Lipophilicity of the Cystic Fibrosis Drug, Ivacaftor (VX-770), and Its Destabilizing Effect on the Major CF-causing Mutation: F508del.

Deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cystic fibrosis (CF)-causing mutation. Recently, ORKAMBI, a combination therapy that includes a corrector of the processing defect of F508del-CFTR (lumacaftor or VX-809) and a potentiator of channel activity (ivacaftor or VX-770), was approved for CF patients homozygous for this mutation. However, clinical studies revealed that the effect of ORKAMBI on lung function is modest and it was proposed that this modest effect relates to a negative impact of VX-770 on the stability of F508del-CFTR. In the current studies, we showed that this negative effect of VX-770 at 10 µM correlated with its inhibitory effect on VX-809-mediated correction of the interface between the second membrane spanning domain and the first nucleotide binding domain bearing F508del. Interestingly, we found that VX-770 exerted a similar negative effect on the stability of other membrane localized solute carriers (SLC26A3, SLC26A9, and SLC6A14), suggesting that this negative effect is not specific for F508del-CFTR. We determined that the relative destabilizing effect of a panel of VX-770 derivatives on F508del-CFTR correlated with their predicted lipophilicity. Polarized total internal reflection fluorescence microscopy on a supported lipid bilayer model shows that VX-770, and not its less lipophilic derivative, increased the fluidity of and reorganized the membrane. In summary, our findings show that there is a potential for nonspecific effects of VX-770 on the lipid bilayer and suggest that this effect may account for its destabilizing effect on VX-809- rescued F508del-CFTR.

Synthesis and Evaluation of Ivacaftor Derivatives with Reduced Lipophilicity.

Mutations in the unique ATP-binding cassette anion channel, the cystic fibrosis conductance regulator (CFTR), lead to the inherited fatal disease known as cystic fibrosis (CF). Ivacaftor enhances channel gating of CFTR by stabilizing its open state and has been approved as monotherapy for CF patients with CFTR gating mutations (e.g., G551D) and as part of combination therapy with lumacaftor for CFTR folding mutations (e.g., ΔF508). However, in the latter context, ivacaftor may destabilize folding-rescued ΔF508-CFTR and membrane-associated proteins and attenuate lumacaftor pharmacotherapy. Here, we tested the hypothesis that the high lipophilicity of ivacaftor may contribute to this effect. We describe the synthesis of three glutamic acid ivacaftor derivatives with reduced lipophilicity that bear different charges at neutral pH (compounds 2, 3, 4). In a cellular ion flux assay, all three restored G551D-CFTR channel activity at comparable or better levels than ivacaftor. Furthermore, unlike ivacaftor, compound 3 did not attenuate levels of folding-rescued ΔF508 at the cell surface. Molecular modeling predicts that the increased polarity of compound 3 allows engagement with polar amino acids present in the binding pocket with hydrogen bonding and ionic interactions, which are collectively higher in strength as compared to hydrophobic interactions that stabilize ivacaftor. Overall, the data suggests that reduced lipophilicity may improve the efficacy of this class of CFTR potentiators when used for folding-rescued ΔF508-CFTR.

Opportunities and Challenges for the Development of MRCK Kinases Inhibitors as Potential Cancer Chemotherapeutics.

Cytoskeleton organization and dynamics are rapidly regulated by post-translational modifications of key target proteins. Acting downstream of the Cdc42 GTPase, the myotonic dystrophy-related Cdc42-binding kinases MRCKα, MRCKß, and MRCKγ have recently emerged as important players in cytoskeleton regulation through the phosphorylation of proteins such as the regulatory myosin light chain proteins. Compared with the closely related Rho-associated coiled-coil kinases 1 and 2 (ROCK1 and ROCK2), the contributions of the MRCK kinases are less well characterized, one reason for this being that the discovery of potent and selective MRCK pharmacological inhibitors occurred many years after the discovery of ROCK inhibitors. The disclosure of inhibitors, such as BDP5290 and BDP9066, that have marked selectivity for MRCK over ROCK, as well as the dual ROCK + MRCK inhibitor DJ4, has expanded the repertoire of chemical biology tools to study MRCK function in normal and pathological conditions. Recent research has used these novel inhibitors to establish the role of MRCK signalling in epithelial polarization, phagocytosis, cytoskeleton organization, cell motility, and cancer cell invasiveness. Furthermore, pharmacological MRCK inhibition has been shown to elicit therapeutically beneficial effects in cell-based and in vivo studies of glioma, skin, and ovarian cancers.

