Sphingosine kinase (SPHK1)

In keratinocytes, MHP (50-250 μM) for 24 hours raises the levels of CAMP mRNA [1]. MHP (100 μM) elevates the levels of CAMP protein in keratinocytes for a 24-hour period [1].

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Background [1]
The ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in keratinocytes (KC). We recently discovered that production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), is stimulated via a NF-κB-dependent mechanism that is activated by S1P when S1P is generated by sphingosine kinase (SPHK) 1.

Objective [1]
We investigated whether pharmacological modulation of SPHK1 activity, using a novel synthetic SPHK1 activator, (S)-Methyl 2-(hexanamide)-3-(4-hydroxyphenyl) propanoate (MHP), stimulates CAMP expression.

Methods [1]
MHP-mediated changes in both S1P and CAMP downstream mediators were analyzed in normal cultured human KC by qRT-PCR, Western immunoblot, ELISA, confocal microscopy for immunohistochemistry, HPLC and ESI-LC/MS/MS, and microbial pathogen invasion/colonization in a human epidermal organotypic model.

Results [1]
Treatment with MHP directly activated SPHK1 and increased cellular S1P content in normal cultured human KC. Because MHP did not inhibit S1P lyase activity, which hydrolyses S1P, augumented S1P levels could be attributed to increased synthesis rather than blockade of S1P degradation. Next, we found that exogenous MHP significantly stimulated CAMP mRNA and protein production in KC, increases that were significantly suppressed by siRNA directed against SPHK1, but not by a scrambled control siRNA. NF-κB activation, assessed by nuclear translocation of NF-κB, occurred in cells following incubation with MHP. Conversely, pretreatment with a specific inhibitor of SPHK1 decreased MHP-induced nuclear translocation of NF-κB, and significantly attenuated the MHP-mediated increase in CAMP production. Finally, topical MHP significantly suppressed invasion of the virulent Staphylococcus aureus into murine skin explants [1].

Enzyme activity assays for sphingosine kinase 1 [1]
SPHK1 activity was assessed as described previously. Briefly, recombinant SPHK1, (2 μg) was incubated with sample reaction buffer (10 mM ATP, 200 mM MgCl2, 200 μM C17-Sphingosine, 5 mM NaF, Na3VO4, 5% Triton X-100) for 30 min. The reaction was terminated by the addition of CHCl3/MeOH/HCl (8:4:3, v/v/v) with 100 pmol C17-sphinganine-1-phosphate as an internal standard. The organic phage separated by addition of CHCl3 was dried using a vacuum system. The dried residue was re-dissolved in MeOH and then injected into the LC-ESI-MS/MS system. The HPLC column effluent was introduced onto an API 3200 Triple quadruple mass (ABCIEX) and analyzed using electrospray ionization in positive mode with multiple reaction monitoring (MRM) to select both parent and characteristic daughter ions specific to each analyte simultaneously from a single injection. The MS/MS transitions (m/z) of 366 → 250 for C17-S1P and 368 → 270 for C17-Sa1P were used as quantifier for the MRM with a dwell time of 100 ms. Data were acquired using Analyst 1.4.2 software and the activity of SPHK1 is expressed as S1P pmol per mg protein per min.

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Enzyme activity assays for sphingosine-1-phosphate lyase [1]
To determine the activity of S1P lyase, KC were treated with Defensamide (MHP)  for 24 h followed by washing with phosphate-buffered saline. Cell lysates (50 μg) were incubated with 10 nmol C17- sphinganine-1-phosphate for 20 min. Lipid extraction was performed by the addition of 100 pmol of (2E)-d5-hexadecenal as the internal standard. Total lipid extracts were derivatized with 5 mM semicarbazide hydrochloride in methanol containing 5% formic acid at 40 °C for 2 h and analyzed by LC-ESI-MS/MS (ABCIEX), as described previously. The activity of S1P lyase is expressed as pentadecanal pmol per mg protein per min.

