Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents.
Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents.
SMYD2 is a catalytic SET domain containing
protein methyltransferase reported to monomethylate lysine residues
on histone and nonhistone proteins.[1] SMYD2
has been proposed as a potential therapeutic target in cancer. Its
overexpression has been reported in cancer cell lines as well as in
esophageal squamous carcinoma, bladder carcinoma, gastric cancer,
and pediatric acute lymphoblastic leukemiapatients.[2−6] In these studies, SMYD2 overexpression often correlated with lower
survival rate and was suggested to be a clinically relevant prognostic
marker. Knockdown of SMYD2 in overexpressing ESCC, bladder, and gastric
cancer cell line models significantly reduced cell proliferation.[2] Initially, SMYD2 was characterized as methylating
H3lysine 36[7] and lysine 4 when interacting
with HSP90a.[8] Methylation of histones by
SMYD2 has been connected to increased transcription of genes involved
in cell-cycle regulation, chromatin remodeling, and transcriptional
regulation.[8] In addition, several studies
have uncovered an important role of SMYD2 methylation activity toward
nonhistone proteins closely connected to cancer. This is in line with
the emerging concept that posttranslational methylation of nonhistone
proteins (e.g., of transcription factors) by protein methyltransferases
can also substantially alter protein function. Thereby, a regulatory
role of lysine methylation can probably be extended to multiple cellular
pathways besides transcriptional regulation and histones.[9,10] So far, the best-characterized example of SMYD2 methylation of a
nonhistone protein is the tumor suppressor transcription factor p53.[11−16] Transcriptional activity of p53 is inhibited by SMYD2-mediated posttranslational
methylation at lysine 370 (K370).[13,17] The structural
basis of p53 methylation by SMYD2 has been characterized by solving
the crystal structure of a ternary complex with the cofactor product S-adenosylhomocysteine and a p53-derived substrate peptide.[16] It has been proposed that methylation at K370
reduces the DNA-binding efficiency of p53 and subsequently prevents
the transcriptional activation of p53 target genes.[13] In the same study, a knockdown of SMYD2 and treatment with
doxorubicin led to an increase in p53-mediated cell-cycle arrest and
apoptosis. In line with these observations, low SMYD2 gene expression
was suggested as a predictive marker for an improved response to neoadjuvant
chemotherapy in breast cancerpatients.[18] Besides p53, several other proteins have been identified as SMYD2
substrates, including the estrogen receptor (ER),[19,20] PARP1,[21] retinoblastoma protein (Rb),[4] and HSP90.[22,23] Mechanistically, methylation
of HSP90 has been connected to the normal physiological role of SMYD2
in muscle biology,[24,25] as well as in cancer.[23] These studies indicate that SMYD2 has many substrates
and various potentially tissue-specific physiological and pathogenic
functions. SMYD2 therefore represents a very attractive target for
further exploration in different disease-relevant models. Nevertheless,
the biology of SMYD2 is still poorly understood and the molecular
contribution of individual substrates to specific knockdown phenotypes
remains largely unknown. For a more unbiased interpretation of biological
experiments, fully profiled chemical probes can substantially contribute
to preclinical target validation.[26,27] Although first
cellular-active probe inhibitors of SMYD2 have been described (Figure ), there is a need
for structurally orthogonal chemical probes to enable cross-validation
studies and thereby rule out unspecific effects.[26] The publication of 1 (AZ505)[12] was the first disclosure of a co-crystal structure of an
inhibitor bound to SMYD2 and paved the way for further studies, leading
to the discovery of 2 (LLY-507),[28] and 3 (A-893),[29] which have
reported significantly improved potency. Analysis of the respective
cocrystal structures reveals that all three inhibitors bind in a similar
fashion, occupying the same binding pockets. Furthermore, the cellular
activity of the known inhibitors is limited, and no data about in
vivo applicability have been published. Here we report the discovery
of a potent and selective aminopyrazoline-based small-molecule inhibitor
(S)-4 (BAY-598).[30,31] We show that (S)-4 has a distinctly
different binding mode compared to previous inhibitors, utilizing
a dichlorophenyl moiety as so far unprecedented chemical motif for
addressing the methyl-lysine binding pocket of SMYD2. For the first
time, we are presenting in vivo xenograft and DMPK data for a SMYD2
inhibitor. In addition to previously described inhibitors, (S)-4 shows very potent cellular activity combined
with reasonable DMPK properties (Figure ). Furthermore, we are indicating the potential
that this inhibitor might offer to the field of protein methyltransferases
in the quest to fully explore the underlying complex biology and therapeutic
potential of SMYD2 by validating AHNAK protein[32] as a new cellular substrate.
Figure 1
Comparison of so far
described SMYD2 inhibitors with (S)-4 discovered in this study. Data from respective references as well
as assay results from this study are presented.
Comparison of so far
described SMYD2 inhibitors with (S)-4 discovered in this study. Data from respective references as well
as assay results from this study are presented.
Results and Discussion
The potential link between SMYD2
and cancer motivated us to screen the Bayer compound collection, with
the aim of identifying small-molecule inhibitors of the enzyme. To
this end, a scintillation proximity assay (SPA) was set up using recombinant
His-tagged SMYD2, a biotinylated p53-derived peptide substrate, and
tritiated S-adenosyl-l-methionine (3H-SAM) (Figure A). Of the three million compounds tested in a primary HTS, we identified
more than 2300 confirmed hits which inhibited SMYD2 with IC50 values below 15 μM. Among the multiple structural clusters
and singletons in the hit list, several offered starting points with
low micromolar potency and tractable chemical matter, and our attention
was drawn to pyrazolines such as compound 5. Initial
hits and later derivatives of the pyrazoline series showed stabilizing
effects in an SMYD2 thermal shift assay (TSA) (Figure B). In addition, binding of compound 6 to SMYD2 was validated by isothermal titration calorimetry
(ITC), which indicated a submicromolar binding constant (Kd = 540 nM, Figure C) and a high enthalpic contribution to the binding energy.
As the latter reduces the likelihood of nonspecific off-target activities,
the ITC data underscored the attractiveness of pyrazoline hit 5.[33,34] It transpired that compounds
of this structural series had been prepared during the course of an
in-house program as antagonists for protease-activated receptor 1
(PAR1), a G-protein-coupled receptor which is highly expressed in
platelets and plays an important role in thrombin signaling and platelet
aggregation.[35,36]
Figure 2
Identification and biophysical validation
of the pyrazoline SMYD2 inhibitor series leading to (S)-4. (A) Scintillation proximity assay (SPA) screening
approach leading to the pyrazoline hit cluster, with representative
hit compound 5 with IC50 = 1.7 μM. (B)
Thermal shift assay (TSA) for SMYD2 protein stabilization for inhibitors
of the aminopyrazoline series [●], with aminopyrazoline hit 5 (ΔTm = 3.3 K) and (S)-4 (ΔTm = 7.1 K) highlighted. Stabilization correlates with SPA potency.
(C) Isothermal titration calorimetry (ITC) of compound 6 indicating a submicromolar binding constant (Kd = 540 nM) and a high enthalpic contribution to the binding
energy.
Identification and biophysical validation
of the pyrazolineSMYD2 inhibitor series leading to (S)-4. (A) Scintillation proximity assay (SPA) screening
approach leading to the pyrazoline hit cluster, with representative
hit compound 5 with IC50 = 1.7 μM. (B)
Thermal shift assay (TSA) for SMYD2 protein stabilization for inhibitors
of the aminopyrazoline series [●], with aminopyrazoline hit 5 (ΔTm = 3.3 K) and (S)-4 (ΔTm = 7.1 K) highlighted. Stabilization correlates with SPA potency.
(C) Isothermal titration calorimetry (ITC) of compound 6 indicating a submicromolar binding constant (Kd = 540 nM) and a high enthalpic contribution to the binding
energy.
Chemistry
The pyrazoline compounds
of interest were synthesized according to previously described procedures.[37] Synthesis of the required intermediates 10a–f started from the commercially available
2-amino-1-phenylethanones 7 (Scheme ). Protection of the amines as the allyl
carbamates 8 was followed by Mannich reaction with formaldehyde
and piperidine, and the resulting product mixture was treated with
hydrazine monohydrate to install the pyrazoline moiety (compounds 9). Subsequent reaction with diphenyl N-cyanocarbonimidate
resulted in intermediates 10a–f.
Scheme 1
Synthesis of Pyrazoline Intermediates 10a–f
Reagents and conditions: (a) allyl
chloroformate, K2CO3 (1.5 M in water), DCM,
rt, overnight; (b) formaldehyde (37 wt % in water), piperidine, EtOH,
rt, overnight; (c) hydrazine monohydrate, EtOH, reflux; (d) diphenyl N-cyanocarbonimidate, i-PrOH, reflux, 1
h, then rt, overnight.
Synthesis of Pyrazoline Intermediates 10a–f
Reagents and conditions: (a) allyl
chloroformate, K2CO3 (1.5 M in water), DCM,
rt, overnight; (b) formaldehyde (37 wt % in water), piperidine, EtOH,
rt, overnight; (c) hydrazine monohydrate, EtOH, reflux; (d) diphenyl N-cyanocarbonimidate, i-PrOH, reflux, 1
h, then rt, overnight.The synthesis of compounds 5, 6, and 12–24 began with 10a (Scheme ). Treatment of 10a with the respective
aniline or amine provided intermediates 11a–i. Deprotection to the 4-aminopyrazolines and further elaboration
led to the desired amides 5, 6, and 12–23 by a sequence of optional reductive
N-alkylation and amide formation. For the hydroxyacetyl derivatives 5, 6, and 12–21, the amide was formed by using acetoxyacetyl chloride, followed
by treatment of the crude amide with methanolic potassium carbonate
to remove the acetyl moiety. The amino acid amide 22 was
prepared by a coupling with 9-fluorenylmethoxycarbonyl (Fmoc) protected
glycine (HATU, NMM, DMF), followed by removal of the Fmoc group with
piperidine. Amide formation with methoxyacetyl chloride gave compound 23. Furthermore, oxoimidazolidine 24 was prepared
by reaction of the N-unsubstituted 4-aminopyrazoline derivative with
chloroacetyl chloride and treatment of the resulting chloroacetamide
with urotropine.
Reagents and conditions: (a) 3-(difluoromethoxy)aniline, n-BuLi, THF, −78 °C; (b) 1,3-dimethylbarbituric
acid, Pd(PPh3)4, THF; (c) acetaldehyde, NaBH4, MeOH, 0 °C; (d) (1) acetoxyacetyl chloride, aqueous
NaHCO3 solution, DCM, (2) K2CO3,
MeOH.The synthesis of compounds 30–34 started from intermediate 10d by addition of the respective aniline derivatives, followed by introduction
of the N-ethylated hydroxyacetamide (Scheme ).
