
>20
Figure 2. X-ray crystal structure of the WDR5−2 complex (PDB code
3SMR). Compound 2 is in magenta.
5
1,2-dimethylpiperazine
1-methyl-1,4-diazepane
morpholine
4.7 0.8
7.0 1.1
6
7
>20
chain of H3 peptides and interacting MLL.11,12,23 The amide
group of 2 forms one direct and one water-mediated (W517)
hydrogen bond with the side chain of S91 and the backbone
nitrogen of C261, respectively. The N-methylpiperazine
moiety, predicted to be significantly protonated at physiological
pH,27 forms a water-mediated (W576) hydrogen bond with the
backbone carbonyl of C261, and it occupies the bottom of the
pocket. The 2-chlorophenyl group lies in a shallow, solvent
exposed, and largely hydrophobic cavity (also normally
occupied by H3 and MLL peptides), surrounded by L321,
A47, D107, and the aliphatic portion of the S49 and S91 side
chains. Due to the flipping of the F133 and F149 side chains,
the nitro group occupies a hydrophobic cleft that is occluded in
most WDR5 structures. These distinct structural features
suggested a systematic optimization of the core benzamide
template through independent variation of the 2-chlorophenyl
(benzamide) moiety at position C-1, the N-methylpiperazine at
C-2, and the nitro substituent at C-5.
8
piperidine
>20
>20
9
1-ethylpiperazine
10
11
13
15
17
18
N1,1-dimethylpyrrolidin-3-amine
N1,1-dimethylpiperidin-4-amine
piperazine
15.8 2.0
>20
>20
>20
>20
>20
pyrrolidin-3-amine
piperidin-4-amine
N1,1,2-trimethylethane-1,2-diamine
ment of fluorescein-labeled H3(1-15) peptide (ARTKQ-
TARKSTGGKA) from its binding pocket in WDR5.23 In this
assay, binding of the fluorescein-labeled peptide to WDR5 (KD
= 458 45 nM) increases the FP signal. Binding compounds
compete with and displace the labeled peptide, resulting in the
reduction of signal. By monitoring the change in FP signal
upon varying the concentration of compounds, Kdisplacement (Kdis)
values can be calculated by fitting the data to a hyperbolic
function as described in the Supporting Information.
We were surprised to see that, despite the available space and
potential for hydrogen bond formation with residue D92 at the
bottom of the pocket, almost all of the N-methylpiperazine
modifications produced less active compounds. Demethylation
(13), replacement (7−8), and ethylation (9) of the nitrogen
proved detrimental, pointing toward the criticality of the water
An efficient two-step synthetic sequence was developed to
first explore the N-methylpiperazine region of the scaffold
(Scheme 1). Commercially available 2-fluoro-5-nitroaniline (3)
was initially reacted with 2-chlorobenzoyl chloride to access 2-
chloro-N-(2-fluoro-5-nitrophenyl)benzamide (4). Subsequent
displacement of the fluorine from this common intermediate
using a variety of secondary amines gave rise to the substituted
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ACS Medicinal Chemistry Letters
Letter
mediated hydrogen bond to C261, and the effect of
conformational and steric constraints within this pocket.
Further emphasizing the importance of the N-methylpiperazine
conformation is ring-opened compound 18, which also showed
a dramatic loss in activity. Only slight expansion of the ring (6)
produced a viable compound equipotent to 2, while ring
contraction (10) resulted in a 2-fold loss of potency. While
efforts at forming hydrogen bonds at the bottom of the pocket
with compounds 11 and 17 failed, methylated compound 5 was
tolerated. However, given the cost and availability of 1,2-
dimethylpiperazine, we opted to retain the original N-
methylpiperazine moiety for the synthesis of subsequent
analogs.
Table 2. Modifications of the 2-Chlorophenyl (Benzamide)
Moiety at C-1
no.
