AU2015100662A4 - Isoliquiritigenin Suppresses human T lymphocyte activation via targeting on cysteine 46 of IKBA Kinase - Google Patents

Abstract

The present invention provides a compound, Isoliquiritigenin, which could inhibit immune suppressive effect resulted from direct interaction with IKK-P C46A in vivo and in vitro. Isoliquiritigenin effectively inhibits the immune-suppressive effects through regulation of T lymphocytes by inhibiting T cells proliferation and division, suppressing expression of CD69 and CD 25 as well as up-regulating CD71 in a dose-dependent manner. The present invention also provides a method of screening therapeutic agents for treating autoimmune diseases.

Description

Isoliquiritigenin Suppresses human T lymphocyte activation via targeting on cysteine 46 of IxBa Kinase FIELD OF INVENTION The present invention relates to a compound, Isoliquiritigenin, which could inhibit mediated immune-suppressive effect resulted from direct interaction with IKK-P C46A in vivo and in vitro. Isoliquiritigenin inhibits mediated immune-suppressive effects through regulation of T lymphocytes, further inhibiting T lymphocyte activation. BACKGROUND OF INVENTION Our bodies have an immune system, which is a complex network of special cells and organs that protects against diseases. A properly functioning immune system is able to detect various agents, known as pathogens, from viruses to parasitic worms, and distinguish them from the body's own healthy tissue. In abnormal immune response, the body cannot distinguish between its own healthy tissue and foreign pathogens, as a result, the body will make autoantibodies that attack normal cells by mistake. At the same time, special cells called regulatory T cells fail to do their job of keeping the immune system in line which leads to a misguided attack on its own body. This causes the damage we know as autoimmune disease. It has been estimated that autoimmune diseases are amongst the top ten leading causes of death among women in all age groups up to 65 years and a small minority of the population suffers from these diseases, which are often chronic, debilitating and life-threatening. Treatment for such diseases is to mitigate inflammation by the activity of anti-inflammatory genes and suppress inflammatory genes in the immune cells which include non-steroidial anti-inflammatory drugs, disease-modfiying anti -inflammatory drugs and biological inhibitors of T cell function. Isoliquiritigenin (ILG; 4,2',4'-trihydrozychalcone, a member of the flavonoids, belongs to the chalcone family that is found in licorice, shallot and bean sprouts, and other Chinese medicinal herbs such as Sinofranchetia chinensis. It has been demonstrated to possess an active component present in plants like Glycyrrhiza and Dalbergia which showed various biological activities including anti-inflammatory, antitumor and antihistamic in vitro and in 1 vivo. The suppressive effect of TLG on human T cell activation as identified has significant potency to be served as a leader compound to develop novel immunomodulatory agents in the future. SUMMARY OF INVENTION In light of the foregoing background, it is an object of the present invention to provide a method for treating autoimmune diseases comprising administering an effective amount of isoliquiritigenin (ILG) to a subject in need thereof. Said treating is mediated in part by T lymphocyte activation. Another object of the present invention is to provide a method for inhibiting T lymphocyte activation comprising contacting TLG to T cells or tissues being affected by autoimmune diseases in a dose dependent manner. In one embodiment, the autoimmune diseases comprise Rheumatoid Arthritis (RA), Graves' Disease, Diabetes Mellitus Type 1, Polymyositis and Dermatomyositis. In a further embodiment, TLG further inhibits one or more of the following activities in relation to T lymphocyte activation: T lymphocyte proliferation, T lymphocyte division, T lymphocyte cycle progression, TL-2 and IFN-y expression of activated T lymphocytes, and/or CD69 and CD25 expression, and up-regulates CD71 expression on T lymphocytes. In yet another embodiment, the effective amount or dose of said ILG being administered to the subject or contacted to the T cells or the tissues in order to effectively inhibit T lymphocyte activation is from 5 [tM to 25 [tM. More specifically, the effective amount or dose of the ILG is from 10 [tM to 25 [tM. In yet another embodiment, T-lymphocyte is human T lymphocyte. In yet another object of the present invention, a method of screening a therapeutic agent as a drug candidate for treating autoimmune diseases is provided. The method comprises exposing the therapeutic agent to an assay comprising IKBa kinase, detecting whether the therapeutic agent binds to cysteine-46 residue of IKBa kinase, detecting whether the therapeutic agent inhibits kinase activity of IKBa kinase and thereby inhibiting T lymphocyte activation. 