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease.

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

meso-4,5-Diphenyl-imidazolidin-2-one.

The crystal structure determination of the title compound, C(15)H(14)N(2)O, confirms the cis relationship between the phenyl groups at the 4- and 5-positions on the imidazolidine ring. The dihedral angle between the two phenyl rings is 48.14 (6)°. In the crystal structure, inter-molecular N-H⋯O hydrogen bonds link mol-ecules into centrosymmetric dimers. These dimers are, in turn, linked into a two-dimensional network via weak N-H⋯π(arene) inter-actions and π-π stacking inter-actions with centroid-centroid distances of 3.6937 (11) Å.

N,1-Bis(4-chloro-2-methyl-benz-yl)-3-methyl-2-oxo-1,2,3,4-tetra-hydro-quinoline-3-carboxamide.

In the title mol-ecule, C(27)H(26)Cl(2)N(2)O(2), the chloro-substituted benzene rings make dihedral angles of 83.29 (9) and 80.81 (9)° with the benzene ring of the tetra-hydro-quinoline group. The dihedral angle formed by the two chloro-substituted benzene rings is 40.87 (12)°. The six-membered N-containing ring is in a half-chair conformation. In the crystal structure, inter-molecular N-H⋯O hydrogen bonds link mol-ecules into centrosymmetric dimers.

Synthesis and properties of molecular probes for the rescue site on mutant cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is a genetic disease caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In vitro experiments have demonstrated that 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol (VRT-532, 1) is able to partially restore the function of mutant CFTR proteins. To help elucidate the nature of the interactions between 1 and mutant CFTR, molecular probes based on the structure of 1 have been prepared. These include a photoreactive aryl azide derivative 11 and a fluorescent dansyl sulfonamide 15. Additionally, a method for hydrogen isotope exchange on 1 has been developed, which could be used for the incorporation of radioactive tritium. Using iodide efflux assays, the probe molecules have been demonstrated to modulate the activity of mutant CFTR in the same manner as 1. These probe molecules enable a number of biochemical experiments aimed at understanding how 1 rescues the function of mutant CFTR. This understanding can in turn aid in the design and development of more efficacious compounds which may serve as therapeutic agents in the treatment of cystic fibrosis.

Copper-catalyzed domino annulation approaches to the synthesis of benzoxazoles under microwave-accelerated and conventional thermal conditions.

Two domino annulation approaches for benzoxazole synthesis have been developed. In the first approach, copper-catalyzed intermolecular cross-coupling of 1,2-dihaloarenes with primary amides initially forms the Ar-N bond of the benzoxazole ring, followed by copper-catalyzed intramolecular cyclization to form the Ar-O bond. Benzoxazoles were formed in good yields for the reaction of 1,2-dibromobenzene, but the reaction was not regioselective for the reaction of 3,4-dibromotoluene. Furthermore, the method is limited by the availability of 1,2-dihaloarenes. As a result of these limitations, an alternative more versatile one-pot domino annulation strategy was developed involving reaction of 2-bromoanilines with acyl chlorides in the presence of Cs2CO3, catalytic CuI, and the non-acylatable ligand 1,10-phenanthroline. Under these conditions initial acylation of the aniline is followed by copper-catalyzed intramolecular cyclization of the resultant 2-haloanilide to form the Ar-O bond of the benzoxazole ring. Optimized conditions using microwave irradiation achieved much shorter reaction times than conventional heating (i.e., 210 degrees C for 15 min versus 95 degrees C for 24 h) and were applied to the synthesis of a small library of benzoxazoles. These copper-catalyzed approaches complement existing strategies for benzoxazole synthesis, which typically utilize 2-aminopheonls as precursors.

A convenient and scalable synthesis of ethyl N-[(2-Boc-amino)ethyl]glycinate and its hydrochloride. Key intermediates for peptide nucleic acid synthesis.

An improved synthesis of ethyl N-[(2-Boc-amino)ethyl]glycinate and its hydrochloride salt is reported. The synthesis is based on the reductive alkylation of Boc-ethylenediamine with ethyl glyoxylate hydrate and furnishes the title compound in near quantitative yield and high purity without chromatography. This compound is suitable, as is, for the synthesis peptide nucleic acid monomers. Further, conversion to the hydrochloride salt provides a stable, nonhygroscopic solid that is a convenient form for handling and storage.