R1
Kdis (μM)
2
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
2-Cl-phenyl
Me
6.9 1.5
>10
3-Cl-phenyl
3-Me-phenyl
2-Cl-3-Me-phenyl
3-OH-phenyl
3-OMe-phenyl
4-F-phenyl
1.8 0.1
1.1 0.1
1.1 0.1
To vary the benzamide moiety, a modified two-step synthetic
sequence was employed (Scheme 2). Commercially available 2-
a
>10
Scheme 2. Synthesis of Compounds 37−54
1.4 0.1
3.1 0.3
0.63 0.1
0.59 0.1
0.68 0.1
0.27 0.1
5.8 0.4
2-Cl-4-F-phenyl
3-Me-4-F-phenyl
3-OMe-4-F-phenyl
2-Cl-3-Me-4-F-phenyl
phenyl
cyclohexyl
>10
1-naphthyl
0.71 0.1
1.5 0.2
5-quinolyl
benzyl
>10
3-pyridyl
>10
>10
2-furanyl
a
Reagents and conditions: (a) R1COCl, pyridine, CH2Cl2, 4 h, rt. (b)
1-Methylpiperazine, N,N-diisopropylethylamine, DMF, 2 h, 80 °C.
versus the series parent 2, 47 is the most significant compound
synthesized in this study.
The same core, two-step sequence used to prepare the
benzamide analogs 37−54 was used once again to introduce
substituents modifying the prototypic nitro group at C-5 to give
compounds 62−69 (Scheme 3). In this instance, however, the
sequence was also initiated with a number of 2-fluoroanilines
(55−57) variably substituted at the 5-position, allowing for
access to final compounds 62−64 and 66−69. Reduction and
hydrolysis were used to generate compounds 65 and 66 from 2
fluoro-5-nitroaniline (3) was used once again, but now it
reacted with a series of different acid chlorides to afford
substituted N-(2-fluoro-5-nitrophenyl)benzamides 19−36. Dis-
placement of the fluorine with N-methylpiperazine proceeded
as before, giving rise to the substituted benzamides 37−54 in
high overall yields.
In general, modifications of the benzamide moiety proved to
have a more variable impact on potency (Table 2). The
fundamental need for an aromatic substituent directly attached
to the carbonyl of the benzamide is made clear by the relative
activity of compounds 37, 49, and 52 versus compound 48.
Reducing the size of this aromatic substituent (54) appears
detrimental, while increasing its size (50) affords a substantial
gain in potency, resulting from greater surface interactions with
the hydrophobic pocket.
a
Scheme 3. Synthesis of Compounds 2 and 62−69
Interestingly, the introduction of a nitrogen into the aromatic
moiety (51 and 53) reduces activity (versus parent compounds
50 and 48, respectively), failing to make any hydrogen bond
interactions. Particularly significant are variable substitutions
made at the 3-position of the aromatic ring (38−42), all of
which, with the exception of 41, resulted in an appreciable
potency gain. Interestingly, further gains in potency were
achievable through monofluorination at the 4-position of 2, 40,
39, 42, and 48, leading to 10-fold (44), 4-fold (47), and 2-fold
(45−46, 43) more active compounds, respectively. While the
4-F substituent may serve to increase potencies by strengthen-
ing the amide-to-S91 hydrogen bond interaction via an electron
withdrawing effect, such a rationale requires further inves-
tigation. It should be noted that, with a 25-fold greater potency
a
Reagents and conditions: (a) 2-chlorobenzoyl chloride, pyridine,
CH2Cl2, 4 h, rt. (b) 1-Methylpiperazine, N,N-diisopropylethylamine,
DMF, 2 h, 80 °C. (c) SnCl2, conc HCl, 1 h, 60 °C. (d) LiOH·H2O,
THF/H2O, 1 h, rt. (e) R1B(OH)2, Pd(PPh3)4 (cat.), Na2CO3,
toluene/H2O (1:1), 16 h, 110 °C.
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ACS Medicinal Chemistry Letters
Letter
and 62 respectively, while compounds 67−69 were accessible
via Suzuki coupling from precursor 64.
Variations of the C-5 position substituent proved disappoint-
ing, producing largely inactive compounds (Table 3). Protic
Table 3. Modifications of the Nitro Group at C-5
no.
R1
NO2
Kdis (μM)
2
62
63
64
65
66
67
68
69
6.9 1.5
6.6 1.1
CO2Me
CF3
>10
Figure 3. X-ray crystal structure of the WDR5−47 complex (PDB
code 4IA9). Compound 47 (gray sticks) binds in the central cavity of
WDR5. Addition of methyl and fluoro substituents at positions 3 and 4
of the 2-chlorophenyl moiety optimizes occupancy of the side cavity
and introduces additional interactions with surrounding hydrophobic
side chains (orange). Color coding: white, carbon; red, oxygen; blue,
nitrogen; green, chlorine; magenta, fluorine.
