The development of highly potent and selective small molecule correctors of Z α-antitrypsin misfolding.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Development of a small molecule that corrects misfolding and increases secretion of Z α -antitrypsin.

Severe α -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α -antitrypsin. The lead compound blocks Z α -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α -antitrypsin threefold in an iPSC model of disease.

Synthesis and Evaluation of Bicyclic Hydroxypyridones as Inhibitors of Catechol -Methyltransferase.

A series of bicyclic pyridones were identified as potent inhibitors of catechol -methyltransferase (COMT). Substituted benzyl groups attached to the basic nitrogen of the core scaffold gave the most potent inhibitors within this series. Rat pharmacokinetic studies showed medium to high levels of clearance for this series, but with high free fraction due to remarkably low levels of protein and tissue binding.

Discovery and Lead-Optimization of 4,5-Dihydropyrazoles as Mono-Kinase Selective, Orally Bioavailable and Efficacious Inhibitors of Receptor Interacting Protein 1 (RIP1) Kinase.

RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species.

Optimization of 8-Hydroxyquinolines as Inhibitors of Catechol O-Methyltransferase.

A series of 8-hydroxy quinolines were identified as potent inhibitors of catechol O-methyltransferase (COMT) with selectivity for the membrane-bound form of the enzyme. Small substituents at the 7-position of the quinoline were found to increase metabolic stability without sacrificing potency. Compounds with good pharmacokinetics and brain penetration were identified and demonstrated in vivo modulation of dopamine metabolites in the brain.

Binding mode and structure-activity relationships around direct inhibitors of the Nrf2-Keap1 complex.

An X-ray crystal structure of Kelch-like ECH-associated protein (Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1 a) was obtained. This X-ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)-1 a, as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently.

Utility of drug depletion-time profiles in isolated hepatocytes for accessing hepatic uptake clearance: identifying rate-limiting steps and role of passive processes.

Drug depletion-time profiles in isolated hepatocytes, as well as microsomes, have become a standard method of assessing hepatic metabolic clearance in vitro. There is a previously described adaptation of the depletion approach to allow determination of hepatic uptake by transporters in addition to metabolism (Drug Metab Dispos 35:859-865, 2007). Dual incubations are performed where one set of incubations undergo conventional methodology, whereas for the second set, cells and media are separated for determination of drug loss from the media.

Differential regulation of sinusoidal and canalicular hepatic drug transporter expression by xenobiotics activating drug-sensing receptors in primary human hepatocytes.

Sinusoidal and canalicular hepatic drug transporters constitute key factors involved in drug elimination from liver. Regulation of their expression via activation of xenosensors, such as aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and nuclear factor E2-related factor 2 (Nrf2), remains incompletely characterized. The present study was therefore designed to carefully analyze expression of major drug transporters in primary human hepatocytes exposed to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) (an AhR activator), rifampicin (RIF) (a PXR activator), phenobarbital (PB) (a CAR activator), and oltipraz (OPZ) (a Nrf2 activator), using mainly reverse transcription-real time polymerase chain reaction assays.

Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line.

Functional expression of both sinusoidal and canalicular hepatic drug transporters was investigated in the highly differentiated human hepatoma HepaRG cell line and also, for comparison, in primary human hepatocytes and in the hepatoma HepG2 cell line. Using RT-qPCR assays, differentiated HepaRG cells were found to display a pattern of transporter expression close to that found in primary human hepatocytes, i.e.

Functional expression of sinusoidal drug transporters in primary human and rat hepatocytes.

Primary hepatocyte cultures are considered as a useful in vitro system for pharmacological/toxicological studies. Although expression of drug-metabolizing enzymes and canalicular drug transporters has been well documented in this cellular model, less information is available about sinusoidal drug transporter activities. This has led us to investigate functional expression of the major sinusoidal transporters in primary human and rat hepatocytes.

Physiological, pharmacological and clinical features of the multidrug resistance protein 2.

Multidrug resistance protein 2 (MRP2, ABCC2) is a drug efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily. MRP2 is present predominantly at the biliary pole of hepatocytes and is also expressed in the kidney and intestine. It plays a major role in hepato-biliary elimination of many structurally diverse xenobiotics, including organic anions and drug conjugates, and therefore most likely contributes to pharmacokinetic parameters of these compounds.