WO2014120790A1 - Petites molécules pour bloquer la voie de signalisation - Google Patents

Petites molécules pour bloquer la voie de signalisation Download PDF

Info

Publication number
WO2014120790A1
WO2014120790A1 PCT/US2014/013627 US2014013627W WO2014120790A1 WO 2014120790 A1 WO2014120790 A1 WO 2014120790A1 US 2014013627 W US2014013627 W US 2014013627W WO 2014120790 A1 WO2014120790 A1 WO 2014120790A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
cells
group
orail
cell
Prior art date
Application number
PCT/US2014/013627
Other languages
English (en)
Inventor
Yousang Gwack
Sonal SRIKANTH
Kyun-do KIM
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO2014120790A1 publication Critical patent/WO2014120790A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Definitions

  • CRAC Ca 2+ -Release-Activated-Ca 2+ channels
  • Ca 2+ entry via CRAC channels triggers the downstream signaling cascades such as calcineurin and a transcription factor, NFAT to turn on the transcription event such as cytokine production in immune cells.
  • activation of the CRAC channel activity is a seminal event in the etiology of autoimmune diseases and blocking this activity can be used to treat undesired autoimmune responses such as those arising with autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, type I diabetes, and inflammatory bowel disease as well as with organ transplantation.
  • CRAC channel-calcineurin-NFAT pathway has been emphasized by the common use of cyclosporin A and FK506 as immunosuppressant that block the calcineurin activity.
  • calcineurin is ubiquitously important for most of cell types, application of cyclosporin A and FK506 can have a broad side effect.
  • CRAC channels are made of homomultimers of Orail and current specifically carried by these homomultimers is detected predominantly only in the immune cells. Hence a specific blocker of CRAC channels is unlikely to have the same negative side effects observed with cyclosporine A and FK506.
  • R is selected from the group consisting of aryl, heteroaryl, substituted aryl, and substituted heteroaryl;
  • R J and R 2 are independently selected from the group consisting of hydrogen and CX 3 or R and R 2 together form a carbonyl or thiocarbonyl;
  • each X is selected from the group consisting of CI, F and Br;
  • L is selected from the group consisting of a covalent bond and Ci-C 6 alkylene
  • R 3 is selected from the group consisting of aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl and substituted heteroaryl;
  • IC 50 of the compound is less than 10 ⁇ .
  • the pharmaceutically acceptable salt thereof is also intended.
  • a method for suppressing an immune response in a mammal in need thereof comprising administering to the mammal a compound of Formula I in an amount sufficient to suppress said immune response.
  • Another aspect of this invention relates to a method for modulating calcium transport through the CRAC channel so as to modulate the immune response of an immune cell by contacting said immune cell with a therapeutically effective amount of a compound of Formula I so as to modulate the immune response. The contacting can be in vitro or in vivo.
  • pharmaceutical compositions useful for suppressing an immune response comprising a compound of Formula I as described herein.
  • FIG. la-c depict the immunosuppressive activity of the compound N-[2,2,2- trichloro- 1 -(2 -naphthylamino)ethyl]-2-furamide (compound 5D).
  • FIG. la shows the IC50 value of the compound N-[2,2,2-trichloro-l-(2-naphthylamino)ethyl]-2-furamide to block Ca2+ entry in T cells as 195 nM.
  • FIG. lb demonstrates that N-[2,2,2-trichloro-l-(2- naphthylamino)ethyl]-2-furamide blocks the cytokine production of T cells and T cell activation/proliferation in FIG. lc.
  • FIG. 2a-c depict development of high throughput screening system using NFAT translocation as readout.
  • HeLa-OSN cells are a cell-line that harbors amplified CRAC currents by overexpression of Orail and STIM1, the components of CRAC channels (a). Comparing plain HeLa cells, these cells show much higher Ca2+ entry levels mediated by CRAC channels. In these cells, also we stably expressed NFAT1-460-GFP to use as readout for the screen.
  • NFAT is a transcription factor that is activated by CRAC channels and translocates from the cytoplasm into the nuclei (b). By measuring NFAT accumulation in the nuclei, we can monitor CRAC channel activity.
  • the known CRAC channel blocker, 2-APB was used as a positive control for the screen.
  • the calculated Z' factor was 0.692 meaning that our design was suitable for high throughput screen (c).
  • FIG. 3 depicts compounds identified in the chemical library screen.
  • FIG. 4 shows the structure of N-[2,2,2-trichloro-l-(l-naphthylamino)ethyl]-2- thiophenecarboxamide and analogues that were tested.
  • FIG. 5a-b demonstrate that the compound 5d can block CRAC current by electrophysiology.
  • the graphs are plotted as current- voltage relation (a) and versus time (b).
  • FIG. 6a-e demonstrate the therapeutic potential of the compound 5d and 5j-4 using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) was further tested.
  • EAE experimental autoimmune encephalomyelitis
  • the disease can be induced by T cell attack of central nervous system (e.g. brain and spinal cord) to mimic multiple sclerosis.
  • N- ⁇ [(6-hydroxy- l-naphthyl)amino]carbonothioyl ⁇ -2-furamide (compound 5J-4) was tested and it was found to have a better suppressive effect on the onset and severity of the diseases considering the reduced disease severity (c) and the infiltrated T cell number to the central nervous system (d and e).
  • the infiltrated T cell number was determined after isolation of mononuclear cells from the spinal cord and the frequency of CD4+ T cells (helper T cells) among the isolated cells was determined by surface staining of CD4 and analysis by flow cytometry.
  • FIG. 7a-c show the development of a high throughput system to screen chemical libraries
  • HeLa cells were retrovirally transduced with vectors encoding Orail and STIM1 (HeLa-O+S) and examined for SOCE after intracellular store depletion with thapsigargin (1 ⁇ ).
  • Orail -mediated SOCE was inhibited by 2-APB (100 ⁇ ). The influx rate, peak, and sustained levels of intracellular Ca 2+ concentrations were calculated,
  • FIG. 8a-f show the identification of compound 5D as a small molecule blocker of CRAC channels.
  • HeLa cells stably expressing Orail, STIM1, and NFATc2 (l-460)-GFP were left untreated (-TG) or treated with thapsigargin (+TG, 1 ⁇ , 30 min) with and without 2-APB. These cells were analyzed by microscopy for nuclear NFAT-GFP (left) or immunoblotting to examine dephosphorylation of NFAT with anti-GFP antibody (right).
  • Orail contains four transmembrane segments (TM1-TM4) with its N- and C-termini facing the cytoplasm, and two extracellular loops (ECl and EC2). Residues L95, VI 02 and El 06 in TM1 directly line the ion permeation pathway and VI 02 has been proposed to form the gate of the CRAC channel (marked in red).
  • the D 110 xD 112 xD 114 motif in the ECl is important for ion selectivity of the channel and mutation of W176 in TM3 makes the channel constitutively open, (f) Compound 5D-mediated block of SOCE induced by various mutants of Orail .
  • Residues deleted in the second extracellular loop (AEC) are described in Methods. Data represent average ⁇ s.e.m from 25-35 fibroblasts and are normalized to the block of wild-type Orail .
  • FIG. 9 shows structural analogues of compound 5 used in this study. Chemical structures of analogues of compound 5 examined for their potency in blocking SOCE. The structures of known CRAC channel blockers, 2-APB and SFK96365 are also presented in a dotted box. Boxed areas highlight divergent side chains.
  • FIG. lOa-g are the characterization of the inhibitory activity of compound 5D on CRAC channels, (a) Comparison of the inhibitory effects of compound 5 (10 ⁇ ) and 5D (1 ⁇ ) on CRAC channel activity in HeLa-OSN cells, (b) Measurement of nuclear translocation of NFAT in HeLa-OSN cells left untreated (-TG) or treated with thapsigargin (+TG, ⁇ , 30 min) in the presence of 100 ⁇ 2-APB or 10 ⁇ compound 5D.
  • Compound 5D blocks currents generated by Orail wl76C . Measurement of currents from HEK293 cells expressing Orail wl76C (in the absence of STIM1). I-Vs were plotted in the absence (black trace), presence (red trace) or after washing out (green trace) of 10 ⁇ compound 5D. Each trace is representative of data obtained from six different cells, (f) Compound 5D marginally blocks currents generated by Orail vl02C .
  • FIG. lla-c demonstrate that compound 5D inhibits SOCE and cytokine production in effector T cells,
  • Na ' ive T cells cultured under TnlV-polarizing conditions and re-stimulated with PMA and ionomycin with different concentrations of compound 5D or DMSO (vehicle) were examined for IL-17A and IFN- ⁇ production,
  • Compound 5D-mediated inhibition of cytokine production by T H 1, T H 2, and T H 17 cells Na ' ive T cells cultured under ⁇ ⁇ 1-, T H 2-, or T H 17-polarizing conditions were stimulated with PMA and ionomycin after 4 days in the presence of different concentrations of compound 5D and examined for cytokine expression using intracellular staining. Data were normalized to cytokine levels of cells treated with DMSO.
  • FIG. 12a-c show that inhibition of CRAC channels reduces cytokine production and differentiation of effector T cells
  • Compound 5D affects T cell differentiation. Na ' ive T cells differentiated under T H 1, T H 2, and T H 17-polarizing conditions in the absence or presence of compound 5D (15 ⁇ ) for four days were re-stimulated with PMA and ionomycin for 6 hrs (without compound 5D) and stained for indicated cytokines, (b)
  • Compound 5D-mediated reduction in the expression of cytokines and transcription factors in T cells Na ' ive T cells differentiated under T H 1, T H 2, and T H 17-polarizing conditions with compound 5D were washed, restimulated with PMA plus ionomycin, and harvested for examination of the mRNA expression levels of IFN- ⁇ (T H 1 cells), IL-4 (T H 2 cells), and IL- 17A (T H 17 cells) (left panel). In addition, the mRNA levels of T-bet, GATA-3, RORyt, and RORa were measured using quantitative PCR under T H 1, T H 2, and T H 17-polarizing conditions, respectively (right panel). *P ⁇ 0.05, ***P ⁇ 0.0005.
  • 13a-c show the expression levels of molecules involved in T H 17 pathogenicity and differentiation in compound 5D-treated T cells
