Process for treating an individual with an esthetic defect of skin by modulating SASPase FLG2 complex

Abstract

The present invention relates to the use of a compound, suitable for modulating the interaction between first and second partner proteins, or between homologues, mutants, or fragments of said proteins, said first and second proteins being SASPase and filaggrin-2, or FLG2, as an active agent for treating and/or preventing aesthetic defects in the skin, and/or in the appendages thereof, linked to an imbalance in the differentiation and/or proliferation of the cells of the epidermis.

Claims

1. A process for treating an individual with an esthetic defect of the skin, wherein said defect is at least one selected from the group consisting of signs of aging of the skin, a disorder of the epidermal barrier function, a skin desquamation disorder, a cicatrization and/or re-epithelialization defect, the process comprising: administering to said individual an active agent comprising a compound that modulates the interaction between a first and a second partner protein, homologs, mutants or fragments of said proteins, wherein said first and second proteins are SASPase and filaggrin-2, or FLG2, and said compound is represented by formula (Ia) or (Ib): ##STR00012## wherein: R.sup.1 is H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkyl, or C(O) R8, wherein R.sup.8 is a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkyl; R.sup.2 is O, OR.sup.9 or OC(O) R9, wherein R.sup.9 is H or a saturated C.sub.1-C.sub.2 alkyl; R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each independently of each other H; NO.sub.2; OH; a fluorine; CF.sub.3; a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkyl; a phenyl; OC(O)CH(Ph).sub.2; O-Ph-X wherein X is H, OH, NO.sub.2, a fluorine, a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkyl or alkoxy; OS(O.sub.2)-Ph-Z wherein Z is a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkyl; R.sup.8OH; OR.sup.9; OC(O)R.sup.9, wherein R.sup.8 is a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkyl and R.sup.9 is H or a saturated C.sub.1-C.sub.2 alkyl; R.sup.10Ph, wherein R.sup.10 is a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkylene; or OC(O)Y.sub.nAr.sub.1(X).sub.m, wherein n is 0 or 1 and m is an integer ranging from 0 to 4, with the proviso that n and m are not simultaneously 0, Y is a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkylene, X is H, OH, NO.sub.2, a fluorine, a linear or branched, saturated or unsaturated C.sub.1-C.sub.4 alkyl or alkoxy, and Ar.sub.1 is ##STR00013## wherein * is a bond with Y and @ is a bond with X, and physiologically acceptable salts thereof.

2. The process of claim 1, wherein R.sup.1 is H, a methyl, an ethyl, a propyl, or C(O) R8, wherein R.sup.8 is a methyl, an ethyl, or a propyl.

3. The process of claim 1, wherein R.sup.2 is O or OR.sup.9.

4. The process of claim 1, wherein R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each independently of each other H; OH; a fluorine; a methyl or an ethyl; a phenyl; OC(O)CH(Ph).sub.2; O-Ph-X, wherein X is H, OH, a fluorine, a methyl, an ethyl, a methoxy, or an ethoxy; OS(O.sub.2)-Ph-Z, wherein Z is a methyl or an ethyl; R.sup.8OH; OR.sup.9; OC(O)R.sup.9, wherein R.sup.8 is a methyl or an ethyl and R.sup.9 is H or a methyl; R.sup.10Ph, wherein R.sup.10 is a methylene or an ethylene, or OC(O)Y.sub.nAr.sub.1(X).sub.m.

5. The process of claim 1, wherein not more than one from among R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is a OC(O)Y.sub.nAr.sub.1X.sub.m.

6. The process of claim 1, wherein n is equal to 0, m is equal to 1, X is a methyl or a methoxy, and Ar.sub.1 is: ##STR00014## wherein * is a bond with Y and @ is a bond with X.

7. The process of claim 1, wherein R.sup.3, R.sup.5, R.sup.7 and R.sup.4 or R.sup.6 are each independently of each other H; NO.sub.2; OH; a fluorine; a methyl or an ethyl; a phenyl; R.sup.8OH; OR.sup.9; OC(O) R9, wherein R.sup.8 is a methyl or an ethyl and R.sup.9 is H or a methyl; R.sup.10Ph, wherein R.sup.16 is a methylene or an ethylene.

8. The process of claim 1, wherein said compound is represented by formula (II): ##STR00015##

Description

FIGURES

(1) FIG. 1: illustrates the evolution of the enzymatic hydrolysis of the fluorescent substrate Dabcyl-QIDRIMEK-Edans by a recombinant SASPase (28 kDa) in the presence of increasing concentrations of active recombinant FLG2 (Hs complex=thioredoxin-His-S.Tag-FLG2.sub.2-213; black histogram) or inactive recombinant FLG2 (AbD complex=thioredoxin-His-S.Tag-FLG2.sub.81-212; gray histogram) expressed in multiples (0; 0.01; 0.02; 0.04; 0.06; 0.16; 0.3; 0.63; 1.25; 2.50; 5.00 and 10.00) of the initial SASPase concentration (0.025 mg/ml). The enzymatic reaction is performed in an acetate/acetic acid buffer. The FIGURE illustrates the meansem of an experiment performed in duplicate.

EXAMPLES

Example 1

(2) Enzymatic Activation of SASPase by FLG2

(3) aMaterials and Methods

(4) Two forms of recombinant filaggrin-2 (FLG2) corresponding, respectively, to the amino acid sequences 2-213, active form, and 81-212, inactive form, of Q5D862 (Uniprot/Swissprot ref.) fused, at their N-terminal end, to a thioredoxin-His-S.Tag (THX-His-S-tag) affinity marker were prepared in a parental plasmid pET32c (Novagen) or pEB6 (HBGX) (FLG2.sub.2-213-S-His-THX and FLG2.sub.81-212-S-His-THX).

(5) These recombinant chimeric proteins are identified hereinbelow under the names FLG2-Hs=FLG2.sub.2-213-S-His-THX (active form) and FLG2-AbD=FLG2.sub.81-212-S-His-THX (inactive form).

(6) A 28-kDa recombinant SASPase corresponding to the amino acid sequence 85 to 343 of the sequence Q53RT3 (Uniprot/Swissprot ref.) was prepared as described by Bernard et al. (J. Invest. Dermatol., 2005, 125: 278-287) (SASPase.sub.85-343).

(7) The SASPase was dissolved in a PBS buffer and used at a final concentration of 0.025 mg/ml in the presence of a final concentration of a colorimetric substrate Dabcyl-QIDRIMEK-Edans of 0.01 mM (Production Jerini Peptide Ref. D17: JPT Peptide Technologies GmbH).

(8) FLG2-Hs and FLG2-AbD are used at concentrations corresponding to 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5 or 10 times the final concentration of SASPase, i.e. 0.025 mg/ml.

(9) SASPase and FLG2-Hs, or FLG2-AbD, are incubated at 37 C. in a 0.1 M acetate/acetic acid buffer at pH 5.5 containing 150 mM of NaCl, in the presence of Dabcyl-QIDRIMEK-Edans.

(10) The hydrolysis of the colorimetric substrate by the activated SASPase leads to the separation of the Edans fluorochrome and of its Dabcyl deactivator (or quencher), and to an increase in the fluorescence of Edans.

(11) Measurement of the fluorescent signal is performed at .sub.ex 340 nm/.sub.ex 490 nm. The fluorescence is read every 10 minutes for 1 hour 30 minutes at 37 C., with a Spectramax M5e (Molecular Devices).

(12) bResults

(13) The results obtained show that the enzymatic activity of the SASPase is significantly increased in the presence of an increasing concentration of active FLG2-Hs, whereas it remains virtually unchanged in the presence of inactive FLG2-AbD (see FIG. 1).

(14) These results show that FLG2 interacts with SASPase, and stimulates its proteolytic activity. Such an activation is obtained at and above an FLG2 concentration equivalent to that of the SASPase.

Example 2

(15) Screening of Agents that Modulate the SASPase-FLG2 Interaction

(16) The SASPase-FLG2 interaction, and the stimulation of the proteolytic activity of SASPase resulting therefrom, were exploited in a process for screening active compounds that are capable of modulating this interaction.

