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Probing Ligand Binding Mechanisms in Insulin-Regulated Aminopeptidases: Computational analysis and free energy calculations of binding modes
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (The Åqvist Lab)ORCID iD: 0000-0002-8195-1099
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

In recent years insulin-regulated aminopeptidase (IRAP) has emerged as a new therapeutic target for the treatment of Alzheimer’s disease and other memory-related disorders. So far, many potent and specific IRAP inhibitors had been disclosed, including peptides, peptidomimetics, and low-molecular-weight sulfonamides. In this thesis, various computational approaches such as docking, molecular dynamics (MD), linear interaction energy (LIE), and free energy perturbations (FEP) are used to understand the molecular basis for the binding of these inhibitors to the IRAP.

By applying MD and LIE, the binding mode of Ang IV and the critical role of its N-terminal tripeptide in the binding to IRAP were described. The stark difference in the binding properties of two stereoisomers of a peptidomimetic inhibitor, HA08 and HA09, was determined using MD simulations and LIE binding affinity estimations. With the help of the FEP method, we discriminate the most probable, between two alternative binding poses for the sulfonamide family of compounds. The binding modes of the HFI family of compounds (competitive inhibitors), and spiro-oxindole compounds (allosteric, uncompetitive inhibitors) were also proposed utilizing a combination of related computational approaches. In this thesis, the specificity of the diverse class of inhibitors and substrates (oxytocin and vasopressin) for IRAP compared to other M1 aminopetidase family members was disclosed as a result of the unique Gly-Ala-Met-Glu-Asn (GAMEN) loop orientation. The different studies performed along this thesis resulted in several proposed binding modes, which were evaluated by different free energy calculation approaches, namely LIE and FEP methods. In all cases, the calculated free energies are in excellent agreement with the experimental data, which strongly supports the final binding models here proposed.

These results of this thesis will be useful in future lead generation and optimization process and hopefully in the development of better cognitive enhancers for the treatment of dementia and other related diseases such as Alzheimer’s disease.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 64
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1870
Keywords [en]
Docking, Molecular Dynamics, Binding free energy, Linear Interaction Energy, Free Energy Perturbation, Insulin-Regulated Aminopeptidase, Angiotensin IV, Oxytocin, Vasopressin, HA08, Aryl sulfonamides, HFI compounds.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-395295ISBN: 978-91-513-0784-8 (print)OAI: oai:DiVA.org:uu-395295DiVA, id: diva2:1361742
Public defence
2019-12-06, B42, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-11-13 Created: 2019-10-16 Last updated: 2019-11-13
List of papers
1. Binding to and Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Macrocyclic Disulfides Enhances Spine Density
Open this publication in new window or tab >>Binding to and Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Macrocyclic Disulfides Enhances Spine Density
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2016 (English)In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 89, no 4, p. 413-424Article in journal (Refereed) Published
Abstract [en]

Angiotensin IV (Ang IV) and related peptide analogues, as well as non-peptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocylic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N-terminal of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09 and of Ang IV in either the extended or γ-turn conformation at the C-terminal to human IRAP were predicted by docking and molecular dynamics (MD) simulations. The binding free energies calculated with the linear interaction energy (LIE) method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-275166 (URN)10.1124/mol.115.102533 (DOI)000370935700003 ()26769413 (PubMedID)
Funder
Swedish Research Council
Available from: 2016-01-31 Created: 2016-01-31 Last updated: 2019-10-16
2. Macrocyclic Peptidomimetics as Inhibitors of Insulin-Regulated Aminopeptidase (IRAP)
Open this publication in new window or tab >>Macrocyclic Peptidomimetics as Inhibitors of Insulin-Regulated Aminopeptidase (IRAP)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Macrocyclic analogues of the linear hexapeptide, angiotensin IV (AngIV) have proved to be potent inhibitors of insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3). Along with higher affinity, macrocycles may also offer better metabolic stability, membrane permeability and selectivity, however predicting the outcome of particular cycle modifications is challenging. Here we describe the development of a series of macrocyclic IRAP inhibitors with either disulphide, olefin metathesis or lactam bridges and variations of ring size and other functionality. The binding mode of these compounds is proposed based on molecular dynamics analysis. Estimation of binding affinities (∆G) and relative binding free energies (∆∆G) with the linear interaction energy (LIE) method and free energy perturbation (FEP) method showed good general agreement with the observed inhibitory potency. Experimental and calculated data highlight the cumulative importance of an intact N-terminal peptide, the specific nature of the macrocycle, the phenolic oxygen and the C-terminal functionality.

National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-395288 (URN)
Available from: 2019-10-16 Created: 2019-10-16 Last updated: 2019-10-16
3. Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures
Open this publication in new window or tab >>Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures
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2016 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 7, no 10, p. 1383-1392Article in journal (Refereed) Published
Abstract [en]

The zinc metallopeptidase insulin regulated aminopeptidase (IRAP), which is highly expressed in the hippocampus and other brain regions associated with cognitive function, has been identified as a high-affinity binding site of the hexapeptide angiotensin IV (Ang IV). This hexapeptide is thought to facilitate learning and memory by binding to the catalytic site of IRAP to inhibit its enzymatic activity. In support of this hypothesis, low molecular weight, nonpeptide specific inhibitors of TRAP have been shown to enhance memory in rodent models. Recently, it was demonstrated that linear and macrocyclic Ang IV-derived peptides can alter the shape and increase the number of dendritic spines in hippocampal cultures, properties associated with enhanced cognitive performance. After screening a library of 10 500 drug like substances for their ability to inhibit IRAP, we identified a series of low molecular weight aryl sulfonamides, which exhibit no structural similarity to Ang IV, as moderately potent IRAP inhibitors:A structural and biological characterization of three of these aryl sulfonamides was performed. Their binding modes to human IRAP were explored by docking calculations combined with molecular dynamics simulations and binding affinity estimations using the linear interaction energy method. Two alternative binding modes emerged from this analysis, both of which correctly rank the ligands according to their experimental binding affinities for this series of compounds. Finally, we show that two of these drug-like IRAP inhibitors can alter dendritic spine morphology and increase spine density in primary cultures of hippocampal neurons.