Reagents and conditions:
(a) aniline derivative, n-BuLi, THF, −78 °C;
(b) 1,3-dimethylbarbituric acid, Pd(PPh3)4,
THF; (c) acetaldehyde, NaBH4, MeOH, 0 °C; (d) (1)
acetoxyacetyl chloride, aqueous NaHCO3 solution, DCM, (2)
K2CO3, MeOH.Compounds 4, 28, and 30–34 were separated into their enantiomers by preparative chiral HPLC
or chiral supercritical fluid chromatography. For compounds 6 and 4, the integrity of the pyrazoline stereocenter
was tested. The compounds were stable to racemization in aqueous solution
at pH 7, as well as in mouse and human plasma at 37 °C, for at
least 48 h. However, clean racemization was obtained under basic conditions
and microwave irradiation (DBU, THF, 90 °C).
Structure–Activity
Relationships
Our initial structure–activity relationship
(SAR) studies of 3-(4-chlorophenyl)pyrazoline derivatives with respect
to SMYD2 inhibition are summarized in Table . For the carboximidamide N-substituent (R3), a meta-substituted phenyl seemed optimal, with the 3-(difluoromethoxy)phenyl
derivative 6 displaying highest potency and binding efficiency[38] (IC50 = 0.8 μM, BEI = 12.5).
Small para substituents are tolerated, such as with the 3-chloro-4-fluorophenyl,
3,4-dichlorophenyl, and 2,2-difluoro-1,3-benzodioxol-5-yl derivatives 5, 12, and 13 (IC50 =
1.7, 3.3, and 2.2 μM, respectively). In contrast, the 4-(difluoromethoxy)phenyl
derivative 14 is more than 15-fold less active than the
corresponding meta-derivative 6. The extended 3-chlorophenethyl
derivative 15 showed no measurable activity (IC50 > 20 μM), implying that the space within the relevant binding
pocket is limited. Nonaromatic derivatives, such as 4,4,4-trifluorobutyl-substituted 16 or the cyclopropylethyl derivative 18, are
less potent than 6 (IC50 = 6.9 and 8.3 μM
for 16 and 18, respectively). Surprisingly,
the corresponding n-butyl derivative 17 is inactive (IC50 > 20 μM). The SAR for PAR1
antagonism at the carboximidamide N-substituent appears to be rather
flat: all compounds 5, 6, and 12–18 are potent PAR1 antagonists, with IC50 values below 100 nM.
Table 1
SAR of 3-(4-Chlorophenyl)pyrazoline
Derivatives: Variation of the Carboximidamide N-Substituent
SMYD2 thermal shift
assay.
Binding efficiency
index: pIC50/MW × 1000.[38]
SMYD2 thermal shift
assay.Binding efficiency
index: pIC50/MW × 1000.[38]With respect to the amide
moiety, it became clear that the presence and orientation of a hydrogen-bond
donor has a large impact on potency toward SMYD2 (Table ). Variation of the amide N-alkyl substituents (R1) revealed that the N-ethyl derivative is preferred: secondary amide 19 (IC50 = 10.9 μM) is about 10-fold less active and
the N-propyl derivative 20 is also less
potent than the corresponding N-ethyl derivative 6 (IC50 = 0.8 μM), whereas the larger cyclopropylmethyl
substituent in 21 resulted in a loss of potency (IC50 > 20 μM). Replacement of the hydroxyacetyl in compound 12 (IC50 = 3.3 μM) by an aminoacetyl group
gave the equipotent derivative 22 (IC50 =
2.8 μM). In contrast, the methoxyacetyl derivative 23, lacking the hydrogen-bond donor, is inactive (IC50 >
20 μM). In compound 24, where the hydrogen-bond
donor is fixed in an oxoimidazolidine ring, there is significantly
reduced potency (IC50 = 6 μM) relative to the N-ethyl derivative 6. On the basis of the available
amide derivatives, with BEI values in the same range (10–11),
an improvement in the binding efficiency was not envisaged. Furthermore,
alterations of the amide moiety did not offer a path forward to selectivity
against PAR1. Compounds 22, 23, and 24 are in a similar potency range for PAR1 antagonism and
greater than 10-fold more potent against PAR1 (IC50 = 130,
30, and 100 nM, respectively) than SMYD2.
Table 2
SAR of
3-(4-Chlorophenyl)pyrazoline Derivatives: Variation of the Amide Moiety
SMYD2 thermal shift
assay.
Binding efficiency
index: pIC50/MW × 1000.[38]
SMYD2 thermal shift
assay.Binding efficiency
index: pIC50/MW × 1000.[38]At this stage, we selected
compound 6 for co-crystal structure determination with
SMYD2 based on its potency and promising biophysical properties. Compound 6 was soaked into crystals of SMYD2 grown in the presence
of SAM. The crystal structure revealed that compound 6 binds into the substrate peptide binding pocket of SMYD2; the observed
binding mode is consistent with the previously established SAR. There
is a very good steric and electrostatic fit of 6 to the
substrate binding site of SMYD2 (Figure A,B). The pyrazoline and the NH of the carboximidamide
form hydrogen bonds to Gly183. The 4-chlorophenyl substituent inserts
into the lysine binding channel and is engaged in π-stacking
interactions with Phe184 and Tyr240. There is a good fit of the 3-(difluoromethoxy)phenyl
substituent into the adjacent hydrophobic pocket-1, with the difluoromethoxy
group pointing into a hydrophobic subpocket. Although racemic 6 was used for soaking, the density maps reveal that only
the S-enantiomer is bound. The S configuration at the pyrazoline provides an optimal exit vector
for the hydroxyacetamide substituent, which occupies pocket-2 and
forms two hydrogen bonds with Thr185 (Figure A). The N′-nitrile
of the carboximidamide contributes to a water-bridged hydrogen bond
with Ser196. On the basis of the crystal structure of compound 6, we envisioned that further exploration of the aniline and
amide moieties would not lead to a significant improvement of potency
and binding efficiency. Therefore, we elected to focus on derivatives
of the 4-chlorophenyl substituent, which were underrepresented in
our compound library.
Figure 3
Binding mode of compound 6 and (S)-4. (A,B) Two different views of the co-crystal structure
of 6 in complex with SMYD2. There is a good steric and
electrostatic fit of 6 into the substrate binding site
of SMYD2, and multiple hydrogen-bond interactions are involved. The
lysine channel is occupied by the 4-chlorophenyl residue of the ligand,
representing a novel lysine channel binding motif. In addition, pockets-1
and -2 are addressed. (C) Visualization of the WaterMap calculations
suggesting an optimal displacement of water molecules by a 3,4-dichlorophenyl
residue. Calculated water sites are shown as spheres and colored based
on their free energy. Only sites within the lysine channel are shown.
WaterMap results are based on the crystal structure of the monochloro
derivative 6 (PDB code 5ARF). The protein surface is depicted in
gray. The modeled ligand structure is shown as colored sticks (chlorine,
green; carbon, yellow; hydrogen, white; nitrogen, blue; oxygen, red;
fluorine, light green). For clarity, protein residues and cofactors
are not shown. (D) Overlay of the crystal structures of compound 6 (magenta) and (S)-4 (yellow,
PDB code 5ARG), showing nearly identical binding modes.
Binding mode of compound 6 and (S)-4. (A,B) Two different views of the co-crystal structure
of 6 in complex with SMYD2. There is a good steric and
electrostatic fit of 6 into the substrate binding site
of SMYD2, and multiple hydrogen-bond interactions are involved. The
lysine channel is occupied by the 4-chlorophenyl residue of the ligand,
representing a novel lysine channel binding motif. In addition, pockets-1
and -2 are addressed. (C) Visualization of the WaterMap calculations
suggesting an optimal displacement of water molecules by a 3,4-dichlorophenyl
residue. Calculated water sites are shown as spheres and colored based
on their free energy. Only sites within the lysine channel are shown.
WaterMap results are based on the crystal structure of the monochloro
derivative 6 (PDB code 5ARF). The protein surface is depicted in
gray. The modeled ligand structure is shown as colored sticks (chlorine,
green; carbon, yellow; hydrogen, white; nitrogen, blue; oxygen, red;
fluorine, light green). For clarity, protein residues and cofactors
are not shown. (D) Overlay of the crystal structures of compound 6 (magenta) and (S)-4 (yellow,
PDB code 5ARG), showing nearly identical binding modes.With the co-crystal structure in hand, we employed molecular
modeling for the prioritization of derivatives. In particular, WaterMap
calculations,[39,40] which estimate the position and
the thermodynamic properties of water molecules in the ligand-free
structure, suggested the introduction of a second substituent at the
3-position of the 4-chlorophenyl group. To test this hypothesis, a
few derivatives were synthesized (see Table ). The unsubstituted phenyl derivative 25 is inactive, while the 4-bromo derivative 26 (IC50 = 1.1 μM) is as potent as the 4-chloro derivative 6, indicating the importance of a hydrophobic substituent
at the 4-position. As predicted by WaterMap calculations, introduction
of a second substituent at the meta position, as exemplified by 3,4-dichloro
derivative 4 (IC50 = 0.08 μM, BEI =
13.5) and 4-chloro-3-methyl derivative 28 (IC50 = 0.08 μM, BEI = 14), resulted in significantly improved potency
(ca. 10-fold greater than 6) and binding efficiency.
As highlighted in Figure C, the two chloro substituents are colocated with two calculated
water sites that have high free energy, suggesting an optimal water
displacement by 3,4-dichlorophenyl and thus a lower binding free energy
than for the 4-chlorophenyl or the unsubstituted phenyl derivative,
consistent with the observed potency difference for these derivatives.
However, introduction of a third substituent leads to a decrease in
potency compared to the 3,4-disubstituted derivatives, as exemplified
by 4-chloro-3,5-dimethyl derivative 27 (IC50 = 0.57 μM). Compound 29, with the bulkier1,3-benzodioxole
moiety, is also less potent (IC50 = 4.2 μM), suggesting
that the SAR at this position is rather steep.
Table 3
SAR of 3-Phenylpyrazoline Derivatives: Exploration of Phenyl Substituents
SMYD2 thermal shift assay.
Binding efficiency index: pIC50/MW ×
1000.[38]
SMYD2 thermal shift assay.Binding efficiency index: pIC50/MW ×
1000.[38]Compound 4 was selected for further biological
and crystallographic studies. The co-crystal was obtained by soaking
(S)-4 into crystals of SAM-bound SMYD2.
Structure determination revealed that (S)-4 features an almost identical binding mode as compound 6, as evidenced in the overlay of both structures (Figure D). In line with the observed S configuration of compound 6 and (S)-4, we observed a greater than 50-fold difference
in the IC50 values of the two enantiomers of 4 [27 nM for (S)-4 vs 1.7 μM for
(R)-4, see Table ]. Introduction of the additional 3-chloro
substituent also resulted in a dramatic decrease in the antagonistic
effect on PAR1 [IC50 = 1.7 μM for (S)-4 and >30 μM for (R)-4]. Although (S)-4 proved to
be the active isomer for both SMYD2 inhibition and PAR1 antagonism,
there is a greater than 50-fold selectivity for SMYD2 relative to
PAR1.
