title: Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening.
abstract: Intracerebroventricular injection of angiotensin IV, a ligand of insulin-regulated aminopeptidase (IRAP), has been shown to improve cognitive functions in several animal models. Consequently, IRAP is considered a potential target for treatment of cognitive disorders. To identify nonpeptidic IRAP inhibitors, we adapted an established enzymatic assay based on membrane preparations from Chinese hamster ovary cells and a synthetic peptide-like substrate for high-throughput screening purposes. The 384-well microplate-based absorbance assay was used to screen a diverse set of 10,500 compounds for their inhibitory capacity of IRAP. The assay performance was robust with Z'-values ranging from 0.81 to 0.91, and the screen resulted in 23 compounds that displayed greater than 60% inhibition at a compound concentration of 10 μM. After hit confirmation experiments, purity analysis, and promiscuity investigations, three structurally different compounds were considered particularly interesting as starting points for the development of small-molecule-based IRAP inhibitors. After resynthesis, all three compounds confirmed low μM activity and were shown to be rapidly reversible. Additional characterization included activity in a fluorescence-based orthogonal assay and in the presence of a nonionic detergent and a reducing agent, respectively. Importantly, the characterized compounds also showed inhibition of the human ortholog, prompting our further interest in these novel IRAP inhibitors.
authors.affiliation: 1 Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University , Uppsala, Sweden .
authors.forename: Karin
authors.initials: K
authors.lastname: Engen
authors.affiliation: 1 Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University , Uppsala, Sweden .
authors.forename: Ulrika
authors.initials: U
authors.lastname: Rosenström
authors.affiliation: 2 Chemical Biology Consortium Sweden, Science for Life Laboratory Stockholm, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Solna, Sweden .
authors.forename: Hanna
authors.initials: H
authors.lastname: Axelsson
authors.affiliation: 1 Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University , Uppsala, Sweden .
authors.forename: Vivek
authors.initials: V
authors.lastname: Konda
authors.affiliation: 3 Drug Discovery and Development Platform, Science for Life Laboratory Stockholm , Solna, Sweden .
authors.forename: Leif
authors.initials: L
authors.lastname: Dahllund
authors.affiliation: 2 Chemical Biology Consortium Sweden, Science for Life Laboratory Stockholm, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Solna, Sweden .
authors.forename: Magdalena
authors.initials: M
authors.lastname: Otrocka
authors.affiliation: 2 Chemical Biology Consortium Sweden, Science for Life Laboratory Stockholm, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Solna, Sweden .
authors.forename: Kristmundur
authors.initials: K
authors.lastname: Sigmundsson
authors.affiliation: 4 Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel , Brussels, Belgium .
authors.forename: Alexandros
authors.initials: A
authors.lastname: Nikolaou
authors.affiliation: 4 Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel , Brussels, Belgium .
authors.forename: Georges
authors.initials: G
authors.lastname: Vauquelin
authors.affiliation: 5 Beijer Laboratory, Division of Biological Research on Drug Dependence, Department of Pharmaceutical Biosciences, Uppsala University , Uppsala, Sweden .
authors.forename: Mathias
authors.initials: M
authors.lastname: Hallberg
authors.affiliation: 2 Chemical Biology Consortium Sweden, Science for Life Laboratory Stockholm, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Solna, Sweden .
authors.forename: Annika
authors.initials: A
authors.lastname: Jenmalm Jensen
authors.affiliation: 2 Chemical Biology Consortium Sweden, Science for Life Laboratory Stockholm, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Solna, Sweden .
authors.forename: Thomas
authors.initials: T
authors.lastname: Lundbäck
authors.affiliation: 6 Science for Life Laboratory Uppsala, Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University , Uppsala, Sweden .
authors.forename: Mats
authors.initials: M
authors.lastname: Larhed
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D003412:Cricetulus; D010122:Cystinyl Aminopeptidase / Q000037:antagonists & inhibitors*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D011480:Protease Inhibitors; D054852:Small Molecule Libraries; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase