Compounds as modulators of a mutant CFTR protein and their use for treating diseases associated with CFTR protein malfunction

Abstract

An exemplary embodiment relates to novel protein modulators capable of altering function of the mutant CFTR protein and their use for treating diseases associated with CFTR protein malfunction. The invention provides compositions, pharmaceutical preparations and methods of correcting the cellular alteration of a mutant CFTR protein wherein the CFTR mutation is a mutation F508-CFTR, or another mutation of class II.

Claims

1. A pharmaceutical composition comprising: a compound of general formula (IIIb) used for the manufacture of a medication for the treatment of diseases associated with CFTR protein malfunction: ##STR00018## its tautomers, E and Z geometric isomers, enantiomers, diastereomers, or pharmaceutically acceptable salts thereof or complexes thereof; wherein any occurrence of Z.sup.1 independently represents the following optional substituents, OR.sub.B, OC(O)R.sub.C, OC(O)OR.sub.B, OC(O)N(R.sub.A)R.sub.A, C(O)R.sub.C, C(O)N(R.sub.A)R.sub.A, C(O)N(OR.sub.B)R.sub.A, C(O)OR.sub.B, C(S)R.sub.C, C(O)C(O)R.sub.C, CH.sub.2OR.sub.B, CH.sub.2CH.sub.2OR.sub.B, CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2OCH.sub.2R.sub.C, CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, SR.sub.D, S(O)R.sub.D, SO.sub.2R.sub.D, SO.sub.2N(R.sub.A)R.sub.A, SO.sub.3R.sub.B, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)C(O)N(R.sub.A)R.sub.A, N(R.sub.A)SO.sub.2R.sub.D, N(R.sub.A)SO.sub.2N(R.sub.A)R.sub.A, N(R.sub.A)R.sub.A, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)N(R.sub.A)R.sub.A, N(R.sub.A)N(R.sub.A)C(O)R.sub.C, NO.sub.2, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, NH.sub.2, SCN, SO.sub.2CN, F, Cl, Br, I, C.sub.nH.sub.2nR.sub.C which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n-2)R.sub.C in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n-4)R.sub.C which is branched or unbranched wherein n is an integer from 2 to 5, PO.sub.3H.sub.2, or OPO.sub.3H.sub.2; wherein R.sub.A, R.sub.A, R.sub.A are each independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, and OH; wherein R.sub.B is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, and CH.sub.2I; wherein R.sub.C is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I, F, Cl, Br, I, and NH.sub.2; wherein R.sub.D is independently selected from the group consisting of: H and lower alkyl group wherein R.sup.5 and R.sup.6 are optional substituents which are independently selected from the group consisting of: OH, NH.sub.2, COOH, Cl, Br, I, CH.sub.3, and C.sub.2H.sub.5; wherein R.sup.7 is an optional substituent which is independently selected from the group consisting of: F, Cl, Br, I, CH.sub.3, and C.sub.2H.sub.5; wherein R.sup.8 is an optional substituent which is independently selected from the group consisting of: NH.sub.2, NHAr, OH, CH.sub.2Ar, C(O)Ar, and OAr; wherein Ar is an aromatic group or heteroaromatic group, and wherein the compound of formula (IIIb) is present in a pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the compound has an effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II, and wherein a mutation F508-CFTR, or another mutation of class II are involved in CFTR protein malfunction.

3. The pharmaceutical composition according to claim 1, wherein the compound is characterized in that it has effect on CFTR-dependent ion transport across a cellular membrane and/or it has the ability to increase the number of mutant CFTR proteins that reach a cell surface.

4. The pharmaceutical composition according to claim 1, wherein the compound is characterized in that it has a stabilizing effect on the structure of the mutant CFTR protein and/or blocks interaction with cellular proteins responsible for the premature degradation of mutant CFTR.

5. The pharmaceutical composition according to claim 1, wherein the compound is characterized in that it has an effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II.

6. A pharmaceutical composition comprising: a compound used for the manufacture of a medication for the treatment of diseases associated with CFTR protein malfunction: ##STR00019## its tautomers, E and Z geometric isomers, enantiomers, diastereomers or its pharmaceutically acceptable salts thereof, or complexes thereof, wherein the compound is present in a pharmaceutical composition.

7. The pharmaceutical composition according to claim 6, wherein the compound has an effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR or another mutation of class II, and wherein the mutation F508-CFTR or another mutation of class II are involved in CFTR protein malfunction.

8. The pharmaceutical composition according to claim 6, wherein the compound is characterized in that it has effect on CFTR-dependent ion transport across a cellular membrane and/or it has the ability to increase mutant CFTR proteins that reach a cell surface.

9. The pharmaceutical composition according to claim 6, wherein the compound is characterized in that it has a stabilizing effect on the structure of the mutant CFTR protein and/or blocks interaction with cellular proteins responsible for premature degradation of mutant CFTR protein.

10. The pharmaceutical composition according to claim 6, wherein the compound is characterized in that it has an effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II.

11. A method comprising: treating a disease associated with CFTR protein malfunction in a patient by administering a therapeutically effective amount of a composition comprising: a compound: ##STR00020## its tautomers, E and Z geometric isomers, enantiomers, diastereomers or its pharmaceutically acceptable salts thereof, or complexes thereof, as a modulator of a mutant CFTR protein to the patient.

12. The method according to claim 11, wherein the CTFR protein malfunction is a mutation of F508-CFTR.

13. A method comprising: treating a disease associated with CFTR protein malfunction in a patient by administering to the patient a therapeutically effective amount of a composition comprising: a compound of general formula (IIIb): ##STR00021## its tautomers, E and Z geometric isomers, enantiomers, diastereomers, or pharmaceutically acceptable salts thereof or complexes thereof; wherein any occurrence of Z.sup.1 independently represents the following optional substituents, OR.sub.B, OC(O)R.sub.C, OC(O)OR.sub.B, OC(O)N(R.sub.A)R.sub.A, C(O)R.sub.C, C(O)N(R.sub.A)R.sub.A, C(O)N(OR.sub.B)R.sub.A, C(O)OR.sub.B, C(S)R.sub.C, C(O)C(O)R.sub.C, CH.sub.2OR.sub.B, CH.sub.2CH.sub.2OR.sub.B, CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2OCH.sub.2R.sub.C, CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, SR.sub.D, S(O)R.sub.D, SO.sub.2R.sub.D, SO.sub.2N(R.sub.A)R.sub.A, SO.sub.3R.sub.B, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)C(O)N(R.sub.A)R.sub.A, N(R.sub.A)SO.sub.2R.sub.D, N(R.sub.A)SO.sub.2N(R.sub.A)R.sub.A, N(R.sub.A)R.sub.A, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)N(R.sub.A)R.sub.A, N(R.sub.A)N(R.sub.A)C(O)R.sub.C, NO.sub.2, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, NH.sub.2, SCN, SO.sub.2CN, F, Cl, Br, I, C.sub.nH.sub.2nR.sub.C which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n-2)R.sub.C in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n-4)R.sub.C which is branched or unbranched wherein n is an integer from 2 to 5, PO.sub.3H.sub.2, or OPO.sub.3H.sub.2; wherein R.sub.A, R.sub.A, R.sub.A are each independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, and OH; wherein R.sub.B is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, and CH.sub.2I; wherein R.sub.C is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I, F, Cl, Br, I, and NH.sub.2; wherein R.sub.D is independently selected from the group consisting of: H and lower alkyl group wherein R.sup.5 and R.sup.6 are optional substituents which are independently selected from the group consisting of: OH, NH.sub.2, COOH, Cl, Br, I, CH.sub.3, and C.sub.2H.sub.5; wherein R.sup.7 is an optional substituent which is independently selected from the group consisting of: F, Cl, Br, I, CH.sub.3, and C.sub.2H.sub.5; wherein R.sup.8 is an optional substituent which is independently selected from the group consisting of: NH.sub.2, NHAr, OH, CH.sub.2Ar, C(O)Ar, and OAr; wherein Ar is an aromatic group or heteroaromatic group.