Table 4
In Vitro Pharmacokinetic Properties of Potent
SMYD2 Inhibitors
ee >95%.
Rat hepatocytes.
Permeability Papp(A–B)/efflux ratio.
Aqueous solubility.
ee >95%.Rat hepatocytes.Permeability Papp(A–B)/efflux ratio.Aqueous solubility.The co-crystal structures of compound 6 and (S)-4 revealed that the pyrazoline-based SMYD2
inhibitors feature a different binding mode to other recently reported
inhibitors. Thus, 1(12) and 2(28) (Figure A) have published IC50 values
of 0.12 μM and <15 nM, respectively, and like (S)-4 they bind to the substrate binding site of SMYD2.
The structures of the three inhibitors are superimposed in Figure B. All ligands occupy
the lysine binding channel and the adjacent hydrophobic pocket-1;
however, (S)-4 employs pocket-2 via
hydrogen-bond interactions with its hydroxyacetyl moiety (cf. Figure A), which are not
present in 1 or 2. 1 and 2, on the other hand, occupy a remote hydrophobic pocket-3
which is not addressed by (S)-4. Figure C provides a view
into the lysine binding channel (the respective motifs are highlighted
in color on the structures in Figure A). 2 binds with its N-alkylpyrrolidine moiety at this position, which is structurally
closely related to the lysine side chain of a methylated substrate
peptide. 1 and (S)-4 address
the lysine channel with aromatic substituents. It is noteworthy that
the 3,4-dichlorophenyl group of 1 and of (S)-4 bind to different positions.
Figure 4
Comparison of (S)-4 (this work) with the recently reported
SMYD2 inhibitors 1(11) and 2.[25] (A) Chemical structures of
the three selected SMYD2 inhibitors. (B,C) Two different views of
an overlay of (S)-4 (yellow) with 1 (green) and 2 (blue). (S)-4 has a distinct binding mode, addressing pocket-2 which is
not occupied by 1 or 2, while only 1 and 2 occupy the distant hydrophobic pocket-3.
Comparison of (S)-4 (this work) with the recently reported
SMYD2 inhibitors 1(11) and 2.[25] (A) Chemical structures of
the three selected SMYD2 inhibitors. (B,C) Two different views of
an overlay of (S)-4 (yellow) with 1 (green) and 2 (blue). (S)-4 has a distinct binding mode, addressing pocket-2 which is
not occupied by 1 or 2, while only 1 and 2 occupy the distant hydrophobic pocket-3.On the basis of 3,4-dichlorophenyl
as a novel lysine channel binding motif, we identified several pyrazoline
derivatives as potent SMYD2 inhibitors (see Table ). Aiming to identify a candidate for in
vivo experiments, we focused on the potent S-enantiomers
of these derivatives, profiling them in pharmacokinetic assays in
vitro. (S)-4 showed moderate stability
upon incubation with rat hepatocytes (CLblood = 2.5 L/h/kg)
as well as moderate apparent permeability (34 nm/s) and a hint of
active transport in the Caco2 assay (efflux ratio = 5). In comparison,
the 4-chloro-3-methylphenyl derivative (S)-28 had similar permeability and efflux (33 nm/s, efflux ratio
= 7) and slightly lower metabolic stability in rat hepatocytes (CLblood = 2.8 L/h/kg). Compounds (S)-30 and (S)-32–34 exhibited
high metabolic stability (CLblood = 1.8–0.3 L/h/kg);
however, these derivatives have very low aqueous solubility (<5
mg/L), thereby limiting their suitability for in vivo experiments.
On the other hand, the methoxyphenyl derivative (S)-31 has high aqueous solubility (163 mg/L) and moderate
permeability with a hint of active transport (62 nm/s, efflux ratio
= 3) in the Caco2 assay; nevertheless, (S)-31 displayed low stability upon incubation with rat hepatocytes
(CLblood = 3.2 L/h/kg), and thus bioavailability is expected
to be low. On the basis of these data, (S)-4 was selected for further in vitro and in vivo studies. The
in vivo pharmacokinetic properties of (S)-4 were first evaluated by a single-dose administration of (S)-4 at 0.4 mg/kg by iv bolus or 0.8 mg/kg
po, respectively, to rats; there was moderate blood clearance (1.6
L/h/kg) and a low bioavailability of 24% (see Supporting Information Figure S1A). These data prompted us to assess
the exposure of (S)-4 following oral
administration. Hence, we treated mice with 10–100 mg/kg po
qd which are well tolerated doses of (S)-4. As a result, unbound IC50 [cellular methylation In-Cell
Western (ICW) assay; see Figure A–C] is covered for ∼9 to ∼12
h at steady state when 10 and 100 mg/kg, respectively, are administered
(see Supporting Information Figure S1B).
Figure 7
AHNAK is a novel substrate
of SMYD2 and methylation can be inhibited by aminopyrazoline inhibitors.
(A) In-Cell Western (ICW) assay. Immunofluorescence-based detection
of AHNAK methylation. Each row represents a different inhibitor tested
in increasing concentrations ranging from 39 nM up to 5 μM.
(S)-4 and 1 are highlighted.
(B) Inhibition of AHNAK methylation in MDA-MB231 cells overexpressing
SMYD2. Methylation signal overlaps with AHNAK protein detection. (S)-4 inhibits concentration dependent the AHNAK
methylation. (C) Good correlation of cellular activity (ICW) to biochemical
activity (SPA) of (S)-4 and pyrazoline
cluster derivatives. (S)-4 has a cellular
IC50 ∼ 60 nM.
Potency, Selectivity, and Inhibition Mode
(S)-4 showed potent in vitro inhibition of SMYD2 with
an IC50 of 27 ± 7 nM in the biochemical SPA assay
(Figure A). To analyze the mode of inhibition and affinity
of (S)-4, our SMYD2 protein preparation
was characterized with respect to its apparent Michaelis–Menten
constants for SAM and the p53 peptide substrate (data not shown).
The Km(app) values of 60 nM and 1 μM,
respectively, were in excellent agreement with the constants previously
reported using a similar assay.[12] Then,
we performed IC50 determinations at increasing concentrations
of one substrate and a fixed amount of the other ([S] = Km(app)) and applied the Cheng–Prusoff relationship[41] as described elsewhere.[42] Increasing the concentration of peptide substrate resulted in a
linear increase in IC50 (Figure B, upper panel), as would be expected for
a competitive mode of inhibition. Fitting these data to the corresponding
Cheng–Prusoff competitive inhibition model revealed a Ki(app) of 8 ± 1 nM (SD). On the other hand,
when SAM concentrations were titrated to saturation, we observed a
decrease in IC50 which converged to a constant value (Figure B, lower panel).
This type of behavior toward SAM corresponds to an uncompetitive mode
of inhibition. Consequently, the IC50 vs [S]/Km(app) plot fits best to the Cheng–Prusoff model
for uncompetitive inhibition, yielding an inhibitor constant αKi(app) of 28 ± 3 nM (SD). Our
data suggest that (S)-4 is a peptide-competitive,
SAM-uncompetitive inhibitor of SMYD2 methyltransferase activity, which
preferably binds to the SMYD2–SAM substrate complex. Interestingly,
SMYD2 has been reported to follow a random sequential Bi Bi mechanism
of substrate binding,[12,17] but the uncompetitive mode of
inhibition of (S)-4 regarding the SAM
cofactor suggests an ordered sequential Bi Bi mode of substrate binding,
where SAM would be required to bind before the peptide substrate.
Similar results have recently been described for inhibitors of the
SAM-dependent arginine methyltransferase PRMT5.[43]
Figure 5
(S)-4 mode of inhibition and selectivity
profile. (A) Activity in the scintillation proximity assay (SPA).
IC50 (n > 10) for SMYD2 inhibition
= 26 ± 7 nM (representative inhibition curve shown). (B) IC50 values obtained from SPA were plotted against the indicated
substrate concentrations (represented as [substrate]/Km(app)). Data were fitted to competitive and uncompetitive
models of the Cheng–Prusoff equation.[41,42] Data points are the mean of eight replicates, error bars indicate
1 × SD. The data indicate that (S)-4 is a peptide-competitive, SAM-uncompetitive inhibitor of SMYD2 methyltransferase
activity. (C) Selectivity profile on a panel of 32 additional methyltransferases
showing high selectivity of (S)-4 for
SMYD2. Only SMYD3 is weakly inhibited by (S)-4 with a > 1 μM IC50.
(S)-4 mode of inhibition and selectivity
profile. (A) Activity in the scintillation proximity assay (SPA).
IC50 (n > 10) for SMYD2 inhibition
= 26 ± 7 nM (representative inhibition curve shown). (B) IC50 values obtained from SPA were plotted against the indicated
substrate concentrations (represented as [substrate]/Km(app)). Data were fitted to competitive and uncompetitive
models of the Cheng–Prusoff equation.[41,42] Data points are the mean of eight replicates, error bars indicate
1 × SD. The data indicate that (S)-4 is a peptide-competitive, SAM-uncompetitive inhibitor of SMYD2 methyltransferase
activity. (C) Selectivity profile on a panel of 32 additional methyltransferases
showing high selectivity of (S)-4 for
SMYD2. Only SMYD3 is weakly inhibited by (S)-4 with a > 1 μM IC50.For further evaluation of selectivity,
(S)-4 was tested on a panel of 32 additional
methyltransferases, including closely related family members SMYD3,
SUV420H1, and SUV420H2. As a result, (S)-4 displayed >100-fold selectivity for SMYD2, with very weak residual
activity toward the closest related methyltransferase SMYD3 (IC50 ∼ 3 μM) (Figure C). In addition to the methyltransferases, (S)-4 was also profiled in the commercially
available KINOMEscan (DiscoveRx)[44] and
LeadProfilingScreen (Eurofins Panlabs) assay panels to fully determine
relative selectivity and specificity for kinases and other primary
molecular targets, including several CNS targets. Overall, we were
able to confirm the high selectivity and specificity of (S)-4 for SMYD2 inhibition (see Supporting Information Tables S1 and S2).
Cellular Methylation Activity
on p53
The ability of (S)-4 to inhibit SMYD2 was tested by monitoring its effects on cellular
p53 methylation using different cellular mechanistic assays. First,
we generated a polyclonal antibody (SY46) for the specific detection
of p53 monomethylation at lysine 370, as described elsewhere.[13] This antibody was then tested on recombinant
p53 protein which had been in vitro methylated by SMYD2 in a Western
blot. Specificity for methylated p53 was confirmed by the exclusive
detection of the in vitro methylated p53 protein isoforms, whereas
the nonmethylated p53 was not detected (Figure A). Endogenous methylation
of p53 protein was characterized by treatment of KYSE-150 esophageal
cancer cells with increasing concentrations of (S)-4 for 5 days. The KYSE-150 cell line model was selected
based on a described SMYD2 gene amplification[2] and a heterozygous R248Q mutation in p53 (COSMIC), leading to p53
protein accumulation without a stress stimulus. After treatment with
(S)-4, a significant reduction of methylation
was detected confirming that p53 is a cellular target of SMYD2-dependent
methylation (Figure B). Nevertheless overall endogenous detection of p53 protein methylation
led to weak signals, hence this method was not useful for the determination
of a cellular IC50. Therefore, we additionally employed
an established system with a transient FLAG-tagged SMYD2 and FLAG-tagged
p53 overexpression in HEK293T cells as benchmark assay. This assay
has been used previously to characterize the structurally unrelated
SMYD2 inhibitor 2.[28] As shown
in Figure C, (S)-4 showed a concentration-dependent decrease
in p53 methylation without affecting p53 total protein levels. A cellular
IC50 of 58 nM was determined (Figure D), which confirms that (S)-4 is the most potent cellular-active SMYD2 inhibitor
known to date (Figure ).
