FOXM1 (Forkhead box protein M1) DNA binding domain: inhibition of FOXM1-DNA binding with IC50 = 22.5 ± 12.3 μM (determined by EMSA displacement assay). [1]

After extensive analysis, it is demonstrated that FDI-6 binds directly to the FOXM1 protein in MCF-7 breast cancer cells, displacing it from its genetic sites and causing hydrolysis as a result. After three hours of FDI-6 treatment, MDA-MB-231 ER breast cancer and PEO-1 breast cancer show significant modulation of CDC25B. Both types of breast cancer are susceptible to FDI-6 in cell viability (GI50=21.8 μM and 18.1 μM, respectively). of. These genes are networked and essential for G2/M transition, chromosomal segregation, and mitotic spindle integration; their absence triggers the cell killer cycle. Significantly, it has been demonstrated that abnormal overexpression of FOXM1 is a major role in the course of cancer and has been suggested as the initial cause of carcinogenesis [1].

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FDI-6 directly binds to FOXM1 protein with 1:1 stoichiometry as confirmed by native mass spectrometry (Figure 3A). It inhibits FOXM1 DBD-DNA interaction in EMSA with IC50 = 22.5 ± 12.3 μM (Figure 2C). In MCF-7 cells, 6 h treatment with 20 μM FDI-6 decreased chromatin-bound FOXM1 by nearly 50% while total FOXM1 protein level remained unchanged (Western blot, Figure 3B-C). ChIP-qPCR showed reduced FOXM1 occupancy at CCNB1 and CDC25B promoters: -0.68 and -0.64 log2 fold decrease, respectively (Figure 3D). RNA-seq revealed that FDI-6 down-regulates 1,552 genes and up-regulates 1,951 genes after 3 h treatment (FDR<0.01, |logFC|>0.3). The down-regulated genes are significantly enriched for FOXM1 promoter occupancy (19-fold enrichment, P<1×10⁻¹⁶), while no enrichment was seen for FOXA1, FOXA2, FOXP2 or GATA1 (Figure 6A). Temporal cluster analysis identified 208 genes (Cluster 5) that are immediately down-regulated (17-fold enrichment for FOXM1 peaks, P=5×10⁻⁸²). FDI-6 also down-regulates established FOXM1 targets including CDKN3 (-0.739), CENPA (-0.61), KIF20A (-1.22), NEK2 (-0.86) after 6 h treatment. In MDA-MB-231 and PEO-1 cells, FDI-6 shows dose-dependent growth inhibition and down-regulates CDC25B expression after 3 h (Figure 5). FDI-6 does not inhibit the 20S proteasome (Supplementary Fig. 7), unlike thiostrepton. The compound exhibits GI50 values of 18.0 ± 3.0 μM in MCF-7 cells after 72 h treatment (Figure 2B inset). [1]

Fluorescence Polarization (FP) assay: A 16 bp dsDNA probe containing FOXM1 recognition motif (T/CAAACA) with 5'-6-FAM label was used. Recombinant FOXM1 DBD (aa 222-360) was titrated with probe (Kd = 224±30 nM). For screening, 400 nM FOXM1 DBD was pre-incubated with compounds (0.46–115 μM, 23 nL transfer) for 15 min, then 50 nM DNA probe added and incubated 30 min before FP reading (488/520 nm) in 1536-well format with assay buffer containing 0.01% Tween-20 and 0.1 mg/mL BSA. Z' factor averaged 0.71. [1]
Electrophoretic Mobility Shift Assay (EMSA): 15 nM FAM-labeled dsDNA consensus probe was incubated with 750 nM FOXM1 DBD and increasing concentrations of FDI-6 (up to 200 μM) for 1.5 h at RT in binding buffer (20 mM Tris pH 7.5, 100 mM KCl, 1 mM EDTA, 0.1 mM DTT, 10% glycerol, 0.01 mg/mL BSA). Samples were run on 6% DNA retardation gel at 40V for 10 min then 120V for 20 min, and fluorescent bands quantified. IC50 was calculated from dose-response curves. [1]
Native mass spectrometry: 4 μM FOXM1 protein was incubated with 50 μM FDI-6, spectra recorded on Synapt HDMS in positive ion mode (capillary 1.7-1.8 kV, cone 40-80 V, source 20°C). Binding stoichiometry determined by mass shift (1:1). [1]
20S Proteasome inhibition assay: Recombinant 20S proteasome was incubated with 10 μM FDI-6 or DMSO control and AMC peptide substrate for 90 min at 37°C; fluorescence (380/460 nm) measured. FDI-6 showed no inhibition compared to MG-132 positive control. [1]