14. The method according to claim 13, wherein the CTFR protein malfunction is a mutation of F508-CFTR.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 indicated effects of different compounds on iodide efflux at 1 M in F508-CFTR HeLa cells.

(2) FIG. 2 indicates iodide efflux in untreated WT-CFTR HeLa cells.

(3) FIG. 3 indicates iodine efflux in F508-CFTR HeLa cells with forskolin alone, forskolin plus genistein or forskolin plus other compounds.

(4) FIG. 4 indicates the impact of correctors on F508-CFTR maturation and cell localization.

(5) FIG. 5 indicates synergistic effects of active compounds on iodide efflux.

(6) FIG. 6 indicates current-voltage relationship for cAMP-dependent chloride currents in HeLa cells.

(7) FIG. 7 indicates effects of different compounds on iodide efflux.

(8) FIG. 8 indicates the effect of 73100 plus 118208 molecules on nasal potential difference (6VTE) in 6F508/M508 mice.

DETAILED DESCRIPTION

(9) Exemplary embodiments will be described. Various modifications, adaptations or variations of the exemplary embodiments described herein may become apparent to those skilled in the art as such are disclosed. It will be understood that all such modifications, adaptations or variations that rely upon the teachings hereof, and through which these teachings have advanced the art, are considered to be within the scope and spirit of the disclosure presented herein.

(10) The methods and compositions of the exemplary embodiments may suitably comprise, consist of, or consist essentially of the components, ingredients, elements, steps and process delineations described herein. The embodiments illustratively disclosed herein suitably may be practiced in the absence of any element, process step, or ingredient which is not specifically disclosed herein.

(11) Unless otherwise stated, all percentages, parts, and ratios expressed herein are based upon weight of the total compositions.

(12) The headings provided herein serve to illustrate, but not to limit the teachings herein in any way or manner.

(13) An exemplary embodiment is a compound of general formula (I):

(14) ##STR00001## its tautomers, E and Z geometrical isomers, optically active forms such as enantiomers, diastereomers and their racemate forms or a mixture of stereoisomeric forms or its pharmaceutically acceptable salts thereof or complexes thereof; wherein Z.sup.1 is independently selected from the group consisting of: C.sub.nH.sub.(2n), which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n2) in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n4) which is branched or unbranched wherein n is an integer from 2 to 5; CRH, C.sub.2H.sub.3R, E or ZC.sub.2HR, C.sub.3H.sub.5R, E or ZC.sub.3H.sub.3R, OCH.sub.2, CH.sub.2O, NRCH.sub.2, CH.sub.2NR; wherein R is independently selected from the group consisting of: H, halogen, NH.sub.2, OH, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, SH, SCN, CH.sub.3, C.sub.2H.sub.5; wherein R is independently selected from the group consisting of: H, CH.sub.3, C.sub.2H.sub.5; wherein R.sup.1 and R.sup.2 are independently selected from the group consisting of aromatic ring or heteroaromatic ring,
as a modulator of a mutant CFTR protein for use in the treatment of cystic fibrosis. R.sup.1 and R.sup.2 are independently selected from the group of sub-formula (Ia):

(15) ##STR00002## wherein A.sub.1,A.sub.2,A.sub.3,A.sub.4,A.sub.5,A.sub.6 is independently selected N or C atoms wherein ring contain 0-3 nitrogen atoms; wherein E.sup.1, E.sup.2, E.sup.3, E.sup.4, E.sup.5 represents optional substituents, which are selected from: OR.sub.B, OC(O)R.sub.C, OC(O)OR.sub.B, OC(O)N(R.sub.A)R.sub.A, C(O)R.sub.C, C(O)N(R.sub.A)R.sub.A, C(O)N(OR.sub.B)R.sub.A, C(O)OR.sub.B, C(S)R.sub.C, C(O)C(O)R.sub.C, CH.sub.2OR.sub.B, CH.sub.2CH.sub.2OR.sub.B, CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2OCH.sub.2R.sub.C, CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, SR.sub.D, S(O)R.sub.D, SO.sub.2R.sub.D, SO.sub.2N(R.sub.A)R.sub.A, SO.sub.3R.sub.B, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)C(O)N(R.sub.A)R.sub.A, N(R.sub.A)SO.sub.2R.sub.D, N(R.sub.A)SO.sub.2NR.sub.A)R.sub.A, N(R.sub.A)R.sub.A, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)N(R.sub.A)R.sub.A, N(R.sub.A)N(R.sub.A)C(O)R.sub.C, NO.sub.2, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, NH.sub.2, SCN, SO.sub.2CN, F, Cl, Br, I, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, C.sub.nH.sub.2nR.sub.C which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n2)R.sub.C in E or Z geometrical conformation which branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n4)R.sub.C which is branched or unbranched wherein n is an integer from 2 to 5; wherein R.sub.A, R.sub.A, R.sub.A are each independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, OH; wherein R.sub.B is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I; wherein R.sub.C is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I, F, Cl, Br, I, NH.sub.2, wherein R.sub.D is independently selected from the group consisting of: H, lower alkyl group;

(16) An exemplary embodiment of the compound is represented by the following structures:

(17) ##STR00003##

(18) An exemplary embodiment of the compound has effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II and where a mutation F508-CFTR, or another mutation of class II are involved in CFTR protein malfunction.

(19) In an exemplary embodiment the CFTR protein malfunction occurs in the protein associated with the disease cystic fibrosis.