Figure 6
Inhibition of cellular methylation of the tumor suppressor protein
p53 by (S)-4. (A) A specific antibody
directed against p53K370me1 (SY46) was generated and tested for specificity
on recombinant p53 (rec. p53) which had been in vitro methylated by
SMYD2, or nonmethylated. (B) Endogenous methylation of p53 protein
was characterized by treatment of KYSE-150 esophageal cancer cells
with increasing concentrations of (S)-4 for 5 days (Co = control). (C) Cellular p53 methylation assay using
transient FLAG-tagged SMYD2 and FLAG-tagged p53 overexpression in
HEK293T cells. Increasing concentrations of (S)-4 reduce the methylation of overexpressed p53 (for this assay
p53K730me1-specific antibody kindly provided by Dr. Shelly Berger
was used). (D) IC50 determination in the cellular p53 methylation
assay.
Inhibition of cellular methylation of the tumor suppressor protein
p53 by (S)-4. (A) A specific antibody
directed against p53K370me1 (SY46) was generated and tested for specificity
on recombinant p53 (rec. p53) which had been in vitro methylated by
SMYD2, or nonmethylated. (B) Endogenous methylation of p53 protein
was characterized by treatment of KYSE-150 esophageal cancer cells
with increasing concentrations of (S)-4 for 5 days (Co = control). (C) Cellular p53 methylation assay using
transient FLAG-tagged SMYD2 and FLAG-tagged p53 overexpression in
HEK293T cells. Increasing concentrations of (S)-4 reduce the methylation of overexpressed p53 (for this assay
p53K730me1-specific antibody kindly provided by Dr. Shelly Berger
was used). (D) IC50 determination in the cellular p53 methylation
assay.
Characterization and Inhibition
of SMYD2-Mediated AHNAK Methylation
To further characterize
the effects of our aminopyrazoline-based inhibitors on the cellular
methylation activity of SMYD2, we generated a polyclonal cell line
derived from KYSE-150 with stable N-terminal 2xc-myc-tagged SMYD2
overexpression to maximize cellular methylation activity. In an immunofluorescence
analysis, SMYD2 was localized mainly in the cytosol (Supporting Information Figure S2A), as reported by others.[22] Surprisingly, the antibody SY46 directed against
methylated p53 showed a very strong signal specifically in the clones
with SMYD2 overexpression in the immunochemistry staining not derived
from p53 protein (Supporting Information Figure S2B,C). We validated the novel SMYD2 substrate giving rise
to the strong immunochemistry staining signal by knockdown and overexpression
experiments in additional cell lines to be derived from AHNAK protein[32] (Figure B and Supporting Information Figure 2D–F). AHNAK methylation has been
very recently reported in a proteomics study by other as an additional
methylation substrate of SMYD2, further confirming our results.[45] Importantly, we used the strong AHNAK methylation
signal to set up an In Cell Western (ICW) assay for cellular optimization
of our lead series (Figure A). The methylation signal was reduced by treatment with increasing
concentrations of (S)-4, derivatives
of the aminopyrazoline series or 1 (Figure A). In addition, (S)-4 specifically reduced methylation of AHNAK without
altering AHNAK protein expression in a Western blot (Figure B). Importantly, IC50 values in the cellular ICW assay for aminopyrazolines correlated
with potency in the scintillation proximity assay (Figure C) and were also comparable
to the p53 methylation assay (see Figure D). Our results of the methylation of AHNAK
and the recent identification of many additional methylation targets[45] clearly point toward additional roles beyond
p53 regulation of SMYD2, and further studies supported by potent and
selective inhibitors as (S)-4 are needed
to fully elucidate the underlying biology.AHNAK is a novel substrate
of SMYD2 and methylation can be inhibited by aminopyrazoline inhibitors.
(A) In-Cell Western (ICW) assay. Immunofluorescence-based detection
of AHNAK methylation. Each row represents a different inhibitor tested
in increasing concentrations ranging from 39 nM up to 5 μM.
(S)-4 and 1 are highlighted.
(B) Inhibition of AHNAK methylation in MDA-MB231 cells overexpressing
SMYD2. Methylation signal overlaps with AHNAK protein detection. (S)-4 inhibits concentration dependent the AHNAK
methylation. (C) Good correlation of cellular activity (ICW) to biochemical
activity (SPA) of (S)-4 and pyrazoline
cluster derivatives. (S)-4 has a cellular
IC50 ∼ 60 nM.
Characterization of (S)-4 in Proliferation and Apoptosis
Assays
To explore the potential effects of (S)-4 on proliferation, we tested a panel of 240 different
cancer cell lines (OncoPanel 240/Eurofins Panlabs). Cell lines were
long-term cultured with (S)-4 for 10
days to allow for a sufficient translation of demethylation of SMYD2
substrates to potential antiproliferative effects, which takes 48–72
h (Supporting Information Figure S2C).
(S)-4 exposure resulted in only limited
responses in a subset of cell lines; an antiproliferative response
with IC50 < 10 μM was seen in 21 cell lines (∼9%),
however, most cell lines (83%) did not reach 50% proliferation inhibition
with 20 μM of (S)-4 (Figure A). In addition, there was no clear preference for a specific
tissue origin of the responding cancer cell line. Thus, SMYD2 inhibition
by (S)-4 has only limited proliferation
effects in a small subset of cancer cell lines under the employed
conditions. On the basis of the observation that p53 protein is methylated
by SMYD2, which should lead to suppression of apoptosis, we were additionally
interested in the effects of (S)-4 in
combination with an apoptotic stimulus. KYSE-150, U2OS, and A2780
cell lines were pretreated with (S)-4 or inactive derivative 25 (see Table ) for 2 days (demethylation phase), followed
by treatment with doxorubicin (apoptotic trigger). (S)-4, but not 25, significantly improved
caspase 3/7 activation in all three tested cell lines without inducing
apoptosis alone (Figure B). Thus, SMYD2 inhibition can enhance apoptotic responses.
Figure 8
Proliferation
and apoptosis effects of (S)-4. (A)
A panel of 240 different cancer cell lines (OncoPanel 240/Eurofins
Panlabs) was long-term cultured with (S)-4 for 10 days and proliferation effects were determined. (S)-4 showed proliferation effects with an IC50 < 10 μM in a subset of cell lines. (B) Effects
on apoptosis induction were determined by caspase 3/7 activation.
KYSE-150, U2OS, and A2780 cell lines were pretreated with (S)-4 or inactive derivative 25 (see Table ) for
2 days, then apoptosis was induced by treatment with doxorubicin.
(S)-4, but not 25, significantly
improving caspase 3/7 activation.
Proliferation
and apoptosis effects of (S)-4. (A)
A panel of 240 different cancer cell lines (OncoPanel 240/Eurofins
Panlabs) was long-term cultured with (S)-4 for 10 days and proliferation effects were determined. (S)-4 showed proliferation effects with an IC50 < 10 μM in a subset of cell lines. (B) Effects
on apoptosis induction were determined by caspase 3/7 activation.
KYSE-150, U2OS, and A2780 cell lines were pretreated with (S)-4 or inactive derivative 25 (see Table ) for
2 days, then apoptosis was induced by treatment with doxorubicin.
(S)-4, but not 25, significantly
improving caspase 3/7 activation.
The First Chemical Probe Suitable for In Vivo Characterization of
SMYD2 Inhibition
Functional validation of novel potential
cancer targets such as the protein methyltransferase SMYD2 relies
on appropriate model systems in vitro as well as in vivo. Additionally,
chemical probe inhibitors also suitable for in vivo applications are
highly desirable. Most reported activities of SMYD2 are not directly
involved in survival signaling of cancer cells. Hence, in vitro proliferation
assays may not adequately cover the full phenotype of SMYD2 inhibition,
and more complex (in vivo) assays are thus required. The research
work characterizing SMYD2 in heart and skeletal muscle cells by knockdown
experiments in vivo[15,24,25] clearly illustrates the complex underlying biology of SMYD2 and
the necessity for chemical probes suitable for in vivo applications.
Therefore, we were interested in establishing if SMYD2 methylation
activity in tumor cells can be inhibited by (S)-4 in vivo. To this end, mice bearing subcutaneous tumor xenografts
(tumor tissue derived from the SMYD2-overexpressing KYSE-150 cell
line) were treated orally with 10, 30, 70, or 100 mg/kg (S)-4, or vehicle (PEG 400/water 8:2), once daily for
3 days. After the treatment period, tumors were harvested and analyzed
ex vivo for methylation of AHNAK by dot-blotting. For detection of
the methylation signals, SY46 methylation antibody was used (see Figure A and Supporting
Information Figure S2A–F). (S)-4 significantly reduced the methylation
with doses starting from 30 mg/kg, with most significant effects in
the 100 mg/kg treated group (P < 0.001, Student’s t test) (Figure A). Treatment with 10 mg/kg (S)-4 resulted in no significant effect on the methylation
level. Exposure at 10 mg/kg is close to the level of the cellular
IC50 for ∼9 h, which may indicate a need for an
even higher exposure as the IC50 to achieve in vivo effects
on demethylation.
Figure 9
In vivo characterization of (S)-4. (A) Demethylation of AHNAK was evaluated ex vivo. Average
methylation signals ± standard deviation per group are shown.
Mice (n = 12 per group) bearing subcutaneous tumor
xenografts (tumor tissue derived from the SMYD2-overexpressing KYSE-150
cell line) were treated as indicated, then the tumors were analyzed
for methylation signals by dot-blotting. (S)-4 significantly reduced the methylation with doses starting
from 30 mg/kg, with most significant effects in the 100 mg/kg group
(P < 0.001, Student’s t test). (B) Tumor area graph summarizing the in vivo tumor efficacy
study with the KYSE-150 xenograft model. Average tumor area ±
standard error of mean per group is plotted as the mean. Treatment
was started at day 4 after tumor inoculation (black arrow), and groups
were treated as indicated. (C) Tumor weight graphs corresponding to
the same experiment shown in (B). Average tumor weight are blotted
as box plot. * Significant (p value <0.05) difference
between vehicle control and treatment group (Dunn’s method).
Group 2: one animal was excluded on the 14th treatment day due to
animal welfare reasons (ulcerated tumor), tumor size 82 mm2. (D) Mouse body weight analysis. Average body weight per group throughout
the experiment is plotted as the mean.
