Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2

Abstract

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.

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.

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 (ΔT_m = 3.3 K) and (S)-4 (ΔT_m = 7.1 K) highlighted. Stabilization correlates with SPA potency. (C) Isothermal titration calorimetry (ITC) of compound 6 indicating a submicromolar binding constant (K_d = 540 nM) and a high enthalpic contribution to the binding energy.

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.

Scheme 2. Synthesis of Compounds 5, 6, and 12–24

Reagents and conditions: (a) aniline derivative, n-BuLi, THF, −78 °C, or alkylamine, 1,4-dioxane, reflux; (b) 1,3-dimethylbarbituric acid, Pd(PPh₃)₄, THF; (c) acetaldehyde, NaBH₄, DCM, 0 °C; (d) (1) acetoxyacetyl chloride, aqueous NaHCO₃ solution, DCM, (2) K₂CO₃, MeOH; (e) skip (for compound 19), or aldehyde derivative, NaBH₄, MeOH, 0 °C; (f) Fmoc-glycine, HATU, NMM, DMF; (g) piperidine, DMF; (h) methoxyacetyl chloride, aqueous NaHCO₃ solution, DCM; (i) chloroacetyl chloride, N,N-diisopropylethylamine, DCM; (j) urotropine, EtOH.

Scheme 3. Synthesis of Compounds 4 and 25–29

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.

Scheme 4. Synthesis of Compounds 30–34

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.

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.

Figure 4

Comparison of (S)-4 (this work) with the recently reported SMYD2 inhibitors 1(11) and 2. (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.

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]/K_m(app)). Data were fitted to competitive and uncompetitive models of the Cheng–Prusoff equation.^, 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.

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.

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.

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 3) for 2 days, then apoptosis was induced by treatment with doxorubicin. (S)-4, but not 25, significantly improving caspase 3/7 activation.

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.