Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00493

We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.

Full text of DOI:10.1021/acsmedchemlett.9b00493

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Letter
Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors
Dai-Shi Su, Junya Qu, Mark Schulz, Chuck W Blackledge, Hongyi Yu, Jenny Zeng, Joelle Burgress,
Alexander Reif, Melissa Stern, Raman Nagarajan, Melissa Baker Pappalardi, Kristen Wong, Alan P.
Graves, William Bennette, Liping Wang, Patricia Elkins, Beth Knapp-Reed, Jeffrey D. Carson, Charles
McHugh, Helai Mohammad, Ryan Kruger, Juan Luengo, Dirk A. Heerding, and Caretha L. Creasy
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00493 • Publication Date (Web): 27 Dec 2019
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Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors
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Dai-Shi Su,*,† Junya Qu,† Mark Schulz,† Chuck W. Blackledge,† Hongyi Yu,† Jenny Zeng,†
Joelle Burgess,† Alexander Reif,† Melissa Stern,‡ Raman Nagarajan,‡ Melissa Baker Pappalardi,‡
Kristen Wong,‡ Alan P. Graves,§ William Bonnette,║, ≠ Liping Wang,║ Patricia Elkins,║ Beth
Knapp-Reed,† Jeffrey D. Carson,║ Charles McHugh,‡ Helai Mohammad,‡ Ryan Kruger,‡ Juan
Luengo,†,≠ Dirk A. Heerding,†, ≠ Caretha L. Creasy‡
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†Medicinal Chemistry, Medicine Design, ‡Oncology R&D, § Data and Computational Sciences, ║
Protein Cellular and Structural Sciences, Medicine Design, Medicinal Science and Technology
GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, 19426, USA
ABSTRACT:
We report herein the discovery of isoxazole amides as potent and selective SET and MYND
Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity
relationship of the high-throughput screening (HTS) lead compound 1 provided potent and
selective SMYD3 inhibitors. The SAR optimization, co-crystal structures of small molecules with
SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The
synthesis, biological and pharmacokinetic profiles of compounds are also presented.
Keywords: SMYD3, MEKK2, inhibitors, isoxazole amides
INTRODUCTION
Lysine methylation represents a fundamental regulatory mechanism that influences protein
function.1-2 Lysine methylation often serves to recruit proteins with lysine specific reader domains
thereby mediating transcriptional changes, as is the case for methylation of histones. However,
lysine methylation can also alter the enzymatic activity of methylated proteins and/or
promote/inhibit other post translational modifications.1 SET and MYND Domain-Containing
Protein 3 (SMYD3) is a protein lysine methyltransferase shown to catalyze methylation of histone
and non-histone proteins including methylation of Histone H4 at lysine 5 and Mitogen-activated
protein kinase kinase 2 (MEKK2) at lysine 260.3-4
MEKK2 methylation by SMYD3 regulates the MEK/ERK kinase signaling pathway in RAS-
driven tumors.4 In both lung and pancreatic models of cancer, abrogation of SMYD3 methylase
activity inhibits tumorigenesis in response to RAS activation suggesting an opportunity to target
the methyltransferase activity of SMYD3 in cancers with elevated RAS activity. Oncogenic RAS
≠ Current address: RN: University of California Davis; WGB: Janssen Pharmaceuticals, Spring
House, PA 19477; JL: Prelude Therapeutics; DAH: retired
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mutations are among the most frequent alterations in cancer; therefore, the development of
compounds which inhibit signaling downstream of RAS represents a unique approach to target
RAS-driven cancers.
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9
Histone H4 is one of the five histone proteins; featuring a main globular domain and a long N-
terminal tail. In addition to being critical to the structure of nucleosomes, histones are highly post-
translationally modified and these epigenetic modifications influence nucleosome positioning and
the recruitment of transcriptional activators and/or inhibitors thereby regulating gene transcription.