In vivo characterization of (S)-4. (A) Demethylation of AHNAK was evaluated ex vivo. Average
methylation signals ± standard deviation per group are shown.
Mice (n = 12 per group) bearing subcutaneous tumor
xenografts (tumor tissue derived from the SMYD2-overexpressing KYSE-150
cell line) were treated as indicated, then the tumors were analyzed
for methylation signals by dot-blotting. (S)-4 significantly reduced the methylation with doses starting
from 30 mg/kg, with most significant effects in the 100 mg/kg group
(P < 0.001, Student’s t test). (B) Tumor area graph summarizing the in vivo tumor efficacy
study with the KYSE-150 xenograft model. Average tumor area ±
standard error of mean per group is plotted as the mean. Treatment
was started at day 4 aftertumor inoculation (black arrow), and groups
were treated as indicated. (C) Tumor weight graphs corresponding to
the same experiment shown in (B). Average tumor weight are blotted
as box plot. * Significant (p value <0.05) difference
between vehicle control and treatment group (Dunn’s method).
Group 2: one animal was excluded on the 14th treatment day due to
animal welfare reasons (ulcerated tumor), tumor size 82 mm2. (D) Mouse body weight analysis. Average body weight per group throughout
the experiment is plotted as the mean.Then the KYSE-150 esophageal xenograft model was used to
evaluate if the observed improved apoptosis induction in the in vitro
setting after treatment with doxorubicin (Figure B) could translate to antitumor efficacy
in vivo. Four groups of tumor-bearing mice were treated as follows:
Group 1 (control group) was only treated with vehicle (Solutol/ethanol/water
1:1:8) iv qd and once at day 4 with the vehicle used for doxorubicin
(saline) iv; group 2 was treated with (S)-4 at 500 mg/kg po qd; group 3 was treated with 10 mg/kg doxorubicin
iv once at day 4; group 4 was treated with a combination of (S)-4 and doxorubicin. There was a slight reduction
in area (Figure B)
and weight (Figure C) of tumors from mice treated with the combination of (S)-4 and doxorubicin relative to tumors from the control
group. Combination treatment only resulted in a minor increase in
treatment-related body weight loss (Figure D). The combination treatment reached a T/C
level (based on tumor weight) of 0.46, which is significant (Figure C). Therefore, combination
of a SMYD2 inhibitor with a chemotherapeutic agent resulted in reduced
cancer cell growth in vivo. In comparison, the monotherapy groups
treated with only doxorubicin or (S)-4 showed no significant antitumor efficacy relative to the vehicle
control group. These data are consistent with the observed limited
cellular proliferation effects of (S)-4 in the cell line panel (Figure A) and indicate that, in contrast to SMYD2 knockdown,[2] a catalytic inhibition may be insufficient to
induce cell death in the KYSE-150 esophageal model. Furthermore, the
monotherapeutic approach with doxorubicin did not result in any antitumor
efficacy in the KYSE-150 xenograft model.
Conclusion
In
summary, we have identified (S)-4 as
a potent, selective, and cell-active, substrate-competitive inhibitor
of SMYD2. Our data show that SMYD2 inhibition can enhance efficacy
of doxorubicin in vivo, which confirms our in vitro observation of
highercaspase 3/7 activation (Figure B). Our results (S)-4 are also in agreement with an earlier study,[13] where an increased apoptosis induction in cells with an
SMYD2 knockdown was observed. Nonetheless, in our initial explorative
in vivo study with (S)-4, effects on
xenografted tumors were only moderate. In addition, high doses of
(S)-4 were needed in vitro as well as
in vivo relative to the concentration needed to achieve effects on
methylation. Therefore, we cannot exclude the possibility that additional,
so-far unexplored activities of SMYD2 might be responsible for the
observed effects. This again underlines the necessity to identify
suitable chemical probes for more extensive target validation campaigns
to fully explore the complex biology of SMYD2 and other targets. In
this regard, (S)-4 will be a highly
valuable tool for the further exploration of SMYD2 biology, not only
for in vitro but also for in vivo studies.
Experimental
Section
General Procedures
All reagents
and solvents were used as purchased, unless otherwise specified. All
final products were at least 95% pure, as determined by analytical
HPLC.
Materials
Intermediate 10a and compounds 5, 6, and 12–24 were synthesized according to the methods described previously.[37]1H NMR were determined to be >95%. 1H NMR spectra were recorded on Bruker Avance III HD spectrometers
operating at 300, 400, or 500 mHz. The chemical shifts (δ) reported
are given in parts per million (ppm), and the coupling constants (J) are in hertz (Hz). The spin multiplicities are reported
as s = singlet, br s = broad singlet, d = doublet, t = triplet, q
= quartet, m = multiplet, and br = broad. The LC/MS analysis was performed
on Waters Acquity UPLCMS SingleQuad with a Acquity UPLC BEH C18 column
(1.7 μm, 50 mm × 2.1 mm) at 60 °C, using water +0.1
vol % formic acid (99%) and acetonitrile as mobile phase at a flow
rate of 0.8 mL/min and a DAD detector (210–400 nm). LC/MS/MS
was performed on a CTC PAL autosampler, an Agilent 1200 HPLC, and
a ABSciex 4000 mass spectrometer.
Assignment of Stereochemistry
For all separated enentiomers, it was assumed based on the co-crystal
structure of 4, that the active enantiomer (SMYD2 inhibition)
has S-configuration.
To
a stirred solution of 2-amino-1-(3,4-dichlorophenyl)ethanone hydrochloride
(1:1) (7d; 116 g, 0.480 mol) in water (500 mL) was added
allyl chloroformate (56.5 mL, 0.530 mol) in DCM (800 mL). The reaction
mixture was cooled to 0 °C, and K2CO3 (207
g, 1.49 mol) in water (1 L) was added dropwise over 1 h. The mixture
was allowed to warm to rt, stirred overnight, then diluted with DCM
(500 mL). The organic phase was washed with saturated aqueous NH4Cl solution (400 mL) followed by brine (500 mL), then dried
overMgSO4, filtered, and concentrated in vacuo. The crude
mixture was purified by dry flash column chromatography (eluent: DCM/heptane
2:1, 3:1, 4:1; DCM; EtOAc) to yield allyl [2-(3,4-dichlorophenyl)-2-oxoethyl]carbamate
(8d; 120 g, 87%) as a white crystalline solid. 1H NMR (400 MHz, CDCl3): δ 4.62 (d, J = 5.5 Hz, 2H), 4.68 (d, J = 4.6 Hz, 2H), 5.25 (dd, J = 1.4, 10.5 Hz, 1H), 5.35 (dd, J = 1.4,
16.9 Hz, 1H), 5.72 (br s, 1H), 5.94 (m, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.79 (dd, J = 2.1, 8.5 Hz, 1H),
8.06 (d, J = 1.8 Hz, 1H). LC-MS (ESI): m/z = 288.1 [M + H]+. To a stirred suspension
of 8d (50.0 g, 0.174 mol) in EtOH (390 mL) was added
formaldehyde solution (20 mL, 0.261 mol; 37 wt % in water) followed
by the dropwise addition of piperidine (26 mL, 0.261 mol) in EtOH
(130 mL) over 30 min. The reaction mixture was stirred overnight;
TLC indicated consumption of 8d. The volatiles were removed
by evaporation to yield an orange oil which was not further purified.
To a solution of this crude material in EtOH (480 mL) was added hydrazine
monohydrate (29.6 mL, 0.609 mol), and the reaction mixture was heated
to reflux for 2.5 h. Then the mixture was allowed to cool to rt, concentrated,
and poured over ice-cooled saturated NH4Cl solution (300
mL). The crude product was extracted with EtOAc (1.5 L), and the combined
organic layers were washed with brine (300 mL) then dried overMgSO4, filtered, and concentrated to yield allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate (9d; 50.0 g, 91%)
as a pale-yellow solid. 1H NMR (400 MHz, [D]6DMSO): δ 3.24 (m, 1H), 3.59 (m, 1H), 4.39–4.54 (m, 2H),
5.08–5.25 (m, 3H), 5.79–5.90 (m, 1H), 7.52 (dd, J = 1.8, 8.3 Hz, 1H), 7.57 (br s, 1H), 7.59 (m, 1H), 7.68
(d, J = 1.8 Hz, 1H), 7.84 (d, J =
8.7 Hz, 1H). LC-MS (ESI): m/z =
314.1 [M + H]+. To a stirred suspension of 9d (50.0 g, 0.159 mol) in i-PrOH (860 mL) was added
diphenyl N-cyanocarbonimidate (38.0 g, 0.159 mol).
The reaction mixture was heated to reflux, at which point the suspension
dissolved into solution; after a further 10 min at reflux, a white
precipitate formed. The mixture was stirred at reflux for a further
1 h then allowed to slowly cool to rt overnight. The precipitate was
collected by filtration and washed with Et2O (2 ×
250 mL). The resulting white solid was allowed to dry to yield 10d (48.6 g, 67%) as a white solid. 1H NMR (400
MHz, [D]6DMSO): δ 4.13 (apparent d, J = 8.2 Hz, 1H), 4.47 (m, 3H), 5.14 (m, 2H), 5.51–5.63 (m,
1H), 5.79–5.90 (m, 1H), 7.23 (d, J = 7.3 Hz,
2H), 7.30 (t, J = 7.8 Hz, 1H), 7.45 (m, 2H), 7.79
(br m, 2H), 7.97 (br s, 1H), 8.19 (d, J = 8.7 Hz,
1H). LC-MS (ESI): m/z = 458.0 [M
+ H]+.
Compound 10f was prepared as described
for 10d, starting from 7f (12.0 g, 55.7
mmol) to obtain 10f (9.1 g, 38% over 4 steps) as white
solid. 1H NMR (400 MHz, [D]6DMSO): δ 4.05–4.08
(m, 1H), 4.43–4.47 (m, 3H), 5.1–5.2 (m, 2H), 5.50–5.55
(m, 1H), 5.80–5.90 (m, 1H), 6.08 (s, 2H), 7.05 (d, J = 7.8 Hz, 1H), 7.21–7.27 (m, 2H), 7.27–7.41
(m, 3H), 7.43–7.50 (m, 2H), 8.16 (d, J = 8.7
Hz, 1H). LC-MS (ESI): m/z = 434.2
[M + H]+.
General Procedure for the Synthesis of Allyl
[1-(N′-Cyano-N-arylcarbamimidoyl)-3-phenyl-4,5-dihydro-1H-pyrazol-4-yl]carbamates 11j–s
To a stirred solution of the respective aniline
derivative (3 equiv) in anhydrous THF (2 mL/mmol) at −78 °C
was added n-BuLi (3 equiv, 2 M in hexane) dropwise
while maintaining the reaction temperature below −65 °C.
The reaction mixture was stirred at −78 °C for 1 h, then
the respective phenyl 4-{[(allyloxy)carbonyl]amino}-N-cyano-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidate 10 (1 equiv) in anhydrous THF (30 mL/mmol) was added dropwise
while maintaining the reaction temperature below −65 °C.