2 In one embodiment, the autoimmune diseases comprise Rheumatoid Arthirits, Graves' Disease, Diabetes Mellitus Type 1, Polymyositis and Dermatomyositis. In another embodiment, said inhibiting of the T lymphocyte activation by said agent is detected by one or more of the following indications: whether T lymphocyte proliferation is inhibited, whether T lymphocyte division is inhibited, whether T lymphocyte cycle progression is inhibited, whether TL-2 and IFN-y expression of activated T lymphocytes is inhibited, and/or whether CD69 and CD25 expression are down-regulated, and whether CD71 expression are up-regulated on T lymphocytes. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the chemical structure of Isoliquiritigenin (ILG). FIG. 2 shows the relative fold of human T lymphocyte proliferation inhibited by different concentrations of ILG (5 [tM, 10 [tM, 25 [M); ctrl: control; P/I: ionomycin. FIG. 3 shows the inhibitory effect of different concentrations of ILG on cell division of human T lymphocyte: flow cytometry result in terms of single-parameter histogram (upper panel); measure of division in terms of % of cells divided and index of division (lower panel). FIG. 4 shows the inhibitory effects of different concentrations of ILG on (A) IFN-y secretion of human T lymphocyte mediated by P/I and (B) IL-2 secretion of human T cells mediated by P/I. FIG. 5 is a series of dual-parameter histograms of the flow cytometry result according to an embodiment of the present invention showing the inhibitory effect of different concentrations of ILG on human T lymphocyte cell cycle progression mediated by P/I: (A) control (ctrl); (B) P/I only; (C) 5 [tM ILG; (D) 10 [tM ILG; (E) 25 [tM ILG; (F) percentage of cells in different phases (GO; S; G2M) in the cell cycle of T lymphocyte under different treatment groups. FIG. 6 is a series of dual-parameter histograms of the flow cytometry result according to an embodiment of the present invention showing the inhibitory effect of different concentrations of ILG on CD69 expression of T lymphocyte: (A) control; (B) P/I only; (C) 5 jaM ILG; (D) 10 jaM ILG; (E) 25 jaM ILG. 3 FIG. 7 is a series of dual-parameter histograms of the flow cytometry result according to an embodiment of the present invention showing the inhibitory effect of different concentrations of ILG on CD25 expression of T lymphocyte: (A) control; (B) P/I only; (C) 5 [iM ILG; (D) 10 [tM TLG; (E) 25 [tM TLG. FIG. 8 is a series of dual-parameter histograms of the flow cytometry result according to an embodiment of the present invention showing the effect of different concentrations of ILG on CD71 expression: (A) control; (B) P/I only; (C) 5 [tM TLG; (D) 10 [tM ILG; (E) 25 [tM TLG. FIG. 9 is a series of flow cytometry histograms showing that TLG dose-dependently inhibits NF-xB nucleus translocation in human T cells: (A) control (Ctrl); (B) ionomycin-treated (P/I); (C) 5 [tM TLG; (D) 10 [tM TLG; (E) 25 [tM TLG. FIG. 10 shows that TLG dose-dependently inhibits IxBa degardation and phosphorylation: western blot results of IxBa and phosphorylated IxBa (P-IxBa) expression in different groups of treatment (ctrl; P/I; 5 [tM TLG; 10 [tM TLG; 25 [tM TLG). FIG. 11 shows that TLG dose-dependently inhibits phosphorylation of IKKa/p and p65: western blot results of phosphorylated IKKa/p (P-IKKa/0) and phosphorylated p65 (P-p65) expression in different groups of treatment (ctrl; P/I; 5 [tM TLG; 10 [tM ILG; 25 [tM TLG). FIG. 12 further shows the effect of ILG on the IKKp activity. Human HEK293 cells transfected with FLAG-IKKO (wt) plasmid were immunoprecipitated (TP) with anti-FLAG antibody, and the IP FLAG-IKKO was incubated with GST-IKBa substrate and ATP in the presence or absence of different concentrations of ILG. IKKp kinase activity was determined by the level of phosphorylated GST IKBa using antibody against p- IfBa. P/I: PMA (20 ng/ml) plus ionomycin (1 [M). FIG. 13 