(17) The process developed is based on the FLG2-SASPase interaction and uses the homogeneous time-resolved fluorescence (or HTRF) technique.

(18) A recombinant SASPase protein (comprising the amino acid sequence 85-343 of Q53RT3) mutated at its active site (D212A of Q53RT3) and fused with the GST (glutathione S-transferase from Schistosoma japonicum) affinity marker, at its C-terminal end, was prepared in the vector pGEX-4-T3 (GenBank U13855) (SASPase.sub.85-343D212A-GST). The D212A mutation was introduced to inactivate the SASPase and to promote a stable interaction with FLG2.

(19) A recombinant FLG2 protein (comprising the amino acid sequence 2-213 of Q5D862) fused to the thioredoxin-His-S.Tag affinity marker, at its N-terminal end, was prepared as follows (FLG2.sub.2-213-S-His-Thx).

(20) The plasmids obtained are used to transform competent E. coli BL21DE3 bacteria. After transformation of the plasmid corresponding to the protein in the bacteria, 4 ml of preculture were incubated overnight at 37 C. with stirring (250 rpm). The preculture was added to 100 mL of culture medium (LB+ampicillin) and the assembly is placed at 37 C. with stirring until an optical density at 600 nm of at least 0.8 is obtained.

(21) The transformed bacteria are then induced in the presence of 1 mM IPTG overnight at 16 C. with stirring, and then centrifuged and lyzed by ultrasonication. The chimeric peptides are purified by affinity for the HIS or GST tag on a suitable resin, and then dialyzed against a pH 8, 1PBS buffer. The chimeric proteins obtained are then assayed by the Bradford method.

(22) Antibodies recognizing, respectively, the affinity markers GST or His-S.Tag, and bearing, respectively, an EuK fluorescence donor group (europium cryptate .sub.ex 337 nm/.sub.em 620 nm) or an XL1665 fluorescence receiver group (.sub.ex 570-630 nm/.sub.em 665 nm) are used to monitor the peptide-SASPase interaction.

(23) Such antibodies are commercially available, for example from the company Cisbio International under the references 61GSTKLB (anti-GST K antibody) or 61HISKLB (anti-6HIS K antibody).

(24) The SASPase-FLG2 interaction was observed by fluorescence energy transfer (FRET) between the fluorescent markers Euk and XL1665. The Euk fluorescence donor was excited at .sub.ex 337 nm and the fluorescence emission of the XL1665 fluorescence acceptor was measured at .sub.em 665 nm.

(25) The Euk fluorescence emission at .sub.em 620 nm, taking place independently of the interaction between SASPase and FLG2, was used to normalize the signal. Thus, for each point, a ratio .sub.em 665/.sub.em 620 is determined.

(26) The screening process was performed in 384-well plates using a Janus robot from Perkin-Elmer in a PBS phosphate buffer at pH 7.4.

(27) The tests are performed in quadruplicate. The positive control (100% signal) is obtained in the presence of the recombinant proteins, DMSO and antibodies labeled with the fluorophores. The negative control (0% signal or background) is obtained in the presence of protein dilution buffer (free of proteins), DMSO and antibodies labeled with the fluorophores.

(28) The DMSO is used at a final concentration of 2.5%.

(29) The recombinant proteins SASPase.sub.85-343D212A-GST and FLG2.sub.2-213-S-His-THX were used at respective final concentrations of 50 nM and 200 nM.

(30) The test compounds were used at final concentrations of 510.sup.9 M; 1.510.sup.8 M; 4.610.sup.8 M; 1.410 M; 4.110.sup.7 M; 1.210.sup.6 M; 1.110.sup.5M; 3.310.sup.5 M and 110.sup.4 M.

(31) A buffer, or dilution solution free of compound, was used as control.

(32) The SASPase.sub.85-343D212A-GST and the test compounds were preincubated at 4 C. for 60 minutes, and FLG2-His-THX was then added, and the assembly was incubated at 4 C. overnight.