The reaction mixture was stirred at −78 °C for 2 h, then
slowly poured over saturated NH4Cl solution (30 mL/mmol
based on 10). The crude product was extracted into EtOAc
(30 mL/mmol); the combined organic layers were washed with brine (15
mL/mmol), dried overMgSO4, filtered, and concentrated.
The crude solid was precipitated from a minimum volume of EtOAc, collected
by filtration, and washed with Et2O to give the corresponding
intermediate 11.
Compound 11j was prepared from 10b (5.0 g, 10.7 mmol)
and 3-(difluoromethoxy)aniline. 11j was not pure after
chromatography and was used as such in further steps (3.8 g, purity
77%; UV, 51%). LC-MS (ESI): m/z =
533.1 [M + H]+.
N-(1-{N′-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-phenyl-4,5-dihydro-1H-pyrazol-4-yl)-N-ethyl-2-hydroxyacetamide
(25) and N-[3-(4-Bromophenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide
(26)
To a stirred solution of 11j (3.8 g, 77%-UV, 5.5 mmol) in degassed THF (150 mL) was added 1,3-dimethylbarbituric
acid (1.8 g, 11.0 mmol), followed by tetrakis(triphenylphosphine)palladium(0)
[Pd(PPh3)4; 0.25 g, 0.22 mmol]. The reaction
mixture was stirred underargon for 12 h, then cautiously quenched
with saturated NaHCO3 solution (150 mL) and extracted into
EtOAc (150 mL). The organic layer was washed with brine (100 mL),
then dried overNa2SO4, filtered, and concentrated.
The crude material was purified by dry flash column chromatography
(eluent: EtOAc, MeOH) to yield 4-amino-3-(4-bromophenyl)-N′-cyano-N-[3-(difluoromethoxy)phenyl]-4,5-dihydro-1H-pyrazole-1-carboximidamide (2.4 g, 95%; UV, 92%). 1H NMR (400 MHz, [D]6DMSO): δ 2.38 (br s,
2H), 4.02 (dd, J = 4.4, 11.5 Hz, 1H), 4.37 (dd, J = 9.9, 11.4 Hz, 1H), 4.81 (dd, J = 4.5,
9.9 Hz, 1H), 6.98 (dd, J = 2.0, 8.1 Hz, 1H), 7.22
(t, J = 74.0 Hz, 1H), 7.20–7.23 (m, 1H), 7.27
(ddd, J = 0.8, 2.0, 8.1 Hz, 1H), 7.40 (t, J = 8.1 Hz, 1H), 7.64–7.70 (m, 2H), 7.98–8.03
(m, 2H), 9.62 (br s, 1H). LC-MS (ESI): m/z = 449.3 [M + H]+. To the amine product (2.30
g, 5.1 mmol) in MeOH (70 mL) at rt was added acetaldehyde (0.27 g,
6.1 mmol). The reaction mixture was stirred at rt for 1 h, and at
40 °C for 1 h, then it was cooled to 0 °C and NaBH4 (0.22 g, 5.7 mmol) was added in small portions. The mixture was
stirred at rt for 1 h, then poured over saturated NaHCO3 solution (10 mL). The volatiles were removed by evaporation, and
the resulting aqueous slurry was extracted with EtOAc (2 × 10
mL). The combined organic layers were washed with brine (100 mL),
then dried overNa2SO4, filtered, and concentrated
to yield a crude black oil. The crude material was purified by dry
flash column chromatography (eluent: DCM, MeOH) to yield 3-(4-bromophenyl)-N′-cyano-N-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-4,5-dihydro-1H-pyrazole-1-carboximidamide (1.11 g, 45%) as the main N-ethylation
product. LC-MS (ESI): m/z = 477.1
[M + H]+. The corresponding debrominated derivative N′-cyano-N-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamide (0.41 g, 19%) was obtained
as a byproduct. LC-MS (ESI): m/z = 398.3 [M + H]+. To the debrominated byproduct (410
mg, 1 mmol) in DCM (15 mL) were added Et3N (0.15 mL, 1.1
mmol), acetoxyacetyl chloride (0.12 mL, 1.1 mmol), and DMAP (126 mg,
1 mmol), and the mixture was stirred at rt for 1 h. EtOAc (15 mL)
was added, and the mixture was washed with saturated aqueous NH4Cl solution (20 mL) and brine (20 mL). The organic phase was
dried overNa2SO4 and concentrated. The crude
product was purified by dry flash column chromatography (eluent: DCM,
MeOH) to yield 504 mg of an impure intermediate, which was dissolved
in MeOH (12 mL), K2CO3 (140 mg, 1.01 mmol) was
added, and the mixture was heated to reflux for 30 min. After cooling,
to the reaction mixture was added saturated aqueous NH4Cl, and the mixture was extracted with EtOAc. The organic phase was
dried overNa2SO4, concentrated, and the residue
purified by flash column chromatography (eluent: DCM, MeOH) and subsequent
preparative HPLC to yield N-(1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-phenyl-4,5-dihydro-1H-pyrazol-4-yl)-N-ethyl-2-hydroxyacetamide
(25; 50 mg, 10%). 1H NMR (300 MHz, [D]6DMSO): δ 1.03 (br s, 3H), 3.16–3.33 (m, 2H),
3.92–4.25 (m, 3H), 4.47 (br t, J = 11.7 Hz,
1H), 4.75 (br t, J = 5.5 Hz, 1H), 6.94–7.03
(m, 1H), 7.19–7.30 (m, 3H), 7.36–7.52 (m, 4H), 7.75–7.84
(m, 2H), 9.77 (br s, 1H). LC-MS (ESI): m/z = 457.2 [M + H]+.To the main product
from the N-ethylation step (1.1 g, 2.3 mmol) in DCM (30 mL) were added
Et3N (0.34 mL, 2.4 mmol), acetoxyacetyl chloride (0.27
mL, 2.4 mmol), and DMAP (282 mg, 2.3 mmol) and the mixture was stirred
at rt for 1 h. EtOAc (30 mL) was added, and the mixture was washed
with saturated aqueous NH4Cl solution (40 mL) and brine
(40 mL). The organic phase was dried overNa2SO4 and concentrated. The crude product was purified by dry flash column
chromatography (eluent: DCM, MeOH) to yield 1.2 g of an impure intermediate.
Then 200 mg of this intermediate were dissolved in MeOH (5 mL), K2CO3 (48 mg, 0.35 mmol) was added, and the mixture
was heated to reflux for 30 min. After cooling, the reaction mixture
was concentrated and the residue was purified by HPLC to yield (N-[3-(4-bromophenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide
(26; 50 mg, 24%). 1H NMR (400 MHz, [D]6DMSO): δ 1.05 (br s, 3H), 3.27–3.36 (m, 2H),
3.98–4.18 (m, 3H), 4.48 (t, J = 11.7 Hz, 1H),
4.72 (t, J = 4.9 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 7.21 (s, 1H), 7.23 (t, J = 74.0
Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H),
7.73 (d, J = 8.3 Hz, 2H), 9.78 (br s, 1H). LC-MS
(ESI): m/z = 535.1 [M + H]+.
Cell
lines were obtained from the American Type Culture Collection (HEK293,
U2OS), the German Collection of Microorganisms and Cell Cultures (KYSE-150),
and the European Collection of Cell Cultures (A2780). They were maintained
in the recommended cell culture media at 37 °C in 5% CO2.
Enzyme Activity and Inhibition Assays
SMYD2 enzyme
kinetics and inhibitory activities of compounds were analyzed using
a scintillation proximity assay (SPA) which measured methylation by
the enzyme of the synthetic, biotinylated peptide Btn-Ahx-GSRAHSSHLKSKKGQSTSRH-amide
(Biosyntan) derived from the C-terminal domain of p53. The SMYD2 full-length
enzyme with an N-terminal 6xHis tag was expressed in Escherichia coli and purified by affinity chromatography
on a Ni-NTASepharose column, followed by size-exclusion chromatography
on a Superdex 200 16/60 column (GE Healthcare). Assays were conducted
in 384-well microtiter plates in a buffer containing 50 mM Tris/HCl
pH 9.0, 1 mM DTT, 0.01% (w/v) BSA, and 0.0022% (v/v) Pluronic, and
a final volume of 5 μL. The SMYD2 concentration in the assay
was 3 nM, while tritiated S-adenosyl-l-methionine
(3H-SAM) and the peptide substrate were present at 60 nM
and 1 μM, respectively, to ensure “balanced” conditions.[46] Apparent Michaelis–Menten constants for
SAM and the p53 peptide were determined by titrating one substrate
to saturation at cosubstrate concentrations of 1 × Km(app). Enzyme kinetics were followed over 2 h by quenching
the reactions as described above at time points 0, 5, 10, 15, 30,
60, 90, and 120 min. Compounds were tested in 11-point, 3.5-fold dilution
series ranging from 0.1 nM to 20 μM. Reactions were run for
30 min and quenched by adding Streptavidin PS SPA imaging beads (PerkinElmer)
to a concentration of 3.12 μg/μL and 25 μM “cold”
SAM. The amount of product was evaluated using a Viewlux (PerkinElmer)
CCD plate imaging device [emission filter 613/55]. The data were normalized
using two sets of control wells for high (= enzyme reaction with DMSO
instead of test compound = 0% = minimum inhibition) and low (= all
assay components without enzyme = 100% = maximum inhibition) SMYD2
activity. IC50 values were calculated by fitting the normalized
inhibition data to a four-parameter logistic equation using either
a Bayer proprietary tool or Genedata Screener analysis software.For mechanism-of-inhibition studies, IC50 determinations
were basically performed as described above, but incubation times
were adjusted to guarantee kinetic linearity at each concentration
of substrate analyzed. For peptide competition studies, IC50 values were determined at six different concentrations of p53 peptide
(0.5, 1, 2, 4, 6, and 12 μM). For SAM competition, 25, 50, 100,
200, 300, and 600 nM 3H-SAM were used. In both cases, the
other cosubstrate was used at a concentration of 1 × Km(app).Calculation of Ki(app) and αKi(app) using
the corresponding Cheng–Prusoff equations:[41]where [S] = substrate concentration, Km(app) = apparent Michaelis–Menten constant, Ki(app) = apparent inhibitor constant for binding to free enzyme,
and αKi(app) = apparent inhibitor
constant for binding to enzyme–substrate complex.
