Micropthalmia-associated transcription factor (MITF) (IC50 = 1.2 μM)

Many target genes of the micropthalmia-associated transcription factor (MITF) are inhibited by ML329, which also prevents the growth of many cell lines that depend on MITF. MITF or other elements of the MITF regulatory network may come into contact with ML329 directly or indirectly. Since MITF is a transcription factor that controls pigmentation and the cell cycle, interfering with ML329 will help identify the precise functions of MITF in melanoma and confirm that blocking MITF function may be a viable melanoma treatment. While ML329 has no effect on the viability of A375 cells, it exhibits particular activity against primary melanocytes that are dependent on MITF. ML329 inhibits the expression of several MITF target genes, including as genes associated to pigmentation and CDK2, a regulator of the cell cycle. As a tool chemical, ML329 will be helpful in clarifying how MITF functions in the establishment of melanocyte lineages and in the advancement of melanoma disease[1].

Effects of the strains from breast milk on intestinal morphology of IUGR rats [2]
As shown in Figure 1a, the intestine from the IUGR newborn rats feeding with ML‐446 were longer than the blank control group (p < 0.01). Meanwhile, the length of pups' intestine fed with ML‐446 was significantly longer than the L. rhamnosus GG group (p < 0.05). The intestinal weight of the IUGR newborn rats' weight fed with ML‐446 was significantly heavier than the blank control group (p < 0.01). These results demonstrated that ML‐446 from breast milk facilitated the IUGR newborn rats' intestinal growth. To obtain further information of the IUGR newborn rats' intestinal development, the villus height in jejunum, ileum, and colon of the IUGR newborn rats were measured. In the jejunum, villus heights of rats' pups fed with ML‐446 were observed longer than the blank control group (p < 0.01) (Figure 1b). Meanwhile, in the ileum, villus heights from ML‐446‐fed pups were extremely significantly longer than that in blank control (p < 0.01) (Figure 1b). In the ileum and colon, ML‐329 exhibited significantly increased villus height of the IUGR rats' pups than the blank control and L. rhamnosus GG group (p < 0.01) (Figure 1b). In general, both ML‐446 and ML‐329 showed better potential effort on intestinal development, especially compared L. rhamnosus GG.

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Colonization ability of the strains from breast milk in the intestine [2]
To further confirm the colonization of the breast milk‐derived ML‐329 and ML‐446 in intestine, the IUGR newborn rats were administered with CFDASE labelled L. rhamnosus GG, ML‐329, and ML‐446. The profile of the CFDASE signal following oral administration of bacteria was determined by using an IVIS Spectrum in vivo imaging system. As shown in epifluorescence images (Figure 2), the fluorescence signals of all the three strains were detected throughout the jejunum, ileum, and colon, suggested that all strains have the ability to colonize in the intestine. In the jejunum, more fluorescence signals were observed when administered with the CFDASE (Solarbio) labelled L. rhamnosus GG than that of the ML‐446 and the ML‐329. In the ileum, higher fluorescence signals were recorded for the ML‐329 compared to the ML‐446. In the colon, high fluorescence signals appeared in all groups. In general, these two strains from breast milk have the ability to colonize in the jejunum, ileum, and colon.

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Effect of bacteria from breast milk on IECs proliferation [2]
To further investigate how the strains ML‐329 and ML‐446 improve intestinal development, the proliferation of IECs were examined using intestinal tissue sections immunostained using antibodies against ki67. For the ki67, the marker for cell proliferation, the expression of ki67 increased significantly during oral administration of ML‐329 than blank control group in the jejunum and colon (p < 0.01). Moreover, the mean density of ki67 increased significantly in the groups of ML‐446 treatment compared with the blank control group in the ileum and colon (p < 0.01) (Figure 3).