  • FIG. 14a-d show that Ca 2+ signalling mediated by Orail is important for NFAT- mediated expression of RORa and RORyt in T R 17 cells,
  • Na ' ive T cells cultured under T H 17-polarizing conditions with compound 5D were transduced with retroviruses encoding RORa or RORyt, and examined for IL-17A production on day 4.
  • Line graph on the right shows average ⁇ s.e.m of IL-17A + population from four independent experiments,
  • CsA Cyclosporin A
  • Na ' ive T cells were cultured under T H 17-polarizing conditions with 5 nM cyclosporine A (CsA) for four days, washed, restimulated with PMA and ionomycin, and examined for production of IL-17A (top) and mRNA levels of RORa or RORyt (bottom). Data are representative of three independent experiments. Transcript analysis shows average ⁇ s.d.m. (c) Expression of constitutively active NFAT rescues compound 5D-mediated inhibition of T R 17
  • Na ' ive T cells cultured under T H 17-polarizing conditions with different concentrations of compound 5D were transduced with retroviruses encoding constitutively active NFATc2 (CA-NFAT), and examined for IL-17A production and RORyt expression on day 4 using intracellular staining (left two panels). The same cells were also used for mRNA expression analysis of RORa or RORyt using quantitative PCR (right two panels), (d) ChlP-PCR analysis of NFAT recruitment and acetylation of histone H3K9/14 at the promoters of RORa or RORyt.
  • CA-NFAT constitutively active NFATc2
  • RORap and RORytp Real-time PCR quantification of RORa and RORyt promoters (RORap and RORytp) sequences after ChIP with antibody to NFATc2 (left two panels) and acetylated H3K9 and H3K14 (H3K9/14Ac, right two panels) in DMSO and compound 5D- treated cells after stimulation with anti-CD3 and anti-CD28 antibodies for 16 hours under TnlV-polarizing conditions. Data were normalized to the mean ChIP recovery of all experiments. *P ⁇ 0.05, **P ⁇ 0.005, ***P ⁇ 0.0005.
  • FIG. 15a-e demonstrate that Orail "7" T cells show a defect in T R 17 differentiation similar to that observed in compound 5D-treated cells, (a) Orail "7" T cells show a defect in IL- 17 production.
  • Naive CD4 + T Cells were stimulated and cultured under T H 17-polarizing conditions, restimulated with PMA and ionomycin after four days, and examined for IL-17A expression, (b) Real-time PCR quantification of the mRNA levels of RORa and RORyt in control and Orail "7" T cells after stimulation with anti-CD3 and anti-CD28 antibodies under T H l7-polarizing conditions for four days, (c) The mRNA expression analyses in Orail "7" T cells cultured under T H 17-polarizing conditions.
  • WT and Orail "7" na ' ive T cells cultured for four days were stimulated with PMA and ionomycin, and analyzed by quantitative RT-PCR for expression of indicated genes,
  • WT and Orail "7" na ' ive T cells differentiated under T H 17-polarizing conditions were transduced with retroviruses encoding CA-NFAT, and examined for mRNA expression of RORa or RORyt (top panels) and IL-17A production (lower panels),
  • ChlP-PCR analysis of NFAT recruitment into the promoters of RORa or RORyt were assessed for mRNA expression of RORa or RORyt.
  • RORap and RORytp Real-time PCR quantification of RORa and RORyt promoter sequences after ChIP recovery with antibody to NFATc2 in control and Orail "7" T cells left unstimulated (na ' ive) or after stimulation with anti-CD3 and anti-CD28 antibodies for 16 hours under T H l7-polarizing conditions. Data were normalized to the mean ChIP recovery of all experiments. **P ⁇ 0.005, ***P ⁇ 0.0005.
  • FIG. 16a-b show the chemical structures of compound 5D analogues and their blocking efficacy on SOCE.
  • FIG. 17a-f show that a structural analogue of compound 5D, 5J-4 ameliorates T R 17- mediated autoimmune disease,
  • Compound 5 J-4 decreases MOG 35 - 55 peptide-responding T cells in the draining lymph nodes after immunization.
  • Cells were isolated from the draining lymph nodes (dLNs) of control and compound 5 J-4-treated mice after 14 days of immunization and MOG 35 - 55 peptide-responding T cells were estimated by thymidine incorporation after incubation with the peptide.
  • the graph shows average ⁇ s.d.m. from triplicates, (e) Compound 5J-4 treatment decreases T R 17 differentiation in vivo.
  • FIG. 19 shows the induction of regulatory T cells by Orail deficiency.
  • Na ' ive CD4 + CD25 ⁇ T cells were purified and stimulated in the indicated conditions, non-skewing, TGF- ⁇ , or TGF- ⁇ with different concentrations of IL-6. After four days of stimulation, Foxp3 expression was measured by intracellular staining in control and Orail -deficient T cells.
  • FIG. 20 shows the induction of regulatory T cells by treatment of a small molecule blocker of Orail, SKF96365. Na ' ive CD4 CD25 T cells were purified and stimulated in the indicated conditions with different concentrations of TGF- ⁇ .
  • FIG. 21 shows the increased natural regulatory T cells in Orail-null mice.
  • Cells from the thymus, lymph nodes, and spleen of control and Orail-null mice were isolated and stained for CD4, CD25, and Foxp3 to determine the population of regulatory T cells. The cells were gated for CD4 + populations.
  • FIG. 22 (right and left panels) show the reduced autoimmune disease symptoms of SKF96365 -treated mice. The EAE symptoms of control and SKF96365 -treated mice were compared (left). SKF96365 was treated in an alternate day between day 5 and 20.
  • SKF96365-treted mice were tolerant for further repetitive immunization to induce autoimmune disease (data not shown).
  • a single injection of SKF96365 at day 5 and one day earlier of immunization (day-1) dramatically ameliorate the symptoms of EAE (right).
  • compositions comprising of the compounds and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the compounds or methods.
  • Consisting of shall mean excluding more than trace elements of other ingredients for claimed compounds and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this invention. Accordingly, it is intended that the processes and compositions can include additional steps and components (comprising) or alternatively include additional steps and compounds of no significance (consisting essentially of) or alternatively, intending only the stated methods steps or compounds (consisting of).
  • Mammals include, but are not limited to, murines, rats, rabbits, simians, bovines, ovines, porcines, canines, felines, farm animals, sport animals, pets, equines, and primates, particularly humans.
  • Subjects include, but are not limited to, mammals, patients, laboratory animals, and the like.
  • treating or “treatment” of a disease, disorder, symptom or condition will depend on the disease, disorder, symptom or condition to be treated, and the mammal to be treated.
  • treatment intends one or more of inhibiting the progression of the manifested disease, disorder, symptom or condition as measured by clinical or subclinical parameters (where the term “inhibiting” or “inhibition” is intended to be a subset of “treating” or “treatment”), arresting the development of the disease, disorder, symptom or condition as measured by clinical or sub-clinical parameters, ameliorating or causing regression of the disease, disorder, symptom or condition as measured by clinical or subclinical parameters, or reducing pain or discomfort for the mammal treated as measured by clinical and/or pharmacological parameters.
  • inhibitor As used herein, “inhibit,” “inhibiting,” “reduce” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result.
  • a "therapeutically effective amount” or an “effective amount” is used synonymously with and intends an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications, or dosages.
  • the term in vitro administration refers to manipulations performed on cells removed from or outside of a subject, including, but not limited to cells in culture.
  • in vivo administration includes all manipulations performed within a subject, including administrations.
  • Aryl or refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring ⁇ e.g., phenyl) or multiple condensed rings ⁇ e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic ⁇ e.g., 2-benzoxazolinone, 2H-l,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the point of attachment is at an aromatic carbon atom.
  • Preferred aryl groups include phenyl and naphthyl.
  • Substituted aryl refers to aryl groups which are substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, amino sulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano
  • heterocyclyloxy heterocyclylthio, substituted heterocyclylthio, nitro, S0 3 H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein.
  • heteroaryl refers to a monovalent, aromatic ring having 6-14 ring carbon atoms and 1-6 ring heteroatoms selected preferably from N, O, S, and P.
  • Nonlimiting examples of heteroaryl include furan, imidazole, pyridine, quinoline, and the like.
  • Substituted heteroaryl refers to heteroaryl groups that are substituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of the same group of substituents defined for substituted aryl.
  • Alkylene refers to a linear or branched saturated divalent hydrocarbon radical.
  • Ci-C6-alkylene means a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of three to six carbon atoms, e.g. methylene, ethylene, 2,2-dimethylethylene, n-propylene, 2-methylpropylene, 1- methyl-ethylene, 2-methyl-ethylene and the like.
  • Cycloalkyl refers to a divalent cyclic or polycyclic alkyl group containing 3 to 15 carbon atoms.
  • Substituted cycloalkyl refers to a cycloalkyl group substituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of the same group of substituents defined for substituted aryl.
  • “Pharmaceutical composition” refers to a composition comprising an active ingredient and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile, e.g., for injectibles.
  • carrier refers to a glidant, diluent, adjuvant, excipient, or vehicle with which the compound is administered. Examples of carriers are described herein and also in “Remington's Pharmaceutical Sciences” by E.W. Martin.
  • diluent refers to chemical compounds that are used to dilute the compound of interest prior to delivery. Diluents can also serve to stabilize compounds. Non- limiting examples of diluents include starch, saccharides, disaccharides, sucrose, lactose, polysaccharides, cellulose, cellulose ethers, hydroxypropyl cellulose, sugar alcohols, xylitol, sorbitol, maltitol, microcrystalline cellulose, calcium or sodium carbonate, lactose, lactose monohydrate, dicalcium phosphate, cellulose, compressible sugars, dibasic calcium phosphate dehydrate, mannitol, microcrystalline cellulose, and tribasic calcium phosphate.
  • binder when used herein relates to any pharmaceutically acceptable film which can be used to bind together the active and inert components of the carrier together to maintain cohesive and discrete portions.
  • binders include hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone, copovidone, and ethyl cellulose.
  • disintegrant refers to a substance which, upon addition to a solid preparation, facilitates its break-up or disintegration after administration and permits the release of an active ingredient as efficiently as possible to allow for its rapid dissolution.
  • disintegrants include maize starch, sodium starch glycolate, croscarmellose sodium, crospovidone, microcrystalline cellulose, modified corn starch, sodium carboxymethyl starch, povidone, pregelatinized starch, and alginic acid.
  • lubricant refers to an excipient which is added to a powder blend to prevent the compacted powder mass from sticking to the equipment during the tabletting or encapsulation process. It aids the ejection of the tablet form the dies, and can improve powder flow.
  • lubricants include magnesium stearate, stearic acid, silica, fats, calcium stearate, polyethylene glycol, sodium stearyl fumarate, or talc; and solubilizers such as fatty acids including lauric acid, oleic acid, and C 8 /Ci 0 fatty acid.
  • glidant as used herein is intended to mean agents used in tablet and capsule formulations to improve flow-properties during tablet compression and to produce an anti-caking effect.
  • Non-limiting examples of glidants include colloidal silicon dioxide, talc, fumed silica, starch, starch derivatives, and bentonite.
  • salts refers to salts that retain the desired biological activity of the above -identified compounds and exhibit minimal or no undesired toxicological effects.
  • examples of such salts include, but are not limited to, salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, methanesulfonic acid, p-toluenesulfonic acid and
  • the compounds can also be administered as pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula— NR+Z— , wherein R is hydrogen, alkyl, or benzyl, and Z is a counterion, including chloride, bromide, iodide, --O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).
  • quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula— NR+Z— , wherein R is hydrogen, alkyl, or benzyl, and Z is a counterion
  • Method aspects of the disclosure relate to a method for suppressing an immune response in a mammal in need thereof comprising administering to the mammal an immunosuppressive amount of a compound of Formula I:
  • R is selected from the group consisting of aryl, heteroaryl, substituted aryl, and substituted heteroaryl;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen and CX 3 or R and R 2 together form a carbonyl or thiocarbonyl;
  • each X is independently selected from the group consisting of F, CI, and Br;
  • L is selected from the group consisting of a covalent bond and Ci-C 6 alkylene
  • R 3 is selected from the group consisting of aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl and substituted heteroaryl and wherein the IC 50 of the compound is less than 10 ⁇ .
  • the IC 50 of the compound refers to the half maximal inhibitory concentration. More specifically, the IC 50 of the compound refers to the half maximal inhibitor concentration required for Compounds of Formula I to block SOCE (store -operated Ca2+ entry) and/or the CRAC channel-calcineurin-NFAT pathway. In certain embodiments, the IC 50 of the compound is about less than 5 ⁇ , about less than 2 ⁇ , about less than 1 ⁇ , about less than 500 nM, or about less than 300 nM.
  • the mammal suffers from an autoimmune disease.
  • Autoimmunity is the failure of an organism to recognize its own constituent parts as self, which allows an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Autoimmune diseases resulting from an immune response against the body's own cells and tissues are well known in the art. In one embodiment, the autoimmune disease is selected from rheumatoid arthritis, multiple sclerosis, type I diabetes, and inflammatory bowel disease.
  • Autoimmune diseases also include, for example, inflammatory hyperproliferative skin diseases, psoriasis, allergic intraocular inflammatory diseases, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (R MS), atherosclerosis, arteriosclerosis, inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adh
  • granulomatosis Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies,
  • the autoimmune disease is multiple sclerosis. In a further embodiment, the autoimmune disease is lupus. [0065] Methods and compositions of this invention are useful for suppressing an immune response in different situations. Therefore, compounds of Formula I can be used as an immunosuppressant in any situation in which suppression of the immune system is desired. In one embodiment, the compound of Formula 1 is administered to a mammal undergoing organ transplantation surgery.
  • a method for modulating calcium transport through the CRAC channel so as to modulate the immune response of an immune cell by contacting (in vitro or in vivo) said immune cell with a compound of Formula I.
  • modulating as used herein can refer to a decrease, increase, reduction, or inhibition of an activity and preferably refers to a decrease or inhibition.
  • An immune cell refers to cells that are active in the immune system and the immune response of a mammal.
  • Immune cells include, for example, leukocytes such as neutrophils, eosinophils, basophils, lymphocyts, monocytes, macrophages, dendritic cells, and more specifically B cells, T cells such as Th cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, gamma delta T cells, plasma B cells, memory B cells, B-l cells, B-2 cells, marginal-zone B cells, and follicular B cells.
  • leukocytes such as neutrophils, eosinophils, basophils, lymphocyts, monocytes, macrophages, dendritic cells, and more specifically B cells
  • T cells such as Th cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, gamma delta T cells, plasma B cells, memory B cells, B-l cells, B-2 cells, marginal-zone B cells, and follicular B cells.
  • the immune cell is a T cell.
  • the activation and/or proliferation of the T cell is reduced.
  • the activation and proliferation of a T cell can be assessed by methods known in the art.
  • One example is a CFSE
  • Kits and protocols to conduct such assays are commercially available (see for e.g. Invitrogen, Cat No. C34554).
  • Some typical immunosuppressants have some level of toxicity due to off-target effects.
  • the term "off-target effect" as used herein describes an undesired, non-specific effect of the administered compound.
  • some immunosuppressants target the CRAC channel-calcineurin-NFAT pathway. While the activities of the CRAC channel can be limited to cells of the immune system, calcineurin is a ubiquitously expressed protein that has important activities in other cell types. Calcineurin is a protein phosphatase also known as protein phosphatase 3, PPP3CA, and calcium-dependent serine-threonine phosphatase, and formerly known as protein phosphatase 2B (PP2B).
  • calcineurin is inhibited by less than about 30%, or by less than about 25% or by less than about 20% of the activity obtained in the absence of a compound of the disclosure.
  • calcineurin is inhibited by less than about 15%, or by less than about 10% of the activity obtained in the absence of a compound of the disclosure. In another embodiment, calcineurin is inhibited by less than about 5% or less than about 1% of the activity obtained in the absence of a compound of the disclosure. In yet another embodiment, there is no detectable inhibition of calcineurin.
  • the inhibition of calcineurin can be determined by methods known in the art. Such methods include those that assess the protein or RNA level of calcineurin in a sample of cells. These can be assessed using standard immunohistochemistry, western blotting, southern blotting, PCR, microarray analysis, or RNA analysis techniques well known in the art. Since the sequence of calcineurin is known, and antibodies are commercially available, these techniques are easily performed by one skilled in the art. The activity and/or inhibition of calcineurin can also be determined by accessing the phosphatase activity of the protein. The protein can be isolated from tissues using standard techniques and a protein phosphatase assay can be performed according to standard protocols.
  • the compound of Formula I specifically inhibits the Orail protein and/or its homologues.
  • the Orail gene encodes for proteins that make up the CRAC.
  • the mammalian Orai family has two additional homo logs, Orai2 and Orai3.
  • Orail is specifically inhibited by the compound of Formula I.
  • compounds of this invention exhibit inhibitory activity towards all Orai homologs. It is also contemplated that inhibitory activity is specific to one homolog and not the others. The inhibition of Orai can be assessed by methods known in the art and as previously stated.
  • the methods of this invention can be used as an adjunct to conventional drug therapy or other therapeutic modalities for immunosuppression, anti- inflammation, or palliative treatments.
  • the therapy can be combined with that of traditional therapies such as hormone replacement, dietary manipulation, steroidal or NSAID anti- inflammatants, TNFa antagonists, the B cell depleting agent rituximab, the anti-IL-6 receptor tocilizumab or the costimulation blocker abatacept.
  • a method for inhibiting Orai activity in a cell by administering to the cell a compound of Formula I or a pharmaceutically acceptable salt thereof in an amount sufficient to inhibit Orai activity.
  • Blocking Orai has been shown to have therapeutic effects in certain diseases. For example, blocking Orai has been shown to inhibit metastasis of cancers in vivo. Accordingly, in one embodiment, inhibition or Orai activity inhibits metastasis in a subject with cancer.
  • Cancers in which Orai has been shown to be important for metastasis include cervical and breast cancer. Therefore, compounds of formula I can be used as therapeutics for the inhibition of metastasis in cervical and breast cancer.
  • aspects of the disclosure relate to methods for screening compounds for their ability to act as immunosuppressants.
