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Abstract

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase that was initially described as an H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has been reported to methylate and regulate several nonhistone proteins relevant to cancer, including mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition, overexpression of SMYD3 has been linked to poor prognosis in certain cancers, suggesting SMYD3 as a potential oncogene and attractive cancer drug target. Here we report the discovery of a novel SMYD3 inhibitor. We performed a thermal shift assay (TSA)-based high-throughput screening (HTS) with 410,000 compounds and identified a novel benzodiazepine-based SMYD3 inhibitor series. Crystal structures revealed that this series binds to the substrate binding site and occupies the hydrophobic lysine binding pocket via an unprecedented hydrogen bonding pattern. Biochemical assays showed substrate competitive behavior. Following optimization and extensive biophysical validation with surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC), we identified BAY-6035, which shows nanomolar potency and selectivity against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibits methylation of MAP3K2 by SMYD3 in a cellular mechanistic assay with an IC₅₀ <100 nM. Moreover, we describe a congeneric negative control to BAY-6035. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe that will foster the exploration of the biological role of SMYD3 in diseased and nondiseased tissues.

Keywords

chemical probe SMYD3 thermal shift assay drug discovery

Introduction

SMYD3 is a lysine-specific protein methyltransferase reported to methylate lysine residues on histone and nonhistone proteins. It is one of five SMYD family members of methyltransferases that are characterized by an MYND zinc finger domain splitting the catalytic SET domain into two parts. The function of the MYND domain has remained elusive and is thought to be involved in mediating protein or DNA binding.¹ SMYD3 gained a lot of interest as a potential therapeutic cancer target initially based on observed abnormal high expression in cancer tissues. Originally SMYD3 was identified as an overexpressed gene in colorectal and hepatocellular carcinomas,^2,3 and a functional oncogenic role in these tissues has been demonstrated in mouse models.⁴ Additional studies reported overexpression of SMYD3 in esophageal squamous cell carcinoma, gastric, ovarian, bladder, pancreatic, and non-small cell lung cancer cell line models.^5–8 As one potential reason of SMYD3 upregulation, researchers identified activation of the RAS oncogene pathway.^9–11 In genetic SMYD3 knockdown experiments in different cancer cell line models, proliferation was significantly reduced, suggesting an essential survival role in these models.^5,8,9,12,13

On a mechanistic level, while SMYD3 has been observed to methylate multiple protein targets, the main biological functionality remains elusive. SMYD3 was initially characterized to di/tri-methylate histone 3 at lysine 4 (H3K4),² a chromatin mark associated with active gene promotors. In several studies that followed, methylation of H3K4 by SMYD3 has been correlated to aberrantly increased transcription of diverse cancer-promoting genes in different cell line models.^5,14–20 More recently, histone 4 at lysine 5 was uncovered to be more efficiently methylated by SMYD3 in vitro. The functional consequences of H4K5 methylation are unknown.²¹ Protein localization of SMYD3 was found to be mainly cytosolic,^22–24 which suggests that SMYD3 substrates might not be limited to histones. In fact, several studies uncovered an important role of SMYD3 methylation activity on the regulation of nonhistone proteins that affect different and important signaling pathways, thereby promoting cancer phenotypes.^9,22,25–28 In contrast to these reported widespread effects on different signaling pathways, organ-specific and whole-body SMYD3 deletion in mice surprisingly does not translate into any obvious phenotype.^4,9 Moreover, SMYD3 was not sufficient to either promote tumorigenesis by itself or accelerate tumorigenesis progression in liver-specific SMYD3 overexpressing transgenic mice.⁴

Exploration of the mechanistic consequences of a catalytic SMYD3 inhibition is hindered by a lack of specific and potent inhibitors openly available for the research community. The first inhibitors for SMYD3 have been described,²⁹ particularly 1 (BCL-121),³⁰ 2 (GSK2807),³¹ 3 (EPZ031686),³² 4 (EPZ028862),³³ and 5 (compound 29)³⁴ ( Fig. 1 ). In a virtual screening approach, the piperidine-4-carboxamide acetanilide 1 was described as an SMYD3 inhibitor, but no data for selectivity or potency on targets have been reported besides a weak binding K_d value of 11.8 µM. Nevertheless, 1 was effective on H4 methylation and proliferation inhibition in cell lines when used with higher two-digit micromolar concentrations. Compound 2 is an analog of the cofactor S-adenosyl methionine (SAM) with an IC₅₀ of 130 nM. However, poor membrane permeability properties limit the usage of 2 as a tool to study the kinetic and catalytic mechanisms of SMYD3. An ultra-high-throughput screening (uHTS) campaign followed by optimization resulted in the very potent and specific substrate-competitive SMYD3 inhibitor 3 (EPZ031686) with a reported IC₅₀ of 3 nM. Based on this series, an isoxazole sulfonamide derivative 4 (EPZ028862) has been reported that combines an improved potency of 1.8 nM with more favorable physicochemical properties potentially suitable for in vivo studies. More recently, a covalent inhibitor 5 has been reported; 5 bears a 4-chloroquinoline warhead and acts via a nucleophilic aromatic substitution reaction within the lysine channel, resulting in SMYD3 inhibition with a reported IC₅₀ of 12 nM.