Epigenetic modifications play an important role in the regulation of many cellular processes
including cell proliferation, differentiation, and cell survival. Global epigenetic dysregulation is
common in cancer and includes global changes in DNA and/or histone methylation, dysregulation
of non-coding RNAs and nucleosome remodeling leading to aberrant activation or inactivation of
oncogenes, tumor suppressors and signaling pathways. However, unlike genetic mutations, these
epigenetic changes can be reversed through selective inhibition of the enzymes involved. Thus,
selective inhibitors of dysregulated methyltransferases could be useful in the treatment of
proliferative diseases such as cancer.
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SMYD3 amplification and/or increased expression has been observed in numerous solid tumors
including those of colorectal, breast, prostate, ovarian, gastric and hepatocellular origin and in
some instances, correlates with poor prognosis.6-9 Studies have reported that direct knockdown of
SMYD3 via siRNA or shRNA decreases cancer cell line proliferation in a number of cancer cell
types including breast, liver, colon and prostate.3, 6, 10-11 Thus, there is evidence to suggest that
inhibition of SMYD3 activity decreases cellular proliferation. Accordingly, compounds that
inhibit SMYD3 activity hold promise as novel therapeutic agents for the treatment of cancer.
While small molecule SMYD3 inhibitors have been reported previously,12-13 this paper reports the
SAR results of a novel class of compounds that exhibit good potency, and excellent selectivity and
pharmacokinetics profiles. Additionally, the synthesis, crystallography, mode of inhibition (MOI)
characterization, and cellular mechanistic activity, of these compounds are described.
RESULTS AND DISCUSSIONS
To identify inhibitors of SMYD3, a high-throughput screen (HTS) was conducted using the GSK
proprietary compound collection and a SMYD3 biochemical SPA format that measures the
inhibition of MEKK2 methylation. Compound 1, an isoxazole amide analog which is closely
related to another recently reported SMYD3 inhibitor, EPZ028862,13 was identified and presented
as a good starting point for our chemistry effort (Table 1). The molecule has reasonable
biochemical potency (IC50 = 5 M), ligand efficiency (LE = 0.35), and physicochemical properties
(Table 1). Compound 1 also possesses an excellent PK profile (vide infra). To facilitate chemistry
efforts, we obtained the co-crystal structure of SMYD3 with compound 1 (Figure 1). Compound
1 is bound in the lysine tunnel of SMYD3 in close proximity to the SAM binding site. The
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cyclopropyl group of compound 1 points to SAM and the piperidine sulfonamide points toward
the substrate peptide binding site. The other key observations are that a hydrogen bond interaction
is made between the amide oxygen of compound 1 and the backbone amine of Thr184. This
interaction mimics that made by the backbone carbonyl of the substrate lysine observed in the
crystal structure of SMYD3 bound to a MEKK2 peptide substrate.14 The piperidine sulfonamide
of compound 1 does not appear to make any significant interactions in the rather large and polar
substrate pocket; therefore, focus was placed on optimization of this portion of the compound as
will be described in more detail below.
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O
O
S
O
N
N
O
N
H
1
Compound
A.
B.