  • one aspect relates to a method for identifying a compound that modulates calcium transport through the CRAC channel of a cell comprising contacting a candidate compound with the cell, and assaying and comparing the CRAC channel activity to the activity of the compounds of Formula I.
  • the method comprises identifying a compound that reduces CRAC channel activity.
  • CRAC channel activity can be measured directly or indirectly.
  • store- operated Ca2+ entry (SOCE) can be measured by methods known in the art and methods described in the examples.
  • NFAT nuclear factor of activated T-cells
  • translocation can serve as a readout for CRAC channel activity.
  • a further method aspect relates to a method for identifying a compound that modulates Orail protein function, or its homologue, in a cell comprising contacting a candidate compound with the cell, and assaying for Orail protein activity.
  • the method comprises identifying a compound that reduces Orail protein activity.
  • Orail protein activity can be measured by methods known in the art and by methods described herein.
  • the phrase "modulates Orai protein function" includes modulation at the RNA or protein level. For example, a compound that reduces the transcription of Orail would be considered one that modulates its protein function. Also, compounds that reduce or change the amount of translation of the protein or inhibit the protein directly or indirectly would be considered one that modulates its protein function.
  • R is a heteroaryl. In a related embodiment, R is
  • R 3 is aryl. In a related embodiment, R 3 is naphthyl. In a yet further embodiment, R 3 is selected from the group consisting of 1-naphthyl and 2- naphthyl. In a specific embodiment, R 3 is 2- naphthyl.
  • R is a heteroaryl and R 3 is aryl.
  • R 1 is hydrogen and R 2 is CX 3 .
  • X is CI.
  • L is a covalent bond.
  • R is a heteroryl, R 3 is aryl, and L is a covalent bond.
  • the compound of Formula I is selected from a compound disclosed below in Table 1 or a pharmaceutically acceptable salt thereof:
  • compositions for use in suppressing an immune response comprising a compound of Formula I as described herein and a carrier or excipient.
  • the composition comprises one or more excipients, e.g., talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc. Coloring and flavoring agents may also be added to preparations, particularly to those for oral administration.
  • Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1 ,2-propylene glycol, polyglycols, dimethylsulfoxide, fatty alcohols, triglycerides, partial esters of glycerine and the like.
  • the compound is present in an amount sufficient to suppress the immune response.
  • R is selected from the group consisting of aryl, heteroaryl, substituted aryl, and substituted heteroaryl;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen and CX 3 or R and R 2 together form a carbonyl or thiocarbonyl;
  • each X is selected from the group consisting of CI, F and Br;
  • L is selected from the group consisting of a covalent bond and Ci-C 6 alkylene
  • R 3 is selected from the group consisting of aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl and substituted heteroaryl, wherein the compound is not a compound tabulated below:
  • R is a heteroaryl. In a related embodiment, R is
  • R 3 is aryl. In a related embodiment, R 3 is naphthyl. In yet a further embodiment, R 3 is selected from the group consisting of 1-naphthyl and 2- naphthyl. In another embodiment, R 3 is 2- naphthyl.
  • R 1 is hydrogen and R 2 is CX 3 .
  • X is CI.
  • Another embodiment provides for a pharmaceutical composition comprising a compound described herein and a carrier, e.g., a pharmaceutically acceptable carrier.
  • Solid pharmaceutical excipients include starch, cellulose, hydroxypropyl cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like.
  • Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • the concentration of the excipient is one that can readily be determined to be effective by those skilled in the art, and can vary depending on the particular excipient used.
  • the compounds of Formula I can be administered to the subject using any method and route suitable for delivery of the compounds, including systemic or localized routes. Routes of administration may be combined, if desired, or adjusted depending upon the pharmaceutical composition and/or the desired effect.
  • the compounds of Formula I may be administered using any medically appropriate procedure, e.g., intravascular (intravenous, intraarterial, intracapillary) administration, injection into the cerebrospinal fluid, intracavity or direct injection.
  • intravascular intravenous, intraarterial, intracapillary
  • injection into the cerebrospinal fluid intracavity or direct injection.
  • administration maybe carried out through the use of an Ommaya reservoir, in accordance with known techniques. (F. Balis et al., Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989).
  • administration may involve administering the compound of Formula I to a desired target tissue, such a brain, spine, etc.
  • a desired target tissue such as a brain, spine, etc.
  • the administration may be by injection or by placement of a composition containing the inhibitor in the desired tissue or organ by surgery, for example.
  • an implant such as a cannula implant, that acts to retain the active dose at the site of implantation may be used.
  • systemic, intraperitoneal, intravascular or subcutaneous protocols are employed, e.g., as described in Pardridge WM (2008) Re-engineering biopharmaceuticals for delivery to brain with molecular Trojan horses. Bioconjug Chem. 19: 1327-38.
  • nanoparticle mediated delivery protocols may be employed, e.g., as described in Tosi G, Costantino L, Ruozi B, Forni F, Vandelli MA (2008) Polymeric nanoparticles for the drug delivery to the central nervous system. Expert Opin Drug Deliv. 5:155-74; and Ulbrich K, Hekmatara T, Herbert E, Kreuter J (2008) Transferrin- and transferrin-receptor-antibody- modified nanoparticles enable drug delivery across the blood-brain barrier (BBB). Eur J Pharm Biopharm. 2008 Sep 5. [Epub ahead of print].
  • intracerebral, ventricular or intrathecal delivery protocols may be employed, e.g., as described in Buchli AD and Schwab ME (2005) Inhibition of Nogo: a key strategy to increase regeneration, plasticity and functional recovery of the lesioned central nervous system.
  • the compound may be formulated to cross the blood brain barrier (BBB).
  • BBB blood brain barrier
  • One strategy for drug delivery through the blood brain barrier (BBB) entails disruption of the BBB, either by osmotic means such as mannitol or leukotrienes, or biochemically by the use of vasoactive substances such as bradykinin.
  • osmotic means such as mannitol or leukotrienes
  • vasoactive substances such as bradykinin.
  • a BBB disrupting agent can be co-administered with the compositions disclosed herein when the compositions are administered by intravascular injection.
  • Methods of administration of the compound through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration.
  • a suitable pharmaceutical preparation for transdermal transmission, absorption promoters or iontophoresis are suitable methods.
  • Iontophoretic transmission may be accomplished using commercially available "patches" which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
  • kits comprising the pharmaceutical composition of any one of claims 1-30 or 43 and instructions for use.
  • the compounds of this invention may typically contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
  • the starting materials useful for making the compounds utilized in this invention are generally known or can be prepared by known methods or obvious modifications thereof.
  • many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif, USA), Emka- Chemce or Sigma (St. Louis, Mo., USA).
  • BtH is benzotriazole and Bt is 1-benzotriazolyl or 2-benzotriazolyl. See also, Katritzky et al, Synthesis, 1998(10): 1421-23 and Sansone et al., Tetrahedron Lett., 51(46), 2010, 6031-33 (each of which is incorporated herein in its entirety by reference).
  • the reactions are carried out for a period of time sufficient to produce a substantial amount of the product, which can be determined using well known methods such as thin layer chromatography and 1 H-nuclear magnetic resonance spectroscopy.
  • the product obtained can be used without purification in a subsequent step, or may be separated using well known methods such as crystallization, precipitation, column chromatography, and distillation.
  • protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.
  • Example 1 Chemical library screen and assays to test for blockers of CRAC.
  • HeLa-OSN cells are a cell-line that harbors amplified CRAC currents by
  • NFAT1-460-GFP was stably expressed for use as a readout for the screen.
  • NFAT is a transcription factor that is activated by CRAC channels and translocates from the cytoplasm into the nuclei (FIG. 2b).
  • CRAC channel activity can be monitored.
  • the known CRAC channel blocker, 2-APB was used as a positive control for the screen.
  • the calculated Z' factor was 0.692 meaning that the design was suitable for high throughput screen.
  • Thirteen chemical compounds were identified from the screen using a chemical library composed of 85,000 compounds (FIG. 3). These are potential immunosuppressants.
  • compound 5 (N-[2,2,2-trichloro-l-(l-naphthylamino)ethyl]-2- thiophenecarboxamide) was found to be the most potent blocker of the CRAC.
  • Compound 5 and analogues of compound 5 (FIG. 4) were tested. Among these, the compound 5d (N-[2,2,2-trichloro-l-(2-naphthylamino)ethyl]-2-furamide) and the compound 5 J (N-[(2-naphthylamino)carbonothioyl]-2-furamide) showed the strongest blocking activity.
  • FIG. 5 demonstrates that the compound 5d can block CRAC current by
  • FIG. 1 demonstrates the immunosuppressive activity of the compound 5d (N-[2,2,2- trichloro-l-(2-naphthylamino)ethyl]-2-furamide).
  • the IC50 value of the compound 5d to block Ca2+ entry in T cells was determined to be 195 nM using single cell fura-2 Ca2+ imaging (FIG. la). It was shown that the compound 5d blocks the cytokine production of T cells (FIG. lb), and T cell activation/proliferation in (FIG. lc).
  • the therapeutic potential of the compound 5d analogue, compound 5J-4 (N- ⁇ [(6-hydroxy-l-naphthyl)amino]carbonothioyl ⁇ -2-furamide) was tested and it showed a better suppressive effect on the onset and severity of the diseases considering the reduced disease severity (FIG. 6c) and the infiltrated T cell number to the central nervous system by H&E staining (FIG. 6d).
