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Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
Karolinska Inst, Dept Med Biochem & Biophys, Div Translat Med & Chem Biol, Sci Life Lab Stockholm,Chem Biol Consortium Swede, Tomtebodavagen 23A, S-17165 Solna, Sweden..
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
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2016 (English)In: Assay and drug development technologies, ISSN 1540-658X, E-ISSN 1557-8127, Vol. 14, no 3, p. 180-193Article in journal (Refereed) Published
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Text
Abstract [en]

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 10M. 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 M 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.

Place, publisher, year, edition, pages
2016. Vol. 14, no 3, p. 180-193
National Category
Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-297122DOI: 10.1089/adt.2016.708ISI: 000374641700005PubMedID: 27078680OAI: oai:DiVA.org:uu-297122DiVA, id: diva2:941150
Funder
The Karolinska Institutet's Research FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research CouncilAvailable from: 2016-06-22 Created: 2016-06-21 Last updated: 2020-03-07Bibliographically approved
In thesis
1. Inhibitors Targeting Insulin-Regulated Aminopeptidase (IRAP): Identification, Synthesis and Evaluation
Open this publication in new window or tab >>Inhibitors Targeting Insulin-Regulated Aminopeptidase (IRAP): Identification, Synthesis and Evaluation
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insulin-regulated aminopeptidase (IRAP) has emerged as a potential new therapeutic target for treatment of cognitive disorders. Inhibition of the enzymatic activity facilitates cognition in rodents. Potent and selective peptide and pseudopeptide based inhibitors have been developed, but most of them suffer from poor pharmacokinetics and blood-brain-barrier penetration. Hence, development of less-complex inhibitors with good pharmacokinetic properties are of great importance.

The aim of this thesis was to identify and optimize new small-molecule based IRAP inhibitors for use as research tools to investigate the cognitive effects of IRAP inhibition. Adaptation of an existing enzymatic assay into a screening compatible procedure allowed the evaluation of 10,500 compounds as IRAP inhibitors. The screening campaign resulted in 23 compounds displaying more than 60% inhibition. Two of these compounds, a spiro-oxindole dihydroquinazolinone and an imidazo[1,5-α]pyridine, were further investigated in terms of structure-activity relationship, physicochemical properties, metabolic stability, and mechanism of inhibition.

Spiro-oxindole dihydroquinazolinone based IRAP inhibitors were synthesized via fast and simple microwave-promoted reactions, either in batch or in a continuous flow approach. The most potent compounds displayed sub-µM affinity, and interestingly an uncompetitive mode of inhibition with the synthetic substrate used in the assay. Molecular modeling confirmed the possibility of simultaneous binding of the compounds and the substrate. Furthermore, the molecular modeling suggested that the S-enantiomer accounts for the inhibitory effect observed with this compound series. The compounds also proved inactive on the closely related enzyme aminopeptidase N. Unfortunately, the spiro-oxindole based inhibitors suffered from poor solubility and metabolic stability.

Imidazo[1,5-α]pyridine based IRAP inhibitors were synthesized via a five step procedure, providing inhibitors in the low-µM range. The stereospecificity of a methyl group proved important for inhibition. The compound series displayed no inhibitory activity on aminopeptidase N. Intriguing, these compounds exhibit a noncompetitive inhibition mechanism with the model substrate. As observed for the spiro-compounds, the imidazopyridines suffered from both poor solubility and metabolic stability.  

In summary, the work presented in this thesis provide synthetic procedures, initial structure-activity relationship, and pharmacological evaluation of two distinct inhibitors classes. The compounds are among the first non-peptidic IRAP inhibitors presented, serving as interesting starting points in the development of research tools for use in models of cognition.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. p. 86
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 285
Keywords
compound screening, insulin-regulated aminopeptidase, IRAP, inhibitors, cognitive disorders, spiro-oxindole, quinazolinone, imidazopyridine, medicinal chemistry, structure-activity relationship, microwave heating
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-406417 (URN)978-91-513-0894-4 (ISBN)
Public defence
2020-04-24, Room B41, BMC, Husargatan 3, Uppsala, 13:15 (Swedish)
Opponent
Supervisors
Available from: 2020-04-03 Created: 2020-03-07 Last updated: 2020-05-19

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Engen, KarinRosenström, UlrikaKonda, VivekHallberg, MathiasLarhed, Mats

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