Figure 1.

Published SMYD3 inhibitors.

Herein, we describe the identification of a novel, selective, and potent probe inhibitor of SMYD3 from a thermal shift assay (TSA) uHTS campaign using a library of 410,000 compounds. We were able to identify and optimize a series of benzodiazepines resulting in the discovery of BAY-6035.³⁵ This has excellent properties as a probe that will enable a more comprehensive chemical biology-based target validation of SMYD3.^36–38 In addition, we identified a structurally closely related compound BAY-444 lacking SMYD3 activity that may be used as a corresponding negative control. Both compounds are freely available via the Structural Genomics Consortium (SGC).

Materials and Methods

Chemistry

Acetylated 4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepines have been described in the literature since 1953, and multiple synthetic routes have been published.^39–42 In our work, we used commercially available building block Int-1 as the starting point for the synthesis (Scheme 1). Amide coupling using T3P affords intermediates such as Int-2a/b. Acylation using various benzoyl chlorides can be accomplished in dichloromethane using pyridine as a base to afford the final test compounds. Overacylation on one or both amide nitrogens was detected with varying intensity, but could be reversed by stirring the crude reaction mixture with aqueous sodium hydroxide solution before purification.

Scheme 1.

Synthesis of acetylated 4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepines.

Initially we used chiral separation to obtain both enantiomers, but once it was determined that the (S)-enantiomer was preferred, we set out to synthesize enantiomerically enriched building blocks for further derivatization. Intermediate (S)-Int-7 was synthesized from (S)-β-alanine according to Scheme 2. Coupling acid Int-3 with the appropriate amine followed by nucleophilic aromatic substitution with a β-amino acid yielded Int-5. Reduction of the nitro group followed by T3P-mediated cyclization produced common intermediate Int-7. Ureas were synthesized either by treating the stable p-nitrophenolcarbamate intermediate Int-8 with amines or by synthesizing the required carbamoyl carbamates in situ and coupling directly with intermediate Int-7. Scheme 2 shows the synthesis of compound 31.

Scheme 2.

Synthesis of compound 31 (BAY-6035).

Ureas bearing a second nucleophilic substituent, such as compounds 15–20, 22, and 23, were synthesized from the appropriately protected diamine, followed by deprotection with hydrochloric acid in dioxane.

Detailed syntheses of test compounds used in this study were described⁴³ and can be found in the supplemental information.

Thermal Shift Assay

We used a TSA to identify compounds that induce a positive shift (stabilization of the protein) of the SMYD3 protein melting temperature (Tm). Thermal melting experiments were performed with a ViiATM Real-Time PCR machine (Thermo Fisher Scientific, Waltham, MA) in a 384-well microliter plate with a final volume of 5 µL. Melting curves were obtained at a protein concentration of 1.5–2.0 µM and 5xSYPRO Orange using buffer containing 20 mM Tris (pH 7.0), 150 mM NaCl, and 1.0 mM DTT. Full-length, untagged SMYD3 protein was used for these measurements. Compounds were diluted in DMSO at a single concentration of 120 μM for primary screening, 100 μM for confirmation test, or for dose–response curves a titration row ranging from 10 up to 200 µM. As a control, 4% DMSO was used. Scans were measured from 25 to 80 °C at a scanning rate of 0.1 °C/s. SMYD1 and SMYD2 proteins were used for selectivity in buffer 20 mM Tris (pH 8.0), 100 mM NaCl, 8.5% glycerol, and 1.0 mM DTT with a protein concentration of 1.4 µM and 8xSYPRO Orange. All TSA data were analyzed using the Genedata Assay Analyzer software.

Purification of SMYD3

Recombinant human SMYD3 (UniProt Q9H7B4, amino acids 1–428) was expressed in Escherichia coli containing an N-terminal TEV-cleavable 6xHis tag. Cell pellets were resuspended in lysis buffer (20 mM Tris [pH 8.0], 500 mM NaCl, 5% glycerol, and 10 mM imidazole) supplemented with complete EDTA-free protease inhibitor tablets. The cell lysate was loaded onto a Ni-NTA (Protino NiNTA) column, eluted with imidazole. The 6xHis tag was subsequently cleaved in solution by TEV. After ion-exchange chromatography (MonoQ10/100GL), SMYD3 was further purified on a Superdex S200 (35/600) column preequilibrated in 20 mM Tris (pH 8.0), 100 mM NaCl, and 1 mM DTT.

Purification of SMYD1 and SMYD2

Recombinant human SMYD1 (UniProt Q8NB12, amino acids 2–490) and SMYD2 (UniProt Q9NRG4, amino acids 2–433) were 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, and 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. Both proteins were further purified on a Superdex S200 column preequilibrated in 20 mM Tris (pH 8), 100 mM NaCl, 5% glycerol, and 1 mM DTT.

SPA for MAP3K2

Inhibitory activities of compounds were evaluated using a scintillation proximity assay (SPA) in which methylation by the enzyme of a biotinylated peptide was measured. The peptide, DYDNPIFEKFGKGGTYPRRYHVSYHGK-Biotin, is derived from MAP3K2 (249–273) and contains the Lys 260 residue previously shown to be methylated by SMYD3.⁹ Assays were conducted in 384-well microtiter plates in quadruplicate in a buffer containing 20 mM Tris (pH 9), 2.5 mM DTT, and 0.01% Triton X-100 in a total volume of 10 µL.