  • the infiltrated T cell number was determined after isolation of mononuclear cells from the spinal cord and the frequency of CD4+ T cells (helper T cells) among the isolated cells was determined by surface staining of CD4 and analysis by flow cytometry (FIG. 6e).
  • Polyclonal rabbit antibody for detection of Orail was generated, affinity-purified (Open Biosystems, Huntsville, AL), and used at 1 : 1000 dilution for immunocytochemistry.
  • Alexa Fluor 568 labeled secondary antibodies were purchased from Invitrogen (Carlsbad, CA) and used at 1 : 1000 dilutions for immunocytochemistry.
  • Plasmids Full-length cDNA of human Orail was subcloned into bicistronic retroviral expression vector pMSCV-CITE-eGFP-PGK-Puro, which allows for simultaneous expression of Orail, GFP and a puromycin resistance gene. Single-point mutants were generated using Quickchange XL site-directed mutagenesis kit (Stratagene) following manufacturer's instructions. All the clones were verified by sequencing.
  • HEK293 cells were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM - Mediatech, Hargrave, VA) supplemented with with 10% fetal bovine serum (Hyclone, Logan, UT), 10 mM HEPES, 10 mM Glutamine and 1% penicillin/streptomycin (Mediatech, Hargrave, VA). Cells were transfected at 80- 90% confluency using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
  • DMEM - Mediatech, Hargrave, VA Dulbecco's modified Eagle's medium
  • fetal bovine serum Hyclone, Logan, UT
  • 10 mM HEPES 10 mM Glutamine
  • penicillin/streptomycin Mediatech, Hargrave, VA
  • phoenix cells stably expressing gag-pol and ecotropic env purchased from ATCC
  • plasmids encoding Orai proteins were transfected with plasmids encoding Orai proteins to produce ecotropic, replication-incompetent retrovirus using calcium phosphate transfection method.
  • Virus-containing supernatant was collected at 2 and 3 days after transfection and immortalized Orail "7" murine embryonic fibroblasts (MEFs) were transduced twice on day 2 and day 3 in the presence of 8 ⁇ g/ml polybrene. Transduction efficiencies were evaluated visually by GFP expression and Orail expression using immunoblotting.
  • HeLa cells stably expressing Orail and STIM1 and NFAT-1-466-GFP were generated by retroviral transduction followed by flow cytometry sorting for bright GFP + cells.
  • Cells were FACS- sorted for selection of high-GFP expressing cells.
  • -5000 cells were plated onto individual 384-well plates coated with poly-L-Lysine (Greiner Bioone) over night. Next day, the cells were washed twice with 2 mM Ca 2+ -containing Ringer solution and bathed in the same solution. Compounds were added using a 384-well pin tool (V&P Scientific, Inc.) at a final concentration of 10 ⁇ .
  • CRAC currents Measurement of CRAC currents by whole cell recording.
  • HEK293T cells were co-transfected with plasmids encoding Orail WT or mutant cDNAs in the presence or absence of STIM1 encoding plasmid at a molar ratio of 1 : 1 using Lipofectamine 2000 (Invitrogen, Carlsbad). Cells were used for experiments 24-48 hrs post transfection. Patch-clamp recordings were performed using an Axopatch 200B amplifier (Molecular Devices, California) interfaced to a Digidata 1320A (Axon Instruments, CA) for stimulation and data acquisition. Currents were filtered at 1 kHz with a 4-pole Bessel filter and sampled at 5 kHz.
  • CD4 + CD25 T cells were purified from single- cell suspensions of spleens and lymph nodes of adult mice. Single-cell suspensions were prepared by mechanical disruption using cell strainer (BD Biosciences). CD4 + CD25 " T cells were isolated by magnetic sorting with CD4 + beads (Invitrogen) followed by CD25 MACS positive selection (Miltenyi Biotech). For effector T cell differentiation, cells were stimulated with 2 ⁇ / ⁇ 1 of anti-CD3 (Bio X cell) and 2 ⁇ / ⁇ 1 of anti-CD28 (Bio X cell) for 48 hours on a plate coated with 0.1 mg/ml of goat anti-hamster (ICN). For T R I
  • CD4 + CD25 T cells were cultured in the presence of 10 ⁇ / ⁇ 1 anti-IL-4 (Bio X cell), and lOng/ml IL-12.
  • CD4 + CD25 were cultured in the presence of 20 ⁇ / ⁇ 1 anti-IFN- ⁇ (Bio X cell), 2.5 ⁇ / ⁇ 1 anti-IL-12 and 10 ng/ml IL-4
  • CD4 + CD25 cells were cultured in the presence of 10 ⁇ g/ml anti-IL-4, 20 ⁇ anti-IFN- ⁇ , 30 ng/ml IL-6 (Peprotech), 10 ng/ml TGF- ⁇
  • differentiated T cells were restimulated with anti-CD3 antibody with or without anti-CD28 antibody, or with ionomycin with or without phorbol myristate acetate (PMA) for Intracellular staining.
  • PMA phorbol myristate acetate
  • T cell proliferation assays Proliferation was analyzed by flow cytometric measurement of carboxy fluorescein succinimidyl ester (CFSE) dilution. Purified T cells were labeled with 5 ⁇ CFSE (Invitrogen) at 37°C for 10 minutes followed by extensive washing with phosphate-buffered saline (PBS). CFSE-labeled T cells were stimulated with anti-CD3 and anti-CD28 for 48 hours.
  • CFSE carboxy fluorescein succinimidyl ester
  • mice were immunized s.c. in the 2 flanks, 100 ⁇ per site. These mice also received i.p. 200 ng of pertussis toxin (List Biological Laboratories) in 200 ⁇ of PBS at the time of immunization and again 2 days later.
  • EAE was scored according to the following clinical scoring system : 0, no clinical signs; 1, loss of tail tone; 2, wobbly gait; 3, hind limb paralysis; and 4, moribund or death.
  • Mice in the test groups were treated i.p. 5D or 5J-4 in 50 ⁇ 1 DMSO on every other day from 0 to 29 days following induction of EAE.
  • Mice in the control group received 50 ⁇ DMSO.
  • T cell analysis Draining lymph nodes were collected 14 days after EAE induction, and cell suspensions were prepared. For proliferation analysis, cells were distributed in a 96- well plate at 1 x 10 6 /ml concentration and cultured in media. Cell suspensions were restimulated with 20 ⁇ g/ml of MOG35-55 for 2 days at 37 °C with 5% C0 2 and humidified atmosphere. All of the cultures were run in triplicate. The 48-h cultures were pulsed with 1 ⁇ [ 3 H]-thymidine (Amersham Biosciences) for an additional 16-18 h. After this treatment, cells were harvested, lysed, and acid precipitated.
  • cells were distributed in a 12-well plate at 1 x 10 6 /ml concentration and cultured Cells cultured for four more days with MOG peptide and exogenous IL-6 and IL-23 or IL-12, in the presence or absence of 5D.
  • a pilot screen using direct measurement of cytoplasmic Ca 2+ concentration ([Ca 2+ ]i) provided a Z' factor of ⁇ 0.5.
  • NFAT translocation as a readout for CRAC channel activity in Drosophila cells
  • ⁇ 20 candidates were selected from a genome-scale RNAi screen while similar screens using direct [Ca 2+ ]i measurement resulted in 500-1,000 candidates. Therefore, we generated HeLa-O+S cells stably expressing NFATc2 (amino acid l-460)-GFP (abbreviated NFAT-GFP) to utilize nuclear translocation of NFAT-GFP as readout (FIG. 8a).
  • Nuclear translocation of NFAT in HeLa-O+S cells triggered by store depletion was specifically suppressed by blocking CRAC channels with 2-APB, La 3+ , and Gd 3+ suggesting that it is predominantly regulated by CRAC channel activity (FIG. 8a and data not shown).
  • a pilot screen utilizing NFAT translocation as readout showed a Z' factor of ⁇ 0.7, providing an ideal platform for high throughput screening (FIG. 7c).
  • a chemical library encompassing -85,000 compounds was screened to identify blockers of NFAT translocation. Each plate included a positive control, 2-APB and plates were automatically scored for colocalization of NFAT-GFP and 4,6-diamidino-2-phenylindole (DAPI) signals.
  • DAPI 4,6-diamidino-2-phenylindole
  • compound 5D N-[2,2,2-trichloro-l-(2-naphthylamino)ethyl]-2-furamide showed an enhanced blocking of SOCE and NFAT translocation (FIG. 10a and b).
  • Modifications in the structure of the thiophene or naphthalene rings of compound 5 reduced the blocking effect suggesting their important role in inhibiting CRAC channel activity.
  • Compound 5D blocked endogenous SOCE, especially sustained Ca 2+ levels of effector T cells in a dose-dependent manner with a half-maximum inhibitory concentration (IC 50 ) of 195 nM (FIG. 8c).
  • Compound 5D inhibits CRAC channel activity by blocking ion permeation.
  • the CRAC channel has unique gating and inactivation mechanisms.
  • ER store depletion induces multimerization and clustering of STIM1 at the ER-PM junctions, which allows for a direct protein interaction between Orail and STIM1.
  • TIRF total internal reflection fluorescence
  • Orail contains four transmembrane segments (TM1-4), cytoplasmic N and C termini, and two extracellular loops ECl and EC2 with TMl lining the pore (FIG. 8e).
  • D 110 AD 112 >AAA) did not affect blocking by compound 5D.
  • deletion of extracellular loop 2 also did not affect block by compound 5D (FIG. 8f).
  • residue VI 02 within TMl may form the gate that opens and closes CRAC channels and its mutation results in STIMl -independent and constitutively active channels.
  • the mutational analysis described herein of this residue demonstrated a very slow and partial block of Orail vl02C and Orail vl02A -evoked currents by compound 5D (FIG. 8f, FIG. lOf and g).
  • T R 17 differentiation exhibits highest sensitivity to CRAC channel inhibition.
  • T H 17 cells showed highest sensitivity to block of SOCE by compound 5D when compared with na ' ive, T H 1 or T H 2 cells (FIG. 11a). Consistent with these results, IL-17 production by T H 17 cells was dramatically reduced when compared to IFN- ⁇ and IL-4 production by T H 1 and T H 2 cells respectively, in the presence of compound 5D (FIG. 1 lb and c).
  • na ' ive T cells were stimulated under T H 1, T H 2, and T H 17-polarized conditions in the presence and absence of compound 5D. In these experiments, compound 5D was included only during the differentiation stage but excluded during restimulation to examine cytokine production.
  • Compound 5D showed the strongest inhibitory effects on T H 17 differentiation when compared to that of T H 1 and T H 2 cells as judged by their production of IL-17A, IFN- ⁇ , and IL-4, cytokines, respectively (FIG. 12a). Further analysis of lineage-specific transcription factors showed a pronounced reduction of mRNA levels of RORa and RORyt under T H 17- polarized conditions even with low concentrations of compound 5D, compared to those of T- bet or GATA-3 expression under T H 1 or T H 2 -polarized conditions (FIG. 12b).
  • IL-23R IL-23 receptor
  • CCR6 integrin aL
  • LFA-1 IL-23 receptor
