| 1. |
- Diwakarla, Shanti, et al.
(författare)
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Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures
- 2016
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 7:10, s. 1383-1392
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Diwakarla, Shanti, et al.
(författare)
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Binding to and Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Macrocyclic Disulfides Enhances Spine Density
- 2016
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Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 89:4, s. 413-424
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Engen, Karin, et al.
(författare)
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Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening
- 2016
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Ingår i: Assay and drug development technologies. - : Mary Ann Liebert Inc. - 1540-658X .- 1557-8127. ; 14:3, s. 180-193
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Tidskriftsartikel (refereegranskat)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 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.
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| 4. |
- Engen, Karin, et al.
(författare)
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Inhibition of Insulin-Regulated Aminopeptidase by Imidazo[1,5-α]pyridines; Synthesis and Evaluation
- 2024
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067.
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Engen, Karin, 1988-
(författare)
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Inhibitors Targeting Insulin-Regulated Aminopeptidase (IRAP) : Identification, Synthesis and Evaluation
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
- Engen, Karin, et al.
(författare)
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Microwave Heated Flow Synthesis of Spiro-oxindole Dihydroquinazolinone Based IRAP Inhibitors
- 2014
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Ingår i: Organic Process Research & Development. - : American Chemical Society (ACS). - 1083-6160 .- 1520-586X. ; 18:11, s. 1582-1588
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
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| 8. |
- Engen, Karin, et al.
(författare)
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Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-oxindole Dihydroquinazolinones as IRAP Inhibitors
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 9. |
- Engen, Karin, et al.
(författare)
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Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors
- 2020
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Ingår i: ChemistryOpen. - : Wiley. - 2191-1363. ; 9:3, s. 325-337
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Seifert, Tina, 1985, et al.
(författare)
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Chroman-4-one- and Chromone-based Sirtuin 2 Inhibitors with Antiproliferative Properties in Cancer Cells
- 2014
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 57:23, s. 9870-9888
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Tidskriftsartikel (refereegranskat)abstract
- Sirtuins (SIRTs) catalyze the NAD+-dependent deacetylation of Nε-acetyl lysines on various protein substrates. SIRTs are interesting drug targets as they are considered to be related to important pathologies such as inflammation and aging-associated diseases. We have previously shown that chroman-4-ones act as potent and selective inhibitors of SIRT2. Herein we report novel chroman-4-one and chromone-based SIRT2 inhibitors containing various heterofunctionalities to improve pharmacokinetic properties. The compounds retained both high SIRT2 selectivity and potent inhibitory activity. Two compounds were tested for their antiproliferative effects in breast cancer (MCF-7) and lung carcinoma (A549) cell lines. Both compounds showed antiproliferative effects correlating with their SIRT2 inhibition potency. They also increased the acetylation level of α-tubulin, indicating that SIRT2 is likely to be the target in cancer cells. A binding mode of the inhibitors that is consistent with the SAR data was proposed based on a homology model of SIRT2.
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