The SMYD3 concentration in the assay was 10 nM, while tritiated S-adenosyl-l-methionine (3H-SAM) and the peptide substrate were present at 335 nM and 15 µM, respectively. Reactions were run for 40 min at a reaction temperature of 23 °C before quenching by adding 10 µL of GuHCl, followed by the addition of 180 µL of 20 mM Tris (pH 8). Samples were analyzed for incorporated radioactivity using 96-well SPA plates. 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) SMYD3 activity. IC₅₀ values were calculated by fitting the normalized inhibition data to a four-parameter logistic equation using the XLFit analysis software (IDBS).

Isothermal Titration Calorimetry

Eight injections of 4 μL each in 180 s intervals were performed. The heat production appears during the first few injections. In the syringe were 20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween 20, 0.3 mM compound concentration and 2.5% DMSO concentration.

SPR Analysis

Surface plasmon resonance (SPR) analysis was performed using a Biacore T200 (GE Health Sciences Inc.) at 25 °C. Approximately 4700 resonance units of biotinylated SMYD3 (amino acids 1–428) was captured onto one flow cell of an streptavidin sensor chip according to the manufacturer’s protocol. A second flow cell was left empty for reference subtraction. SPR analysis was performed in 20 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% Tween-20 supplemented with 2% DMSO. Five concentrations of BAY-6035 (25, 74, 222, 667, and 2000 nM) were prepared by serial dilution. Kinetic determination experiments were performed using single-cycle kinetics with a contact time of 60 s, off time of 300 s, and flow rate of 30 μL/min. To favor complete dissociation of compound for the next cycle, a regeneration step (300 s, 40 μL/min buffer), a stabilization period (120 s), and two blank cycles were run between each cycle. Kinetic curve fittings were done with a 1:1 binding model and the Biacore T200 Evaluation software (GE Health Sciences Inc.). The experiments were performed in triplicate in the presence of 50 μM SAM.

X-Ray Crystallography

Purified SMYD3 was crystallized at 293 K using the hanging-drop vapor diffusion method. One microliter of the protein solution at ~10 mg/mL was mixed with 1 µL of the reservoir solution containing 8% PEG 3350, 50 mM magnesium formate, and 100 mM Tris (pH 7.5). No additional SAM was added. Needle-shaped crystals were soaked in mother liquor with compound concentrations of 3–6 mM. Soaked crystals were flash-frozen in liquid nitrogen. Crystallographic data were collected at Bessy Synchrotron (Berlin, Germany). Data reduction was performed with XDS, phasing was performed by molecular replacement using PHASER, and refinement was carried out using COOT and REFMAC5 as implemented in the CCP4 software package. The coordinates of the Smyd3 complexes of compounds 6, 15, and 31 (BAY-6035) have been deposited with the Protein Data Bank (PDB) entry codes 7O2B, 7O2A and 7O2C, respectively. Data collection and refinement statistics are listed in Supplemental Table S2 .

Cellular SMYD3 Assay

HeLa cells (ATCC CCL-2) were seeded in 12-well plates (1 × 10⁵ cells/mL) in DMEM (Wisent) supplemented with 10% fetal bovine serum (Wisent), penicillin (100 U/mL), and streptomycin (100 µg/mL). On the next day, cells were co-transfected with 0.1 µg/well untagged SMYD3 wild-type or catalytic inactive SMYD3 mutant (deletion in amino acids 205–211 in the catalytic SET domain) and 0.9 µg/well HA-tagged MAP3K2 using Xtreme gene HP transfection reagent (Roche, Basel, Switzerland), following the manufacturer’s instructions. After 4 h, media was removed and the cells were treated with inhibitors or a DMSO control. After 20 h, media was removed and the cells were lysed in 100 µL of total lysis buffer (20 mM Tris-HCl [pH 8], 150 mM NaCl, 1 mM EDTA, 10 mM MgCl₂, 0.5% TritonX-100, 12.5 U/mL benzonase [Sigma-Aldrich (St. Louis, MO)], and complete EDTA-free protease inhibitor cocktail [Roche]). After 1 min of incubation at room temperature, sodium dodecyl sulfate (SDS) was added at a final concentration of 1%. Total cell lysates were analyzed on 4%–12% Bis-Tris Protein Gels (Invitrogen) with MOPS buffer (Invitrogen) and blotted for 1.5 h (80 V) onto a polyvinylidene fluoride membrane (GE Healthcare Amersham Hybond, Fisher Scientific) in Tris-glycine transfer buffer containing 20% MeOH and 0.05% SDS. Blots were blocked for 1 h in blocking buffer (5% milk in phosphate-buffered saline with Tween [PBST]–0.1% Tween 20 PBS) and then incubated with primary antibodies: anti-HA tag (1:500; Santa Cruz Biotechnologies, Dallas, TX, cat. sc-7392), anti-SMYD3 (1:2000; Abcam, Cambridge, UK, cat. ab183498), anti-MAP3K2 (1:1000; CST, Danvers, MA, cat. 19607), and anti-MAP3K2-K260me3 (1:2000) in blocking buffer overnight at 4 °C. The antibody anti-MAP3K2-K260me3 was produced in-house with rabbit antisera generated using the DNPIFEKFGK(me3)GGTYPRRYHV KLH-conjugated peptide at the University of Toronto, Division of Comparative Medicine facility and affinity purified ( Suppl. Fig. S1 ). After five washes with PBST, the blots were incubated with goat anti-rabbit (IR800 conjugated; LiCor, cat. 926-32211) and donkey anti-mouse (IR 680; LiCor, cat. 926-68072) antibodies (1:5000) in Odyssey Blocking Buffer (LiCor) for 30 min at room temperature and washed five times with PBST. The signal was measured with an Odyssey scanner (LiCor, Lincoln, NE) at 800 and 700 nm.

