Negative analog of SB203580

The synthesis of melanin pigments, or melanogenesis, is regulated by the balance of a variety of signal transduction pathways. Among these pathways, p38 MAPK signaling was found to be involved in stress-induced melanogenesis and to be activated by alpha-melanocyte-stimulating hormone (alpha-MSH) and ultraviolet irradiation. Previous studies have shown that alpha-MSH-stimulated melanogenesis can be inhibited by blocking p38 MAPK activity with SB203580, a pyridinyl imidazole compound. Consistent with this, we observed that pyridinyl imidazoles (SB203580 and SB202190) inhibited both basal and alpha-MSH-induced melanogenesis in B16 melanoma cells. However, SB202474, which has no ability to inhibit p38 MAPK activity and is usually used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from the repression of melanogenesis by pyridinyl imidazole compounds was also confirmed by small interfering RNA experiments. Interfering with p38 MAPK expression surprisingly stimulated melanogenesis and tyrosinase family protein expression. Although the molecular mechanism(s) by which p38 promotes the degradation of melanogenic enzymes remain to be determined, the involvement of the ubiquitin-proteasome pathway was demonstrated by co-treatment with the proteasome-specific inhibitor MG132 and the relative decrease in the ubiquitination of tyrosinase in cells transfected with p38-specific small interfering RNA [1].

Tyrosinase Assay [1]
Tyrosinase enzyme activity was estimated by measuring the rate of l-DOPA oxidation as previously described with slight modifications. Briefly, the cells were treated with p38-specific siRNA or control siRNA for 72 h in Dulbecco's modified Eagle's medium containing 2% (v/v) fetal bovine serum ± α-MSH. At the end point, the cells were solubilized with phosphate buffer (pH 6.8) containing 1% Triton X-100. The cells were then disrupted by freezing and thawing, and the lysates were clarified by centrifugation at 10,000 × g for 10 min. After protein quantification and adjustment of protein concentrations with lysis buffer, 80 μl of each lysate (each containing the same amount of protein) were aliquoted into the wells of a 96-well plate, and 20 μl of 5 mm l-DOPA were then added to each well. The absorbance was measured spectrophotometrically at 475 nm following a 20-min incubation period at 37 °C. The measurement was repeated five times.

Cell Culture and Reagents [1]
B16-F0 murine melanoma cells were purchased from the American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium with 4 mm l-glutamine + 7% heat-inactivated fetal bovine serum and antibiotics at 37 °C in a 5% CO2 atmosphere. For the induction studies, the cells were plated, and 24 h later, the medium was removed, and the cells were then cultured in Dulbecco's modified Eagle's medium with 2% heat-inactivated fetal bovine serum and antibiotics with or without pharmacological treatment in the absence of phenol red. Normal human melanocytes (NHM) isolated from several independent Caucasians foreskins were maintained in M-254 medium with human melanocyte growth supplements and used for passages 2–6. NHM were treated with compounds for 5 days before analysis because melanogenesis is a much longer process in these cells than in B16 melanoma cells.

[1]. p38 regulates pigmentation via proteasomal degradation of tyrosinase. J Biol Chem. 2010;285(10):7288-7299.

MAPKs are evolutionarily conserved enzymes connecting cell surface receptors to critical regulatory targets within cells. p38 MAPK is a stress-regulated protein kinase belonging to the MAPK superfamily in addition to c-Jun N-terminal kinase and extracellular signal-regulated kinases. p38 MAPK has been shown to play a key role in the regulation of cellular responses to external stress signals. It is activated by diverse stimuli that include ultraviolet light, irradiation, heat shock, osmotic stress, and proinflammatory cytokines. Pharmacological inhibitors have accelerated studies concerning the role of p38 MAPK in many different cellular processes, including proliferation, differentiation, apoptosis, cellular senescence, transcriptional regulation, and cytoskeletal reorganization. The two pyridinyl imidazole compounds, SB202190 and SB203580, have been shown to inhibit p38 MAPK, but they have no effect on related kinases such as extracellular signal-regulated protein kinase end c-Jun N-terminal kinase. In contrast, SB-202474, which has no effect on p38, is commonly included as a negative control when SB203580 and SB202190 are used to study the involvement of p38 MAPK. From these studies emerged some sporadic evidence that pyridynil imidazole compounds have unexpected targets in addition to p38, and consequently they were found to be too nonspecific to assess the physiological roles of p38 MAPK.
A number of studies have reported the involvement of p38 MAPK in melanogenic differentiation. However, the role of p38 MAPK in controlling melanogenesis is not fully understood. We observed here that pyridinyl imidazoles inhibited both basal and α-MSH-induced melanogenesis. At first, we suspected that the p38 MAPK pathway might be involved in melanogenesis suppression. However, SB-202474, which has no ability to inhibit p38 MAPK activity and is widely used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from melanogenesis repression by pyridinyl imidazole compounds was also confirmed by siRNA experiments. In fact, p38 MAPK siRNA surprisingly stimulated melanogenesis. From these results, we conclude that the p38 MAPK pathway is not involved in inhibition by SB202190 and SB203580 and that pyridinyl imidazole compounds have a target molecule(s) besides p38 MAPK that might be important for pigmentation. Previous studies described repression of melanogenesis by p38 activity inhibition using the p38 inhibitor SB203580. It is our concern that no one study regarding melanogenesis included SB-202474 as a negative control or siRNA as a positive control. [1]

C17H17N3O

279.34

279.137

C, 73.10; H, 6.13; N, 15.04; O, 5.73

172747-50-1

5162

Solid powder

1.2±0.1 g/cm3

520.7±50.0 °C at 760 mmHg

183.9±20.4 °C

0.0±1.3 mmHg at 25°C

1.597

4.35

1

3

4

21

311

0

CCC1=C(C2=CC=NC=C2)NC(=N1)C3=CC=C(C=C3)OC

MYKGURNPAUBQLJ-UHFFFAOYSA-N

InChI=1S/C17H17N3O/c1-3-15-16(12-8-10-18-11-9-12)20-17(19-15)13-4-6-14(21-2)7-5-13/h4-11H,3H2,1-2H3,(H,19,20)

4-[5-ethyl-2-(4-methoxyphenyl)-1H-imidazol-4-yl]pyridine

SB-202474; SB202474; sb 202,474; 172747-50-1; 4-[5-ethyl-2-(4-methoxyphenyl)-1H-imidazol-4-yl]pyridine; SB-202474; CHEMBL278724; 4-(5-ethyl-2-(4-methoxyphenyl)-1H-imidazol-4-yl)pyridine; SB 202474

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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