Intrinsically disordered protein c-Myc (specifically binds to the bHLH-LZ domain, region c-Myc^375-485) [1]
Disrupts c-Myc/Max heterodimerization [1]

10074-A4 has 15.1 μM IC50 inhibitory action against HL-60 cells[1]. In HL-60 cells, 10074-A4 (25-50 μM; 24 hours) dose-dependently stops the cell cycle in the S phase. The c-Myc target genes CDK4 and CCND2 had their mRNA levels inhibited by 10074-A4 [1]. It is possible to characterize 10074-A4's binding behavior to c-Myc370-409 as a "ligand cloud" because it can bind to the peptide chain at several locations. The c-Myc370-409 peptide's structure is still dynamic in its bound state. Different locations throughout the c-Myc370-409 chain bind to the 10074-A4 ligand with varying intensities [2].

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Binding Affinity to c-Myc^370-409 (Circular Dichroism): The apparent Kd of 10074-A4 binding to the c-Myc^370-409 peptide was measured by monitoring CD spectra changes. The value was determined to be 128 ± 46 μM using the Hill equation. [1]
Binding Affinity to c-Myc^370-412 (Surface Plasmon Resonance): In a direct binding SPR assay using biotinylated c-Myc^370-412 immobilized on a SA chip, 10074-A4 showed concentration-dependent binding with a Kd value of 36.3 ± 9.0 μM. [1]
Cell Growth Inhibition (MTT Assay): In HL-60 human promyelocytic leukemia cells (which overexpress c-Myc), 10074-A4 inhibited cell proliferation in a dose-dependent manner with an EC50 of 15.1 ± 2.3 μM after 72 hours of treatment. [1]
Cell Cycle Analysis: Treatment of HL-60 cells with 10074-A4 for 24 hours at concentrations below its EC50 resulted in S-phase arrest. A significantly higher percentage of cells in the S-phase and a lower frequency of cells in the G2/M-phase were observed in a dose-dependent manner. [1]
Inhibition of c-Myc Transcriptional Activity (qPCR): Treatment of HL-60 cells with 10074-A4 decreased the mRNA expression levels of c-Myc target genes, CCND2 (encoding cyclin D2) and CDK4 (encoding CDK4), as measured by quantitative real-time PCR. [1]
Binding Site Analysis: The study references previous work identifying that 10074-A4 binds to the c-Myc^375-485 region within its disordered bHLH-LZ domain. [1]

Cell viability assay [1]
Cell Types: HL-60 Cell
Tested Concentrations: 25 μM, 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: The cell cycle was arrested in S phase.

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CD Spectroscopy Binding Assay: Samples of c-Myc^370-409 peptide (20 μM) were prepared in buffer (10 mM potassium phosphate, 100 mM potassium chloride, pH 7.4) in the absence and presence of 10074-A4 at various concentrations. Compounds were added from stock solutions in ethanol. CD spectra were recorded using a 1-mm path-length quartz cuvette at 25°C on a spectropolarimeter. For each experiment, the corresponding concentration of compound was used as a blank. The apparent Kd was calculated using the Hill equation. [1]
Surface Plasmon Resonance (SPR) Direct Binding Assay: SPR experiments were performed at 25°C using a Biacore T200 instrument. C-terminal biotinylated c-Myc^370-412 peptide was immobilized onto an SA chip to a level of approximately 500 response units (RU). The running buffer was 1X PBS-P with 5% DMSO. Serial concentrations of 10074-A4 were injected over the surface at 10 μL/min for 120 seconds with a regeneration time of 240 seconds. After each injection, the system was washed with 50% DMSO. Data were analyzed with Biacore T200 Evaluation Software, and Kd values were obtained from steady-state fitting of the response-concentration plot. [1]
MTT Cell Proliferation Assay: HL-60 cells (2×10⁴ cells/well) were plated into 96-well plates and treated in triplicate with or without 10074-A4 (from DMSO stock, final DMSO concentration 0.2%). After 72 hours, 20 μL of 5 mg/mL MTT was added to each well and incubated for 3 hours. The medium was removed, 200 μL DMSO was added, and the plate was shaken for 5 minutes. Viable cells were assessed by spectrophotometry at 570 nm using a microplate reader. Results were expressed as percentage inhibition, and EC50 was calculated using the Hill equation. [1]
Flow Cytometric Cell Cycle Analysis: HL-60 cells (1×10⁶ cells/well) were plated into 6-well plates and treated in triplicate with or without 10074-A4 for 24 hours. Cells were harvested, washed with PBS, fixed in 70% ice-cold ethanol overnight at 4°C. Fixed cells were washed, resuspended in PBS containing 50 μg/mL propidium iodide and 50 μg/mL RNase A, and incubated in the dark for 30 minutes at 37°C. Cell cycle distribution was analyzed using a flow cytometer. [1]
Quantitative Real-Time PCR (qPCR): HL-60 cells (4×10⁴ cells/well) were plated into 96-well plates and treated in triplicate with or without 10074-A4 for 24 hours. Total RNA was harvested, and qPCR was performed using TaqMan gene expression assay primers for CCND2, CDK4, and ACTB (β-actin, internal control) with a commercial kit. The fold changes in gene expression were calculated relative to DMSO-treated controls using the threshold cycle (CT) method. [1]

[1]. Structure-based Inhibitor Design for the Intrinsically Disordered Protein c-Myc. Sci Rep. 2016 Mar 2;6:22298.

[2]. Ligand clouds around protein clouds: a scenario of ligand binding with intrinsically disordered proteins. PLoS Comput Biol. 2013;9(10):e1003249.

Background and Role: 10074-A4 is a known small-molecule inhibitor of c-Myc/Max dimerization. It was previously identified through high-throughput screening (yeast two-hybrid system) and shown to bind to the c-Myc^375-485 region within the disordered bHLH-LZ domain of c-Myc. In this study, it was used as a reference compound for comparison with newly discovered c-Myc binders. [1]
Virtual Screening Reference: Both the R and S forms of 10074-A4 were used for a compound similarity search using Phase software to find analogues. The docking scores for the R and S forms with the three predicted binding cavities (Apo1, Apo2, Holo1) were -5.086, -3.734, -4.347 and -5.253, -3.966, -4.122 kcal/mol, respectively. It was classified as a "multi-conformational-affinity" compound, as the differences among its three docking scores were small. [1]
Mechanism of Action: 10074-A4 binds directly to the intrinsically disordered bHLH-LZ domain of c-Myc, stabilizing the disordered monomer and preventing its heterodimerization with Max. This disruption of c-Myc/Max complex formation inhibits c-Myc transcriptional activity, leading to cell cycle arrest (S-phase) and reduced proliferation of c-Myc-overexpressing cancer cells. [1]

C18H14CL2N2O3S

409.28

408.01

312631-87-1

2836204

White to off-white solid powder

4.095

1

4

4

26

555

0

C1C(=O)N(C(=O)S1)CC(CN2C3=C(C=C(C=C3)Cl)C4=C2C=CC(=C4)Cl)O

SCNHANGMZXFWOH-UHFFFAOYSA-N

InChI=1S/C18H14Cl2N2O3S/c19-10-1-3-15-13(5-10)14-6-11(20)2-4-16(14)21(15)7-12(23)8-22-17(24)9-26-18(22)25/h1-6,12,23H,7-9H2

3-[3-(3,6-dichlorocarbazol-9-yl)-2-hydroxypropyl]-1,3-thiazolidine-2,4-dione

10074-A4 10074A4 10074 A4

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~125 mg/mL (~305.41 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.08 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.08 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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