(33) The specific antibodies for each of the chimeric proteins were then added, at a final dilution of 1/400 in a KF (potassium fluoride) buffer (Acros ref. 20135).

(34) The mixture was incubated overnight at 4 C. before reading the fluorescence (.sub.ex 337 nm/.sub.em 665 nm, and normalization at k.sub.em 620 nm) using a Pherastar reader (BMG).

(35) The calculation of the result is performed as follows.

(36) The mean of the ratios .sub.em 665/.sub.em 620 for the positive controls is determined: S.

(37) The mean of the ratios .sub.em 665/.sub.em 620 for the negative controls is determined: B.

(38) The mean of the ratios .sub.em 665/.sub.em 620 for each point of the concentration range of the test compounds is determined: C.

(39) For each point of the concentration range of the test compounds, an activity percentage A is determined via the formula: A=(CB)/(SB).

(40) A dose-response curve was established with the software GraphPad Prism V.5.02 using the equation of a sigmoidal dose-response curve with a variable gradient coefficient, according to the indications in the software.

(41) The IC.sub.50 (concentration inhibiting 50% of the effect) or the EC.sub.50 (concentration activating 50% of the effect) are determined by taking the mid-height concentration, between the bottom and the plateau phase of the curve obtained.

(42) A decrease of A is indicative of the presence of compounds that are capable of preventing or destabilizing the interaction between SASPase and FLG2.

(43) An increase of A is indicative of the presence of compounds that are capable of stabilizing or promoting the interaction between SASPase and FLG2.

(44) The screening of a molecule databank HBGXS10 (ChemDiv Discovery Chemistry Collection Public Database (Order Number: 4477-2039), ChemDiv, Inc. San Diego, USA) made it possible to identify the compound, referred to as M7, of formula (II) below:

(45) ##STR00011##

(46) This compound led to an inhibition of the SASPase-FLG2 interaction and has an IC.sub.50 of about 2 M.

(47) The screening protocol of the invention thus proves to be particularly advantageous for identifying novel active compounds that are capable of positively or negatively modulating the SASPase-FLG2 interaction.

(48) The novel compounds may advantageously be used with regard to esthetic defects or pathological disorders of the skin and its integuments resulting from an imbalance in epidermal cell proliferation and/or differentiation, and in particular aging of the skin and skin dryness.

Example 3

Effect of Compound M7 on the Differentiation and/or Proliferation of Epidermal Cells

(49) aMaterials and Methods

(50) A model of reconstructed human epidermis Episkin J6 (reference 09-EPIS-015) was cultured by immersion in Episkin differentiation medium, 3.5 ml under insert in an oven at 37 C., 5% CO.sub.2, the medium being changed every 2 days.

(51) The compound M7 was tested at concentrations of 10 M, 1 M and 0.5 M by incubation of the epidermal model with a differentiation culture medium supplied by EpiSkin, comprising the compound at the test concentration or a buffer solution as control (DMSO at 0.1% final).

(52) The effect of the compound M7 on the differentiation and/or proliferation of epidermal cells is evaluated by measuring the thickness of the stratum corneum, and measurement of the expression and/or activation of transglutaminase I (TGI), caspase 14, SASPase, corneodesmosin, desmoglein 1 or protein Ki67.

(53) The thickness of the stratum corneum was estimated by histological observation on slices.

(54) The activation, expression and maturation of TGI, filaggrin, caspase 14, SASPase, corneodesmosin and desmoglein 1 were determined by western blotting.

(55) The activation, expression and maturation of Ki67 were determined by immunofluorescence on frozen slices.

(56) For the western blotting, the soluble proteins, TGI, caspase 14, corneodesmosin and SASPase were extracted from two Episkin nacelles with a manual potter in 1 ml of native + buffer (TBS, 1M NaCl, 0.1% Tween 20). The extract is centrifuged at 14 000 rpm, at 4 C. for 10 minutes. The supernatant obtained is filtered through a 0.45 m Millipore filter (millex) and the proteins are then quantified by the BCA method (Pierce). The protein concentrations are equilibrated to 625 g/ml final.