(20) A further exemplary embodiment is a modulator according to the above, for use in the treatment of cystic fibrosis wherein it has effect on CFTR-dependent ion transport across cellular membrane and/or it has the ability to increase the number of mutant CFTR proteins that reach the cell surface.

(21) An exemplary embodiment is used in the treatment of cystic fibrosis wherein it has stabilizing effect on the structure of the mutant CFTR protein and/or blocks the interaction with cellular proteins responsible for the premature degradation of mutant CFTR

(22) An exemplary embodiment is used in the treatment of cystic fibrosis wherein it has effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II.

(23) An exemplary embodiment are compounds, modulators of a mutant CFTR protein, of general formula (II):

(24) ##STR00004##
its tautomers, E and Z geometrical isomers, optically active forms such as enantiomers, diastereomers and their racemate forms or a mixture of stereoisomeric forms or its pharmaceutically acceptable salts thereof or complexes thereof; wherein Q.sup.1 and Q.sup.2 are independently selected from the group consisting of: C, CH, N, NH; wherein A is a fused five-membered ring having 0-3 independently selected heteroatoms wherein the heteroatoms comprise nitrogen, sulfur or oxygen; wherein R.sup.4, R.sup.5 and R.sup.6 represent optional substituents, which are independently selected from: OR.sub.B, OC(O)R.sub.C, OC(O)OR.sub.B, OC(O)N(R.sub.A)R.sub.A, C(O)R.sub.C, C(O)N(R.sub.A)R.sub.A, C(O)N(OR.sub.B)R.sub.A, C(O)OR.sub.B, C(S)R.sub.C, C(O)C(O)R.sub.C, CH.sub.2OR.sub.B, CH.sub.2CH.sub.2OR.sub.B, CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2OCH.sub.2R.sub.C, CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, SR.sub.D, S(O)R.sub.D, SO.sub.2R.sub.D, SO.sub.2N(R.sub.A)R.sub.A, SO.sub.3R.sub.B, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)C(O)N(R.sub.A)R.sub.A, N(R.sub.A)SO.sub.2R.sub.D, N(R.sub.A)SO.sub.2N(R.sub.A)R.sub.A, N(R.sub.A)R.sub.A, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)N(R.sub.A)R.sub.A, N(R.sub.A)N(R.sub.A)C(O)R.sub.C, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, NH.sub.2, SCN, SO.sub.2CN, F, Cl, Br, I, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, which may be optionally preceded by: C.sub.nH.sub.(2n1)R.sub.C which is branched or unbranched wherein n is an integer from 1 to 4; C.sub.nH.sub.(2n3)R.sub.C in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n5)R.sub.C which is branched or unbranched wherein n is an integer from 2 to 5; wherein Z.sup.2 is selected from: a single bond, N(R.sub.A), S, S-alkil-, O, O-alikil-, C(O), S(O), OC(O), C(O)N(R.sub.A), OC(O)N(R.sub.A), C(O)O, SO.sub.2, SO.sub.2N(R.sub.A), N(R.sub.A)SO.sub.2, N(R.sub.A)SO.sub.2N(R.sub.A), CH.sub.2O, N(R.sub.A)C(O), N(R.sub.A)C(O)O, N(R.sub.A)C(O)N(R.sub.A), C(O)C(O), N(R.sub.A)C(O)O, N(R.sub.A)N(R.sub.A), N(R.sub.A)N(R.sub.A)C(O), C(O)N(R.sub.A)N(R.sub.A), CH.sub.2N(R.sub.A), CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2N(R.sub.A), CH.sub.2OCH.sub.2, CH.sub.2N(R.sub.A)CH.sub.2, C.sub.nH.sub.2n which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n2) in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n4) which is branched or unbranched wherein n is an integer from 2 to 5; wherein R.sup.7 are independently selected from the group consisting of: H, aromatic ring or heteroaromatic ring; wherein Z.sup.3 is selected from: a single bond, double bond, N(R.sub.A), S, S-alkyl-, O, O-alkyl-C(O), C(S), OC(O), C(O)N(R.sub.A), OC(O)N(R.sub.A), C(O)O, SO.sub.2, SO.sub.2N(R.sub.A), N(R.sub.A)SO.sub.2, N(R.sub.A)SO.sub.2N(R.sub.A), CH.sub.2O, N(R.sub.A)C(O), N(R.sub.A)C(O)O, N(R.sub.A)C(O)N(R.sub.A), C(O)C(O), N(R.sub.A)C(O)O, N(R.sub.A)N(R.sub.A), N(R.sub.A)N(R.sub.A)C(O), C(O)N(R.sub.A)N(R.sub.A), CH.sub.2N(R.sub.A), CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2N(R.sub.A), CH.sub.2OCH.sub.2, CH.sub.2N(R.sub.A)CH.sub.2, C.sub.nH.sub.2n which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n2) in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n4) which is branched or unbranched wherein n is an integer from 2 to 5; wherein R.sup.8 is selected from: H, O, S, aromatic ring or heteroaromatic ring; C.sub.nH.sub.(2n+1) which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n1) in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n3) which is branched or unbranched wherein n is an integer from 2 to 5; wherein R.sub.A, R.sub.A, R.sub.A are each independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, OH; wherein R.sub.B is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I; wherein R.sub.C is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I, F, Cl, Br, I, NH.sub.2; wherein R.sub.D is independently selected from the group consisting of: H, lower alkyl group; wherein the 5-membered ring A is moiety selected from the group consisting of:

(25) ##STR00005## and wherein compound 6-[(4-oxo-1H-quinolin-3-yl)carbonylamino]-1H-indole-4-carboxylic acid ethyl ester is excluded, as a modulator of a mutant CFTR protein, for use in the manufacture of a medicament for the treatment of diseases associated with CFTR protein malfunction.

(26) An exemplary embodiment of the compound described above has the general formula (IIa) or (IIb):

(27) ##STR00006##
wherein Q.sup.1, Q.sup.2, Q.sup.3, Q.sup.4, Q.sup.5 represent optional substituents which are independently selected from the group consisting of: OR.sub.B, OC(O)R.sub.C, OC(O)OR.sub.B, OC(O)N(R.sub.A)R.sub.A, C(O)R.sub.C, C(O)N(R.sub.A)R.sub.A, C(O)N(OR.sub.B)R.sub.A, C(O)OR.sub.B, C(S)R.sub.C, C(O)C(O)R.sub.C, CH.sub.2OR.sub.B, CH.sub.2CH.sub.2OR.sub.B, CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2OCH.sub.2R.sub.C, CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, SR.sub.D, S(O)R.sub.D, SO.sub.2R.sub.D, SO.sub.2N(R.sub.A)R.sub.A, SO.sub.3R.sub.B, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)C(O)N(R.sub.A)R.sub.A, N(R.sub.A)SO.sub.2R.sub.D, N(R.sub.A)SO.sub.2N(R.sub.A)R.sub.A, N(R.sub.A)R.sub.A, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)N(R.sub.A)R.sub.A, N(R.sub.A)N(R.sub.A)C(O)R.sub.C, NO.sub.2, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, NH.sub.2, SCN, SO.sub.2CN, F, Cl, Br, I, C.sub.nH.sub.2nR.sub.C which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n2)Rc in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n4)Rc which is branched or unbranched wherein n is an integer from 2 to 5; PO.sub.3H.sub.2, OPO.sub.3H.sub.2.

