ABSTRACT
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 α1-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.
Subject(s)
Drug Design , Protein Folding , alpha 1-Antitrypsin/metabolism , Crystallization , Drug Development/methods , Drug Evaluation, Preclinical , Endoplasmic Reticulum/metabolism , Gene Library , Hepatocytes/metabolism , Humans , Models, Molecular , Protein Conformation , alpha 1-Antitrypsin/geneticsABSTRACT
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.
Subject(s)
Benzamidines/chemistry , Kallikreins/antagonists & inhibitors , Netherton Syndrome/drug therapy , Serine Proteinase Inhibitors/chemistry , Amino Acid Sequence , Benzamidines/pharmacology , Binding Sites , Drug Evaluation, Preclinical , Humans , Isomerism , Models, Molecular , Molecular Structure , Mutation , Protein Binding , Serine Peptidase Inhibitor Kazal-Type 5/genetics , Serine Proteinase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z' value 0.8) and provided several tractable hit series for further investigation.