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Effect of bacteria from human breast milk on IECs differentiation [2]
As shown in Figure 4a,b, there were no significant differences between the IUGR newborn rats treated with two strains and the blank control group in the numbers of goblet cells in the jejunum. But the numbers of goblet cells were increased significantly in rats treated with ML‐329 (p < 0.01) and ML‐446 (p < 0.05) in the ileum. Meanwhile, the numbers of goblet cells increased significantly in the groups of ML‐329 and ML‐446 treatment compared with the blank control group in the colon (p < 0.05). Additionally, the mean density of lysozyme was significantly increased in the jejunum of IUGR newborn rats treated with ML‐446 and ML‐329 compared with blank control group (p < 0.01) (Figure 4c,d). Moreover, the mean density of lysozyme was significantly (p < 0.05) increased in the jejunum of ML‐446 treated IUGR newborn rats compared with L. rhamnosus GG (Figure 4d). Meanwhile, the expression of lysozyme increased significantly (p < 0.01) in the ileum of ML‐329 treated rats than that of L. rhamnosus GG and blank control rats (Figure 4d). These results suggested that treatment of the IUGR newborn rats with ML‐446 and ML‐329 results in promoting the differentiation of goblet cells and Paneth cells in the intestine, and showed the potential regulating ability of intestinal development.

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Effect of bacteria from breast milk on expression and activity of the Wnt and Notch signalling pathways [2]
To further elucidate the mechanism of breast milk‐derived ML‐446 and ML‐329 in promoting intestinal development, the expression and activity of proteins in Wnt and Notch signalling pathways were examined by RT‐qPCR and western blot, which play important roles in driving the IECs proliferation and differentiation. [2]
In the jejunum and colon, the expression of lrp5 gene were upregulated with ML‐446 treated in compassion with blank control group (p < 0.05) (Figure 5a). In the ileum, the expression of wnt (p < 0.05) was upregulated when treated with ML‐329 and ML‐446 compared with blank control group (Figure 5b). In comparison to blank control group, there were significant (p < 0.05) upregulation of β‐catenin gene expression in the jejunum treated with ML‐329 and ML‐446 (Figure 5c). In the ileum, β‐catenin gene expression was also significant (p < 0.05) increased in ML‐446 group when comparing with blank control group (Figure 5c). Correspondingly, immunoblot analysis revealed that the abundance of β‐catenin protein was markedly (p < 0.05) increased in the ileum treated with ML‐329 and ML‐446 when compared with blank control group (Figure 5d,e). Correspondingly, immunoblot analysis revealed that the abundances of β‐catenin, one key protein for regulating of Wnt signalling pathway, were markedly (p < 0.05) increased in the intestine treated with ML‐329 and ML‐446 when compared with blank control group (Figure 5d,e). [2]
The expression of notch gene and abundances of activated notch protein in ML‐329 and ML‐446 gavaged rats were investigated. The expression of notch gene was downregulated in the colon of ML‐446‐treated rats compared with that of the blank control group (p < 0.05) (Figure 5f). Meanwhile, the abundances of activated notch protein in ML‐329 and ML‐446 were lower when compared with that of the blank control group in jejunum, ileum, and colon (p < 0.05) (Figure 5g,h).

ML329 was tested for stability in PBS/1% DMSO (Figure 1A), human serum and mouse serum. ML329 was also tested for solubility and GSH/DTT adduct formation (Figures 1B/1C). All results indicate that ML329 is very stable in PBS/1% DMSO, the GSH/DTT adduct assays, and in the different serums. Experimental procedures for the analytical assays are provided in Appendix D [1].