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Staphylococcus aureus invasion assay [1]
Staphylococcus (S.) aureus invasion assay was performed, as we described previously. Briefly, full-thickness pieces of murine skin treated with Defensamide (MHP)  or vehicle were harvested from hairless mice (10-week-old female, hr/hr, n=5). Epidermal permeability barrier was attenuated by topical application of oxazolone (0.1%) once every other day five times. Mice were treated with Defensamide (MHP)  two times daily for the last 3 days, during the oxazolone treatment, before harvesting skin. Skin was placed on a filter paper, dermis side down, and maintained at the air–medium interface in KC growth medium. S. aureus in PBS or PBS was epicutaneously applied (20 μl/cm−2), followed by incubation for 24 hours at 37 °C in 5% CO2. Gram staining was performed to assess S. aureus invasion

Cell Viability Assay[1]
Cell Types: Keratinocytes
Tested Concentrations: 100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased CAMP protein levels in keratinocytes.

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ELISA for CAMP quantifications [1]
CAMP content of cell lysates of KC previously incubated with (S)-Methyl 2-(hexanamide)-3-(4-hydroxyphenyl) propanoate (Defensamide (MHP) ) is determined by ELISA kit in accordance with the manufacturer’s instructions.

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Immunofluorescence [1]
Immunofluorescence was performed, as described previously, NF-κB [14] and murine CAMP (mCAMP). KC were treated with Defensamide (MHP)  or vehicle for 30 min. NF-κB distribution was assessed using anti- NF-κB p65 and anti-rabbit IgG conjugated with fluorescein isothiocyanate. Cells were counterstained with the nuclear marker histone H4. Sections fixed with formalin (5 μm) were incubated for 1 hour in blocking buffer (4% BSA, 0.5% cold water fish gelatin in PBS), and then incubated with anti-mCAMP overnight at 4°C. The sections were then incubated for 1 hour at room temperature with goat anti-rabbit Alexa Fluor 488 followed by counterstaining with propidium iodide.

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Measurement of intracellular levels of sphingosine-1-phosphate [1]
To assess the levels of cellular S1P, KC were incubated with Defensamide (MHP)  and washed with phosphate-buffered saline followed by extraction of total S1P, as we reported previously. S1P was derivatized with o-phthalaldehyde (OPA) reagent and then quantitated using an HPLC system equipped with a fluorometrical detector system, as described previously. S1P levels were expressed as pmol per mg protein.

[1]. Sphingosine kinase 1 activation enhances epidermal innate immunity through sphingosine-1-phosphate stimulation of cathelicidin production. J Dermatol Sci. 2015 Sep;79(3):229-34.

Researchers further investigated whether the MHP-induced increase in S1P activates the S1P → NF-κB activation → C/EBP activation → CAMP mechanism, which we recently identified, and showed that MHP stimulates increased CAMP production through this S1P-dependent, downstream signaling pathway. Specifically, we demonstrate that SPHK1 activation also efficiently enhances cutaneous antimicrobial defense through stimulation of CAMP production in KC. To assess the clinical relevance of MHP, we then showed that topical MHP enhances antimicrobial defense in murine skin exposed to virulent S. aureus. Since MHP does not increase human β-defensin production, these studies show that CAMP plays an important role in suppression of S. aureus invasion and colonization. Activation of S1P production could also be valuable in several other clinical settings; i.e., not only to correct CAMP deficiency in atopic dermatitis, but also to enhance wound healing, or to boost innate immunity in chronically ill or immune compromised patients. Three small molecules, stilbenoid, isoflavone, and MHP, are useful chemicals to enhance innate immunity and antimicrobial defense. Hence, we name MHP as “Defensamide”.[1]

C16H23NO4

293.36

293.163

C, 65.51; H, 7.90; N, 4.77; O, 21.81

1104874-94-3

56652925

White to off-white solid powder

3.013

2

4

9

21

324

1

CCCCCC(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)OC

GBTVWZMJUZJPBU-AWEZNQCLSA-N

InChI=1S/C16H23NO4/c1-3-4-5-6-15(19)17-14(16(20)21-2)11-12-7-9-13(18)10-8-12/h7-10,14,18H,3-6,11H2,1-2H3,(H,17,19)/t14-/m0/s1

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: ≥ 2.5 mg/mL (8.52 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 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 (8.52 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.52 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.)