Figure 1. A. Structure of Compound 1; B. The crystal structure of Compound 1 (green carbons)
bound to SMYD3 overlaid with a MEKK2 peptide (pink carbons) from PDB 5HQ8.14 The co-
factor SAM is colored with orange carbons and crystallographic waters are represented as red
spheres. Key hydrogen bond interactions between Compound 1 and SMYD3 are depicted with
yellow dashes. The binding site surface is colored by electrostatic potential. An alternate
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conformation observed for the sidechain of Glu192 has been omitted for clarity. All crystal
structure figures were generated with PyMOL (The PyMOL Molecular Graphics System, Version
2.0.7 Schrodinger, LLC)
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The initial SAR study around the left-hand side of the molecule, such as the replacement of
cyclopropyl, isoxazole and amide moiety, was largely not fruitful in terms of improving the
potency. Although the small methyl and ethyl isoxazoles showed similar potency as 1, they
provided no obvious advantage over cyclopropyl analogs. Modifications to the amide
functionality that presumably disrupted the H-bond between the amide oxygen and the backbone
NH of Thr184 were not tolerated for replacement and is in concert with the observation of the co-
crystal structure. On the basis of co-crystal analysis, we shifted our focus to the right-hand side of
the molecule. Gratifyingly, the potency of 1 can be improved 10-fold by introduction of a basic
amine group, like compound 3. The crystal structure of this compound bound to SMYD3 was
solved, and it was observed that the relatively small ethyl amine can bind in two conformations
(Figure 2). To improve potency, larger groups and constraints were explored to more optimally
direct the basic amine toward polar residues in the substrate pocket. Simply extending the amine
side chain by one carbon (4) further improved the biochemical potency 10-fold and showed an
EC50 potency of about 0.5 M in a cellular mechanistic assay developed to quantify inhibition of
MEKK2 methylation. Conversion of the primary amine to pyrrolidine (5 and 6) or piperidines (7-
9) resulted in similar biochemical IC50 values and a slight improvement in cellular potency in some
instances (presumably due to the enhancement of permeability). However, introduction of a ring
constraint in the linker to form cyclohexane amines improved the potency up to 15-fold in the
biochemical assay (10 and 11). The trans isomer 10 is more potent than the cis analog 11 in the
biochemical and cellular assays. Further extending the amine by insertion of methylene on the
right-hand side of compound 10 to form a cyclohexyl methyl amine (12) caused a loss of potency;
however, inserting a methylene group in the left-hand side of molecule 10 to form methyl
cyclohexyl amine (13) retained potency. Introduction of an aromatic phenyl ring as a linker
between the sulfonamide and amine provided compounds 14 – 18. A crystal structure of compound
14 with SMYD3 reveals that the sulfonamide oxygens make water mediated hydrogen bond
interactions with SMYD3 residues Ile214 and Met242 (Figure 3). The phenyl ring projects the
aminomethyl group toward a highly charged, solvent rich region of the SMYD3 substrate binding
site. The basic amine of compound 14 makes water mediated hydrogen bond interactions with
Lys297 and Glu294. The overall loss of potency (14-18) compared to compound 13 may be due
to the relative hydrophobicity and rigidity imposed by the aromatic phenyl.
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Table 1: SAR
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O
O
S
O
N
R
10
11
12
13
14
15
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17
18
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N
O
N
H
MEKK2 SPAa
IC50 (nM)
MEKK2meb
EC50 (nM)
CLNDd
(µM)
AMPe
(nm/sec)
Comp #
R
1
2
Me
Et
5010
7940
398
NDc
ND
ND
55
120
240
<10
306
375
3
4
CH2CH2NH2
CH2CH2CH2CH2
NH2
25
494
385
410
<10
50
5
6
126
1517
N
40
611
406
24
N
H
7
8
9
32
50
183
300
617
515
474
502
6.9
56
52
NH
N
100
N
10
11
1.6
10
71
329
420
<10
<10
NH2
NH2
160
12
13
25
237
74
479
297
<10
<10
NH2
NH2
2.5
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79
204
597
179
373
919
295
130
302
122
26
52
200
160
130
79
NH2
N
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N
16
N
100
N
ascintillation proximity assay; bmethylation of MEKK2 cellular mechanistic assay;
cnot determined; dCLND: Chemiluminescent nitrogen detection; eAMP: Artificial Membrane
Permeability
Figure 2. The crystal structure of Compound 3 (green carbons) bound to SMYD3. A second
conformation of the ethylamine of Compound 3 observed in the crystal structure is colored with
blue carbons, the co-factor SAM is colored with orange carbons and crystallographic waters are
represented as red spheres. Key hydrogen bond interactions are depicted with yellow dashes and
the binding site surface is colored by electrostatic potential. An alternate conformation observed
for the sidechain of Glu192 has been omitted for clarity.