Selectivity
Assays
The effect of (S)-4 on
the methyltransferase activity of G9A, EHMT1, SUV39H1, SUV39H2, SETDB1,
SETD7, SETD8, SUV420H1, SUV420H2, PRMT1, PRMT3, PRMT6, PRMT8, PRDM9,
SETD2, and SMYD3, and MLL1, MLL3, EZH2, and PRMT5 complexes, as well
as DNMT1 and BCDIN3D, was assessed using the SPA method.[28] For DNMT3A/3L, DNMT3B/3L, ASH1L, DOT1L, NSD1, NSD2, NSD3, METTL21A, and METTL21D,
a trichloroacetic acid (TCA) precipitation method in filter plates
were employed.[47]
PAR1 Assay
The
identification of PAR1 antagonistic effects and the quantification
of those effects, as well as the resulting IC50 values,
were performed as described previously.[36]
Biophysical Validation by ITC/TSA
Thermal shift assays
(TSA) were carried out with the ThermoFluor system (Johnson &
Johnson Pharmaceutical Research and Development). Melting curves were
obtained at a protein concentration of 1.5 μM, 8xSypro Orange
(Invitrogen), and a ligand concentration of 100 μM, using buffer
containing 20 mM Tris pH 8.0, 100 mM NaCl, 8.5%. Scans were measured
from 25 to 95 °C at a scanning rate of 2 °C/min. TSA data
were analyzed using ThermoFluor++ (version 1.3.7) software.Isothermal titration calorimetry (ITC) measurements were carried
out using a MicroCal iTC200 system. In the experiments, compound (400
μM) was titrated into SMYD2 protein (67 μM) dissolved
in 25 mM Tris pH 8.5, 50 mM NaCl, 0.0022% Pluronic F-127, and 2% DMSO
at 25 °C. After an initial injection of 0.2 μL, which was
not considered for data fitting, 10 injections of 4 μL each
in 150 s intervals were performed. The stirring speed of the protein
in the cell was 500 rpm. The protein concentration was determined
from UV absorbance measurements at 280 nm. Data were evaluated using
ORIGIN software, as supplied with the instrument.
Purification
and Crystallization of SMYD2
Recombinant humanSMYD2 (UniProt
Q9NRG4, amino acids 2–433) was expressed in insect cells (Sf9)
containing an N-terminal TEV-cleavable 6xHis tag. Cell pellets were
resuspended in lysis buffer (40 mM Tris pH 8, 500 mM NaCl, 0.1% IGEPAL,
5 mM imidazole, 1 mM DTT) supplemented with complete EDTA-free protease
inhibitor tablets and 50 U/mL benzonase. The cell lysate was loaded
onto a Ni-NTA column, eluted with imidazole, and concentrated using
an ultracentrifugal filter unit. Then, SMYD2 was gel-filtrated on
a Superdex S200 column equilibrated in 20 mM Tris pH 8, 100 mM NaCl,
5% glycerol, and 1 mM DTT. The 6xHis tag was cleaved with TEV protease
in solution at 6 °C overnight. Uncleaved SMYD2 and TEV protease
were separated from the cleaved product by a second Ni-NTA affinity
step. The cleaved SMYD2 protein was further purified by a second gel-filtration
step using a Superdex 200 column equilibrated in 20 mM Tris pH 8,
150 mM NaCl, 5% glycerol, and 1 mM Tris(2-carboxyethyl)phosphine (TCEP).
The protein was concentrated to 15.5 mg/mL (313 μM) (UV–vis)
using an ultracentrifugal filter unit and shock-frozen in liquid nitrogen.
For crystallization, the cofactor SAM was added to a final concentration
of 3.8 mM as follows: SAM stock solution (100 mM in DMSO, 1.2 μL)
was added to concentrated SMYD2 solution (30 μL) and incubated
at 4 °C for 2 h. Crystals grew at 20 °C within 3 d using
the hanging drop method. Drops were made from the SMYD2/SAM solution
(1 μL) and reservoir solution [20–24% (w/v) PEG 3350,
100 mM HEPES pH 7.0; 0.8 μL]. Thirty minutes after drop setup,
a seed solution (0.2 μL) was added. The seed solution was made
from SMYD2:SAM crystals (obtained with the same reservoir conditions
in a previous experiment), which were crashed manually (using Seed
Beads, Hampton Research), diluted in reservoir solution, shock-frozen,
and stored at −80 °C. For complex formation, a crystal
was transferred into a new drop reservoir solution (1.5 μL).
A stock solution of the respective inhibitor (100 mM in DMSO) was
10-fold diluted with reservoir solution. Over the course of 2 h, this
diluted stock solution (1.5 μL) was added in three steps of
0.5 μL to the drop containing the SMYD2:SAM crystal, resulting
in a final concentration of 5 mM inhibitor in the soaking drop. The
crystal was soaked in this drop at 20 °C for 4 days in the case
of (S)-4 and for 1 h for compound 6.
Crystallographic Data Collection, Structure
Determination, and Refinement
The soaked crystal was briefly
immersed in cryo buffer (0.1 M HEPES pH 7.0, 22% PEG 3350, 20% glycerol,
2 mM inhibitor) and shock-frozen in liquid nitrogen. Diffraction data
sets were collected using beamline 14.1 at the Helmholtz-Zentrum Berlin
at 100 K using a wavelength of 0.91841 Å and a PILATUS detector.
The diffraction images were processed using the program XDS.[48] The crystals belonged to space group P212121 with one molecule
per asymmetric unit. The crystal form described here was first solved
for an SMYD2:SAM crystal in the absence of an inhibitor, using the
Molecular Replacement method (program Phaser[49] from the CCP4 program suite[50] and PDB
entry 3TG5 as
search model). The data sets described here were then solved by rigid
body refinement using the SMYD2:SAM structure as starting model and
the program Refmac[51] from the CCP4 program
suite. 3D models for compound 6 and (S)-4 were generated using the program Discovery Studio,
and parameter files for crystallographic refinement and model building
were generated using the software PRODRG.[52] (S)-4 was manually built into the
electron-density maps using Coot,[53] followed
by several cycles of refinement with Refmac and rebuilding in Coot.
For the data collection and refinement statistics, see Supporting
Information Table S3.
WaterMap
Calculations
WaterMap calculations were based on the crystal
structure of the 4-chlorophenyl derivative 6 (PDB code 5ARF). The SMYD2–compound 6 complex structure was prepared using the Protein Preparation
Wizard functionality in Maestro.[54−57] Preparation involved assignment
of bond orders, addition of hydrogens, creation of zero-order bonds
to metals, deletion of water molecules beyond 5 Å from heteroatoms,
assignment of protonation states according to pH 7.0, and optimization
of the hydrogen-bonding network and restrained minimization. Here,
and for all further minimizations and simulations, the OPLS 2005 force
field[58,59] was used.Water sites and corresponding
free energies were calculated using WaterMap.[39,40] The calculation involved the following (default) settings: water
molecules within 10 Å of the ligand were investigated, the ligand
as well as any crystallographic water molecules were removed prior
to simulation, the protein was truncated beyond 20 Å from the
ligand, and the simulation time was 2 ns. In brief (see ref (40) for details), the different
stages of the calculation were solvation of the system in an orthorhombic
box of TIP4P water[60] with a minimum distance
between box edge and solute of 10 Å and a series of minimizations
and short simulations to equilibrate the system, followed by a 2 ns
production simulation. All nonwater heavy atoms were harmonically
restrained during all minimizations and simulations using a force
constant of 5 kcal mol–1 Å–2. Coordinates were saved every 1.5 ps, yielding a total of 1334 snapshots
for further analysis. Water molecules were subsequently clustered
such that nonoverlapping spheres (i.e., hydration sites) with radius
1 Å were obtained. Thermodynamic properties of these sites (i.e.,
enthalpies, entropies, and thus free energies) were approximated as
follows: Enthalpies were estimated as the difference between the average
interaction energy of the water molecule with the rest of the system
and the average interaction energy in bulk water. Entropies were estimated
using inhomogeneous solvation theory.[61]
Antibody Generation
To detect SMYD2-mediated methylation,
we used a lysine monomethylation specific rabbit polyclonal antibody
(SY46). The antibody was generated (Eurogentec) against a p53 peptide
containing the monomethylated K370 epitope, as described elsewhere.[13] Antibody has been purified against unmethylated
p53 peptide. Using this antibody in cellular systems revealed that
it also recognized methylated AHNAK, which is also methylated by SMYD2
(see Supporting Information Figure S2).
In Vitro Methylation of Recombinant p53
Recombinant p53
protein (BD Pharmingen no. 556439) was incubated with full-length
SMYD2 protein (in-house production) and SAM in reaction buffer (50
mM Tris pH 9, 1 mM DTT, 0.01% BSA, 0.0022% Pluronic) at 30 °C
overnight.
Western Blot
Cells were lysed in
RIPA buffer Pierce (Thermo Fisher Scientific) with benzonase (Merck)
and protease inhibitors (Roche Diagnostics). Proteins were separated
by SDS-PAGE and blotted onto nitrocellulose membranes (Invitrogen).
Membranes were blocked in 5% milk PBS-T (phosphate buffered saline
with 0.1% Tween 20) and immunoprobed with antibodies raised against
histone H3 (Cell Signaling Technology no. 4499S), p53 (BD Biosciences
no. 554294), AHNAK (Santa Cruz Biotechnology, no. sc-390743, 1:500),
SMYD2 (abcam, no. ab108217, 1:1000), beta-actin (abcam, no. ab8224,
1:1000), or with the methylation specific antibody SY46 (Eurogentec).
The secondary antibodies used were goat antimouse/rabbit IRDye 800
CW (LI-COR Biosciences) and Alexa Fluor 680 goat antimouse/rabbit
IgG. Bands were detected and quantified with Odyssey Fc Software (LI-COR
Biosciences). Western blot assay for p53 methylation in HEK293T cells
was performed as described elsewhere with an antibody directed against
p53K370me1 kindly provided by Dr. Shelley Berger.[13,28]
Caspase 3/7 Activation Assay
Effects on apoptosis induction
were measured with the Caspase-Glo 3/7 Assay System (Promega). In
brief, 10000 cells/well were treated for 2 d with compound 25 or (S)-4 (to allow for demethylation
of p53. Then, caspase activation was induced by doxorubicin (Sigma)
(1.25 μM for U2OS and A2780 cells, 2.5 μM for KYSE-150
cells) treatment for 24 h. The apoptotic index was determined by normalization
of caspase 3/7 activation signals to proliferation effects, measured
by alamarBlue (Thermo Fisher Scientific).