Keywords
Insulin-regulated aminopeptidase, aryl sulfonamides, molecular dynamics, ligand interaction energy simulations, dendritic spines, hippocampal neurons
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-307715 (URN)10.1021/acschemneuro.6b00146 (DOI)000385994000011 ()
Funder
Swedish Research Council
Available from: 2016-11-22 Created: 2016-11-21 Last updated: 2019-10-16Bibliographically approved
4. Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Aryl Sulfonamides
Open this publication in new window or tab >>Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Aryl Sulfonamides
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2018 (English)In: ACS OMEGA, ISSN 2470-1343, Vol. 3, no 4, p. 4509-4521Article in journal (Refereed) Published
Abstract [en]

The insulin-regulated aminopeptidase (IRAP) is a membrane-bound zinc metallopeptidase with many important regulatory functions. It has been demonstrated that inhibition of IRAP by angiotensin IV (Ang IV) and other peptides, as well as more druglike inhibitors, improves cognition in several rodent models. We recently reported a series of aryl sulfonamides as small-molecule IRAP inhibitors and a promising scaffold for pharmacological intervention. We have now expanded with a number of derivatives, report their stability in liver microsomes, and characterize the activity of the whole series in a new assay performed on recombinant human IRAP. Several compounds, such as the new fluorinated derivative 29, present submicromolar affinity and high metabolic stability. Starting from the two binding modes previously proposed for the sulfonamide scaffold, we systematically performed molecular dynamics simulations and binding affinity estimation with the linear interaction energy method for the full compound series. The significant agreement with experimental affinities suggests one of the binding modes, which was further confirmed by the excellent correlation for binding affinity differences between the selected pair of compounds obtained by rigorous free energy perturbation calculations. The new experimental data and the computationally derived structure-activity relationship of the sulfonamide series provide valuable information for further lead optimization of novel IRAP inhibitors.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2018
National Category
Biochemistry and Molecular Biology Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-358572 (URN)10.1021/acsomega.8b00595 (DOI)000434352800025 ()30023895 (PubMedID)
Funder
Swedish Research Council
Available from: 2018-09-17 Created: 2018-09-17 Last updated: 2019-10-16Bibliographically approved
5. Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Benzopyran-based Inhibitors
Open this publication in new window or tab >>Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Benzopyran-based Inhibitors
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Inhibition of the insulin-regulated aminopeptidase (IRAP) improves memory and cognition in animal models. The enzyme has been recently crystallized and several series of inhibitors reported. We herein focused on one series of benzopyran-based inhibitors of IRAP, known as HFI series, and developed a robust computational model to explain the SAR and potentially guide the optimization of this scaffold. Our binding model positions the benzopyran ring in the catalytic binding site, coordinating the Zn+2 ion through the oxygen in position 3 of the, in contrast to previous hypothesis. The whole series of HFI compounds was systematically simulated using molecular dynamics in this binding orientation and binding affinity estimated with the linear interaction energy (LIE) method. The agreement with experimental affinities supports the binding mode proposed, which was further challenged by rigorous free energy perturbation calculations. Here, we found excellent correlation between experimental and calculated binding affinity differences, both between selected compound pairs and also for recently reported experimental data concerning the site directed mutagenesis of residue Phe544. The computationally derived structure-activity relationship of the HFI series and the demonstrated involvement of Phe544 in the binding of this scaffold provide valuable information for further lead optimization of novel IRAP inhibitors.

National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-395290 (URN)
Available from: 2019-10-16 Created: 2019-10-16 Last updated: 2019-10-16
6. 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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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3) has been identified as a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of the enzymatic activity by angiotensin IV (Ang IV) has been demonstrated to improve memory and learning in rats. The vast majority of the published inhibitors are peptides or pseudo-peptides often exhibiting high potencies but poor pharmacokinetic properties. Herein, a series of small non-peptide IRAP inhibitors are reported that are derived from a spiro-oxindole dihydroquinazolinone screening hit (pIC50 5.8). To obtain the target compounds either a fast and simple three-component reaction, or alternatively a two-step one-pot synthetic procedure was employed.  Incorporation of various substituents at the oxindole-moiety could be performed by rapid microwave-assisted Suzuki-Miyaura cross-couplings in a reaction time of only one minute. The efforts led to a small improvement of potency (pIC50 6.6 for the most potent compound) and increased solubility in general, but attempts to enhance the metabolic stability were unproductive. A deeper understanding of the structure-activity relationships and of the mechanism of action of this series of IRAP inhibitors was obtained. Moreover, computational modeling and MD simulations of potential binding poses allowed us to strongly suggest that the S-configuration of the spiro-oxindole dihydroquinazolinones is the preferred stereochemistry when inhibiting IRAP.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-395294 (URN)
Available from: 2019-10-16 Created: 2019-10-16 Last updated: 2019-10-28

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