  • Inhibition of CRAC channel activity by compound 5D did not significantly alter mRNA expression levels of other transcription factors including c-Maf, Runt-related transcription factor 1 (Runxl), aryl hydrocarbon receptor (AHR), interferon-regulatory factor 4 (IRF-4), suppressor of cytokine signalling 3 (SOCS3), ⁇ , basic leucine zipper transcription factor ATP-like (BATF), or hypoxia-inducible factor 1 (HIF1), all of which are known to play an important role in T R 17 differentiation (FIG. 13b).
  • Runxl Runt-related transcription factor 1
  • AHR aryl hydrocarbon receptor
  • IRF-4 interferon-regulatory factor 4
  • SOCS3 suppressor of cytokine signalling 3
  • BATF basic leucine zipper transcription factor ATP-like
  • HIF1 hypoxia-inducible factor 1
  • Orail-NFAT-RORa/yt axis plays a crucial role in T R 17 differentiation.
  • Previous studies have predicted putative NFAT binding elements on the RORyt promoter.
  • expression of a constitutively active NFATc2 mutant influenced expression of RORyt during early stages of T H 17 differentiation.
  • NFAT family of transcription factors are directly activated by the increase in [Ca 2+ ]i, their role in regulating expression of RORa and RORyt was examined.
  • Na ' ive T cells were stimulated and cultured under T H 17-polarizing conditions in the presence of cyclosporine A (CsA), a calcineurin blocker that also inhibits nuclear translocation of NFAT.
  • CsA cyclosporine A
  • chromatin immunoprecipitation (ChIP) experiments were performed from na ' ive T cells stimulated and cultured under T H 17-polarizing conditions with and without compound 5D. These experiments demonstrated a direct recruitment of NFATc2, the predominant NFAT family member in na ' ive T cells, into the promoters of RORyt and RORa under T H 17- polarizing conditions, which was markedly reduced in compound 5D-treated cells (FIG. 14d, left two panels).
  • FIG. 14d left two panels
  • T R 17 differentiation of CD4 + T cells from Orail "7" mice was examined. Deficiency of Orail drastically decreased the expression levels of IL-17, RORa and RORyt (FIG. 15a and b). Transcript analysis showed a pronounced reduction in the mRNA expression levels of cytokines and receptors including IL-17A, IL-17F, IL-22, and IL-23R in Orail "7" T cells (FIG. 15c). Previously, we showed that Orail " " na ' ive T cells do not have any defect in proliferation after TCR stimulation. Therefore, together with the results from compound 5D-treated cells (Fig. 13c), reduced T H 17 differentiation in Orail "7” cells suggests that Orail -mediated Ca 2+ entry regulates specific signalling pathways essential for T R 17 differentiation, which is independent of T cell proliferation.
  • T R 17 cells T R 17 cells (FIG. 14c and 15d).
  • Previous studies of transcriptional regulation of RORa and RORyt promoters have suggested an important role for STAT3 and c-Rel transcription factors.
  • Durant et al used genome -wide ChIP and parallel sequencing (ChlP-seq) experiments from wild-type and STAT3 -deficient T cells cultured under T H 17-polarizing condition and identified recruitment of STAT3 to both RORa and RORyt promoters (See, for example, Durant et al, Immunity 32, 605:615 (2010)). Consequently, expression levels of RORa and RORyt were significantly reduced in ST AT3 -deficient T cells. More recently, using ChIP and luciferase reporter assays, Ruan et al have shown a direct regulation of RORyt expression by c-Rel. Furthermore, the authors also observed reduced expression of RORa transcripts in c- Rel deficient T cells.
  • halofuginone was shown to activate the amino acid starvation response (AAR) pathway.
  • AAR amino acid starvation response
  • Halofuginone influenced the functions of T R I , T R 2, and T H 17 cells at high concentrations; however, it selectively suppressed cytokine production by T R 17 cells at low concentrations and ameliorated the symptoms of EAE when administered in vivo.
  • CRAC channel blockers can be considered as general immunosuppressants taking into account their broad role in various immune cells.
  • Plasmids Full-length cDNA of human Orail subcloned into bicistronic retroviral expression vector pMSCV-CITE-eGFP-PGK-Puro, which allows for simultaneous expression of Orail, GFP and a puromycin resistance gene has been described previously 1 .
  • Single-point mutants were generated using Quickchange XL site-directed mutagenesis kit (Stratagene) following manufacturer's instructions. All the clones were verified by sequencing.
  • TIRF total internal reflection fluorescence
  • WT Orail cDNA was fused in frame with pEGFP to have a C-terminal GFP -tag.
  • STIMl-mCherry plasmid has been described previously 1 ' 2 .
  • Orail Q 108 LD 110 ⁇ A 108 AA 110 and D 110 AD 112 >A 110 AA 112 were generated by introducing a Notl site in the primers.
  • Orail AEC2 clone was generated by deletion of amino acids L 202 -P 231 within the second extracellular loop by introduction of a Notl restriction enzyme site.
  • HEK293 cells were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM - Mediatech, Hargrave, VA) supplemented with with 10% fetal bovine serum (Hyclone, Logan, UT), 10 mM HEPES, 10 mM Glutamine and 1% penicillin/streptomycin (Mediatech, Hargrave, VA). Cells were transfected at 60- 70% confluency using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
  • DMEM - Mediatech, Hargrave, VA Dulbecco's modified Eagle's medium
  • fetal bovine serum Hyclone, Logan, UT
  • 10 mM HEPES 10 mM Glutamine
  • penicillin/streptomycin Mediatech, Hargrave, VA
  • phoenix cells stably expressing gag- pol and ecotropic env purchased from ATCC were transfected with plasmids encoding Orai cDNAs to produce ecotropic, replication-incompetent retrovirus using calcium phosphate transfection method.
  • Virus-containing supernatant was collected at 2 and 3 days after
  • transfection and immortalized Orail " murine embryonic fibroblasts (MEFs) or primary T cells were transduced in the presence of 8 ⁇ g/ml polybrene. Transduction efficiencies were evaluated visually by GFP expression.
  • HeLa cells stably expressing Orail, STIM1 and NFATc2-l-460-GFP were generated by retroviral transduction with viruses encoding each cDNA and antibiotic selection.
  • Cells were FACS-sorted for selection of GFP hlgh population.
  • -5000 cells were plated onto individual 384-well plates coated with poly-L-Lysine (Greiner Bioone). Next day, the cells were washed twice with 2 mM Ca 2+ -containing Ringer solution and bathed in the same solution. Compounds were added using a 384-well pin tool (V&P Scientific, Inc.) at a final concentration of 10 ⁇ .
  • Peak-basal Ca 2+ ratio was calculated after subtracting the ratio value after store depletion, before reintroduction of Ca 2+ containing Ringer's solution from the maximal value of 340/380 ratio after reintroduction of Ca 2+ containing Ringer's solution.
  • sustained Ca 2+ was calculated as the 340/380 ratio at the time point of 800s after subtracting basal Ca 2+ ratio.
  • TIRFM Total internal reflection fluorescence microscopy
  • the angle of the incident light at the interface between the glass coverslip and the aqueous medium was controlled by independently adjusting the position of each laser beam before passing through a 60x oil-immersion objective (NA 1.49, Olympus).
  • NA 1.49, Olympus The emission was filtered either at D525/50 or 660/50 nm filter (Chroma) and captured by a Hamamatsu ORCA cooled CCD (Roper Scientific) camera. Acquisition and image analysis were performed using Slidebook (Intelligent Imaging Innovations, Inc.) and OriginPro8.5 software.
  • CRAC currents Measurement of CRAC currents by whole-cell recording.
  • HEK293 cells were co-transfected with plasmids encoding Orail WT or mutant cDNAs in the presence or absence of STIM1 encoding plasmid at a molar ratio of 1 : 1 using Lipofectamine 2000 (Invitrogen, Carlsbad). Cells were used for experiments 24-48 hrs post transfection. Patch-clamp recordings were performed using an Axopatch 200B amplifier (Molecular Devices, California) interfaced to a Digidata 1320A (Axon Instruments, CA) for stimulation and data acquisition. Currents were filtered at 1 kHz with a 4-pole Bessel filter and sampled at 5 kHz.
  • CD4 + T cells were purified from single-cell strained suspensions prepared by mechanical disruption of spleens and lymph nodes of adult mice after magnetic sorting with CD4 + beads (Invitrogen).
  • CD4 CD25 " naive T cells were selected by CD25 MACS positive selection (Miltenyi Biotech).
  • effector T cell differentiation cells were stimulated with 2 ⁇ g/ml of anti-CD3 (Bio X cell) and anti-CD28 (Bio X cell) antibodies for 48 hours on a plate coated with 0.1 mg/ml of goat anti-hamster (MP Biomedicals).
  • CD4 CD25 T cells were cultured with 10 ⁇ g/ml anti-IL-4 (Bio X cell), and 10 ng/ml IL-12 for T R I differentiation, 20 ⁇ g/ml anti-IFN- ⁇ (Bio X cell), 2.5 ⁇ g/ml anti- IL-12 and 10 ng/ml IL-4 (Peprotech) for T H 2 differentiation, and 10 ⁇ g/ml anti-IL-4, 20 ⁇ / ⁇ 1 anti-IFN- ⁇ , 30 ng/ml IL-6 (Peprotech), 3 ng/ml TGF- ⁇ (Peprotech) and 10 ng/ml IL-23 (R&D Systems) for T H 17 differentiation.
  • differentiated T cells were restimulated with 20 nM phorbol myristate acetate (PMA) and 1 ⁇ ionomycin for cytokine analysis.
  • PMA phorbol myristate acetate
  • mice EAE induction in mice. All animals were maintained in pathogen-free barrier facilities and used in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles.
  • MOG35-55 peptide N- MEVGWYRSPFSRVVHLYRNGK-C, Genscript
  • CFA complete Freund's adjuvant
  • mice were also injected i.p.
  • EAE pertussis toxin
  • T cell analysis Draining lymph nodes were collected 14 days after EAE induction, and cell suspensions were prepared. For proliferation analysis, cells were distributed in a 96- well plate at 1 x 10 6 cells/ml concentration and cultured in media. Cell suspensions were restimulated with 20 ⁇ g/ml of MOG35-55 for 2 days at 37°C with 5% C0 2 and humidified atmosphere. All the cultures were run in triplicates. After 48 h, cultures were pulsed with 1 ⁇ [ 3 H]-thymidine (Amersham Biosciences) for an additional 16-18 h. After this treatment, cells were harvested, lysed, and acid precipitated.
  • [ 3 H]-thymidine incorporation was determined by liquid ⁇ -scintillation counting (Beckman).
  • cells were distributed in a 12-well plate at 1 x 10 6 cells/ml concentration and cultured with 20 nM PMA and 1 ⁇ ionomycin for 5h.
  • the total RNA of draining lymph nodes was extracted with TRIzol reagent (Gibco-BRL) following the manufacturer's instructions.