Cell-Based Assay for the
Detection of SMYD2Methylation Activity
For the detection
of SMYD2 cellular methylation activity, an In-Cell Western (ICW) assay
was established. For the ICW, KYSE-150 cells stably transfected with
a construct expressing wild-type N-terminal 2xc-myc-tagged SMYD2 (NCBI
reference sequence: NP_064582.2) were used. For further detection
and validation experiments of AHNAK methylation, we additionally generated
stable HeLa and MDA-MB-231 cell lines using the same construct. For
conducting the ICW assay, 5000 SMYD2-engineered KYSE-150 cells/well
were seeded in 96-well plates (Sigma) and cultivated for 24 h at 37 °C
in 5% CO2. Nontransfected KYSE-150 cells were used as a
control for maximal inhibition of methylation activity. Cells were
grown in 49% RPMI 1640 and 49% Ham’s F12 media supplemented
with 2% heat-inactivated FCS. For the determination of SMYD2 inhibitory
activity, cells were treated for 72 h in the presence of test compound
(at a final concentration range of 3.9 × 10–8 to 5 × 10–6 M) or with DMSO. Media were removed,
and 3.7% (w/v) formaldehyde in PBS was added for 20 min. After two
washes with PBS, 0.25% (v/v) Triton X-100 in PBS was added for 15
min of permeabilization. After one wash with PBS, cells were blocked
in 5% (w/v) nonfat dry milk in PBS for 1 h. Fixed cells were exposed
to primary methylation antibody (SY46, 1:200) in 5% nonfat dry milk
in PBS for 24 h. One row of cells on each plate was not exposed to
methylation antibody (SY46) and was reserved for background control
measurements. The wells were washed three times with PBS, then secondary
IR800-conjugated antibody (LI-COR, no. 926–32211, 1:1000) and
DNA-intercalating dye DRAQ5 (Thermo Fisher Scientific, no. 62251,
1:1000) were added for 3 h in blocking buffer. After five washes with
PBS, the fluorescence in each well was measured on an Odyssey scanner
(LI-COR) at 800 nm (SY46 methylation signal, 764 nm excitation) and
700 nm (DRAQ5 signal, 683 nm excitation). Fluorescence intensity was
quantified and normalized to background and DRAQ5 signals. The normalized
data were further analyzed by four-parameter logistic regression analysis
using a Bayer proprietary tool to determine the IC50 value
for each tested compound. For IC50 determinations, C0 (= no inhibition) was defined as the signal
measured for the DMSO-treated controls. Ci (maximal inhibition) was defined as the signal measured for nontransfected
KYSE-150 cells.
Proliferation Panel
For the characterization
of proliferation effects, the OncoPanel 240 (Eurofins Panlabs) was
used.
Mouse Tumor Xenograft Model
Animal experiments were
conducted in accordance with the German animal welfare laws, approved
by local authorities and in accordance with the ethical guidelines
of Bayer AG. Eight-week-old female BALB/c nude mice obtained from
Charles River Laboratories (Germany) were acclimated for at least
for 24 h before tumor cell injection. KYSE-150 cells (4 × 106) were resuspended in 100% Matrigel (100 μL) and injected
subcutaneously into the right flank region of the mice; 4 days aftertumor cell inoculation, mice were randomized into four treatment groups.
Treatment was started at day 4 aftertumor inoculation. Group 1 (n = 12) was treated with vehicle only (Solutol/ethanol/water
1:1:8) iv qd and once at day 4 with saline iv; group 2 (n = 6) was treated with 500 mg/kg (S)-4 po qd; group 3 (n = 12) was treated with 10 mg/kg
doxorubicin iv once at day 4; group 4 (n = 6) was
treated with a combination of (S)-4 and
doxorubicin. Tumors were measured three times per week for 14 days.
Tumor weight is assumed to be log normally distributed, thus all tumor
weights were logarithmically transformed (base = 2) prior to statistical
inference. One-sided Dunnett’s comparison of log 2 tumor weights
of all treatment groups with the vehicle group were carried out on
an overall significance level of 0.05. The results were transformed
from the log 2 scale to the original scale so that the difference
from the pairwise comparisons and the respective 95% confidence intervals
calculated on the log 2 scale correspond to T/C ratios of geometric
mean values plus respective 95% confidence intervals on the original
tumor weight scale (mg). Data was analyzed with R 3.0.1.
Ex Vivo
Methylation Detection by Dot Blot
Eight-week-old female BALB/c
nude mice obtained from Charles River Laboratories (Germany) were
acclimated for at least for 24 h before tumor cell injection. SMYD2-engineered
KYSE-150 cells (4 × 106) were resuspended in 100%
Matrigel (100 μL) and injected subcutaneously into the right
flank region of the mice. Treatment was started when tumors reached
a tumor area of 60–70 mm2 (day 7 after inoculation).
Mice (n = 12 per group) were treated with 10, 30,
70, or 100 mg/kg (S)-4 po qd for 3 days,
or vehicle (PEG 400/water 8:2) po qd for 3 days. After the treatment
period, tumor samples were immediately frozen in liquid nitrogen and
stored at −80 °C. Frozen tumors were mechanically homogenized
using TissueLyser and stainless steel beads (Qiagen), and proteins
were extracted as described for the Western blot method. Whole protein
lysate (50 μg per sample) was transferred with the Dot-Blot
system MiniFold-1 (Whatman) onto a nitrocellulose membrane (Invitrogen).
Membrane were blocked in 5% milk PBS-T and immunoprobed with the SY46
methylation antibody. The secondary antibody used was goat antirabbit
IRDye 800 CW (LI-COR, no. 926–32211, 1:1000). Bands were detected
and quantified with Odyssey Fc software (LI-COR Biosciences).
In
Vivo Rat PK
Pharmacokinetic properties of (S)-4 were determined by administering the test compound
as indicated. Blood samples were withdrawn at different time points,
and plasma was separated by centrifugation. The samples were analyzed
by LC-MS/MS.
Metabolic Stability in Rat Hepatocytes
Liver cells were distributed in Williams’ medium E containing
5% fetal calf serum (FCS) to glass vials at a density of 1.0 ×
106 vital cells/mL. The test compound was added to a final
concentration of 1 μM. During incubation, the hepatocyte suspensions
were continuously shaken and aliquots were taken at 2, 8, 16, 30,
45, and 90 min, to which an equal volume of cold MeOH was immediately
added. Samples were frozen at −20 °C overnight, then centrifuged
(15 min, 3000 rpm), and the supernatant was analyzed with an Agilent
1200 HPLC system with LC-MS/MS detection. The half-life of a test
compound was determined from the concentration–time plot. The
intrinsic clearances were calculated from the half-life, together
with additional parameters (liver blood flow, quantity of liver cells
in vivo and in vitro). The hepatic in vivo blood clearance (CL) and
the maximal oral bioavailability (Fmax) were calculated using the following parameters: liver blood flow,
4.2 L/h/kg rat; specific liver weight, 32 g/kg rat body weight; liver
cells in vivo, 1.1 × 108 cells/g liver; liver cells
in vitro, 0.5 × 106/mL.
Caco2 Drug Permeability
Assay
Caco-2 cells (purchased from DSMZ Braunschweig, Germany)
were seeded at a density of 4.5 × 104 cell per well
on 24-well insert plates, 0.4 μm pore size, and grown for 15
days in DMEM medium supplemented with 10% fetal bovine serum, 1% GlutaMAX
(100×, GIBCO), 100 μg/mL penicillin, 100 μg/mL streptomycin
(GIBCO), and 1% nonessential amino acids (100×). Cells were maintained
at 37 °C in a humified 5% CO2 atmosphere. Medium was
changed every second to third day. Before running the permeation assay,
the culture medium was replaced by an FCS-free Hepes-carbonate transport
buffer (pH 7.2). For assessment of monolayer integrity, the transepithelial
electrical resistance (TEER) was measured. Test compounds were dissolved
in DMSO and added either to the apical or basolateral compartment
in a final concentration of 2 μM. Before and after incubation
at 37 °C, samples were taken from both compartments. Analysis
of compound content was performed following precipitation with methanol
and LC/MS/MS analysis. Permeability (Papp) was calculated in the apical to basolateral (A → B) and
basolateral to apical (B → A) directions. The apparent permeability
was calculated using following equation:where Vr = volume of medium
in the receiver chamber, P0 = measured
peak area of the test drug in the donor chamber at t = 0, S = surface area of the monolayer, P2 = measured peak area of the test drug in the
acceptor chamber after incubation for 2 h, and t =
incubation time. The efflux ratio basolateral (B) to apical (A) was
calculated by dividing Papp (B–A)
by Papp (A–B). In addition, the
compound recovery was calculated. Reference compounds were analyzed
in parallel as assay controls. All samples were analyzed by LC/MS/MS.
Authors: Miles A Fabian; William H Biggs; Daniel K Treiber; Corey E Atteridge; Mihai D Azimioara; Michael G Benedetti; Todd A Carter; Pietro Ciceri; Philip T Edeen; Mark Floyd; Julia M Ford; Margaret Galvin; Jay L Gerlach; Robert M Grotzfeld; Sanna Herrgard; Darren E Insko; Michael A Insko; Andiliy G Lai; Jean-Michel Lélias; Shamal A Mehta; Zdravko V Milanov; Anne Marie Velasco; Lisa M Wodicka; Hitesh K Patel; Patrick P Zarrinkar; David J Lockhart Journal: Nat Biotechnol Date: 2005-02-13 Impact factor: 54.908
Authors: Jing Huang; Laura Perez-Burgos; Brandon J Placek; Roopsha Sengupta; Mario Richter; Jean A Dorsey; Stefan Kubicek; Susanne Opravil; Thomas Jenuwein; Shelley L Berger Journal: Nature Date: 2006-11-15 Impact factor: 49.962
Authors: A Pimkhaokham; Y Shimada; Y Fukuda; N Kurihara; I Imoto; Z Q Yang; M Imamura; Y Nakamura; T Amagasa; J Inazawa Journal: Jpn J Cancer Res Date: 2000-11
Authors: Jeffrey Mowat; Alexander H M Ehrmann; Sven Christian; Carolyn Sperl; Stephan Menz; Judith Günther; Roman C Hillig; Marcus Bauser; Wolfgang Schwede Journal: ACS Med Chem Lett Date: 2022-02-18 Impact factor: 4.345
Authors: Léa Bouché; Clara D Christ; Stephan Siegel; Amaury E Fernández-Montalván; Simon J Holton; Oleg Fedorov; Antonius Ter Laak; Tatsuo Sugawara; Detlef Stöckigt; Cynthia Tallant; James Bennett; Octovia Monteiro; Laura Díaz-Sáez; Paulina Siejka; Julia Meier; Vera Pütter; Jörg Weiske; Susanne Müller; Kilian V M Huber; Ingo V Hartung; Bernard Haendler Journal: J Med Chem Date: 2017-05-01 Impact factor: 7.446
Authors: Michael J Thomenius; Jennifer Totman; Darren Harvey; Lorna H Mitchell; Thomas V Riera; Kat Cosmopoulos; Alexandra R Grassian; Christine Klaus; Megan Foley; Elizabeth A Admirand; Haris Jahic; Christina Majer; Tim Wigle; Suzanne L Jacques; Jodi Gureasko; Dorothy Brach; Trupti Lingaraj; Kip West; Sherri Smith; Nathalie Rioux; Nigel J Waters; Cuyue Tang; Alejandra Raimondi; Michael Munchhof; James E Mills; Scott Ribich; Margaret Porter Scott; Kevin W Kuntz; William P Janzen; Mikel Moyer; Jesse J Smith; Richard Chesworth; Robert A Copeland; P Ann Boriack-Sjodin Journal: PLoS One Date: 2018-06-01 Impact factor: 3.240
Figure 1
Figure 2
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Figure 3
Figure 4
Figure 7
Figure 5
Figure 6
Figure 8
Figure 9