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Figure 3. The crystal structure of Compound 14 (green carbons) bound to SMYD3. The co-
factor SAM is colored with orange carbons and crystallographic waters are represented as red
spheres. Key hydrogen bond interactions are depicted with yellow dashes and the binding site
surface is colored by electrostatic potential. A glycerol molecule that has partial occupancy with
the water which is in hydrogen bonding distance to the isoxazole and amide nitrogens of
Compound 14 and alternate conformations observed for the sidechains of Glu192 and Glu294
have been omitted for clarity.
Based on the excellent cellular potency, we focused on compound 13. While the basic amine
group may be key to improving the potency in compound 13, a primary amine has been known to
have some undesirable liability and associates with a very poor permeability (AMP <10 nm/sec,
compound 13). Aiming to modulate the physiochemical properties, the amine group was
substituted to form simple secondary amines (19-22) and a tertiary amine (23) that proved to be
less effective both in terms of potency and permeability (Table 2). Introduction of branched alkyl
groups on the amine (24-26) maintained good potency in cells and improved the permeability.
Incorporating fluorinated alkyl groups (27-30) improved the biochemical and cellular potency
further; in particular, compound 28 has an EC50 of 39 nM in the MEKK2me assay. A crystal
structure of compound 28 with SMYD3 suggests that this compound makes many of the same
interactions as compound 14 though the basic amine of compound 28 is stabilized by coming into
close proximity to the negatively charged sidechains of Glu192 and Asp241 rather than making
water mediated interactions with the protein (Figure 4). Compound 28 further displaces several
crytallographically observed water molecules with the trifluorobutane that projects the terminal
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CF3 group to bind in a hydrophobic pocket formed by the sidechains of Leu290 and Cys333 and
the aliphatic carbons of residues Glu294, Lys297 and Lys329.
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To balance both the potency and physicochemical properties, we substituted the amine with the
benzyl group (31, Table 2). Although compound 31 suffered with poor solubility (CLND) and
permeability, it showed a potency of 22 nM in the MEKK2me cellular assay. Substitution of the
phenyl ring with fluorine retained excellent potency (compounds 32- 34). The meta or ortho
fluorine substitutions provided compounds (33 and 34) with balanced potency, solubility, and
permeability. Replacement of fluorine with chlorine delivered compound 35 with excellent
potency in the MEKK2me cellular assay (EC50 = 11 nM). The other chlorine analogs (36-37) were
also very potent. Substitution of the phenyl ring with a methyl group (38-40) was less effective
compared to fluorine or chlorine. Replacement of the phenyl ring with pyridine (41-43) caused a
loss of potency; however, substitution of the pyridine ring with chlorine (44), methyl (45), and
fluorine (46-48) regained the potency.