Results

Screening for SMYD3 Inhibitors Using a High-Throughput TSA

The proposed link between SMYD3 function and cancer motivated us to screen for small-molecule inhibitors of the enzyme. To this end, we developed an SMDY3 TSA to identify compounds that induce a positive shift (stabilization of the protein) of the protein melting temperature (Tm). Based on our experiences with SMYD2, a closely related family member of SMYD3, and the identification of BAY-598,⁴⁴ we focused on the development of a TSA uHTS for lead identification. With the inhibitor series leading finally to BAY-598, we observed a very good correlation between TSA stabilization and catalytic inhibition. This prompted us to change the screening strategy for SMYD3 from an enzymatic assay toward a binding/stabilization-based TSA. We screened a core library of ~410000 small molecules to identify compounds that stabilize SMYD3. For the primary screen, compounds were measured at a single concentration of 120 µM. Compounds stabilizing more than 1.2 K were subsequently reconfirmed in triplicate retests. This approach identified more than 3900 confirmed SMYD3 hits. To further characterize the primary hits, we also tested for stabilization of the closely related SMYD1 and SMYD2 proteins. This resulted in 1238 confirmed and specific stabilizers of SMYD3 ( Fig. 2A ). Within these confirmed stabilizers, multiple structural clusters and singletons offered potential starting points for tractable chemical matter. Therefore, we had to employ additional assay readouts to further prioritize our chemical optimization activities on the most promising cluster(s). We employed dose-dependent TSA measurements to uncover concentration-dependent stabilization. Examining dose–response relationships in the TSA allows distinguishing between effects owing to specific binding and nonspecific effects (e.g., by partial unfolding of the protein, which might promote nonspecific interactions).

Figure 2.

TSA screening. (A) The screening cascade resulted in 1238 primary hits. A benzodiazepine cluster was selected for further optimization. RFU, relative fluorescence units. (B) The first SAR for the benzodiazepine cluster was derived from primary screening. The catalytic inhibition of SMYD3 was confirmed using an SPA. n.d., not determined.

Based on the dose-dependent TSA results, our attention was drawn to a cluster of benzodiazepine-based compounds, which showed a first structure–activity relationship (SAR) ( Fig. 2B ). All members from the benzodiazepine cluster showed concentration-dependent stabilization in the TSA, with 6 showing the best stabilization with 4.1 K. In order to validate if the observed stabilization of SMYD3 translates to catalytic inhibition, an SPA was established. The SPA measures methylation of a peptide derived from the previously validated SMYD3 substrate MAP3K2.⁹ All tested benzodiazepine hits from the hit list were able to catalytically inhibit SMYD3 in the SPA, with 6 having the lowest IC₅₀ of 12 µM. Therefore, stabilization of the benzodiazepine hits in the TSA directly translated into catalytic inhibition of SMYD3. Together, these observations prompted us to prioritize this cluster for further optimization.

X-Ray Crystallography of Compound 6 (Hit)

In order to guide further optimization, we selected 6 for co-crystal structure determination with SMYD3 by x-ray crystallography based on its potency and ability to stabilize SMYD3 in the TSA setup. The crystal structure clearly revealed an unambiguous difference density for 6 bound to the peptide substrate binding site of SMYD3, while the cofactor binding site is populated with a well-occupied SAM molecule obviously derived from the protein purification procedure. Compound 6 binds with its benzodiazepine-one core to the center of the peptide pocket, while the ligand’s butyl moiety occupies the lysine channel ( Fig. 3A ). The o-fluoro-benzoic acid amide points toward the opening of the funnel-shaped active site of the enzyme. The electron density unambiguously reveals the (S)-configuration for the bound enantiomer. Binding of the (R)-distomer would clearly require a relocation of the Met 190 side chain, which in the determined complex forms a van der Waals contact (4.0 Å) between the sulfur and the ligand’s asymmetric carbon atom. Interestingly, almost all accessible heteroatoms embedded in 6 are engaged with H bonds to the protein backbone, suggesting an efficiently balanced and well-placed inventory of polar interactions. In addition, the fluorine substituent of 6 forms a close contact (3.1 Å) to the sulfur of Cys 186 ( Fig. 3B ), which is likely beneficially contributing to the binding affinity.⁴⁵ Encouraged by this attractive chemical starting point, we embarked on a hit-to-lead optimization campaign and explored the SAR of this newly identified inhibitor class.