(28) An exemplary embodiment of the compound may be represented by the following structure:

(29) ##STR00007##

(30) An exemplary embodiment of the compound has effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II and where a mutation F508-CFTR, or another mutation of class II are involved in CFTR protein malfunction.

(31) In an exemplary embodiment the CFTR protein malfunction occurs in the protein associated with the disease cystic fibrosis.

(32) A further exemplary embodiment is a modulator according to the above, for use in the treatment of cystic fibrosis wherein it has effect on CFTR-dependent ion transport across cellular membrane and/or it has the ability to increase the number of mutant CFTR proteins that reach the cell surface.

(33) An exemplary embodiment is used in the treatment of cystic fibrosis wherein it has stabilizing effect on the structure of the mutant CFTR protein and/or blocks the interaction with cellular proteins responsible for the premature degradation of mutant CFTR

(34) An exemplary embodiment is used in the treatment of cystic fibrosis wherein it has effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II.

(35) An exemplary embodiment is a compound of general formula (III):

(36) ##STR00008##
its tautomers, E and Z geometrical isomers, optically active forms such as enantiomers, diastereomers and their racemate forms or a mixture of stereoisomeric forms or its pharmaceutically acceptable salts thereof or complexes thereof; wherein Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.6, Z.sup.7 represents optional substituents, which are selected from substituents consisting at least one atom selected from the group consisting of: C, N, S, O, H, P, F, Cl, Br, I; wherein R.sup.4 represents optionally substituted moiety of formula (IIIa):

(37) ##STR00009## wherein R.sup.5 and R.sup.6 are optional substituents which are independently selected from the group consisting of: OH, NH.sub.2, COOH, Cl, Br, I, CH.sub.3, C.sub.2H.sub.5; and having a general formula (IIIb):

(38) ##STR00010## wherein R.sup.7 is an optional substituent which is independently selected from the group consisting of: F, Cl, Br, I, CH.sub.3, C.sub.2H.sub.5; wherein R.sup.8 is an optional substituent which is independently selected from the group consisting of: NH.sub.2, NHAr, OH, CH.sub.2Ar, C(O)Ar, OAr; wherein Ar is an aromatic group or heteroaromatic group; wherein Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.6, Z.sup.7 represent optional substituents which are independently selected from the group consisting of: OR.sub.B, OC(O)R.sub.C, OC(O)OR.sub.B, OC(O)N(R.sub.A)R.sub.A, C(O)R.sub.C, C(O)N(R.sub.A)R.sub.A, C(O)N(OR.sub.B)R.sub.A, C(O)OR.sub.B, C(S)R.sub.C, C(O)C(O)R.sub.C, CH.sub.2OR.sub.B, CH.sub.2CH.sub.2OR.sub.B, CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2CH.sub.2N(R.sub.A)R.sub.A, CH.sub.2OCH.sub.2R.sub.C, CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, SR.sub.D, S(O)R.sub.D, SO.sub.2R.sub.D, SO.sub.2N(R.sub.A)R.sub.A, SO.sub.3R.sub.B, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)C(O)N(R.sub.A)R.sub.A, N(R.sub.A)SO.sub.2R.sub.D, N(R.sub.A)SO.sub.2N(R.sub.A)R.sub.A, N(R.sub.A)R.sub.A, N(R.sub.A)C(O)R.sub.C, N(R.sub.A)C(O)OR.sub.B, N(R.sub.A)N(R.sub.A)R.sub.A, N(R.sub.A)N(R.sub.A)C(O)R.sub.C, NO.sub.2, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, NH.sub.2, SCN, SO.sub.2CN, F, Cl, Br, I, C.sub.nH.sub.2nRc which is branched or unbranched wherein n is an integer from 1 to 5; C.sub.nH.sub.(2n2)R.sub.C in E or Z geometrical conformation which is branched or unbranched wherein n is an integer from 2 to 5; C.sub.nH.sub.(2n4)R.sub.C which is branched or unbranched wherein n is an integer from 2 to 5, PO.sub.3H.sub.2, OPO.sub.3H.sub.2; wherein R.sub.A, R.sub.A, R.sub.A are each independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, OH; wherein R.sub.B is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I; wherein R.sub.C is independently selected from the group consisting of: H, lower alkyl group, CN, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I, F, Cl, Br, I, NH.sub.2; wherein R.sub.D is independently selected from the group consisting of: H, lower alkyl group, as a modulator of a mutant CFTR protein, for use in the manufacture of a medicament for the treatment of diseases associated with CFTR protein malfunction.

(39) An exemplary embodiment of the compound is represented by the following structure:

(40) ##STR00011##

(41) An exemplary embodiment of the compound has effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II and where a mutation F508-CFTR, or another mutation of class II are involved in CFTR protein malfunction.

(42) In an exemplary embodiment the CFTR protein malfunction occurs in the protein associated with the disease cystic fibrosis.

(43) A further exemplary embodiment is a modulator according to the above, for use in the treatment of cystic fibrosis wherein it has effect on CFTR-dependent ion transport across cellular membrane and/or it has the ability to increase the number of mutant CFTR proteins that reach the cell surface.

(44) An exemplary embodiment is used in the treatment of cystic fibrosis wherein it has stabilizing effect on the structure of the mutant CFTR protein and/or blocks the interaction with cellular proteins responsible for the premature degradation of mutant CFTR

(45) An exemplary embodiment is used in the treatment of cystic fibrosis wherein it has effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II.

(46) An exemplary embodiment is a compound of general formula (IV):

(47) ##STR00012##
its esters, ethers, tautomers, E and Z geometrical isomers, optically active forms such as enantiomers, diastereomers and their racemate forms or a mixture of stereoisomeric forms or its pharmaceutically acceptable salts thereof or complexes thereof; wherein E.sup.1, E.sup.2 represent substituents which are independently selected from: H, CH.sub.3, C.sub.2; wherein E represents optional substituent selected from: Cl, F, Br, I, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2Br, CH.sub.2I, optionally substituted lower alkyl group;

(48) An exemplary embodiment of the compound is represented by the following structure:

(49) ##STR00013##

(50) An exemplary embodiment of the compound has effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II and where a mutation F508-CFTR, or another mutation of class II are involved in CFTR protein malfunction.