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Table 2: SAR (continued)
R1
N
5
6
7
8
R2
O
S
O
O
N
9
N
O
N
10
11
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H
MEKK2 SPAa MEKK2meb
CLNDd
(µM)
AMPe
(nm/sec)
Comp #
R1
R2
IC50 (nM)
EC50 (nM)
13
19
20
21
22
23
H
H
H
Me
Et
2.5
73
297
297
295
143
234
126
<10
<10
<3
5.0
32
32
32
<1
180
205
177
95
H
H
Pr
35
H
Bu
Me
68
Me
H
199
<10
24
25
16
40
156
173
434
367
61
H
H
H
140
128
72
10
48
180
160
26
27
20
CF3
< 1
CF3
H
Me
Et
3.2
< 1
20
39
9
140
-
28
29
30
CF3
CF3
261
758
14
53
410
H
H
H
< 1
1.3
< 1
22
19
38
1
3
-
-
31
32
33
F
F
277
200
H
2.5
22
297
320
34
F
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Cl
Cl
H
H
< 1
8.0
11
29
6
4
440
180
35
36
9
10
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H
32
31
6
200
37
Cl
H
H
20
10
54
53
10
21
140
170
38
39
H
< 1
38
15
-
40
N
H
H
H
158
40
NDc
55
26
194
52
45
55
64
41
42
43
N
25
71
N
Cl
H
H
H
< 1
5.0
< 1
26
48
42
14
61
220
89
44
45
46
N
N
F
141
190
N
F
H
H
< 1
10
27
58
12
-
110
66
47
48
N
F
N
ascintillation proximity assay; bmethylation of MEKK2 cellular mechanistic assay;
cnot determined; dCLND: Chemiluminescent nitrogen detection; eAMP: Artificial Membrane
Permeability
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Figure 4. The crystal structure of Compound 28 (green carbons) bound to SMYD3 overlaid with
EPZ028862 (magenta carbons) from PDB 5V37. The co-factor SAH is colored with orange
carbons and crystallographic waters are represented as red spheres. Key hydrogen bond
interactions between Compound 28 and SMYD3 are depicted with yellow dashes and the binding
site surface is colored by electrostatic potential. The teal dashes depict measured distances between
the amine of Compound 28 and the sidechains of Glu192 and Asp241. While there are two
conformations observed in the crystal structure for the cyclopropyl group of Compound 28, only
one is depicted for clarity.
Applying the SAR learned, we revisited the cyclohexyl amine of 10 since it showed similar cellular
potencies vs 13 (Table 1). Substituting the amine group with trifluorobutane provided compound
49 (Figure 5). Compound 49 exhibits IC50 potencies of 6.3 and 44 nM in biochemical and cellular
MEKK2me assays, respectively. At higher doses (>400 nM), greater than 90% inhibition of
MEKK2 methylation was achieved with compound 49 in cells.
O
S
O
O
N
N
O
N
N
CF3
H
H
isomer 1
Figure 5. Structure of compound 49
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To evaluate the mode of inhibition of compounds in this series, 7, 28 and 49 were assessed by
examining the effect of substrate concentration on the inhibition of SMYD3 utilizing the Cheng-
Prusoff relationship for competitive, non-competitive and uncompetitive inhibition.15 IC50 values
for these compounds showed no change with varying MEKK2 concentration indicative of MEKK2
noncompetitive inhibition (Figure 6A-C) while IC50 values decreased with increasing SAM
concentration in a curve-linear fashion indicative of uncompetitive behavior with respect to SAM
binding yielding an αKi value of 17 ± 1 nM, 6 ± 0.5 nM and 18 ± 2 nM for 7, 28 and 49, respectively
(Figure 6D-F). Additionally, to confirm mode of inhibition, initial velocity data was plotted as a
function of varying substrate at different concentrations of compound 49 yielding a Ki of 18 ± 3
nM (Supplementary Figure 1A & B). The double-reciprocal plot versus SAM resulted in a set of
parallel lines consistent with an uncompetitive mode of inhibition (α=0.13, Supplementary Figure
1C) while the double-reciprocal plot versus MEKK2 yielded a set of lines that converged to the
left of the y-axis consistent with a non-competitive inhibitor (α=0.93, Supplementary Figure 1D).
F-test analysis supported the mode of inhibition deduced by the double-reciprocal patterns.
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Figure 6. Mode of inhibition against SMYD3 by Isoxazole series compounds. IC50 values were
determined following a 60-minute SMYD3 reaction and fitting the data to a 3-parameter dose-
response equation. Top Panel. Data represents independent experiments showing IC50 values
plotted as a function of [FL MEKK2]/Kmapp for compound 7 (A), 28 (B) and 49 (C, n=1) where
IC50=Ki for noncompetitive inhibition. Bottom Panel. Data represents independent experiments
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showing IC50 values plotted as a function of [SAM]/Kmapp fit to an equation for uncompetitive
inhibition αKi =IC50/(1+(Km/[S])) for compound 7 (D), 28 (E) and 49 (F, n=1).