SK-MEL-5 TRPM-1 Luciferase Reporter Assay (Primary Assay AID Nos.: AID 493177, AID 493073, AID 493102, AID 540348, AID 624290, AID 624259, AID 624316, AID 624363, AID 624440, AID 624426, AID 624430, AID 651588, AID 651753) [1]
The TRPM1 luciferase promoter construct was transfected into the SK-MEL-5 melanoma cell line and a stable cell line was generated. This promoter is exquisitely sensitive to MITF over-expression and suppression and contains three canonical E-box motifs within the cloned promoter fragment (17). On day 0, cells were plated at 2,000 cells per well into white, opaque 384 well plates in phenol red-free media. On day 1, cells were treated with compounds or positive control for 24 hours. On day 2, 20 uL of SteadyGlo (Promega) was added per well and luminescence signal was determined with the Perkin-Elmer EnVision plate reader. Primary HTS data were analyzed in Genedata Screener Assay Analyzer. All values were normalized against DMSO treated samples and the positive control (18 μM parthenolide, CID 6473881). For the HTS, the average of two replicates was used to rank order activity and to choose compounds for retests. For dose studies, percent (%) activity was determined for each concentration and the concentration response curves (CRCs) were generated with Genedata Screener's Condoseo.

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SK-MEL-5 Cell Cytotoxicity Assay (SA 1: AID Nos.: AID 493240, AID 540347, AID 624289, AID 624315, AID 624366, AID 624427, AID 624429, AID 624428, AID 651586) [1]
SK-MEL-5 cells were treated with compounds for 24 hours, and then cell viability was measured using the CellTiter-Glo Assay, a luciferase-based reagent that measures cellular ATP levels. The compounds were tested at different concentrations to determine IC50 values. Compounds that were active in the primary assay and toxic below 30 μM at 24 hours were considered for probe development. Data were normalized against DMSO in Genedata Screener's Assay Analyzer. Curves were generated with Genedata Screener's Condoseo and showed percent (%) activity for the individual doses.

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A-375 Cell Cytotoxicity Assay (SA1: AID Nos.: AID 540335, AID 540346, AID 624489, AID 624324, AID 624364, AID 624368, AID 624488, AID 624490, AID 624492, AID 651591) [1]
A375 cells were treated with compounds for 24 hours, and then cell viability was measured using the CellTiter-Glo Assay, a luciferase-based reagent that measures cellular ATP levels. The compounds were tested at different concentrations to determine IC50 values. Compounds that were active in the primary assay and were not toxic below 30 μM at 24 hours were considered for probe development. Data were normalized against DMSO in Genedata Screener's Assay Analyzer. Curves were generated with Genedata Screener's Condoseo and showed percent (%) activity for the individual doses.

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MALME-3M Cell Cytotoxicity Assay (SA1: AID Nos.: AID 493191, AID 540339, AID 624299, AID 624362, AID 651584, AID 651585) [1]
MALME-3M cells were treated with compounds for 24 hours, and then cell viability was measured using the CellTiter-Glo Assay, a luciferase-based reagent that measures cellular ATP levels. The compounds were tested at different concentrations to determine IC50 values. Compounds that were active in the primary assay and toxic below 30 μM at 24 hours were considered for probe development. Data were normalized against DMSO in Genedata Screener's Assay Analyzer. Curves were generated with Genedata Screener's Condoseo and showed percent (%) activity for the individual doses.

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qPCR Assay for MITF Expression (SAI: AID 651773) [1]
SK-MEL-5 cells were treated with compounds for 24 hours. Next, cells were lysed with DNase I. Lysed cells were delivered to a RT-PCR plate and the plates were then processed for reverse transcription to create cDNA. qPCR was performed by transferring cDNA from the RT-PCR plate to a qPCR plate containing PCR master mix, FAM Taqman probe/primer set for the target gene (human MITF), VIC Taqman probe/primer set for a house keeping gene (human GAPDH) and water. qPCR plates were cycled using a real-time PCR instrument. Using the instrument software, a cycle call was generated when each well enters log phase amplification (Ct). The delta Ct value was determined by subtracting the Ct value of the control gene (GAPDH) from the Ct value of the target gene (MITF) in each well. The delta delta Ct value of each compound treatment was determined by averaging the delta Ct values of the mock well on each plate and subtracting that average from the delta Ct value of each compound well. The compounds were tested at different concentrations to determine IC50 values. Data were normalized against DMSO in Genedata Screener's Assay Analyzer. Curves were generated with Genedata Screener's Condoseo and showed percent (%) activity for the individual doses.