Figure 3.

Crystal structures of 6 at 2.0 Å resolution and of 15 at 1.57 Å resolution (A) Compound 6 is bound to the peptide substrate binding site (red circle) of SMYD3. The cofactor binding site (blue circle) is populated with a well-occupied SAM molecule. (B) All accessible heteroatoms embedded in 6 are engaged with either H bonds to the protein backbone or a polar fluorine-sulfur contact. (C) Only the (S)-enantiomer 15 was identified as a bound ligand in the obtained complex structure. (D) While the benzodiazepine core of 15 and 6 superimposes well, the butylamine side chain of 6 approaches the end of the lysine channel in a slightly different conformation than the cyclopropyl moiety of 15.

Structure−Activity Relationships

We first turned our attention to the variation of the amine side chain, exploring interactions in the lysine binding channel. Replacing the n-butyl chain with shorter or longer chains did not increase potency. Small cycloalkyl rings were the most beneficial for potency, and we explored compounds bearing the cyclopropylethyl and cyclobutylethyl side chains in more detail. In comparison with the original n-butyl-substituted racemic hit compounds, replacement by cyclopropylethyl or cyclobutylethyl substituents to address the lysine channel yielded a potency improvement by one order of magnitude. The benzoic acid analogs 13 and 14 ( Table 1 ) showed an activity of approximately 1–2 µM with binding efficiency values⁴⁶ of 14.27 and 14.72, respectively. This is in agreement with the structural features of the channel allowing the accommodated ligand to establish van der Waals contacts to the surrounding, mostly aromatic residues in the range of 3.4–4.0 Å. Parallel chemistry efforts producing a library of other amide analogs did not yield analogs with improved potency.

Table 1.

Compound	Stereoisomer	SMYD3TSA ΔTm [K]	SMYD3SPA IC₅₀ [M]	logD (pH 7.5)	Kinetic Solubility (pH 6.5)[mg/L]
13	(rac)	3.90	1.97 E-6	2.10	95.50
14	(rac)	4.77	1.85 E-6	2.40	87.50
15	(S)	9.31	2.45 E-8	1.40	317.75
16	(R)	5.43	2.01 E-7	1.40	320.40
17	(S)	7.42	2.52 E-7	1.40	374.00
18	(S)	8.46	5.42 E-8	1.40	> 413.50
19	(S)	5.91	8.32 E-7	1.40	329.60
20	(S)	7.56	2.59 E-7	1.40	293.90
21	(S)	7.34	4.59 E-7	1.70	>427.50
22	(S)	6.77	3.96 E-7	1.30	251.70
23	(S)	6.10	4.93 E-7	1.30	257.20
24	(S)	8.20	1.77 E-7	1.50	>455.60
25	(S)	5.26	1.51 E-6	2.20	n.d.
26	(S)	3.34	5.56 E-6	2.40	345.10
27	(S)	5.63	1.03 E-6	2.20	312.50
28	(S)	5.63	1.08 E-6	1.50	254.00
29	(S)	5.60	2.33 E-6	2.10	230.90
30	(S)	6.16	7.63 E-7	2,00	294.30
31 BAY 6035	(S)	8.34	8.8 E-8	1.90	362.60
32	(R)	5.15	2.53 E-6	n.d.	n.d.
33	(S)	3.90	2.64 E-6	n.d.	n.d.
34 BAY 444	(R)	n.d.	>1,00E-4	n.d.	n.d.

n.d., not determined.

The presence of three negatively charged amino acids (Glu 192, Asp 241, and Glu 294) in close neighborhood to the benzoic acid side chain in the crystal structure of 6 inspired us to synthesize compounds with a basic amine substituent. We turned our attention to urea derivatives, which allowed us to use a wider variety of commercially available and suitably protected diamines. As hypothesized, compounds that present a basic amine in the acyl substituent were more potent than the benzoic acid analogs. Testing the separate enantiomers 15 and 16 showed that the (S)-enantiomer 15 was preferred by approximately one order of magnitude over the corresponding distomer 16 (24 nM vs 190 nM), as was expected from the crystal structure of SMYD3 in complex with 6.

Despite using a racemic mixture for crystallization experiments, only the (S)-enantiomer 15 was identified as a bound ligand in the obtained complex structure ( Fig. 3C ). While the benzodiazepine core of 15 and 6 superimposes well, the butylamine side chain of 6 approaches the end of the lysine channel in a slightly different conformation than the cyclopropyl moiety of 15 ( Fig. 3D ). Interestingly, given the quite rigid architecture introduced by the oxazole moiety of 4 (PDB entry 5V37), the location of the cyclopropyl moiety of 4 more closely resembles the conformation of the butylamine tail in 6 than our cyclopropyl substituent in 15 ( Suppl. Fig. S2 ). Indeed, the basic amine that was introduced in order to address one of the carboxylic acid moieties of the neighboring amino acids forms a charge-reinforced H bond to Glu 192 ( Fig. 3C ). Here, the introduced aminopiperidine moiety adopts a twist boat-like conformation rather than a pure chair geometry.