(51) In an exemplary embodiment the CFTR protein malfunction occurs in the protein associated with the disease cystic fibrosis.

(52) A further exemplary embodiment is a modulator according to the above, for use in the treatment of cystic fibrosis wherein it has effect on CFTR-dependent ion transport across cellular membrane and/or it has the ability to increase the number of mutant CFTR proteins that reach the cell surface.

(53) An exemplary embodiment is used in the treatment of cystic fibrosis wherein it has stabilizing effect on the structure of the mutant CFTR protein and/or blocks the interaction with cellular proteins responsible for the premature degradation of mutant CFTR

(54) An exemplary embodiment is used in the treatment of cystic fibrosis wherein it has effect on mutant CFTR protein, wherein said CFTR mutation is a mutation F508-CFTR, or another mutation of class II.

(55) FIG. 1

(56) The effects of different compounds on iodide efflux at 1 M in F508-CFTR HeLa cells.

(57) (a) bar graph showing the peak amplitudes of Fsk/Gsk dependent iodide effluxes in cells treated by the different drugs as in A. Values are mean of 3 independent experiments. *p<0.05, **p<0.01.

(58) (b) chemical structures of active correctors identified in silico

(59) (c) examples of iodide efflux curves obtained in HeLa cells stably transfected with F508-CFTR and treated for 24 hours with 10 M with different compounds. CFTR dependent response was induced by 10 M Forskolin (Fsk)+30 M Genistein (Gsk) as indicated by the horizontal bar above the traces.

(60) (d) EC50 was determined for active compounds of pocket 2: 407882 and 73100 and one of pocket 1: 130813, for 118208 EC50 could not be precisely determined since the maximum of iodide efflux was not reached even at 100 M (also shown).

(61) FIG. 2

(62) To test whether the compounds exhibit potentiator activity independent of their effect on CFTR trafficking, we examined iodide efflux in untreated WT-CFTR HeLa cells. Compounds were added along with forskolin and their effects were compared to that of forskolin alone or forskolin plus genistein. Unlike genistein, all tested molecules induced an I.sup. efflux greater than that of forskolin alone.

(63) FIG. 3

(64) Potentiation was also tested in F508-CFTR HeLa cells treated for 2 hours with miglustat to rescue F508-CFTR. I.sup. efflux was stimulated either with forskolin alone, with forskolin plus genistein or forskolin plus the different compounds. As shown in the Figure, only genistein was able to increase efflux, demonstrating the absence of potentiation activity by The drugs.

(65) FIG. 4

(66) Impact of identified correctors on F508-CFTR maturation and cell localization.

(67) (a) Effects of different compounds on CFTR processing. Representative immunoblots of WT-CFTR and F508-CFTR proteins of the proteins from HeLa cells treated with 1 M of the different compounds for 24 hours with Mab 24-1. The positions of the mature (band C) and immature (band B) forms of CFTR are indicated.

(68) (b) Comparison of relative intensity (C/B+C) for WT-CFTR, F508-CFTR alone and F508-CFTR after correction with our molecules.

(69) (c) Effects of the different compounds used at 1 M on CFTR localization. Confocal imaging showing the plasma membrane localisation of WT-CFTR and intracellular localisation of F508-CFTR. The effect of drugs is illustrated in panels c to f. Bars: 20 M. Arrows indicate staining of CFTR at the plasma membrane.

(70) FIG. 5

(71) Synergistic effect of active compounds on iodide efflux tested at 1 M.

(72) (a) Iodide efflux in response to 1 M Forskolin (Fsk)+30 M Genistein (Gsk) as indicated by the horizontal bar above the traces, for cells treated for 24 h with a tested compounds alone and in combination as fallows (a) 407882, 118208, (b) 118208, 73100 (c) 407882, 37173. (d) bar graph showing the peak amplitudes of Fsk/Gst dependent iodide effluxes in cells treated by the different drugs as in. Values are mean of 3 independent experiments. *p<0.05, **p<0.01

(73) FIG. 6

(74) (a)/(b) Current-voltage relationship for cAMP-dependent chloride currents in HeLa cells treated with 407882(12) plus 118208(6) compounds at 1 M.

(75) FIG. 7

(76) The effects of different compounds on iodide efflux at 1 M in an epithelial serous cell line derived from a F508 CF patient (CF-4KM) cells. The concentration-dependence has been shown for the most potent molecule 407882

(77) FIG. 8

(78) The effect of73100 plus 118208 molecules on nasal potential difference (V.sub.TE) in F508/F508 mice. Basal V.sub.TE values and V.sub.TE changes induced by perfusion of nasal epithelium with 100 M amiloride, V.sub.TEamil were similar in mice treated with the two molecules or with liposomes alone. Perfusion of low Cl.sup. solution in 3 out of 5 mice hyperpolarized V.sub.TE by more than 2 mV (V.sub.TEamil-lowCl) i.e. the threshold value established by us as significant effect of treatment. The CFTR-related current unmasked by CFTR inhibitor I.sub.Inh172 represents about 30% of (V.sub.TEamil-lowCl) (data not shown).

(79) For a better understanding of some exemplary embodiments, the examples of the subject matter are disclosed below.

EXAMPLES

(80) Materials and Methods

(81) Antibodies

(82) The following antibodies were used: MAB25031 (clone 24-1, R&D systems, USA) and MM13-4 (Upstate,) monoclonal antibodies (mAb) for CFTR detection; Fluorescent secondary antibodies Alexa 594 and 488 were purchased from Molecular Probes (Cergy Pontoise, France)

(83) Cell Culture

(84) Stably transfected HeLa cells (with pTracer plasmid alone as a control (pTracer) or expressing WT-CFTR (spTCF-WT), F508-CFTR s(pTCF-F508del) were provided by Pascale Fanen (Inserm U.468, Crteil, France) and grown as described in Bobadilla J L, Macek M, Jr., Fine J P, Farrell P M. Cystic fibrosis: a worldwide analysis of CFTR mutationscorrelation with incidence data and application to screening. Hum Mutat, 2002 June; 19(6):575-606. Briefly, HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 g/ml streptomycin and 250 g/ml zeocin. Cultures were done at 37 C. in a humidified incubator with 5% CO.sub.2. The expression of WT-CFTR and F508-CFTR in these cells was verified by immunoprecipitation and immunocytochemistry throughout the study. Treatments with different molecules (at 1 and 10 M) and vehicle were done when cells reached 75% confluence.

(85) CF-KM4 cell line, obtained by transformation of primary cultures of CF tracheal gland serous cells homozygous for the F508 mutation by using the wild-type SV40 virus, were grown as described elsewhere in the art (Antigny, F. et al. Calcium homeostasis is abnormal in cystic fibrosis airway epithelial cells but is normalized after rescue of F508del-CFTR. Cell calcium 43, 175-83(2008)).