shows computational docking predicting the molecular target of ILG at (A) Cys-46 and (B) Cys-46 being replaced by alanine of IKK-P. FIG. 14 is the result of competitive binding experiments which are used to elucidate the binding site of ILG at IKK-3. FIG. 15 shows the effect of TLG on the activity of IKKp C46A. The immunoprecipitated complexes were subjected to an in vitro kinase assay and analyzed. The levels of phosphate 4 IxBa, kinase-mutation IKK3 incorporated into FLAG-tagged are shown in the upper panel, and the levels; FLAG-tagged IKKp are shown in the lower panels. FIG. 16 shows that the inhibitory effect of ILG is abolished in IKKp3C 4 6 A transgenic mice in terms of the thickness of the ear edema: 'CtrlWT': normal mice with wild-type IKK-p; 'ILGO.75mg/ear_WT': normal mice with wild-type IKK-p challenged by 2,4-dinitro-1 fluorobenzene (DNFB) followed by 0.75mg/ear TLG treatment; 'DEX 0.025mg/ear_WT': normal mice with wild-type IKK-p challenged by DNFB followed by 0.025mg/ear of dexamethasone (DEX) treatment; 'CtrlHomo': homozygous IKK C 4 6 A mutant mice; 'ILG 0.75mg/ear _Homo': homozygous IKK C 4 6 A mutant mice challenged by DNFB followed by 0.75mg/ear of ILG treatment; 'DEXO.025mg/earHomo': homozygous IKK C 4 6 A mutant mice challenged by DNFB followed by 0.025mg/ear of DEX treatment. FIG. 17 shows the influences of ILG and dexamethasone (DEX) on the immune organ, including thymus and spleen in terms of their weight. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As used herein and in the claims, "comprising" means including the following elements but not excluding others. The present invention relates to a compound, Isoliquiritigen (ILG), which could inhibit mediated immune-suppressive effect resulted from direct interaction with IKK-0 C46A in vivo and in vitro. ILG inhibits mediated immune-suppressive effects through regulation of T lymphocytes by further inhibiting T lymphocyte activation. The regulation of T lymphocytes can be indicated by the regulation of cell proliferation, cell division, cell cycle progression, and up- or down-regulation of the expression of different cytokines or surface markers involved in the cell cycle progression or division of the T lymphocytes. The present invention has demonstrated that ILG inhibits T lymphocyte activation in a doses-dependent manner, and the effective amount or dose of ILG which effectively inhibits the T lymphocyte activation is determined or verified by measuring different indicators according to different embodiments of the present invention. In some embodiments, it is indicated that the effective amount or dose of ILG to inhibit T lymphocyte activation is from 5 to 25 [tM. In other embodiments, the effective amount or dose of ILG to inhibit T lymphocyte activation is from 5 10 to 25 [tM. More specifically, the most effective amount or dose of TLG to inhibit T lymphocyte activation is about 25 [tM according to an embodiment of the present invention. In the present invention, it is determined that TLG regulates the immune response via suppressing NF-fB signaling. Molecular docking results predict that IKK-p Cystiene-46 (IKKpC46) is likely the target of ILG. Competition assay and kinase assay are further conducted to validate the virtual docking results. Moreover, IKKC46A transgenic mice are generated, and used to demonstrate whether the IKKpC46 is involved in the suppressive effect of ILG in vivo. Collectively, the present invention identifies IKKpC46 as the target of suppressive effect of TLG on human T cell activation, and it has the significant potency to be served as a leader compound to develop the novel immunomodulatory agents in the future. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof Example 1 This example exhibits the effect of TLG on human T lymphocyte proliferation generated by PMA plus 1 [tM ionomycin (P/I). Isolation and cultures of human T cells Human peripheral blood T lymphocytes are isolated from buffy coat according to the previous method. In brief, the buffy coat provided by Macao Blood Transfusion Centre is mixed with normal saline, and then added to 50 ml centrifuge tube containing Ficoll-Pague plus (Amersham Biosciences). The mixture is separated to several layers after centrifuged at 350 g for 35 min. The layer of mononuclear cells is collected, and purified by magnetic activated cell sorting (MACs) pan T cell kit. 20 ng/ml PMA plus 1 aM ionomycin (P/I) is employed in each