SMYD3 (SET and MYND domain-containing protein 3) [1]

BCI-121 efficiently lowers the proliferation of different cancer cell types by substantially inhibiting chromatin recruitment and SMYD3-substrate interaction. After 72 hours, BCI-121 dramatically decreased the HT29 (46% reduction) and HCT116 (54% reduction) cells' ability to proliferate, as well as the expression levels of the SMYD3 target genes. Histone H4 is preferentially methylated by SMYD3, and in vitro SMYD3-mediated H4 methylation is inhibited by BCI-121. Cancer cells treated with BCI-121 had much less growth potential, which accumulated in the S phase of the cell cycle. Targeted methyl marks (H4K5me and H3K4me2) were reduced and cell proliferation was inhibited in a dose-dependent manner in response to BCI-121 treatment. In cancer cell lines overexpressing SMYD3, BCI-121 has antiproliferative characteristics and typically resembles the effects of RNAi targeting SMYD3. BCI-121 inhibits SMYD3 from recruiting to the promoters of its target genes, an event linked to decreased gene expression, according to experiments conducted in cancer cells [1].

---

In CRC cell lines (HT29 and HCT116) expressing high levels of SMYD3, treatment with BCI-121 (100 μM) for 48 hours significantly reduced the global levels of SMYD3-targeted histone methyl marks, specifically H3K4me2, H3K4me3, and H4K5me, while the non-targeted mark H3K27me3 was unaffected. [1]
BCI-121 treatment (100 μM for 48 hours) decreased the mRNA expression levels of SMYD3 target genes (cMET, TERT, WNT10B, CDK2) in HT29 and HCT116 colorectal cancer cells. [1]
BCI-121 inhibited the proliferation of HT29 and HCT116 cells in a dose- and time-dependent manner. At 100 μM, it reduced proliferation by 46% in HT29 cells and by 54% in HCT116 cells at the 72-hour time point. [1]
The antiproliferative effect of BCI-121 was specific to cancer cell lines with high SMYD3 expression. Treatment only affected proliferation in CRC cell lines (like HT29, HCT116) and other cancer cell lines (lung A549, pancreatic Capan-1, prostate DU145, ovarian OVCAR-3) with high SMYD3 levels, but not in those with low SMYD3 expression (e.g., CRC LS174T cells). [1]
In HT29 colorectal cancer cells, BCI-121 treatment (100 μM for 48 hours) affected cell cycle progression, leading to a significant increase in the S-phase population, suggesting a role for SMYD3 in regulating the S/G2 transition. [1]
In synchronized HCT116 cells, treatment with BCI-121 (48 hours) caused cells to fail to exit S phase, as demonstrated by increased BrdU incorporation at both 4 and 8 hours post-thymidine release compared to untreated cells. [1]
In OVCAR-3 ovarian cancer cells, treatment with BCI-121 (100 μM for 72 hours) reduced SMYD3 occupancy at the promoter regions of its target genes (cMET, WNT10B, CDK2), as shown by Chromatin Immunoprecipitation (ChIP) assays. This correlated with a significant reduction in the mRNA expression of these target genes. [1]
In HCT116 colorectal cancer cells, treatment with BCI-121 (100 μM for 72 hours) reduced SMYD3 occupancy at the promoter regions of its target genes (cMET, WNT10B, CDK2), as shown by Chromatin Immunoprecipitation (ChIP) assays. [1]
In HT29 cells, BCI-121 treatment for 48 hours induced a concentration-dependent (1, 10, 30, 60, 100 μM) reduction in cell proliferation, a decrease in global levels of H4K5me and H3K4me2, and a reduction in MEK-ERK signaling activity (pERK levels). The non-targeted mark H3K27me3 was not affected. [1]
The dose-dependent antiproliferative effect of BCI-121 (72-hour treatment) was observed in HCT116 cells (high SMYD3 expression) but not in LS174T cells (low SMYD3 expression). [1]

In vitro methylation assay: Recombinant GST-SMYD3 protein was pre-incubated with either 100 μM BCI-121 or its dilution buffer for 30 minutes at room temperature. Following this, a mixture of calf thymus histones and radiolabeled SAM-³H (S-adenosylmethionine) was added to the reaction and incubated for 45 minutes at 30°C. The reaction mixture was then resolved by SDS-PAGE. Autoradiography revealed that the presence of BCI-121 significantly decreased SMYD3-mediated methylation of histone H4 compared to the control. [1]
Surface Plasmon Resonance (SPR) binding assay: To investigate the mechanism of inhibition, SPR experiments were performed. SMYD3-GST fusion protein was immobilized on a sensor chip via an anti-GST antibody. The binding affinity (KD) of an H4 histone peptide to SMYD3 was first determined to be 1.18 × 10⁻⁵ M. To test inhibition, the H4 peptide (at 72.03 μM) was injected alone or mixed with BCI-121 at molar ratios of 1:1 (peptide:compound) and 1:2.5. BCI-121 inhibited histone H4 binding to SMYD3 by 36.5% and 51.0%, respectively, indicating that the compound acts as a competitive inhibitor at the histone binding site. [1]
Molecular Modeling: Docking calculations predicted that BCI-121 binds in the inner part of the lysine channel of the SMYD3 substrate binding pocket. The 4-carboxamide moiety of its piperidine ring forms hydrogen bonds with Ser202 and Tyr239, while the rest of the molecule engages in hydrophobic interactions within the narrow pocket. [1]