(86) Immunoblot Experiments

(87) Cells cultured in 75 cm.sup.2 flasks were washed twice with ice cold PBS, scraped in 2 ml PBS and centrifuged at 600 g for 5 min. The pellets were suspended in 300 l RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% TritonX-100, 1% Na deoxycholate, 0.1% SDS, pH 7.5) at 4 C. for 30 min with agitation After centrifugation at 15000 g for 30 min the supernatants were processed for immunoblot experiments as previously described in the art (Baudouin-Legros, M. et al. Control of basal CFTR gene expression by bicarbonate-sensitive adenylyl cyclase in human pulmonary cells. Cellular physiology and biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology 21, 75-86(2008)) with slight modifications.

(88) The samples were resolved by 8% SDS-PAGE, transferred onto PVDF membranes and analysis was performed following manufacturer's recommendations for the Odyssey infrared imaging system (LI-COR Biosciences, NE, USA). Blot membranes were blocked with Odyssey buffer (ScienceTec, Paris, France) for 1 hour and hybridized using monoclonal anti CFTR Mab24-1 (1/1000). The proteins were visualized by incubation with secondary antibodies (1/10000) and detected using ECL technique as described in, Bensalem, N. et al. Down-regulation of the anti-inflammatory protein annexin A1 in cystic fibrosis knock-out mice and patients. Molecular & cellular proteomics: MCP 4, 1591-601(2005).

(89) Immunofluorescence Staining

(90) HeLa cells grown on glass coverslips were treated as above and as described in Lipecka, J. et al., Distribution of ClC-2 chloride channel in rat and human epithelial tissues. American journal of physiology. Cell physiology 282, C805-16(2002). Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton in PBS. Cells were blocked with 1% bovine serum albumine in PBS/Triton and incubated at 4 C. overnight with the primary antibodies, 24-1 (1:300). After washing and blocking with 5% normal goat serum, cells were incubated with the secondary antibodies. Glass coverslips were mounted using the Vectashield mounting medium (Vector laboratories) and examined by confocal laser microscopy (Zeiss, LSM 510).

(91) Iodide Efflux Experiments

(92) CFTR chloride channel activity was assayed by measuring iodide (.sup.125I) efflux from transfected CHO cells as described previously in the art, Marivingt-Mounir, C, et al., Synthesis, SAR, crystal structure, and biological evaluation of benzoquinoliziniums as activators of wild-type and mutant cystic fibrosis transmembrane conductance regulator channels. J Med Chem, 2004. 47(4): p. 962-72. Marivingt-Mounir, C, et al., Synthesis, SAR, crystal structure, and biological evaluation of benzoquinoliziniums as activators of wild-type and mutant cystic fibrosis transmembrane conductance regulator channels. J Med Chem, 2004. 47(4): p. 962-72. Cells grown for 4 days in 96-well plates were washed twice with 2 ml of modified Earle's salt solution containing 137 mM NaCl, 5.36 mM KCl, 0.4 mM Na.sub.2HPO.sub.4, 0.8 mM MgCl.sub.2, 1.8 mM CaCl.sub.2, 5.5 mM glucose, and 10 mM HEPES, pH 7.4. Cells were then incubated in the same medium containing 1 mM KI (1 mCi of Na.sup.125I/ml, NEN Life Science Products) for 30 min at 37 C. After washing, cells were incubated with 1 ml of modified Earle's salt solution. After 1 min, the medium was removed to be counted and was quickly replaced by 1 ml of the same medium. This procedure was repeated every 1 min for 8 min. The first three aliquots were used to establish a stable baseline in efflux buffer alone. Medium containing cocktail aiming to increase intracellular cAMP (10 M forskolin and 30 M genistein) was used for next aliquots in order to activate CFTR chloride channels. At the end of the incubation, the medium was recovered, and cells were solubilized in 1 N NaOH. The radioactivity was determined using a g-counter (LKB). The total amount of .sup.125I (in cpm) at time 0 was calculated as the sum of cpm counted in each 1-min sample plus the cpm in the NaOH fraction. The fraction of initial intracellular .sup.125I lost during each time point was determined, and time-dependent rates of .sup.125I efflux were calculated according to the art in, Becq, F., et al., Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel J Biol Chem, 1999. 274(39): p. 27415-25, from ln(.sup.125I.sub.t1/.sup.125I.sub.t2)/(t.sub.1t.sub.2), where

(93) .sup.125It is the intracellular .sup.125I at time t; and

(94) t.sub.1 and t.sub.2 are successive time points.

(95) Curves were constructed by plotting rate of .sup.125I efflux versus time. Data are presented as the meanS.E. of n separate experiments.

(96) Differences were considered statistically significant using the Student's t test when the p value was less than 0.05.

(97) Whole Cell Patch-Clamp Recordings

(98) Technique for patch-clamp recordings in the whole cell configuration has been described, such as in Hinzpeter, A. et al. Association between Hsp90 and the ClC-2 chloride channel upregulates channel function. American journal of physiology. Cell physiology 290, C45-56(2006) and Tanguy, G. et al. CSN5 binds to misfolded CFTR and promotes its degradation. Biochimica et biophysica acta 1783, 1189-99(2008). Stably transfected cells were plated in 35 mm cell culture plastic Petri dishes that were mounted on the stage of an inverted microscope. Patch-clamp experiments were performed at room temperature with an Axopatch 200A amplifier controlled by a computer via a digitdata 1440 interface (Axon Intruments, USA). Pipettes were pulled from hard glass (Kimax 51) using a Setter micropipette puller and their tips were fire-polished. Current recordings were performed using the nystatin-perforated patch clamp configuration. Nystatin stock solution (50 mg/ml) was prepared daily in DMSO. The stock solution was diluted (1:250) in the internal solution which was then sonicated during 1 minute. The internal solution contained the following (in mM): 131 NaCl, 2 MgCl.sub.2, and 10 Hepes-Na.sup.+, pH 7.3, adjusted with NaOH. The bath solution contained (in mM): 150 NaCl, 1 CaCl.sub.2, 1 MgCl.sub.2, 35 sucrose and 10 Hepes-Na.sup.+, pH 7.3, adjusted with NaOH.

(99) Currents were recorded by application of regular voltage pulses of 60 mV amplitude during 1 second, from a holding potential of 0 mV, with an interval of 3 seconds.

(100) To establish I-V curves, regular voltage pulses were interrupted by series of 9 voltage jumps (1-s duration each), toward membrane potentials between 100 and +80 mV. CFTR Cl-currents were activated with 200 m 8-(4-chlorophenylthio)-cAMP sodium salt (CPT-cAMP) plus 100 M 3-isobutyl-1-methylxanthine (IBMX).