  • draining lymph nodes were collected 7 days after EAE induction, and cell suspensions were prepared.
  • Cells were distributed in a 12- well plate at 1 x 10 6 cells/ml concentration and cultured for four more days with the MOG peptide (20 ⁇ g/ml) together with exogenous IL-6 and IL-23 or IL-12, in the presence or absence of compound 5D.
  • cDNA was synthesized from total RNA using oligo(dT) primers and Superscript III First-Strand cDNA synthesis kit (Invitrogen). Real-time PCR was performed using an iCycler IQ5 system (Biorad) and SYBR Green dye (Sigma) using the primers described in Table 2. Threshold cycles (C T ) for all the candidate genes were normalized to the C T values for beta-actin housekeeping gene control to obtain AC T . The specificity of primers was examined by melt-curve analysis and agarose gel
  • Chromatin immunoprecipitation After culturing naive T cells under T R 17- polarizing conditions with plate-coated anti-CD3 and anti-CD28 antibodies for 16 hours, cells were fixed for 8 min at room temperature with 1 :37 dilution of 37.1 % formaldehyde
  • Immunocomplexes were captured and washed twice for 5 min each with low salt washing buffer, high salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, PH 8.1, 500 mM NaCl), LiCl washing buffer (0.25 M LiCl, 1% NP-40, 0.1% deoxycholate, 1 mM EDTA, 10 mM Tris 8.1), and TE buffer (Tris-HCl 10 mM, EDTA 1 mM).
  • PBMC culture Human PBMC culture. Mononuclear cells were obtained from the CFAR Virology core Laboratory at UCLA that were prepared from buffy coats from healthy, unidentified adult donors using FicollPAQUE gradients. PBMCs were activated with anti-CD3/CD28 beads (Miltenyi Biotech) and cultured in T cell media (DMEM containing 20% Fetal bovine serum and 1% Pen-Strep) supplemented with 20 U/mL IL-2 (Peprotech), 10 ng/mL IL- ⁇ ⁇ , and 10 ng/mL IL-23 (eBioscience) in the presence or absence of 20 ⁇ of compound 5D. Cells were expanded with fresh media, cytokines and compound 5D every alternate day.
  • DMEM containing 20% Fetal bovine serum and 1% Pen-Strep
  • CD4-FITC-positive cells were gated for analysis.
  • CD4-FITC OKT4, eBioscience
  • IL-17A-APC eBio64CAP17, eBioscience
  • IFN-y-PECy7 45. B3, eBioscience
  • Example 3 Immunosuppression in mammals with Systemic lupus erythematosus (SLE)
  • Systemic lupus erythematosus is caused by complex communications between hypersensitive, self-reactive innate and adaptive immune cells.
  • CRAC Ca 2+ -re lease-activated Ca 2+
  • This hypothesis can be tested by blocking the activity of Orail that is crucial for functions of various innate and adaptive immune cells including mast, B, and T cells.
  • new findings described herein that blocking the activity of CRAC channels induces enhanced survival and differentiation of regulator T cells that are key players in immune tolerance.
  • Triggering of Ca 2+ signalling though CRAC channels by receptor stimulation is a key step for proliferation and cytokine production of immune cells.
  • Applicants have recently identified and named Orail as a long-sought pore component of the CRAC channel.
  • T cells expressing Foxp3 generated by blocking Orail were functionally active based on the suppression assays to test the functions of regulatory T cells.
  • the signaling pathways that influence regulatory T cell development by blocking of Orail should influence the downstream of Ca 2+ signaling pathways such as calcineurin or a transcription factor, nuclear factor of activated T cells (NFAT). It is contemplated that lower recruitment levels of NFAT polarize T cells into regulatory T cells because abundant levels of nuclear NFAT is required for differentiation of other T cell subsets. If the promoter of Foxp3 is not as sensitive to the decreased levels of NFAT as other promoters, this will polarize T cells into cells expressing Foxp3 in a passive manner.
  • Ca 2+ signaling pathways such as calcineurin or a transcription factor, nuclear factor of activated T cells (NFAT). It is contemplated that lower recruitment levels of NFAT polarize T cells into regulatory T cells because abundant levels of nuclear NFAT is required for differentiation of other T cell subsets. If the promoter of Foxp3 is not as sensitive to the decreased levels of NFAT as other promoters, this will polarize T cells into cells expressing Foxp3 in
  • this hypothesis can be checked by measuring expression levels of NFAT and recruitment of NFAT into the Foxp3 promoter using chromatin immunoprecipitation in Orail -deficient T cells or with small molecule treatment.
  • chromatin immunoprecipitation may actively influences other transcription factors such as STAT5, Runxl, c-Rel, p65, or SMAD2/3 that are crucial for the expression of Foxp3 and TGF- ⁇ signaling.
  • This alternate possibility can be checked by chromatin immunoprecipitation, quantitative PCR, and immunoblotting.
  • na ' ive wild-type and Orail - deficient T cells were stimulated in combination of transforming growth factor (TGF)-P and/or IL-6 that are critical regulators for differentiation of Tregs and Thl7 cells.
  • TGF transforming growth factor
  • the decreased levels of Ca 2+ signaling by suppression of Orail should influence the downstream of Ca 2+ signaling pathways such as calcineurin or a transcription factor, nuclear factor of activated T cells (NFAT). It is contemplated that lower recruitment levels of NFAT turn T cells into regulatory T cells because abundant levels of nuclear NFAT is required for differentiation of other T cell subsets (e.g. Thl7). The essential role of NFAT in diverse T cell differentiations has been emphasized. However, how these promoters are differentially sensitive to reduced levels or Ca 2+ entry or NFAT nuclear accumulation has not been studied.
  • lowered Ca 2+ signaling may positively influence other transcription factors such as STAT5, Runxl, c-Rel, p65, or SMAD2/3 that are crucial for the expression of Foxp3 and TGF- ⁇ signaling in addition to NFAT. This possibility can be checked by determining the expression levels of these transcription factors and chromatin
  • the decreased levels of Ca 2+ signaling in regulatory T cells provides strong resistance to cell death, therefore increases the number of Tregs.
  • the cell death levels of Orail -deficient Tregs can be checked, and the number of Tregs after treatment of a small molecule blocker for Orail can be determined.
  • ex vivo analysis of cell death shows a clear correlation with in vivo cell death assays.
  • the cell survival rate using ex vivo analysis of culturing the lymphocytes or splenocytes with or without cytokines can be checked.
  • the frequency of Tregs will be determined by CD4, CD25, and Foxp3 staining at 24 and 48 hours.
  • the survival of nTregs after adoptive T cell transfer can be measured to prove that the increased population of nTregs is due to the increased levels of resistance to cell death.
  • nTregs can be purified from the control and Orail -deficient mice, and transfered into the immune-compromised mice after labeling with CFSE. At week 1 and 2, the mice can be dissected to check the survival by CFSE cells and proliferation rate. These studies will provide an insight to develop an enhanced method using transfer of regulatory T cells.
  • Amelioration of autoimmune diseases by blocking Orail activity was expected because Orail plays a major role in effector T cell functions.
  • EAE experimental autoimmune encephalomyelitis
  • a great reduction in EAE symptoms after treatment of SKF96365 for 15 days during the onset of EAE was observed (Fig. 22, left).
  • a single injection of SKF96365, a known CRAC channel inhibitor not typically used in vivo because of known side-effects, at the early immunization process greatly reduces the symptoms (right).
  • mice will be treated at the initial stage of the disease (3-4 weeks of age). After treatment of SKF96365 and compounds of Formula I as described herein, the mice can be analyzed for the symptoms of lupus (e.g. production of autoantibodies and inflammatory cytokines (e.g. TNF-a, MCP-1, IL-6, IL-12, IFN- ⁇ , and IL- 10), splenomegaly, and spontaneous lymphocyte activation. In addition, these mice will be dissected and analyzed for the population of regulatory T cells.
  • lupus e.g. production of autoantibodies and inflammatory cytokines (e.g. TNF-a, MCP-1, IL-6, IL-12, IFN- ⁇ , and IL- 10)
  • splenomegaly e.g. TNF-a, MCP-1, IL-6, IL-12, IFN- ⁇ , and IL- 10
  • spontaneous lymphocyte activation e.g. TNF-a, MCP-1,
  • mice The generation and analysis of Orail -deficient lupus-prone mice can be performed according to the following experimental plan.
  • a mouse model of lupus in a setting of Orail deficiency can be generated.
  • Applicants have used a conventional Orail knockout mouse model to have a preliminary data.
  • the low survival rate of these mice delays progress.
  • an Orail knockout mouse model where the Orail gene is conditionally targeted in T cells was generated.
  • the TLR7.Tg.6 strain displays increased TLR7 expression, accumulation of anti-RNA autoantibodies, upregulation of type I IFN gene signature and an autoimmune syndrome resembling human SLE.
  • lupus phenotypes can be analyzed together with determination of regulation of regulatory T cell populations.
  • the striking effect of the Orail blocker in autoimmune disease such as EAE may be explained by two hypotheses. First, naive T cells may preferentially differentiate into regulatory T cells when Orail is blocked.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des procédés et des compositions qui permettent de supprimer l'activité du canal Ca2+ activé par la libération de Ca2+ (CRAC) à l'aide de nouveaux composés de la formule (I). Il s'est avéré que les composés de la formule (I) modulent le transport du calcium à travers le canal CRAC. La fonction du canal CRAC est importante pour la réponse immunitaire et est largement limitée aux cellules du système immunitaire. Par conséquent, selon un aspect, l'invention concerne un procédé qui permet de supprimer l'immunité chez un mammifère qui en a besoin et qui comporte l'administration d'un composé de la formule (I) en une quantité suffisante pour inhiber l'activité du canal CRAC. L'invention concerne également des compositions pharmaceutiques et des nécessaires comportant les composés de la formule (I).
PCT/US2014/013627 2013-01-30 2014-01-29 Petites molécules pour bloquer la voie de signalisation WO2014120790A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361758648P 2013-01-30 2013-01-30
US61/758,648 2013-01-30
US201361911421P 2013-12-03 2013-12-03
US61/911,421 2013-12-03