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Additionally, the ability of compounds 7 and 28 to inhibit SMYD3 methylation of a histone H4
substrate was investigated. Inhibition of SMYD3 methylation on recombinant histone H4
decreased roughly 10-fold in comparison to MEKK2 resulting in IC50 values of 167 nM and 67
nM for compounds 7 and 28, respectively (n=3).
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To assess whether inhibition of SMYD3 methyltransferase activity would affect MAPK signaling
as previously suggested by knockout (KO) and knock down (KD) studies,4 the inhibition of
ERK1/2 phosphorylation was assessed in A549 cells following exposure to compounds 7, 10 and
49. No inhibition of ERK1/2 phosphorylation was observed (Figure 7). Thus, while Compounds
7, 10 and 49 inhibit methylation of MEKK2 by SMYD3 in both biochemical and cellular assays,
the inability of these inhibitors to affect ERK1/2 phosphorylation suggests that methylation of
MEKK2 does not alter MEKK2 activity toward ERK1/2. These data suggest that the decrease in
ERK1/2 phosphorylation observed upon SMYD3 knockout (KO) and knock down (KD) studies is
not due to alteration of MEKK2 methylation and thus SMYD3 may have other methyltransferase
independent activities which contributed to the effects on MAPK signaling observed in KO/KD
studies.
Figure 7. Inhibition of SMYD3 methyltransferase activity does not affect ERK1/2 activation
state. Following treatment of A549 cells for 72 hours with Compound 7, 10 and 49, the cells
were serum starved for 24 hours and then treated with 25 ng/µl EGF for 15 minutes to stimulate
MAPK signaling. Trametinib, a MEK1/2 kinase inhibitor, is included as a positive control.
Lysates were immunoblotted with antibodies against the proteins indicated. Total ERK1/2,
SMYD3 and tubulin are included as controls.
To evaluate the ability of compounds from this series to be used for in vivo studies, several
compounds were examined in rat pharmacokinetic experiments (Table 3). In general, compounds
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from this series exhibited low blood clearance with high volume of distribution and long t1/2.
Bioavailability was high with reasonable to good oral exposures. While some compounds have a
relatively high volume of distribution, overall the PK profile is favorable.
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Table 3. Pharmacokinetics of Selected Compounds.a
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compounds
1b
CL
6
Vd
1.2
t1/2
2.5
2.7
0.2
1.7
12.8
3.6
7.3
1.7
10.8
8.6
DNAUCpo
2142
123
-
F
79
27
-
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14c
37
311
68
20
90
36
71
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20
4.5
2.3
17 c
28 c
31c
35 d
34 d
46 d
49d
6.7
295
676
149
365
87
-
18.9
16.1
14.7
9.4
79
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70
65
59
86
12.0
12.5
578
967
aSprague-Dawley rats. CL: mL/min/Kg; Vd: L/kg; DNAUCpo: (ng*h/mL)/(mg/kg); t1/2:
iv, hour; F: %.
biv: 1 mg/kg, po: 5 mg/kg, n=3.
civ: 0.5 mg/kg, po: 2 mg/kg, n=3.
div: 1 mg/kg, po: 2 mg/kg, n=3.
Compound 49 is selective against >350 kinases and 30 histone methyltransferases (<25% and
<12% inhibition was observed at 10M versus kinase and HMT panels, respectively,
supplementary Table S2 & S3). The balanced and clean profile, excellent potency, and
pharmacokinetic properties makes compound 49 an excellent compound for use in evaluating the
biological role of SMYD3 activity in both cells and animals.
