DOI: 10.1002/cmdc.201200311 Asymmetric 4-Aryl-1,4-dihydropyridines Potentiate Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Michele Giampieri, [a] Nicolas Vanthuyne, [b] Erika Nieddu, [a] Marco T. Mazzei, [a] Maria Anzaldi, [a] Nicoletta Pedemonte, [c] Luis J. V. Galietta, [c] Christian Roussel, [b] and Mauro Mazzei* [a] Introduction Cystic fibrosis (CF) is a genetic pathology caused by mutations in the cAMP-activated cystic fibrosis transmembrane conduc- tance regulator (CFTR) Cl À ion channel. Patients mainly suffer from pulmonary infection and inflammation, pancreatic insuffi- ciency, and male sterility. [1, 2] CFTR is a very complex plasma membrane protein composed of 1480 amino acids arranged in two transmembrane domains, each containing six a-helices, two nucleotide binding domains (NBD1 and NBD2), and a regu- latory domain. [3] At this point, more than 1900 mutations have been discovered, with the deletion of the phenylalanine resi- due at position 508 (DF508 CFTR) being the most common and one of the most severe mutations in CF patients (http:// www.genet.sickkids.on.ca/cftr/app). The severity of disease for each CF patient depends largely on the specific set of mutations that affect the CFTR gene. De- pending on the type of molecular defect, researchers have or- ganized the CFTR mutations into five classes: lack of protein synthesis (class I), CFTR protein maturation defect (class II), channel gating defect (class III), decrease in single channel con- ductance (class IV), and decreased CFTR synthesis (class V). [4–6] Compounds able to repair class II CFTR mutations (among which the most relevant is DF508) are defined as “correctors”, whereas compounds able to ameliorate the gating defect in class III mutations (which include G551D and G1349D) are de- fined as “potentiators”. [7–10] Among these latter potential thera- peutics, great interest has been placed on VX-770, [11, 12] a 4-qui- nolone derivative very recently approved by the USFDA and marketed as Kalydeco (http://www.cff.org/treatments/Pipeline). Notably, when cells with the DF508 mutation are incubated at low temperature, the mutant protein may escape from degra- dation and reach the plasma membrane. [13] However, the “res- cued DF508 CFTR” has decreased channel activity and low membrane stability. [14] These defects can be corrected by the same potentiators that are effective on G551D and G1349D mutations. [15] The precise mechanism of action of potentiators is still un- clear. Mutations of class III allow the normal trafficking of the CFTR protein to the plasma membrane, but impair channel gating by delaying channel opening and decreasing the stabili- ty of the open channel conformation. [8, 9] Potentiators such as genistein, [16] benzoflavones, [17] phenylglycines, [18] and others are thought to have one or more binding sites at the NBD1/NBD2 level, the occupancy of which by the potentiator molecule leads to partial restoration of channel activity. In this regard, many theoretical studies have been performed, particularly Some of the genetic mutations that cause cystic fibrosis (CF) impair the gating of the cystic fibrosis transmembrane conduc- tance regulator (CFTR) Cl À ion channel. This defect can be cor- rected with pharmacological tools (potentiators) that belong to various chemical families, including the 1,4-dihydropyridines (DHPs). A small set of asymmetric 4-aryl-DHPs was synthesized, and each racemic couple was tested in a functional assay car- ried out on cells expressing the G1349D, DF508, and G551D mutants. The most active racemates were subjected to chiral separation by HPLC, and the pure enantiomers were tested to evaluate any gains in activity. Although three enantiomers demonstrated high potency (K d values less than 0.09, 0.1, and 0.5 mm in G1349D, DF508, and G551D, respectively), in general, the screening of pure enantiomers did not produce a great di- versity in potency values. It is probable that the degree of DHP asymmetry considered in our analysis is still insufficient with respect to that allowed in a putative DHP binding site in CFTR, so that the site could equally accommodate both enantiomers. [a] Dr. M. Giampieri, Dr. E. Nieddu, Dr. M. T. Mazzei, Dr. M. Anzaldi, Prof. M. Mazzei Department of Pharmacy, University of Genova Viale Benedetto XV, 3, 16132 Genova (Italy) E-mail : mauro.mazzei@unige.it [b] Dr. N. Vanthuyne, Prof. C. Roussel Aix-Marseille University, ISM2-UMR-CNRS 7313, Chirosciences Avenue Escadrille Normandie-NiØmen, 13397 Marseille Cedex 20 (France) [c] Dr. N. Pedemonte, Dr. L. J. V. Galietta Laboratory of Molecular Genetics, Giannina Gaslini Institute Largo Gerolamo Gaslini, 5, 16147 Genova (Italy) ChemMedChem 0000, 00, 1 – 10  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &1& These are not the final page numbers! ÞÞ MED