Peter D. J. Grootenhuis

Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that impair the function of CFTR, an epithelial chloride channel required for proper function of the lung, pancreas, and other organs. Most patients with CF carry the F508del CFTR mutation, which causes defective CFTR protein folding and processing in the endoplasmic reticulum, resulting in minimal amounts of CFTR at the cell surface. One strategy to treat these patients is to correct the processing of F508del-CFTR with small molecules. Here we describe the in vitro pharmacology of VX-809, a CFTR corrector that was advanced into clinical development for the treatment of CF. In cultured human bronchial epithelial cells isolated from patients with CF homozygous for F508del, VX-809 improved F508del-CFTR processing in the endoplasmic reticulum and enhanced chloride secretion to approximately 14% of non-CF human bronchial epithelial cells (EC50, 81 ± 19 nM), a level associated with mild CF in patients with less disruptive CFTR mutations. F508del-CFTR corrected by VX-809 exhibited biochemical and functional characteristics similar to normal CFTR, including biochemical susceptibility to proteolysis, residence time in the plasma membrane, and single-channel open probability. VX-809 was more efficacious and selective for CFTR than previously reported CFTR correctors. VX-809 represents a class of CFTR corrector that specifically addresses the underlying processing defect in F508del-CFTR.

High throughput P450 inhibition screens in early drug discovery.

This review of high throughput (HT) P450 inhibition technologies and their impact on early drug discovery finds the field at a mature stage. The relationship between P450 inhibition and drug-drug interactions is well understood. A wide variety of P450 inhibition detection technologies are readily available off-the-shelf, but what seems still to be missing is a general agreement on how much weight one should give to the various types of early discovery HT P450 inhibition data. Method-dependent potency differences are a cause of concern, and to resolve this issue the authors advocate calibration of the HT methods with a large set of marketed drugs.

In silico prediction of drug safety: despite progress there is abundant room for improvement

Predictive models for drug safety are crucial for helping to avoid costly late-stage failures. We review recent work on models for genotoxicity, liver toxicity, CYP450 inhibition and cardiotoxicity. These models have improved somewhat in recent years, and research has expanded into new frontiers, such as the prediction of liver toxicity. However, much more needs to be done.:

Efficient Screening of Cytochrome P450 BM3 Mutants for Their Metabolic Activity and Diversity toward a Wide Set of Drug-Like Molecules in Chemical Space

In the present study, the diversity of a library of drug-metabolizing bacterial cytochrome P450 (P450) BM3 mutants was evaluated by a liquid chromatography-mass spectrometry (LC-MS)-based screening method. A strategy was designed to identify a minimal set of BM3 mutants that displays differences in regio- and stereoselectivities and is suitable to metabolize a large fraction of drug chemistry space. We first screened the activities of six structurally diverse BM3 mutants toward a library of 43 marketed drugs (encompassing a wide range of human P450 phenotypes, cLogP values, charges, and molecular weights) using a rapid LC-MS method with an automated method development and data-processing system. Significant differences in metabolic activity were found for the mutants tested and based on this drug library screen; nine structurally diverse probe drugs were selected that were subsequently used to study the metabolism of a library of 14 BM3 mutants in more detail. Using this alternative screening strategy, we were able to select a minimal set of BM3 mutants with high metabolic activities and diversity with respect to substrate specificity and regiospecificity that could produce both human relevant and BM3 unique drug metabolites. This panel of four mutants (M02, MT35, MT38, and MT43) was capable of producing P450-mediated metabolites for 41 of the 43 drugs tested while metabolizing 77% of the drugs by more than 20%. We observed this as the first step in our approach to use of bacterial P450 enzymes as general reagents for lead diversification in the drug development process and the biosynthesis of drug(-like) metabolites.

Stability constants for some divalent metal ion/crown ether complexes in methanol determined by polarography and conductometry

Stability constants in methanol at 25.0°C were evaluated for the complexes of the divalent cations Ca2+, Ni2+, Zn2+, Pb2+, Mg2+, Co2+ and Cu2+ with the macrocyclic polyethers 15-crown-5 (15C5), 18-crown-6 (18C6), dicyclohexyl-18-crown-6 (DC18C6) and dibenzo-24-crown-8 (DB24C8). The log K values of the 1:1 complexes were generally in the range 2.1–4.2, which is low in comparison to the values of the corresponding crown ether/alkali metal ion complexes. M2L complexes were observed for the systems Pb2+/18C6, Pb2+/DC18C6, Ca2+/DC18C6 and Cu2+/D18C6, whereas ML2 complexes were found for Ca2+/18C6 and Cu2+/18C6. Within the series of complexes studied, there was no clear relationship between cation diameter and hole size.