We synthesized a range of urea-containing compounds. The replacement of the benzoic acid amide by a urea-linked aliphatic heterocycle was in general associated with a potency improvement, but all replacements of the aminopiperidine moiety were less potent than compound 15. Piperazine, substituted and unsubstituted; 3-amino pyrrolidine (R and S enantiomers); and aminopiperidine connected via the exocyclic nitrogen atom (with differing alkyl substituents) lost approximately one order of magnitude in potency. Only azepine was a suitable replacement for the aminopiperidine with potency below 100 nM. The basicity of the amine moiety at first seemed important for binding. We confirmed the predicted pKa values of 15 and 18 spectrophotometrically to be 9.4 and 8.8, respectively. None of the compounds with an attenuated basicity (calculated pKa <7) (25–28) had potencies below 1 µM. While compound 15 shows good potency, solubility, and plasma stability, Caco-2 permeability measurements indicated to us that the compound has very low permeability (P_app A–B of 1.1 nm/s). All basic compounds with potencies below 500 nM were tested and showed low permeability and strong efflux.

We hypothesized that the low permeability and strong efflux could be attributed to the presence of the basic amine introduced for the sake of potency optimization, so we subsequently searched for alternative options. We focused on small aliphatic ring systems and found that while difluoropiperidine (29) and difluoropyrrolidine (30) did not have sufficient potency, the bridged azabicyclo[3.1.0]hexane-substituted urea (31) (BAY-6035) had a potency below 100 nM ( Suppl. Fig. S3 ) and stabilized SMYD3 by 8.3 °C in the TSA. Encouraged by these promising properties, we proceeded to determine the crystal structure of BAY-6035 in complex with SMYD3. As expected, the conformation of the benzodiazepine core and the cyclopropyl-ethyl moiety are well conserved compared with 15, while the azabicyclo[3.1.0]hexane substituent is well defined in the difference electron density ( Fig. 4 ). We confirmed that the (S)-methyl enantiomer is indeed the preferred isomer by synthesizing distomer 32. To explore the importance of the hydrogen bond to the backbone oxygen of Tyr 239, we also synthesized methylated analogs 33 and 34 (BAY-444). As expected, BAY-444 was not active in the SMYD3 biochemical assay ( Suppl. Fig. S3 ). We included BAY-444 in the following cellular experiment as a negative control.

Figure 4.

X-ray structure of 31 (BAY-6035) at 1.6 Å.

Cellular Activity of SMYD3 Inhibitors

In order to characterize whether the activity of our series in the biochemical assay is translated to inhibition of SMYD3-dependent methylation in cells, we transiently overexpressed HA-tagged MAP3K2 and untagged SMYD3 in HeLa cells and determined the levels of MAP3K2 trimethylation at lysine 260 (Fig. 6A, Suppl. Fig. S1). To establish the background MAP3K2 methylation, we used a catalytic inactive mutant of SMYD3 lacking the amino acids 205–211 in the catalytic SET domain. Treatment with 1.1 µM of the neutral compound BAY-6035 showed a very robust reduction of the cellular MAP3K2-K260me3 signal to 14% in comparison with the DMSO-treated control ( Fig. 5A ). In contrast, the two more basic inhibitors, 15 and 18, showed less potent inhibition of MAP3K2-K260 methylation. Methylation was reduced to ~31% and ~53%, respectively, at the highest used concentration of 10 µM. Interestingly, 18, with the lower pKa of 8.8, was slightly more active in cells. This indicates that the basic properties of 15 and 18 might limit the cellular permeation. Consequently, the very good in vitro potency of 15 and 18 in the SPA is not translated to comparable potent cellular inhibition of SMYD3 methylation activity. In contrast, less potent in vitro BAY-6035 inhibited SMYD3-dependent MAP3K2 methylation in cells, in a dose-dependent manner, with an IC₅₀ of 183 ± 13 nM, which was only ~2-fold weaker in comparison with the biochemical assay ( Fig. 5B ). Consistent with the lack of biochemical activity, treatment with the negative control BAY-444 up to 10 µM had no detectable effect on MAP3K2 methylation ( Fig. 5A ).

Figure 5.

SMYD3 inhibitor compounds reduce MAP3K2 methylation in cells. HeLa cells were transfected with SMYD3 (wild-type or catalytic mutant) and HA-tagged MAP3K2 and treated with the indicated compounds for 20 h. MAP3K2 K260 trimethylation levels were determined by Western blot and normalized to total HA-MAP3K2 levels. Mut, catalytic inactive SMYD3 mutant (deletion in amino acids 205–211 in the catalytic SET domain). (A) BAY-6035 showed strongest inhibition of SMYD3-dependent MAP3K2 methylation. Overexpression of a catalytically inactive SMYD3 protein did not lead to a detectable MAP3K2me3 level (n = 1). (B) BAY-6035 inhibited SMYD3-dependent MAP3K2 methylation in a dose-dependent manner. The graph represents the nonlinear fits of MAP3K2-K260me3 signal intensities normalized to HA(MAP3K2). The results are mean ± SEM of three technical replicates.

Figure 6.