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qPCR Assay for TRPM1 Expression (SAI: AID 651770) [1]
Protocol is the same as 2.1.5 except the following primers and probes were used: FAM Taqman probe/primer set for the target gene (human TRPM1), VIC Taqman probe/primer set for a house keeping gene.

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qPCR Assay for CDK2 Expression (SAI: AID 651772) [1]
Protocol is the same as 2.1.5 except the following primers and probes were used: FAM Taqman probe/primer set for the target gene (human CDK2), VIC Taqman probe/primer set for a house keeping gene.

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qPCR Assay for DCT Expression (SAI: AID 651771) [1]
Protocol is the same as 2.1.5 except the following primers and probes were used: FAM Taqman probe/primer set for the target gene (human DCT), VIC Taqman probe/primer set for a house keeping gene.

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qPCR Assay for MLANA Expression (SAI: AID 651795) [1]
Protocol is the same as 2.1.5 except the following primers and probes were used: FAM Taqman probe/primer set for the target gene (human MLANA), VIC Taqman probe/primer set for a house keeping gene.

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Cell proliferation assay of primary human melanocytes (SAI: AID 651920) [1]
Primary human neonatal melanocytes were isolated from discarded foreskins by gentle dispase treatment and grown in Ham's F10 media supplemented with 7% FBS, penicillin/streptomycin/glutamine, 0.1 mM 1-methyl-3-(2-methylpropyl)-7H-purine-2,6-dione (IBMX), 50ng/mL 12-tetradecanoylphorbol 13-acetate (TPA), 1 μM Na3VO4 and 1 μM N(6),2′-O-dibutyryladenosine 3′:5′ cyclic monophosphate (dbcAMP). Cells were plated at 4,000 cells per well of a 384 well plate. On the following day, 10 nL of compound was added per well and incubated for 24 hours. The compounds were tested at different concentrations to determine IC50 values. At the end of compound treatment, cell viability was measured with CellTiter-Glo and luminescence measured with the PerkinElmer EnVision plate reader. Data were normalized against DMSO in Genedata Screener's Assay Analyzer. Curves were generated with Genedata Screener's Condoseo and showed percent (%) activity for the individual doses.

Animals' treatments and IUGR newborn rat models [2]
The IUGR newborn rat models were established by maternal nutrition restriction as previously described (Ding et al., 2016). In brief, the pregnant rats that were individually housed were randomly divided into normal diet group and food restricted group. The pregnant rats in the normal diet group were fed with standard rat chow diet and water without restriction (22–28 g of daily food intake), and 50% amount of food were provided to rats in the food restricted group from the first day of pregnancy. All rats in the two groups were provided standard rat chow diet and water at the delivery day. The IUGR model rats were selected from the newborn rats in the food restricted group according to the criterion that the body weight was two standard deviations less than that of the normal diet group. After successfully modelling, all IUGR newborn rats were randomly assigned to four groups with nine in each group. The blank control group was breast feeding and orally administered with 0.01 mol/L PBS, and the positive control group was breast feeding and orally administered with L. rhamnosus GG, whereas the remaining groups were breast feeding and orally administered with ML‐329, ML‐446, respectively. The IUGR rats were gavaged with 100 μl of PBS or the strains suspended in PBS (1 × 107 CFU) of the same volume from days 1 to 10.

[1]. A Small Molecule Inhibitor of the MITF Molecular Pathway. Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-2012 Dec 13.

[2]. Lactobacillus derived from breast milk facilitates intestinal development in IUGR rats. J Appl Microbiol. 2022 Aug;133(2):503-514.

4-[(1,4-dioxo-2-naphthalenyl)amino]benzenesulfonamide is a member of 1,4-naphthoquinones.