experiment with different time intervals for different purpose. Cell proliferation assay Human T lymphocytes (1x 10 5 /well) are cultured in 96-well plates in triplicate in RPMI 1640 medium plus 10% FBS, and then stimulated with 20 ng/ml PMA plus 1PM ionomycin in the presence or absence of the compounds for 72 h. Before the cells are collected, BrdU is added to the cells at final concentration of 10 [tM, and incubated for another 14 h. Finally, BrdU is 6 determined by ELISA method according to the manual, and data is obtained from three independent experiments. Results ILG dose-dependently blocks human T cell proliferation generated by PMA plus ionomycin (P/I) from 5 to 25 pM, with 25 pM of ILG inhibiting T cell proliferation the most and 5 pM of TLG inhibiting T cell proliferation the least as shown in FIG. 2. Example 2 This example studies the effect of TLG on human T lymphocyte proliferation and division generated by P/I. Flow cytometery assay for detection of cell division The division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used according to the previous method. In brief, the cells are incubated with prewarmed PB S/0.1% BSA at a final concentration of 1x 106 cells/mL. 2 [tL of 5 mM stock CFSE solution is added to the cells at final working concentration of 10 [M. After being incubated with the dye at 37'C for 10 min, the cells are added ice-cold culture media and incubated for 5 min to quench the staining. The cells are washed by fresh media and pretreated by ILG for 2 h, followed by stimulated with PMA/ionomycin for another 5 days. CFSE data is analyzed by flow cytometer. Results FIG. 3 shows that TLG significantly reduces numbers of cell division starting from 10 pM, and it is almost totally blocked at 25 pM of ILG. Example 3 This example shows the effect of ILG on IL-2 and IFN-y secretion of activated human T cells mediated by P/I. Enzyme-linked immunosorbent assay (ELISA) for detection of IL-2 and IFN-y Human T cells (1x10 5 /well) are incubated in the presence or absence of ILG at different 7 concentrations. For stimulated with PMA plus inomycin, the cells are pretreated with TLG for 2 h at different concentrations, and then stimulated with 20 ng/ml PMA plus 1 [iM ionomycin for another 48 h. Finally, the culture supernatants are re-collected, and then concentration of TL-2 and IFN-y in the supernatants is evaluated by ELISA method according to the manufacturer's instructions. Results As illustrated in FIG. 4, TLG significantly and dose-dependently suppresses both of two cytokines expression which can be highly induced by P/I in the T cell. Example 4 This example shows the effect of TLG on human T cell cycle progression mediated by P/I. Procedure - Flow cytometry assay for detection of cell cycle analysis Human T lymphocytes (10 6 /well) are pre-treated with TLG for 2 h followed by stimulated with or without PMA (20 ng/ml) plus ionomycin (1 M) for 72 h. After collected, washed by PBS and fixed by 70% ethanol, the cells are stained by PI (Propidium Iodide, BD Pharmingen,San Diego, USA) for 30 min at room temperature, and then the cell cycle is analyzed by flow cytometer. Results As shown in FIG. 5, cycling of P/I-mediated cells was progressing from GO/GI to S and G2 M phase, while it was blocked at the GO/GI phase by TLG at 25 pM. Example 5 This example shows the effect of ILG on CD69, CD25 and CD71 expressions on human T cells surface. Flow cytometry assay is used for detection of CD69, CD25 and CD71 expression Human T cells (1x 10 6 /well) are pre-treated with TLG for 2 h, followed by stimulated with PMA (20 ng/ml) plus ionomycin (1 [M). To determine the expression of CD69, the cells are stimulated with PMA plus ionomycin for 24 h; to evaluate the expressions of CD25 and 8 CD71, the cells are incubated with stimulators for 48 h. After collected, the cells are stained with indicated antibodies, and incubated for 30 min at room temperature avoiding from light, and then fixed with 4% paraformaldehyde (PFA). On the next day, samples are analyzed on FACS Calibur Flow Cytometer using CellQuest software. The separate tubes of cells stained with single-color antibodies for each of the flourochromes are severed as the compensation standards. Results As shown in FIG. 6, FIG. 7 and FIG. 8, the expression of CD69, CD25 and CD71 is produced to 56.7%, 36.8% and 51.3% on the human T cells stimulated by P/I, while TLG reduced expression of CD69 and CD25 to 1 2

