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.

Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro‐Oxindole Dihydroquinazolinones as IRAP Inhibitors

“…The flasks were kept at 37 °C, 8 % CO 2 , 70 % relative humidity and with shaking at 130–135 rpm in an Infors Multitron incubator. The cells were transfected with 1.1 μg IRAP overexpressing plasmid/10 6 cells or 0.7 μg APN overexpressing plasmid (kindly provided Dr. Alexandros Nikolau at Vrije Universiteit Brussels, Brussels, Belgium)/10 6 cells in the presence of 2 μg polyethyleneimine/10 6 cells (Polyethyleneimine, linear, MW‐25 000, Cat. No.…”

“…Details for the assay used to evaluate the inhibitory activity of the compounds have already been published . In short:…”

“…The promising in vivo results encouraged us to commence a screening campaign applying an IRAP activity assay based on natural expression of the target in Chinese Hamster Ovary (CHO) cells to identify novel drug‐like IRAP inhibitors. A compound library of approximately 10 500 compounds was screened and a limited number of hit families were identified . The basic structure activity relationship (SAR) of an arylsulfonamide‐based class of inhibitors from this screen was recently reported, and we could also demonstrate that these compounds can increase the number of mushroom‐shaped dendritic spines, a morphology associated with memory enhancement …”

“…Herein, we report the synthesis and initial SAR of a new class of small‐molecule IRAP inhibitors, comprising a spiro‐oxindole dihydroquinazolinone scaffold, originating from a hit compound ( 1 ) exhibiting a pIC 50 value of 5.8 . Compound 1 is relatively lipophilic with a measured logD of 3.4 and suffers from poor solubility and metabolic stability (Tables and ).…”

Institutional Profile: the National Swedish Academic Drug Discovery & Development Platform at Scilifelab

“…The end products are well characterized proteins and cells fit for purpose for use in drug discovery projects within the DDD Platform. This includes material for assay development, screening, mode of action studies and structure determination as exemplified in these references [9,10]. In addition, development and establishment of technologies and methods relevant for protein expression, purification and characterization is performed within this facility.…”