Cell proliferation assay (GI50): MCF-7, MDA-MB-231 and PEO-1 cells were treated with FDI-6 at various concentrations for 72 h, and cell growth inhibition was measured. GI50 values were determined from dose-response curves; for MCF-7, GI50 = 18.0 ± 3.0 μM. [1]
Western blot: MCF-7 cells treated with 40 μM FDI-6 or DMSO for 6 h. Total lysate and chromatin-bound fractions were isolated. Chromatin isolation involved cell lysis in Buffer A, nuclear pelleting, lysis in Buffer B, and final chromatin pellet sonication. Proteins resolved on 8-16% Tris-Glycine gels, transferred, probed with anti-FOXM1 (1:1000), anti-actin (1:5000) or anti-H3 (1:2000), and detected with IR secondary antibodies. Quantification normalized to actin (total) or H3 (chromatin). [1]
Chromatin immunoprecipitation (ChIP): MCF-7 cells treated with FDI-6 (20 μM) or DMSO for 6 h, cross-linked with 1% formaldehyde, lysed, sonicated (Bioruptor, 15 min high power), and immunoprecipitated with 3 μg anti-FOXM1 antibody (Sc-502) coupled to Protein-A dynabeads. After washes, DNA eluted and purified. qPCR performed with primers for CCNB1 and CDC25B promoters, normalized to intergenic control. Enrichment values calculated. [1]
RNA sequencing: MCF-7 cells treated with FDI-6 (20 μM) for 0, 3, 6, 9 h (triplicates). RNA isolated, TruSeq libraries prepared, 100 bp single-end reads sequenced. Reads aligned to hg19 with TopHat2, counted with HTSeq, differential expression analyzed with edgeR (FDR<0.01). Temporal clustering using MFuzz (fuzzy c-means) with 9 clusters. Promoter binding enrichment assessed using ENCODE ChIP-seq peaks for FOXM1, FOXA1, FOXA2, FOXP2, GATA1. [1]

No significant toxicity or off-target effects were reported; FDI-6 does not inhibit the 20S proteasome, unlike thiostrepton. The compound shows a good concordance between its biochemical IC50 (22.5 μM) and cellular GI50 (18.0 μM), suggesting minimal off-target cytotoxicity. [1]

[1]. Suppression of the FOXM1 transcriptional programme via novel small molecule inhibition. Nat Commun. 2014 Nov 12;5:5165.

FDI-6 (3-amino-N-(4-fluorophenyl)-6-(thiophen-2-yl)-4-(trifluoromethyl)thieno[2,3-b]pyridine-2-carboxamide TFA salt) was synthesized by reacting 6-(thiophen-2-yl)-2-thioxo-4-(trifluoromethyl)-1,2-dihydropyridine-3-carbonitrile with 2-chloro-N-(4-fluorophenyl)acetamide in ethanol with K2CO3 under reflux, followed by preparative HPLC purification. Its structure was confirmed by 1H NMR, HRMS (calcd. m/z 438.0352 [M+H]+, found 438.367). The compound is water-soluble and has a molecular scaffold amenable to optimization. It selectively inhibits FOXM1 without affecting other forkhead factors (FOXA1, FOXA2, FOXP2) or GATA1, as shown by promoter binding enrichment analysis. The transcriptional signature of FDI-6 significantly overlaps with FOXM1 siRNA knockdown (P=6×10⁻⁵), supporting its on-target mechanism. Gene ontology analysis revealed down-regulation of mitosis, nuclear division, and cell cycle pathways. FDI-6 provides a chemical probe to study FOXM1 occupancy and transcriptional consequences, with potential therapeutic applications in cancers overexpressing FOXM1. [1]

C19H11F4N3OS2

437.43

437.028

313380-27-7

5175738

Light yellow to yellow solid powder

6.671

2

9

3

29

603

0

ZATJMMZPGVDUOM-UHFFFAOYSA-N

InChI=1S/C19H11F4N3OS2/c20-9-3-5-10(6-4-9)25-17(27)16-15(24)14-11(19(21,22)23)8-12(26-18(14)29-16)13-2-1-7-28-13/h1-8H,24H2,(H,25,27)

3-Amino-N-(4-fluorophenyl)-6-thiophen-2-yl-4-(trifluoromethyl)thieno[2,3-b]pyridine-2-carboxamide

FDI-6 FDI6FDI 6 NCGC-00099374 NCGC 00099374NCGC00099374

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 : ~41.67 mg/mL (~95.26 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.72 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (5.72 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 25.0 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.)