.

3 % and 5

.

4 %, respectively. Interestingly, we noticed that ILG gave the opposite effect on CD71, and it slightly up-regulated CD71 expression from 5 1.

3 % to 5 7

.

2 % Example 6 This example demonstrates the effect of TLG on IKK--NF-fB signaling pathway Intercellular NF-fB expression was analyzed according to our previous method(18). In brief, the cells were pretreated by TLG for 2 h and incubated with PMA plus ionomycin before fixed and permeabilized by cytofix buffer and methanol, respectively. Subsequently, the cells were harvested and washed by PBS for three times, followed by incubated with the buffer containing NF-fB antibody for 60 min in the dark. Finally, the cells were washed by stain buffer and analyzed by flow cytometer. To evaluate the phosphorylation form of IxBa, the human T lymphocytes (4x 10 6 /well) were pre-treated by ILG at different concentrations with 100 [tg/ml N-acetyl-leucyl-leucyl norleucinal (ALLN) (Calbiochem, USA) for 60 min, and the cells were then incubated with PMA (20 ng/ml) plus ionomycin (1 [M) for another 60 min. For determination of IKB-a, P IKKa/Q, P-p65 and f-actin from whole cellular proteins, the human T lymphocytes (4x 10 6 /well) were stimulated with PMA plus ionomycin for 1 h after pre-incubated with different concentrations of ILG for 120 min. The T lymphocytes were harvested and lyzed with lysis buffer (Sigma, USA) with 1 xprotease inhibitor mix (Roche, USA) to prepare the whole cellular lysates. The whole cellular proteins were then subjected to electrophoresis in 10% SDS/PAGE and to immunoblotting according to the previous method(18). 9 Results The results were shown in Figure 9 & 10, it indicated that P/I stimulation could elevate the expression level of NF-fB in T cell nuclear, and ILG obviously and dose-dependently suppressed the nucleus expression of NF-fB, and IKBc phosphorylation and degradation as well. Furthermore, the results manifested that ILG treatment completely abolished IKKa/p phosphorylation and phospho-Ser 5 36 p65 levels in the human T cells (Fig. 11). Example 7 This example demonstrates the effect of ILG on IKK-3 wild type (wt) activity. TKK kinase assay for detection of the inhibitory effect of ILG on activity of IKK-p wt IKB-a substrate provided by Enzo life science, Flag-IKK- recombinant protein, and ATP are incubated with or without TLG at 30'C for 30 min. The mixture was analyzed by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), and then electro-transferred onto nitrocellulose membranes. The nitrocellulose membranes are incubated with P-IxBa (Ser32/36) for overnight at 4'C after blocked by 5% dried milk for 60 min. Next day, the membranes are further incubated with HRP-conjugated secondary antibodies for 60 min, and developed using ECL Western Blotting Detection Reagents (Life Technologies). Results As shown in FIG 12 the substrate of IKK-p, phosphorylation form of IKBa, is strongly suppressed by ILG than dihydromyricetin (DMY), the reference compound, suggesting that ILG inhibits IKBa-NF-KB signaling of human T lymphocytes via regulating IKK-3 activity. Example 8 This example demonstrates the molecular target of ILG on wild-type and mutant IKK-p of the present invention based on computational docking. Computational docking for prediction of the molecular target of ILG The initial 3D structure of TLG is built using the Molecule Builder module incorporated in MOE software. The structure was then subjected to energy minimization and partial charges calculation with Amber99 force field. The crystal structure of wild-type inhibitor of KB 10 kinase 0 (IKK-) was retrieved from Protein Data Bank (PDB ID code 3RZF (6)). On the basis of the wild type protein structure, the structure of IKK-3 with C46A mutant is obtained by performing single point mutation with Rotamer Explorer in MOE software. To prepare the protein for molecular docking, the protein structure is subjected to partial charges calculation and energy minimization with Amber99 force field. Energy