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Inhibitors Targeting Insulin-Regulated Aminopeptidase (IRAP): Identification, Synthesis and Evaluation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
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 [en]
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: urn:nbn:se:uu:diva-406417ISBN: 978-91-513-0894-4 (print)OAI: oai:DiVA.org:uu-406417DiVA, id: diva2:1412784
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
List of papers
1. Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening
Open this publication in new window or tab >>Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening
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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
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.

National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-297122 (URN)10.1089/adt.2016.708 (DOI)000374641700005 ()27078680 (PubMedID)
Funder
The Karolinska Institutet's Research FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council
Available from: 2016-06-22 Created: 2016-06-21 Last updated: 2020-03-07Bibliographically approved
2. Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors
Open this publication in new window or tab >>Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors
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2020 (English)In: ChemistryOpen, ISSN 2191-1363, Vol. 9, no 3, p. 325-337Article in journal (Refereed) Published
Abstract [en]

Insulin‐regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non‐peptide IRAP inhibitors derived from a spiro‐oxindole dihydroquinazolinone screening hit (pIC50 5.8). The compounds were synthesized either by a simple microwave (MW)‐promoted three‐component reaction, or by a two‐step one‐pot procedure. For decoration of the oxindole ring system, rapid MW‐assisted Suzuki‐Miyaura cross‐couplings (1 min) were performed. A small improvement of potency (pIC50 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S‐configuration of the spiro‐oxindole dihydroquinazolinones accounts for the inhibition of IRAP.

Keywords
enzymes, inhibitors, insulin, preclinical profiling, regulated aminopeptidases, spiro compounds
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-406132 (URN)10.1002/open.201900344 (DOI)000522554000007 ()32154052 (PubMedID)
Available from: 2020-03-05 Created: 2020-03-05 Last updated: 2020-05-07Bibliographically approved
3. Inhibition of Insulin-Regulated Aminopeptidase by Imidazo[1,5-α]pyridines; Synthesis and Evaluation
Open this publication in new window or tab >>Inhibition of Insulin-Regulated Aminopeptidase by Imidazo[1,5-α]pyridines; Synthesis and Evaluation
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Inhibition of Insulin-regulated Aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign identifying novel small-molecule based compounds acting as inhibitors of the enzymatic activity IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of imidazo[1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an pIC50 values of 6.0. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound´s metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn2+ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding.

Keywords
Insulin-regulated aminopeptidase, IRAP, inhibitors
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-406413 (URN)
Available from: 2020-03-07 Created: 2020-03-07 Last updated: 2020-03-07
4. Microwave Heated Flow Synthesis of Spiro-oxindole Dihydroquinazolinone Based IRAP Inhibitors
Open this publication in new window or tab >>Microwave Heated Flow Synthesis of Spiro-oxindole Dihydroquinazolinone Based IRAP Inhibitors
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2014 (English)In: Organic Process Research & Development, ISSN 1083-6160, E-ISSN 1520-586X, Vol. 18, no 11, p. 1582-1588Article in journal (Refereed) Published
Abstract [en]

A fast and convenient synthetic route towards spiro-oxindole dihydroquinazolinones as novel and drug-like insulin-regulated aminopeptidase (IRAP) inhibitors is reported. The synthesis is performed using a MW heated continuous flow system employing 200 mm X 3 mm i MW absorbing silicon carbide (SiC) or MW transparent borosilicate tubular reactors. A three-component MW-flow reaction to build up the spiro compounds (9 examples, 4087% yield), using the SiC reactor, as well as a SuzukiMiyaura cross-coupling reaction (71%), employing the borosilicate reactor, are presented with residence times down to 168 s. The continuous MW-flow routes provide a smooth and scalable synthetic methodology towards this class of IRAP inhibitors.

National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-240315 (URN)10.1021/op500237k (DOI)000345552100043 ()
Available from: 2015-01-07 Created: 2015-01-07 Last updated: 2020-03-07Bibliographically approved

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