saspase-flg2 complex, modulators and uses thereof
A use, technology of compounds, applied in the field of cosmetology, prophylaxis and/or treatment of aesthetic defects or pathological disorders of the skin and/or its coverings, therapeutic or dermatological care, achieving good efficacy, well tolerated results
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Embodiment 1
[0339] Enzyme activation of SASPase caused by FLG2
[0340] a- Materials and Method
[0341] Two forms of recombinant filaggrin-2 (FLG2) were prepared in the parent plasmid pET32c (Novagen) or pEB6 (HBGX), respectively corresponding to Q5D862 (Uniprot / Swissprot ref.) fused to thiol at its N-terminus The amino acid sequence 2-213 (active form) and the amino acid sequence 81-212 (inactive form) of the affinity tag of reductin-His-S.Tag (THX-His-S-tag) (FLG2 2-213 -S-His-THX and FLG2 81-212 -S-His-THX).
[0342] These recombinant chimeric proteins are hereinafter referred to as FLG2-Hs=FLG2 2-213 -S-His-THX (active form) and FLG2-AbD=FLG2 81-212 -S-His-THX (inactive form) said.
[0343] The 28-kDa recombinant SASPase corresponding to the amino acid sequence 85 to 343 of the sequence Q53RT3 (Uniprot / Swissprot ref.) was in accordance with Bernard et al. (J. Invest. Dermatol., 2005, 125:278-287) (SASPase 85-343 ) Is prepared as described.
[0344] Dissolve SASPase in PBS buffer, and in the...
Embodiment 2
[0353] Screening of reagents regulating SASPase-FLG2 interaction
[0354] The SASPase-FLG2 interaction, and thus the stimulation of SASPase proteolytic activity, is used in a method for screening active compounds capable of modulating the interaction.
[0355] The established method is based on the FLG2-SASPase interaction and uses homogeneous time-resolved fluorescence (or HTRF) technology.
[0356] Recombinant SASPase protein (containing the amino acid sequence 85-343 of Q53RT3), mutated at its active site (D212A of Q53RT3), and at its C-terminus, and GST (from Schistosoma japonicum) glutathione S transferase ) The affinity tag is fused and prepared in the vector pGEX-4-T3 (GenBank U13855) (SASPase 85-343D212A -GST). The D212A mutation was introduced to inactivate SASPase and promote stable interaction with FLG2.
[0357] The recombinant FLG2 protein (including the amino acid sequence 2-213 of Q5D862) fused to the thioredoxin-His-S.Tag affinity tag at its N-terminus was prepared ...
Embodiment 3
[0388] The effect of compound "M7" on the differentiation and / or proliferation of epidermal cells should
[0389] a- Materials and Method
[0390] The reconstructed human epidermis Episkin J6 (No. 09-EPIS-015) model was immersed in an oven inserted at 37°C (containing 5% CO 2 ) Is cultured in 3.5ml Episkin differentiation medium, and the medium is changed every 2 days.
[0391] by using The provided differentiation medium (including the test concentration of the compound or the buffer solution (0.1% final concentration of DMSO) as a control) culture the epidermal model, and test the compound "M7" at the concentration of 10 μM, 1 μM, and 0.5 μM.
[0392] Evaluate compounds by measuring the thickness of the stratum corneum and measuring the expression and / or activation of transglutaminase I (TGI), caspase 14, SASPase, corneal chain protein, desmoside glycoprotein 1, or protein Ki67 The effect of "M7" on the differentiation and / or proliferation of epidermal cells.
[0393] The thickne...
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