minimization was terminated when the root mean square gradient falls below 0.05kcal/(mol- A). The prepared proteins and ligand are introduced for molecular docking. The docking site is identified by using Site Finder in MOE software. The identified binding site including residue Cys46 is chosen as the binding site for molecular docking according to our experiment. In molecular docking, the Triangle Matcher placement method and London dG scoring function were used. A total of 30 docking poses were generated for the ligand and the pose with the best binding mode was selected for further analysis. Results As shown in FIG. 13, the hydrophobic groups of ILG form hydrophobic interactions with the side chain of Phe26, Gly27, Val29, Cys46, Leu50, Leul78, Cys179, and Thr180 in the wild type IKK-. The polar groups of ILG exhibit polar interactions with the side chain of Arg47, Gln48, Glu49, Lys53, Asn54, Arg57, Argl44, Glul77, and Glul81 (FIG. 13A). In addition, ILG forms hydrogen bonds with the backbone of Gln48 and the side chain of Lys53, Arg57, and Leul78. In the C46A mutant IKK-,, the binding mode of ILG is changed due to the C46A mutation of the protein. The hydrophobic groups of ILG form hydrophobic interactions with the side chain of Phe26, Gly27, Val29, Ala46, Leu50, Leul78, Cys179, and Thr180 in the mutant IKK-P. The polar groups of ILG exhibit polar interactions with the side chain of Arg47, Gln48, Glu49, Lys53, Asn54, Arg57, Argl44, Glul77, and Glul8l. In addition, ILG forms hydrogen bonds with the backbone of Gln48 and Glul77 and the side chain of Arg57, and Leul78 (FIG. 13B). Collectively, the molecular docking results suggest that IKKp Cys 46 is likely the molecular target of ILG. Example 9 This example further demonstrates Cys46 is the binding site of ILG on IKK-p. 11 Competitive binding assay Anti-FLAG precipitates from BEK 293 expressing FLAG-IKKO are incubated with ILG or DMY for 1h and then 100 [tM DMY-biotin is added. Subsequently, SDS Loading dye is added to the above mixture, and the proteins are separated by SDS-PAGE and transferred to nitro-cellulose membranes. After blocking with BSA and washing with PBST, the membranes are incubated with streptavidin horseradish peroxidase for 1h and developed with enhanced chemiluminescence. Finally, the membranes are incubated with anti-flag antibody to evaluate the expression of Flag-IKKO. Transfection FLAG-IKKO C46A to HEK293 cell line The transfection assay is preformed according to the manufacturer's instruction of lipofectamine LTX (invitrogen, USA). In brief, HEK293 cells were seed in 1.5 ml of DMEM growth media plus 10% FBS at 5x10 5 cells per well. Five hundred 1d Opti-MEM Reduced Serum Media containing 1.25 [tg of DNA is added to the cells to be transfected, and then 1.25 1d of PLUS is added into the above diluted Opti-MEM:DNA solution, gently mixed and incubated for another 5 min at room temperature. Subsequently, lipofectamine LTXTM Reagent is added into the above solution, and then mixed gently and incubated 30 minutes at room temperature to form DNA-lipofectamine LTX Reagent complexes. Finally, 500 1d of the DNA-lipofectamine LTX Reagent complexes is directly added to each well containing cells and mixed gently. The cells are incubated at 37 0 C in a CO 2 incubator for 24 h. IKK-0 kinase assay for detection of the effect of ILG on the activity of IKK-p C46A IKB-a substrate provided by Enzo life science, Flag-IKK- recombinant protein, and ATP are incubated with or without ILG at 30'C for 30 min. The mixture is analyzed by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), and then electro-transferred onto nitrocellulose membranes. The nitrocellulose membranes are incubated with P-IxBa (Ser32/36) for overnight at 4'C after blocked by 5% dried milk for 60 min. Next day, the membranes are further incubated with HIRP-conjugated secondary antibodies for 60 min, and developed using ECL Western Blotting Detection Reagents (Life Technologies). Results 12 Biotinylated-DMY (biotin-DMY) is utilized as a probe to explore the direct interaction between DMY and IKK-3 protein in this example. Both biotinylated-DMY and DMY are found to share the same binding site on IKKp Cys46. Considering the virtual computational docking results of TLG on IKKP (FIG. 13A and B), this probe is utilized to conduct the competition assay. As shown in FIG. 14, the signal of DMY-biotin could be easily found, while the DMY-biotin signal is significantly reduced by ILG, and more TLG less DMY-biotin signal, suggesting that the binding sites of TLG and DMY-biotin are overlapped, and Cys-46 probably is the binding site of TLG on IKKp. As shown in FIG. 15, In vitro IKKp kinase assay explored that IKKp C46A has not shown the response to the effect of ILG and DMY (Figure 5B). On the contrary, berberine, which is reported to target on IKKP Cys-179, could suppress the IKKP C46A activity. The results further validated the speculation that Cys-46 is the target of ILG and DMY, while not the target of berberine. Example 10 This example shows the inhibitory effect of ILG on IKKp3C 4 6 A transgenic mice. Delay-type hypersensitivity (DTH) animal model is used for detection of the inhibitory effect on IKKP C 4 6 A transgenic mice IKKP C 4 6 A transgenic mice and IKKPwt mice are sensitized through topical application onto their shaved abdomens of 20 tl of a 0.5% DNFB in 4 : 1 acetone/olive oil mixture on days 0 and 1(sensitization phase). Five days after sensitization, the mice are challenged on day 6 through application of 20 tl of a 0.5% DNFB in 4 : 1 acetone/olive oil mixture to the left inner and outer surfaces of the mice(elicitation phase). Ear thickness measurements of both the treatment and control/blank groups are taken with an electronic digital caliper at 24, 48 and 72 h after challenge and the response quantitated as the difference in the thickness of the challenged ear. And the weight of ear patches (6 mm punches) were measured 72 h post challenge. Results Immune-suppressive effect of TLG is less potent in homozygous IKK C 4 6 A mutant mice than IKK3 wt mice, as shown in Table 1. The reference compound, dexamethasone, could 13 significantly suppress edema (FIG. 16) and simultaneously induce thymus atrophy. Interestingly, in the present invention, TLG can slightly promote thymus weight in both IKK 0 wt and IKKpC46A mutant mice (FIG. 17). 24h 48h 72h Group n ATS. E. M. % I AT S. E. M. %I AT S. E. M. % Modelwt 10 209.0±10.16 256.0±16.28 315.0+12.22 ILG 0.75 mg/earwt 10 141.0+6.403*** 32.5 180.0+ 15.09** 29.7 253.3±19.65 19.6 DEX 0.025 10 153.0±6.333*** 26.8 165.6+ 17.41** 35.3 157.1 30.45*** 50.1 mg/ear wt ModelIKK$C 4 6 A 10 219.0+13.37 273.0+20.71 331.0+10.48 ILG 0.75 10 206.0+11.08 5.9 266.0+ 14.92 2.6 309.0+15.6 6.6 mg/earIKK@C46A mg/ea I. C 46 A 10 140.0+8.692*** 36.1 150.0+ 13.82*** 45.1 153.0 20.82*** 53.8 Table 1. The effect of ISO on ear edema of DTH mice induced by DNFB (72h treatment) The above results confirm that Cys-46 of IKK3 is the molecular target of TLG to mediate suppressive effect on T cell activation in vivo and in vitro. The exemplary embodiments of the present invention are thus fully described. Although the description referred to particular embodiments, it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details. Hence this invention should not be construed as limited to the embodiments set forth herein. CONCLUSION In summary, Isoliquiritigenin is shown to be a potent agent to inhibit immune-suppressive effect mediated by T lymphocyte activation which is associated with both wild-type and mutant IKK-P expression. ILG effectively inhibits T cells proliferation and division, suppresses expression of CD69 and CD 25 as well as up-regulates CD71 in a dose-dependent manner. The identification of IKK-p C46 as the target of ILG to exert suppressive effect on human T cell activation in the present invention has significant potency to be served as a leader compound to develop immunomodulatory agents in the future. 14