CONCLUSION
While other small molecule SMYD3 inhibitors have been reported and a similar isoxazole analog
of this series has been exemplified,12-13 the compounds disclosed here represent a detailed SAR
study of this class. In particular, a close analog of this class, EPZ028862, showed excellent
inhibition of SMYD3 and selectivity against SMYD2.13 Some of the analogs reported herein are
more potent than previously reported inhibitors and have a better PK profile, including higher
bioavailability, longer t1/2, and/or lower clearance. While all reported inhibitors bind in the same
binding site of SMYD3 and the ends of each inhibitor reach the same binding elements, they do
so through a different trajectory. EPZ028862 generally makes similar interactions as the
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compounds reported here, though in contrast the basic amine of compound 28 is observed to come
in close proximity with the negatively charged Asp241 and Glu192 sidechains while the basic
amine of EPZ028862 is stabilized by solvent mediated interactions (Figure 4).13 Furthermore, the
trifluorobutane group of compound 28 extends deeper into a hydrophobic region of the substrate
binding site.
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In summary, the compounds disclosed in this paper represent a novel structural class of SMYD3
inhibitors. Modification of the HTS hit, compound 1, with the structure guided SAR study led to
compound 49, a potent, selective, orally bioavailable small molecule inhibitor of SMYD3 which
can serve as a useful in vitro and in vivo tool molecule to investigate the role of SMYD3
methyltransferase activity in cancer and other disorders.
ASSOCIATED CONTENT
Supporting Information
ADME/PK methods, cellular assay conditions, biochemical assay conditions and MOI data,
crystallography methods and data, kinase and HMT panels screening conditions and results,
synthesis and data for compound 49. This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
Coordinates have been deposited in the Protein Data Bank with accession codes 6PAF, 6P6K, 6P7Z
and 6P6G.
AUTHOR INFORMATION
Corresponding Author
*Phone: 610-917-5879, Email: dai-shi.x.su@gsk.com
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Patrick Stoy and Mr. Guosen Ye for supporting the early chemistry hit discovery
effort.
ABBREVIATIONS USED
SMYD3: SET and MYND Domain-Containing Protein 3
MEKK2: Mitogen-activated protein kinase kinase 2
SAM: S-Adenosyl methionine
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SAH: S-Adenosyl- l-homocysteine
H4K5: histone 4 lysine 5
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ERK: extracellular signal–regulated kinases
siRNA: small interfering RNA
shRNA: short hairpin RNA
DNA: deoxyribonucleic acid
PK: pharmacokinetics
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IC50: half maximal inhibitory concentration
CLND: Chemiluminescent nitrogen detection
AMP: Artificial Membrane Permeability
EGF: Epidermal growth factor
MOI: mode of inhibition HTS: high throughput screening
SAR: Structure activity relationship
MAPK: mitogen-activated protein kinase
ADME: absorption, distribution, metabolism, and excretion
CL: clearance
Vd: volume distribution
DNAUC: dose normalized area under curve
HMT: histone methyltransferase
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Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors
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Dai-Shi Su,*,† Junya Qu,† Mark Schulz,† Chuck Blackledge,† Hongyi Yu,† Jenny Zeng,† Joelle Burgess,†
Alexander Reif,† Melissa Stern,‡ Raman Nagarajan,‡ Melissa Pappalardi,‡ Kristen Wong,‡ Alan P. Graves,§
William G. Bonnette,║, ≠ Liping Wang,║ Patricia A. Elkins,║ Beth Knapp-Reed,† Jeffrey Carson,║ Charles
McHugh,‡ Helai Mohammad,‡ Ryan Kruger,‡ Juan Luengo,†,≠ Dirk A. Heerding,†, ≠ Caretha L. Creasy‡
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†Medicinal Chemistry, Medicine Design, ‡Oncology R&D, § Data and Computational Sciences, ║ Protein
Cellular and Structural Sciences, Medicine Design, Medicinal Science and Technology GlaxoSmithKline,
1250 South Collegeville Road, Collegeville, PA, 19426, USA
Table of Contents Graphic (TOC)
≠ Current address: RN: University of California Davis; WGB: Janssen Pharmaceuticals, Spring House, PA
19477; JL: Prelude Therapeutics; DAH: retired
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