Publications (1)

Publication Number Publication Date
WO2014120790A1 true WO2014120790A1 (fr) 2014-08-07

Family

ID=51262900

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/013627 WO2014120790A1 (fr) 2013-01-30 2014-01-29 Petites molécules pour bloquer la voie de signalisation

Country Status (1)

Country Link
WO (1) WO2014120790A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11897856B1 (en) 2023-10-12 2024-02-13 King Faisal University N-(Naphthalen-1-ylcarbamothioyl)furan-2-carboxamide as an eco-friendly insecticidal agent against Spodoptera littoralis (boisd.)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008148108A1 (fr) * 2007-05-24 2008-12-04 Calcimedica, Inc. Protéines de canal de calcium et leur utilisation
WO2010127558A1 (fr) * 2009-05-05 2010-11-11 中国科学院上海药物研究所 Composés de benzoylurée substitués, leurs procédés de préparation et leurs utilisations
WO2011063277A1 (fr) * 2009-11-20 2011-05-26 Amgen Inc. Protéines de liaison à un antigène anti-orai1 et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008148108A1 (fr) * 2007-05-24 2008-12-04 Calcimedica, Inc. Protéines de canal de calcium et leur utilisation
WO2010127558A1 (fr) * 2009-05-05 2010-11-11 中国科学院上海药物研究所 Composés de benzoylurée substitués, leurs procédés de préparation et leurs utilisations
WO2011063277A1 (fr) * 2009-11-20 2011-05-26 Amgen Inc. Protéines de liaison à un antigène anti-orai1 et leurs utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARTA M. REINOSO GARCIA ET AL.: "Heavy metal complexation by N-acyl(thio) urea-functionalized cavitands: synthesis, extraction and potentiometric studies", TETRAHEDRON, vol. 60, no. 49, 2004, pages 11299 - 11306 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11897856B1 (en) 2023-10-12 2024-02-13 King Faisal University N-(Naphthalen-1-ylcarbamothioyl)furan-2-carboxamide as an eco-friendly insecticidal agent against Spodoptera littoralis (boisd.)

Similar Documents

Publication Publication Date Title
Loreto et al. Mitochondrial impairment activates the Wallerian pathway through depletion of NMNAT2 leading to SARM1-dependent axon degeneration
Jan et al. Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity
Schmidt et al. Exchange protein directly activated by cAMP (epac): a multidomain cAMP mediator in the regulation of diverse biological functions
Pasciuto et al. Dysregulated ADAM10-mediated processing of APP during a critical time window leads to synaptic deficits in fragile X syndrome
Kim et al. Calcium signaling via Orai1 is essential for induction of the nuclear orphan receptor pathway to drive Th17 differentiation
JP6870988B2 (ja) 神経細胞増幅及び中枢神経系障害治療のためのセレブロンの活性化因子の使用方法
Nakamura et al. PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking
Yamashiro et al. AUTS2 governs cerebellar development, purkinje cell maturation, motor function and social communication
Zapata et al. Epilepsy and intellectual disability linked protein Shrm4 interaction with GABABRs shapes inhibitory neurotransmission
Qin et al. CDYL deficiency disrupts neuronal migration and increases susceptibility to epilepsy
US20160271157A1 (en) Beta-catenin
De La Rossa et al. Paradoxical neuronal hyperexcitability in a mouse model of mitochondrial pyruvate import deficiency
JP2024037770A (ja) Ｓａｒｍ１阻害剤
CN109415342A (zh) 用于治疗纤维化的wnt抑制剂
Chen et al. Atypical cadherin Dachsous1b interacts with Ttc28 and aurora B to control microtubule dynamics in embryonic cleavages
Stagni et al. Long-term effect of neonatal inhibition of APP gamma-secretase on hippocampal development in the Ts65Dn mouse model of Down syndrome
JP6918839B2 (ja) 概日時計の乱れに関連するマイクロバイオームの調節異常を処置するための方法及び医薬組成物
JP6285962B2 (ja) 老年性認知症の早期予測・診断のための末梢血細胞におけるＴＲＰＣ６のｍＲＮＡレベルの使用
Wen et al. Inhibitory gene expression of the Cav3. 1 T-type calcium channel to improve neuronal injury induced by lidocaine hydrochloride
Abramov et al. Targeted stabilization of Munc18‐1 function via pharmacological chaperones
EP2797590B1 (fr) Inhibiteur de canal d'ion trpm-4 pour le traitement ou la prévention de la neurodégénérescence
WO2014120790A1 (fr) Petites molécules pour bloquer la voie de signalisation
Onori et al. RHEB/mTOR-hyperactivity causing cortical malformations drives seizures through increased axonal connectivity
Mich et al. Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent
Suknovic et al. The ULK1 kinase, a necessary component of the pro-regenerative and anti-aging machinery in Hydra

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14746613

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14746613

Country of ref document: EP

Kind code of ref document: A1