Cell Proliferation Assay (WST-1 and Cell Counting): Cells were seeded and treated with BCI-121 or DMSO (vehicle control). For the WST-1 assay, after 48, 72, or 96 hours of treatment, WST-1 reagent was added to each well and incubated for up to 1 hour at 37°C. Absorbance was then measured. For cell counting, cells were counted with a hemocytometer at various time points (24, 48, 72, and 96 hours) to determine the number of proliferating cells. [1]
Immunoblot Analysis (Western Blot): To assess protein levels and histone marks, whole cell extracts or nuclear-enriched fractions were prepared from cells treated with BCI-121. Proteins were separated by SDS-PAGE and transferred for immunoblotting. Primary antibodies against SMYD3, H3K4me2, H3K4me3, H4K5me, H3K27me3, total H3, pERK, total ERK, and loading controls (β-Actin, β-Tubulin, Lamin A/C) were used, followed by HRP-conjugated secondary antibodies and chemiluminescent detection. [1]
Quantitative Real-Time PCR (qPCR): Total RNA was extracted from BCI-121-treated cells and reverse-transcribed into cDNA. Gene expression levels for SMYD3 target genes (cMET, TERT, WNT10B, CDK2) were quantified using SYBR Green PCR Master Mix on a real-time PCR system. Relative quantification was performed using the ddCT method with β-actin as the housekeeping gene for normalization. [1]
BrdU Incorporation Assay: Cells were synchronized using a double thymidine block method. During the synchronization, cells were treated with BCI-121 or DMSO. At 4 and 8 hours after the final thymidine release, BrdU was added to the medium and incubated for 2 hours. Cells were then fixed, and BrdU incorporation was detected using an anti-BrdU primary antibody and a fluorescent secondary antibody. The percentage of BrdU-positive cells was quantified by fluorescence microscopy. [1]
FACS Analysis: For cell cycle analysis, cells treated with BCI-121 or DMSO were collected, fixed with ethanol, and stained with propidium iodide. Cell cycle distribution was then measured using a flow cytometer. [1]
Chromatin Immunoprecipitation (ChIP): Cells treated with or without BCI-121 were cross-linked. Chromatin was immunoprecipitated using an anti-SMYD3 antibody or control IgG. The precipitated DNA was then analyzed by real-time PCR with primers specific for the promoter regions of SMYD3 target genes (cMET, WNT10B, CDK2) that contain SMYD3 binding sites. Quantification was performed using the Input % method. [1]

[1]. A SMYD3 Small-Molecule Inhibitor Impairing Cancer Cell Growth. J Cell Physiol. 2015 Oct;230(10):2447-2460.

SMYD3 is a histone lysine methyltransferase overexpressed in several cancers (colorectal, lung, pancreatic, prostate, and ovarian) and plays an oncogenic role. [1]
BCI-121 (a piperidine-4-carboxamide acetanilide compound) was identified through a virtual high-throughput screening of ~260,000 molecules, targeting the histone binding site of the SMYD3 catalytic domain. It was one of 15 compounds selected for experimental testing based on docking scores and visual inspection. [1]
The study demonstrates that the response to BCI-121 correlates with basal SMYD3 expression levels in cancer cells, suggesting that SMYD3 expression could be a predictive biomarker for patient stratification in a potential SMYD3-targeted therapy. [1]
This research provides the first evidence that SMYD3 is involved in ovarian cancer cell growth, as both genetic ablation and BCI-121 treatment impaired proliferation of ovarian cancer cell lines. [1]
The study provides proof-of-principle that SMYD3 is a druggable target and that small-molecule inhibitors like BCI-121 could be developed as novel therapeutic agents. It is hypothesized that BCI-121's ability to arrest cells at the S/G2 boundary could potentially enhance the effects of conventional chemotherapy. [1]

339.058

432529-82-3

432529-82-3

795875

Light yellow to yellow solid powder

1.5±0.1 g/cm3

576.1±50.0 °C at 760 mmHg

302.2±30.1 °C

0.0±1.6 mmHg at 25°C

1.619

1.8

2

3

4

20

348

0

NC(C1CCN(CC(NC2=CC=C(Br)C=C2)=O)CC1)=O

KSUYPIXCRPCPGF-UHFFFAOYSA-N

InChI=1S/C14H18BrN3O2/c15-11-1-3-12(4-2-11)17-13(19)9-18-7-5-10(6-8-18)14(16)20/h1-4,10H,5-9H2,(H2,16,20)(H,17,19)

4-(Aminocarbonyl)-N-(4-bromophenyl)-1-piperidineacetamide

BCI-121 BCI 121 BCI121

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~293.93 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (8.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.75 mg/mL (8.08 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.75 mg/mL (8.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)