Characterization of BAY-6035. Confirmation of binding activity by (A) ITC assay and (B) SPR. (A) ITC of BAY-6035 indicating a submicromolar binding constant (K_d = 133 nM) and a high enthalpic contribution to the binding energy. (B) SPR analysis of BAY-6035 binding to SMYD3 in the presence of SAM (50 µM). A representative sensogram (solid green) is shown with the kinetic fit (black dots). The steady-state response (black circles) is shown with the steady state being 1:1. (C) Mode of action analysis of BAY-6035. IC₅₀ values were determined for compound BAY-6035 at varying concentrations of 3H-SAM (up to 3.5 µM) and a fixed peptide MAP3K2 (249–273) concentration (50 µM), or at varying concentrations of substrate MAP3K2 (up to 150 µM) and a fixed SAM concentration (3.5 µM). The experiments were performed in triplicate. (D) Selectivity profiling with a set of methyltransferase enzymes.

Full Characterization of BAY-6035 as a Novel Chemical Probe for SMYD3

The good combination of the biochemical and cellular potencies of BAY-6035 prompted us to further characterize this compound. We analyzed the binding of BAY-6035 to SMYD3 in isothermal titration calorimetry (ITC) ( Fig. 6A ) and SPR ( Fig. 6B ) experiments. Binding of BAY-6035 to SMYD3 showed in the ITC a submicromolar binding constant (K_d = 133 nM) and a significant enthalpic contribution to the binding energy, which indicates favorable van der Waals and hydrogen bonding interactions ( Fig. 3C ). Interestingly, with a Gibbs binding free energy of ΔG = −9.2 kcal/mol, an enthalpic contribution of ΔH = −4.2 kcal/mol, and an entropic contribution of –TΔS = −5.0 kcal/mol, the overall enthalpic and entropic drivers for ligand binding appear to be almost balanced. This suggests that in addition to the favorable polar contact inventory, there was a beneficial contribution of the displacement of water molecules from the buried peptide binding site of SMYD3 into the bulk water. In SPR experiments, BAY-6035 showed a comparable K_d of 97 nM, confirming specific binding to SMYD3. In more detail, BAY-6035 showed a fast on-rate (k_on) of 3.1 ± 0.3 × 10⁵ M⁻¹ s⁻¹, which favors a good target engagement even under a limited exposure ( Fig. 6B ). The off-rate (k_off) is rapid (3.0 ± 0.2 × 10⁻² s⁻¹), resulting in a predicted short half-life of the drug–target complex. With the rapid on/off kinetics of BAY-6035, a continuous drug exposure might be needed to achieve efficient SMYD3 inhibition. Next, we were interested in investigating potential substrate/co-substrate competitive behavior of BAY-6035. Increasing the ³H SAM concentration up to 3.5 µM (10-fold K_m) in the SPA did not shift the IC₅₀ of BAY-6035, indicating a non-SAM-competitive mode of action (Fig. 6C). In contrast, increasing the concentration of the substrate peptide up to 150 µM (10-fold K_m) shifted the IC₅₀ of BAY-6035 4-fold, confirming a peptide competitive mode of action. A peptide competitive mode of action of the cluster was already apparent based on the binding mode obtained in the crystallization experiment ( Fig. 3 ).

To further characterize BAY-6035, we tested the selectivity against a set of 34 protein methyltransferases, including several that contained a SET domain and three DNA methyltransferases. BAY-6035 showed highly selective inhibition of SMYD3 with no activity on other methyltransferases ( Fig. 6D ), including the closely related SMYD2 enzyme. Additionally, BAY-6035 was tested on a set of >30 different kinases and showed no significant inhibitory activity (IC₅₀ >20 µM) ( Suppl. Table S2 ). Finally, we used BAY-6035 in some initial experiments to explore if inhibition of SMYD3 leads to inhibition of proliferation in 13 selected cell line models, including two non-cancer-derived models. In all tested cell lines, SMYD3 inhibition with BAY-6035 up to 20 µM did not impact cellular proliferation ( Suppl. Table S3 ). A general acute cytotoxic effect of SMYD3 inhibition by BAY-6035 can therefore be excluded.

Discussion

SMYD3 and other protein methyltransferases have recently emerged as potential therapeutic cancer targets. Validation of SMYD3-associated cancer phenotypes has so far been mainly based on correlative studies of increased protein level in cancer tissues or knockdown/knockout experiments. For a more unbiased interpretation of observed effects and comprehensive validation as a therapeutic drug target, fully profiled chemical probes are highly desirable.³⁸ In this study, our aim was to generate a novel SMYD3 probe inhibitor, which should be openly accessible to the research community. This probe may enable more cross-validation studies based on catalytical inhibition of the enzymatic function(s) of SMYD3. We decided to switch our screening approach to a binding/stabilization-based TSA for this target protein, based on previous experiences with the closely related SMYD2 enzyme.⁴⁴ Using a TSA had several advantages in comparison with assays based on catalytical activity, including being low cost, easy to establish, and fast and having relatively low protein requirements.⁴⁷ During the hit validation phase, the TSA was crucial for the further selection process. Dose-dependent TSA stabilization was used to further narrow down our primary hit list toward more specific hits. We finally prioritized the benzodiazepine hit cluster, which showed clear concentration-dependent stabilization effects in the TSA. Primary hit 6 from this cluster was selected for crystallization experiments based on the higher crystallization likelihood of stabilizers.⁴⁸ After focused lead optimization efforts, we were able to optimize this cluster. Stabilization of SMYD3 in the TSA translated to low nanomolar activities in our SPA. Overall, the direct comparison of the TSA and SPA activity data across the entire lead structure series showed a good correlation ( Suppl. Fig. S4 ). Based on the very good correlation (R² = 0.93) for more potent TSA stabilizers (>2 K), the TSA could even have been used exclusively as the primary assay for optimization. In summary, the discovery of BAY-6035 proved that using TSA as the primary screening assay was very efficient for the identification of SMYD3 inhibitors.