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Micropthalmia-associated transcription factor (MITF) is a lineage restricted basic helix-loop-helix leucine zipper transcription factor that is essential for melanocyte development, function and survival. 15% of human melanomas have MITF gene amplification (1). In addition, a vast majority of melanomas are dependent upon MITF for survival. We set out to identify small molecule inhibitors of MITF activity that would allow for better molecular characterization of MITF's role in melanoma. Using an MITF-dependent melanoma cell line, SK-MEL-5, in a cell-based luminescence assay, we measured the promoter activity of a MITF target gene, melastatin (TRPM-1), in a high throughput screen (HTS). 331,578 compounds from the NIH MLPCN compound library were screened. Of these, 3,206 compounds were active (a hit rate of 0.96%). A chloronaphthoquinone (CID 1716436/SID 22416871) was identified in the primary HTS as an inhibitor of TRPM-1 promoter activity. It had potent activity upon retesting in the primary assay, as did several closely related analogs. Structure activity relationship (SAR) studies were performed to improve potency and to minimize deleterious properties. These efforts generated a probe (CID 12387471/ML329) with improved chemical properties and selectivity. In particular, ML329 was not prone to nucleophilic glutathione addition, whereas the initial hit underwent adduct formation. ML329 was tested in two MITF-dependent melanoma cell viability assays, SK-MEL-5 and MALME-3M plus a MITF-independent cell line, A375. ML329 showed specific activity against the MITF-dependent cells, primary melanocytes but no effect on the viability in A375 cells. ML329 reduced the expression of multiple MITF target genes, including pigment-related genes and the cell cycle regulator CDK2. As a tool compound, ML329 will be useful in elucidating the role of MITF in melanocyte lineage development and in melanoma disease progression.[1]

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Aim: The intestinal microbiota contributes to infant's intestine homeostasis. This study aimed to analyse how probiotics derived from breast milk promote infant intestinal development in rat pups. Methods and results: The effect of potential probiotics derived from breast milk on development of intrauterine growth retardation (IUGR) newborn rats' intestine was investigated. Limosilactobacillus oris ML-329 and Lacticaseibacillus paracasei ML-446 exhibited good hydrophobicity percentages (p < 0.05). ML-446 showed a significant effect on intestinal length and weight (p < 0.05). Meanwhile, the villus height of the IUGR newborn rats fed with ML-329 was significantly higher compared with those fed with Lacticaseibacillus rhamnosus GG (p < 0.05). Moreover, ML-329 and ML-446 both significantly stimulated the proliferation and differentiation of intestinal epithelial cells by increasing the number of ki67-positive cells, goblet cells, and lysozyme-positive Paneth cells (p < 0.05) through Wnt and Notch pathway. Conclusions: The proliferation and differentiation stimulating effects of ML-329 and ML-446 on IECs in the jejunum, ileum, and colon were mediated by activating the Wnt pathway with increased expression of wnt, lrp5, and β-catenin genes and accumulation of β-catenin, and by downregulating the Notch signalling pathway with decreased expression of the activated notch protein. Significance and impact of the study: Lactobacillus could facilitate IUGR rat pups' intestinal development and enhance the proliferation of Paneth cells and goblet cells. These findings provide further insights into promotion of the intestinal development by breast milk-derived beneficial microbes in early life of the IUGR newborn rats. [2]

C₁₆H₁₂N₂O₄S

328.34

328.052

C, 58.53; H, 3.68; N, 8.53; O, 19.49; S, 9.76

19992-50-8

12387471

White to off-white solid powder

3.563

2

6

3

23

627

0

O=S(C1=CC=C(NC(C2=O)=CC(C3=C2C=CC=C3)=O)C=C1)(N)=O

CSYFFQVEQXEIAB-UHFFFAOYSA-N

InChI=1S/C16H12N2O4S/c17-23(21,22)11-7-5-10(6-8-11)18-14-9-15(19)12-3-1-2-4-13(12)16(14)20/h1-9,18H,(H2,17,21,22)

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)

Solubility in Formulation 1: 5 mg/mL (15.23 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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