Insights into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mutants through crystallography, docking, molecular dynamics, and free energy calculations

Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug‐like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti‐inflammatory drugs (NSAIDs). Interestingly, single active‐site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X‐ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free‐energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free‐energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active‐site mutations. Our findings confirm that use of MD and binding free‐energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate. Proteins 2016; 84:383–396.

CFTR Modulators for the Treatment of Cystic Fibrosis

Publisher Summary This chapter focuses on cystic fibrosis transmembrane conductance regulator (CFTR) modulators for the treatment of cystic fibrosis (CF). It summarizes the recent progress made in the discovery of CFTR potentiators and correctors for the treatment of CF, with focus on compounds that have been most extensively characterized or for which chemical optimization has been reported.CF is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), an epithelial chloride-and bicarbonate-selective ion channel activated by cyclic AMP-dependent protein kinase A (PKA). Current therapies to treat CF lung disease, including mucolytics, antibiotics, and anti-inflammatory agents, target the downstream disease consequences that are secondary to the loss of CFTR function. Increasing CFTR function may provide clinical benefits to CF patients with systemic therapies that appear to be well tolerated. However, more studies are required to study the effects of long-term use of CFTR modulators in a greater diversity of CF patients in terms of their genetic background, age, and disease stage; CFTR drug discovery is still at an early stage.

Pharmacological Rescue of Mutant CFTR Function for the Treatment of Cystic Fibrosis

The vast majority of morbidity and mortality in cystic fibrosis (CF) patients is due to lung disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). CFTR is a PKA-regulated anion channel localized in the apical membrane of bronchial epithelia where it controls salt and fluid regulation to facilitate bacteria clearance. There are over 1000 disease-causing mutations in the gene encoding CFTR and, depending on the type of mutation, the cell surface density and/or functional activity of CFTR in the apical membrane is reduced. A therapeutic strategy for the treatment of lung disease in CF patients is to use pharmacological agents that increase mutant CFTR-mediated anion secretion. High-throughput screening strategies have identified multiple chemotypes that increase mutant CFTR-mediated anion secretion. These chemotypes can be grouped into two classes based on their mode of action. The first class is known as CFTR correctors because they correct the processing and trafficking of CFTR to increases its cell surface density. The second class is known as CFTR potentiators as they potentiate the amount of anion secretion through CFTR at the cell surface. In vivo analysis of CFTR activity in CF patients indicates that it is correlated with the severity of lung disease and supports the hypothesis that CFTR modulators that restore mutant CFTR activity to >10% of wild-type-CFTR would improve lung function. The use of high-throughput screening and medicinal chemistry optimization to improve the efficacy, potency, and pharmaceutical properties of the multiple potentiator or corrector scaffolds identified to date offers a promising approach for the treatment of CF by directly targeting the root cause of the disease.

Chapter Twenty-Four – Case History: Kalydeco® (VX-770, Ivacaftor), a CFTR Potentiator for the Treatment of Patients with Cystic Fibrosis and the G551D-CFTR Mutation

Abstract Cystic fibrosis (CF) is caused by mutations in the CFTR gene of an epithelial ion channel, the CF transmembrane conductance regulator (CFTR). We initiated efforts to discover potentiators of mutated CFTR. Potentiators are small molecules that increase the flow of ions through activated CFTR in the cell membrane. In-house HTS campaigns led to the identification of multiple hits. Extensive medicinal chemistry efforts driven by phenotypic assays led to the discovery of VX-770 (ivacaftor). VX-770 was found to be a potent, selective, orally bioavailable potentiator of G551D -CFTR, displaying excellent pharmacokinetic and safety profiles in rodents and nonrodents. Due to poor aqueous solubility, extensive formulation efforts were required and resulted in a spray-dried dispersion of VX-770 suitable for clinical development. After successful clinical trials, ivacaftor was approved by the FDA in 2012 for the treatment of people with CF who have a G551D mutation in the CFTR gene.