BAY-6035 represents a novel structurally orthogonal chemical probe, which will be valuable for further target validation and exploration of additional undiscovered functional roles of SMYD3. In a first report using a chemical probe for SMYD3 target validation, 4 (EPZ028862) was reported to validate RNA interference-based proliferative phenotypes of SMYD3.³³ Surprisingly, none of the previously described proliferation effects could be confirmed using 4. The first experiments with BAY-6035 could independently confirm these observations ( Suppl. Table S3 and data not shown). BAY-6035 structurally significantly deviates from 4 as it occupies the substrate binding site by making use of different chemical features ( Suppl. Fig. S4 ): the oxazole moiety occupies the lysine channel with the attached cyclopropyl moiety in a different orientation than in BAY-6035, while the aza-bicyclo-octane core of 4 overlaps partly with the space occupied by the benzodiazepine core of BAY-6035. In contrast to BAY-6035, 4 possesses a basic amine, while in our probe optimization campaign the presence of the latter appeared detrimental to permeability. The differences between reported knockdown phenotypes of SMYD3 and the results using inhibitors already indicate the necessity of using chemical probes as an additional target validation approach. SMYD3 function(s) might reside afar from proliferation regulation, and chemical probes could be essential to enable the exploration of the underlying biology in more complex model systems (e.g., in vivo experiments in mice). To rule out possible compound-specific effects that are not related to SMYD3 inhibition, different structurally orthogonal chemical probes, including closely related negative controls, are required. BAY-6035 and BAY-444 are now available for the scientific community to further explore SMYD3 biology. Surprisingly, none of the previously described proliferation effects could be confirmed. In the same study, the authors showed by using CRISPR-Cas 9 that silencing the SMYD3 gene leads also to no observable effect in the proliferation of cancer cell lines. First experiments with BAY-6035 could confirm these observations ( Suppl. Table S3 and data not shown). This indicates already the necessity of chemical target validation and the urgent need for chemical probes for more comprehensive mechanistic validation studies. SMYD3 function(s) might reside far from proliferation regulation and chemical probes could be essential to enable the exploration of the underlying biology in more complex model systems (e.g., in vivo experiments in mice). To rule out possible compound-specific effects that are not related to SMYD3 inhibition, different structurally orthogonal chemical probes, including closely related negative controls, are required. BAY-6035 and BAY-444 are now available for the scientific community to further explore SMYD3 biology.

Supplemental Material

sj-pdf-1-jbx-10.1177_24725552211019409 – Supplemental material for Discovery of the SMYD3 Inhibitor BAY-6035 Using Thermal Shift Assay (TSA)-Based High-Throughput Screening

Supplemental material, sj-pdf-1-jbx-10.1177_24725552211019409 for Discovery of the SMYD3 Inhibitor BAY-6035 Using Thermal Shift Assay (TSA)-Based High-Throughput Screening by Stefan Gradl, Holger Steuber, Joerg Weiske, M. Szewczyk, Norbert Schmees, Stephan Siegel, Detlef Stoeckigt, Clara D. Christ, Fengling Li, Shawna Organ, Megha Abbey, Steven Kennedy, Irene Chau, Viacheslav Trush, Dalia Barsyte-Lovejoy, Peter J. Brown, Masoud Vedadi, Cheryl Arrowsmith, Manfred Husemann, Volker Badock, Marcus Bauser, Andrea Haegebarth, Ingo V. Hartung and Carlo Stresemann in SLAS Discovery

Footnotes

Acknowledgements

We thank Elisa Gibson and Taraneh Hajian for the support of SMYD3 protein purification. In addition, we thank Tina Stromeyer for her assistance in the crystallization experiments and Albine Bolotokova for her help in managing the compounds. Simon Holton is acknowledged for helpful discussions and critical reading of the manuscript.

Supplemental material is available online with this article.

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: S.G., H.S., J.W., N.S., S.S., D.S., C.D.C., M.H., V.B., M.B., A.H., I.V.H., and C.S. were or are employees and stockholders of Bayer AG. S.G., H.S., J.W., N.S., S.S., C.D.C., M.H., V.B., and C.S. are listed as inventors of a patent application related to SMYD3 inhibitors.⁴³

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Canada Foundation for Innovation, Genentech, Genome Canada through Ontario Genomics Institute (OGI-196), EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant 875510), Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda, and Wellcome (106169/ZZ14/Z).

ORCID iDs

M. Vedadi

V. Badock

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