Br
>10
>10
>10
>10
NH2
CO2H
cyclopropyl
2-furanyl
4-pyridyl
3.5 0.3
2.2 0.1
groups such as amine (65) and acid (66) were not tolerated
within the hydrophobic cleft, although the ester (62) was
accepted. While this was known from our previous work,23 we
opted to conduct the optimization outlined in this letter using
the nitro group at C-5, both for ease of synthesis and to limit
any potential liability arising from ester hydrolysis in eventual
cell-based studies. While smaller substituents (63−64, 67) also
failed to produce active compounds, the introduction of larger
aromatic groups such as 2-furan (68) or 4-pyridine (69) did
allow for a modest 2- or 3-fold potency increase. This
observation also opens the possibility of further enlarging the
C-5 position substituent to more fully occupy the hydrophobic
cleft, and potentially engage Y191.
through the synthesis of compound 47. Having begun to
establish the parameters of moieties tolerated within the WDR5
binding site regions, we intend to leverage this information for
the continued structure-based design and development of new
compounds for antagonism of the WDR5−MLL interaction.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic experimental procedures, analytical and spectral
characterization data of all synthesized compounds, and details
of biochemical assay methods. This material is available free of
charge via the Internet at http://pubs.acs.org.
We had previously shown that positioning of the benzamide
ring in the shallow side cavity of the binding site can be shifted
depending on the type and position of its substituents, with
potentially significant impact on activity.23 To better under-
stand the increased potency of compound 47, we solved its
structure in complex with WDR5 (PDB code 4IA9) (Figure 3).
As expected, the compound occupies the central cavity of
WDR5 and recapitulates interactions previously observed with
less potent analogs (PDB codes 3SMR and 3UR4), including
direct and water mediated hydrogen bonds with S91 and C261,
opening-up of a cavity occupied by the nitro group, and
aromatic stacking with F133.23 The conformation of side chains
within 4 Å of the compound is almost perfectly conserved
(RMSD = 0.15 Å). The scaffold sits in the pocket, adopting a
conformation closer to that observed for the 2-chlorophenyl
analog (RMSD = 0.33 Å, PDB code 3SMR) versus that of the
3-methoxyphenyl analog (RMSD = 1.47 Å, PDB code 3UR4).
The new structure clearly shows that the 3-methyl and 4-fluoro
substituents bring hydrophobic interactions with side-chains
lining the binding pocket and that the section of the ring facing
D107 remains unsubstituted, suggesting avenues for further
optimization.
AUTHOR INFORMATION
Corresponding Author
*D.S.: tel, +1 (416) 946-0132; fax, +1 (416) 946-0880; e-mail,
david.smil@utoronto.ca. M.V.: tel, +1 (416) 432-1980; fax, +1
(416) 946-0588; e-mail, m.vedadi@utoronto.ca.
■
Author Contributions
§These authors contributed equally to this work. Y.B. and M.G.
designed and synthesized compounds, and contributed to
editing of the paper. E.K. and G.A.W. performed all FP assays
(Table 1, 2 and 3). T.H. purified protein for assays. G.P. and
K.T.N. contributed to the design of compounds. H.W. and L.D.
purified and co-crystallized WDR5 with compound 47. A.D.
determined the crystal structure of WDR5-47. G.S. performed
binding assays (Supplementary Figure 1). M.S. contributed to
the design of compounds, analyzed the WDR5-47 co-structure
(Figure 2 and Figure 3), and wrote the paper. C.H.A., P.J.B. and
R.A. contributed to experimental design and editing of the
paper. M.V. and D.S. reviewed data, contributed to overall
experimental design, and wrote the paper. M.V. was the
principal investigator, designed experiments, analysed data and
supervised the assay and compound evaluation process. D.S.
designed and synthesized compounds, and supervised all
chemistry efforts.
Overall, by independently modifying the benzamide, C-2,
and C-5 position substituents on parent compound 2, we were
ultimately able to realize a greater than 25-fold gain in potency
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ACS Medicinal Chemistry Letters
Letter
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The Structural Genomics Consortium is a registered charity
(number 1097737) that receives funds from the Canadian
Institutes for Health Research, Genome Canada, the Canadian
Foundation for Innovation, the Ontario Innovation Trust, the
Ontario Ministry for Research and Innovation, the Wellcome
Trust, AbbVie, Eli Lilly Canada, GlaxoSmithKline, Novartis,
Pfizer, and Takeda. Ontario Institute for Cancer Research
funding is provided by the Government of Ontario through the
Ontario Ministry of Economic Development and Innovation.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We would like to thank Dr. Ahmed Aman and Dr. Taira Kiyota
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