(101) When maximal stimulation was reached, cells were bathed with 5 M of the specific CFTRinhibitor, CFTR.sub.inh-172, added to the CPT-cAMP solution. CFTR-currents were defined as the differences in current amplitudes recorded during maximum stimulation by CPT-cAMP and after inhibition by CFTR.sub.inh-172.

(102) Nasal Potential Difference (NPD) Measurements

(103) The method for nasal potential measurement was adapted and miniaturised from the technique developed for young children as shown in Sermet-Gaudelus, I. et al. Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis. Thorax 65, 539-44(2010). Mice were anesthetized by an intraperitoneal injection of ketamine (133 mg/kg; IMALGENE 1000, MERIAL, France) and xylazine (13.3 mg/kg; Rompun 2%, BayerPharma, France). Mice were positioned on a 45 tilt board and a paper pad was placed under the nose to avoid mice quelling. A subcutaneous needle was connected to an Ag.sup.+/AgCl reference electrode by an agar bridge. A double-lumen polyethylene catheter (0.5 mm diameter) was inserted into one nostril 4 mm depth. One lumen perfused by a Ringer solution (in mM: 140 NaCl, 6 KCl, 10 Hepes, 10 Glucose, 1 MgCl2, 2 CaCl2, pH adjusted to 7,4 with NaOH) at 0.15 mL/h is connected to a measuring Ag.sup.+/AgCl electrode. The two Ag.sup.+/AgCl electrodes were connected to a high-impedance voltmeter (LOGAN research Ltd, United Kingdom). The second lumen perfused solution with the following sequence: (1) Ringer solution, (2) Ringer solution containing amiloride (inhibitor of Na.sup.+ conductance, 100 M), (3) Low Chloride Ringer solution, to unmask Cl.sup. conductances (in mM: 140 Na gluconate, 6 K gluconate, 10 Hepes, 10 Glucose, 1 MgCl.sub.2, 6 Ca-gluconate, pH adjusted to 7.4 with NaOH), (4) Low Chloride Ringer solution containing CFTR inhibitor-172 (5 M, Calbiochem, Germany) to evaluate the participation of CFTR. Each solution was perfused at least 3 minutes, and 30 seconds stability was required before perfusion switch. Steady state transepithelial potential, V.sub.TE, V.sub.TEAmil (difference between V.sub.TE and transepithelial potential recorded after perfusion of amiloride-containing solution), V.sub.TEamilLowCl (difference between V.sub.TE and transepithelial potential recorded after perfusion with Low Cl.sup. plus amiloride-containing solution) and V.sub.TEamilLowClInh-172 (difference between V.sub.TE and after addition of CFTR inhibitor to the previous solution) were the means of 30 values recorded during stability.

(104) MTT Cell Viability Assay

(105) To determine cell viability the typical MTT assay was used. HeLa cells were cultured in a 96-well plate and exposed to varying concentrations of compounds disclosed herein for 24 h. After washing, MTT solution and medium were then introduced. After incubation, the resultant formazan crystals were dissolved in dimethyl sulfoxide and the absorbance intensity measured by a microplate reader at 570 nm.

(106) Yellow MTT is reduced to purple formazan in living cells. The absorbance of this colored solution can be quantified by measuring at a certain wavelength by a spectrophotometer. This conversion can be directly related to the number of viable (living) cells.

(107) Virtual ScreeningIdentification of Modulator Compounds

(108) A database of a low molecular weight compounds was used in the virtual screening process as a source of hits. Molecular docking program Dock 6.1 was used to test a conformational space of small molecules inside two potential binding sites on the protein surface. Subsequently, all selected ligands and whole complexes were fully minimized in force field. At each step, a set of scoring functions was used for rating of potential complexes and appropriate molecules were selected for experimental tests.

(109) TABLE-US-00001 118208/NOP1.6/Pok 1c 2-[(3-nitrophenyl)methylsulfanyl]-3,7- dihydropurin-6-one 407882/NOP2.6/Pok 2f 2-[hydroxy(phenyl)phosphoryl]ethyl- phenylphosphinic acid 130813/NOP1.2/Pok1b 4-((6-chloro-2-methoxy-9- acridinyl)amino)-2-((4-methyl-1- piperazinyl)methyl)phenol 73100/Pok2e 2-(9H-fluoren-2- ylcarbamoyl)terephthalic acid
Results
Effect of Drugs on Iodide, I, Efflux

(110) To test drug correction of F508-CFTR trafficking and function we evaluated halide permeability by a macroscopic assay using a robotic cell-based methodology using the I.sup. efflux technique. In the first series of experiments, the potential corrector effects were tested by 24 hour pre-treatment of F508-CFTR HeLa cells with all compounds at 1 M followed by measurements of cAMP-dependent radiolabel iodide efflux. Treatments with compounds 130813 and 118208 on pocket 1 and 73100 and 407882 on pocket 2, lead to significant increase of cAMP-stimulated radiolabel iodide efflux (FIG. 1a), the most potent being 407882. At this low dose (1 M) the increase in the cAMP-stimulated efflux was lower than that observed using 100 M of the known corrector miglustat(27). Examples of I.sup. efflux stimulation after treatment with each of the four active compounds are illustrated in FIG. 1b. cAMP-stimulated I.sup. efflux was completely prevented when experiments were performed in the presence of the CFTR channel blocker CFTR.sub.inh-172.

(111) We further tested the effect of the four compounds in a wide range of concentrations and determined EC.sub.50 for pocket 1 compound 130813, and two pocket 2 compounds 407882 and 73100 at 1 M, 10 M and 844 nM, respectively (FIG. 1c). The EC.sub.50 for pocket 1118208 could not be precisely determined since the maximum iodide efflux was not reached even at 100 M (FIG. 1d). Notably, the effect of compound 407882 could be increased by 3-fold when used at 10 M, reaching a stimulated efflux comparable to the value observed for WT-CFTR (FIG. 2).

(112) To test whether the compounds exhibit potentiator activity independent of their effect on CFTR trafficking, we examined iodide efflux in untreated WT-CFTR HeLa cells. Compounds were added along with forskolin and their effects were compared to that of forskolin alone or forskolin plus genistein. Unlike genistein, all tested molecules induced an I.sup. efflux greater than that of forskolin alone (FIG. 2). Potentiation was also tested in F508-CFTR HeLa cells treated for 2 hours with miglustat to rescue F508-CFTR. I.sup. efflux was stimulated either with forskolin alone, with forskolin plus genistein or forskolin plus the different compounds. As shown in FIG. 3, only genistein was able to increase efflux, demonstrating the absence of potentiation activity by our drugs.