Claims (5)

1. A method for treating autoimmune diseases mediated at least in part by T lymphocyte activation comprising administering isoliquiritigenin to a subject in need thereof with said autoimmune diseases in a dose-dependent manner.

2. The method of claim 1, wherein said method further comprises: (i) contacting said isoliquiritigenin to T cells expressing IBa kinase in the subj ect; and (ii) targeting cysteine-46 residue of the IBa kinase in the T cells, wherein the (ii) targeting cysteine-46 residue of the IBa kinase in the T cells further inhibits one or more of the following activities in relation to the T lymphocyte activation: (a) T lymphocyte proliferation; (b) T lymphocyte division; (c) T lymphocyte cycle progression; (d) IL-2 and IFN-y expression of activated T lymphocytes; and/or (e) CD69 and CD25 expression; and up-regulates CD71 expression on T lymphocytes such that said isoliquiritigenin effectively inhibits T lymphocyte activation which mediates in part the autoimmune diseases.

3. A method of inhibiting human T lymphocyte activation associated with autoimmune diseases in a dose-dependent manner comprising: (i) contacting isoliquiritigenin to T cells expressing IBa kinase; and (ii) targeting cysteine-46 residue of the IBa kinase in the T cells, wherein the (ii) targeting cysteine-46 residue of the IBa kinase further mediates one or more of the following activities in relation to the T lymphocyte activation: (a) T lymphocyte proliferation; (b) T lymphocyte division; (c) T lymphocyte cycle progression; (d) IL-2 and IFN-y expression of activated T lymphocytes; (e) CD69 and CD25 expression; and/or (f) CD71 expression.

4. A method of screening a therapeutic agent as a drug candidate for treating autoimmune diseases comprising: 15 (a) exposing said agent to an assay comprising IBa kinase; (b) detecting whether said agent binds to cysteine-46 residue of IKBa kinase; (c) detecting whether said agent inhibits kinase activity of IKBa kinase and thereby inhibiting T lymphocyte activation by contacting said agent to T cells upon said binding in step (b); and (d) identifying a drug candidate that performs said binding action of step (b) and said inhibition action of step (c), wherein said inhibiting T lymphocyte activation by contacting said agent in (c) is verified by one or more of the following indications: (e) whether T lymphocyte proliferation is inhibited; (f) whether T lymphocyte division is inhibited; (g) whether T lymphocyte cycle progression is inhibited; (h) whether IL-2 and IFN-y expression of activated T lymphocytes is inhibited; and/or (i) whether CD69 and CD25 expression are down-regulated, and whether CD71 expression are up-regulated on T lymphocytes.

5. The method of any one of claims I to 4, wherein said autoimmune diseases comprises Rheumatoid Arthirits, Graves' Disease, Diabetes Mellitus Type 1, Polymyositis and Dermatomyositis, and the dose of said isoliquiritigenin or therapeutic agent being administered or contacted to said subject or the T cells thereof is from 5 [tM to 25 [M. 16