(57) The extracts are deposited on a 10-20% Criterion gel (ref.: 345-0044 Bio-Rad) according to the supplier's protocol, and are then transferred onto a PVDF membrane via the semi-dry method.

(58) The western blotting immunochemical detection is performed according to a standard protocol carried out with primary antibodies specific for the target and secondary antibodies coupled to HRP. Revelation is performed with the kits ECL; ECL plus; ECL advanced (Amersham) according to the necessary sensitivity and according to the supplier's protocol. The photon imaging apparatus used is the Fluor S Multilmager (Bio-Rad).

(59) For TGI, primary antibody: Covalab pab 0061 at 1/100 produced in rabbits; secondary antibody: HRP-coupled anti-rabbit antibody at 1/4000: Bio-rad 170-6515

(60) For caspase 14, primary antibody: Santa Cruz sc-5628 at 1/500 produced in rabbits; secondary antibody: HRP-coupled anti-rabbit antibody at 1/400: Bio-rad 170-6515

(61) For corneodesmosin, primary antibody: Abnova 1041-M01 at 1/5000 produced in mice; secondary antibody: HRP-coupled anti-mouse antibody at 1/4000: Bio-rad 170-6516

(62) For SASPase, primary antibody: Covalab 7H9a105 at 1/2000 produced in mice; secondary antibody: HRP-coupled anti-mouse antibody at 1/4000: Bio-rad 170-6516

(63) For the western blotting of filaggrin and desmoglein 1, these proteins are extracted from the pellet obtained during the extraction of the soluble proteins. The pellets are ground with an electric potter in 200 l of whole Laemmli buffer (0.0625 mM Tris; 2% SDS; 200 mM DTT; 10% glycerol) and then heated at 90 C. for 10 minutes and finally centrifuged for 10 minutes at 14 000 rpm at room temperature.

(64) The supernatants obtained are assayed for proteins via the Bradford method (cytoskeleton). The total protein concentrations are equilibrated to 1 mg/ml final. A western blotting protocol identical to the previous one is applied.

(65) For filaggrin, primary antibody: Abcam ab24584 at 1/1000 produced in rabbits; secondary antibody: HRP-coupled anti-rabbit antibody at 1/4000: Bio-rad 170-6515

(66) For desmoglein 1, primary antibody: Progen 61002 at 1/100 produced in mice; secondary antibody: HRP-coupled anti-mouse antibody at 1/4000: Bio-rad 170-6516

(67) The immunofluorescence labeling on frozen slices is performed using a standard protocol, with a PBS buffer; 0.2% BSA for the steps of washing and incubation of the primary and secondary antibodies, and with a PBS buffer; 5% BSA for the step for blocking the nonspecific sites.

(68) For the detection of Ki67, primary antibody: Novocastra mm1 at 1/20 produced in mice; secondary antibody: AlexaFLuor A488-coupled anti-mouse antibody at 1/1000 (Molecular Probes A11017). The visualization and image capture apparatus is a Leica optical microscope.

(69) bResults

(70) The application of compound M7 led to thickening of the stratum corneum, which was observable on histological slices by optical microscopy, at and above the lowest concentration tested.

(71) An increase in the expression and activation of TGI, caspase 14, SASPase, corneodesmosin and desmoglein was observed in the Episkin epidermis cultured in the presence of M7.

(72) The expression of these proteins is combined with differentiation of the cells in the epidermis.

(73) A decrease in the activation and expression of the protein Ki67 was also observed. This protein is associated with stoppage of the proliferation of epidermal cells.

(74) The thickening of the stratum corneum and the increase in the expression and activation of the proteins transglutaminase I, caspase 14, SASPase, corneodesmosin and desmoglein are characteristics of late differentiation of the epidermis.

(75) The compound M7 thus proves to have the property of modulating the proliferation and differentiation of epidermal cells.

(76) Such a compound thus proves to be particularly advantageous for preventing or treating pathological disorders or esthetic defects of the skin and/or its integuments involving an imbalance in epidermal cell differentiation and/or proliferation.