(113) Effect of Drugs on CFTR Maturation

(114) The efficacy of the four compounds as correctors for F508-CFTR trafficking was further validated by immunoblotting. We assumed that detection of a fully glycosylated band C suggests correct processing of F508-CFTR. A representative immunoblot is shown in FIG. 4a. Anti-CFTR antibodies detect two bands in proteins derived from WT-CFTR cells, (line WT-CFTR in FIG. 4a). The diffuse band of approximately 170 kDa (band C) corresponds to a mature, fully glycosylated protein that has processed through the Golgi apparatus. The band below of about 145 kDa corresponds to the immature core-glycosylated protein located in the endoplasmic reticulum. In F508-CFTR expressing cells, only the immature protein is detectable (line F508 in FIG. 4a). Band C was clearly detectable in cells treated with 1 M of compound 407882 as compared to untreated cells, whereas the signal at 170 kDa was not different from DMSO treatment in cells treated with 1 M of compounds 118208, or 130813 or very slightly increased in cells treated by 1 M of compound 73100. None of the compounds modified total protein expression. The relative abundance of mature CFTR, expressed as the ratio of band C to band C+band B is shown in FIG. 4b. Only compound 407882 increased significantly the relative abundance of mature CFTR.

(115) Effect of Drugs on CFTR Immunolocalization

(116) FIG. 4c shows typical CFTR staining at the plasma membrane in WT-CFTR expressing HeLa cells whereas F508-CFTR was found throughout the cytoplasm. Treatment of cells for 24 hours with 1 M of 407882 resulted in a clear CFTR staining at or near the plasma membrane, indicating rescue of F508-CFTR trafficking in agreement with immunoblot experiments. When cells were treated by each of the three other compounds, 118208, 73100 or 130813, a discrete punctuate staining at the plasma membrane was observed in a small fraction of cells, as illustrated for compound 118208 in FIG. 4c.

(117) Combined Effect of Compounds Binding to Different Pockets.

(118) If two compounds are able to correct F508-CFTR by binding to the same protein conformation but at different surface cavities their effects could be additive or synergistic. We tested this hypothesis by two independent types of assays, namely iodide efflux and patch clamp. The results from iodide permeability tests (FIG. 5) showed that combined treatment of cells with compounds 118208 plus 407882 (FIG. 5a) or with 118208 plus 73100 (FIG. 5b) at 1 M of each, leads to greater cAMP-dependent anion fluxes than those observed with any of the molecules alone. In this series of experiments the compound 37173 (FIG. 5c) was used as a control as it did not induce any cAMP-stimulated iodide efflux at the same concentration. As shown in FIG. 5c, co-treatment of F508-CFTR HeLa cells with 37173 plus 407882 induced cAMP-stimulated iodide efflux with an amplitude similar to 407882 treatment alone. By contrast, co-treatment with compounds 118208 and 407882 induced iodide efflux with an amplitude equal to the sum of effluxes induced by each compound, whereas a slight synergistic effect was observed after treatment by 118208 plus 73100.

(119) The activity of the different compounds was also evaluated in patch-clamp experiments. FIG. 6a summarizes the mean values of current amplitudes recorded at 60 mV in the different experimental conditions. CFTR-related current density (I.sub.F508-CFTR; pA/pF) is defined as cAMP-stimulated current minus the current recorded after inhibition by CFTR inh-172 at 5 M, and normalized to cell capacitance. I.sub.F508-CFTR was very low in untreated cells and stimulated by around 3-fold when cells were cultured at 27 C. for 24 hours before recordings. Treatment of cells for 24 h with 1 M of either 118208, 407882 or 73100 alone, did not increase current amplitude as compared to their respective controls (data not shown). However, 24 h pre-treatment with 1 M of 118208 plus 407882 or 118208 plus 73100 showed a significant increase in I.sub.F508-CFTR. Examples of linear I/V plots from cells pretreated by 118208 plus 407882 before stimulation, in the presence of cptcAMP+IBMX and after inhibition by CFTRinh-172 are shown in FIG. 6b.

(120) Effects of 407882 and 118208 on CF-4KM Cells

(121) The effects of the four molecules active in HeLa cells were next tested on CFTR-dependent iodide efflux in an epithelial serous cell line derived from a F508 CF patient (CF-KM4) expressing low amounts of endogenous F508-CFTR. In these epithelial cells compounds 407882 and 118208 were still able to induce significant cAMP-dependent iodide efflux (FIG. 7). However, it must be noted that 2 molecules correcting F508-CFTR in Hela cells (130813 and 73100) were not active in this cell line.

(122) Effects of 73100 Plus 118208 on Nasal Potential Difference in F508 Mice.

(123) Our results in cells suggested that the pairs of molecules acting on different pockets display additive correcting effects. To test if these molecules are active in vivo, nasal potential difference (V.sub.TE) was monitored (as in Sermet-Gaudelus, I. et al., Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis. Thorax 65, 539-44(2010)) in F508/F508 mice treated intranasally for 24 hours with 30 l of 73100 plus 118208 molecules (0.1 mol each) embedded in liposomes (5:1) or with liposomes alone. In F508 mice, basal V.sub.TE values and V.sub.TE changes induced by perfusion of nasal epithelium with 100 M amiloride, V.sub.TEamil were similar in mice treated with the two molecules or with liposomes alone. By contrast, perfusion of low Cl.sup. solution in 3 out of 5 mice hyperpolarized V.sub.TE by more than 2 mV (V.sub.TEamil-lowCl) i.e. the threshold value established by us as significant effect of treatment (manuscript in preparation). The CFTR-related current unmasked by CFTR inhibitor I.sub.Inh172 represents about 30% of (V.sub.TEamil-lowCl) (data not shown).

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(125) Of course these methods are exemplary and alterations thereto are possible by those having skill in the relevant technology.

(126) Thus the example embodiments and arrangements achieve improved capabilities, eliminate difficulties encountered in the use of prior methods and systems, and attain the desirable results described herein.

(127) In the foregoing description, certain terms have been used for brevity, clarity and understanding. However, no unnecessary limitations are to be implied therefrom because such terms are used for descriptive purposes and are intended to be broadly construed.

(128) Moreover the descriptions and illustrations herein are by way of examples and the inventive scope is not limited to the features shown and described.

(129) Further, it should be understood that features and/or relationships associated with one embodiment can be combined with features and/or relationships from other embodiments. That is, various features and/or relationships from various embodiments can be combined in further embodiments. The inventive scope of the disclosure is not limited to only the embodiments shown or described herein.

(130) Having described the features, discoveries and principles of the exemplary embodiments, the manner in which they are utilized and carried out, and the advantages and useful results attained, the new and useful arrangements, combinations, methodologies, structures, devices, elements, combinations, operations, processes and relationships are set forth in the appended claims.