| 1. |
- Al-Amin, Rasel A., Researcher, 1983-, et al.
(author)
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Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ
- 2022
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In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:22, s. e129-e129
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Journal article (peer-reviewed)abstract
- Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.
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| 2. |
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| 3. |
- Borhade, Sanjay R, et al.
(author)
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Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Arylsulfonamides
- 2014
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In: ChemistryOpen. - : Wiley. - 2191-1363. ; 3:6, s. 256-263
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Journal article (peer-reviewed)abstract
- The inhibition of insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) by angiotenesin IV is known to improve memory and learning in rats. Screening 10 500 low-molecular-weight compounds in an enzyme inhibition assay with IRAP from Chinese Hamster Ovary (CHO) cells provided an arylsulfonamide (N-(3-(1H-tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sulfonamide), comprising a tetrazole in the meta position of the aromatic ring, as a hit. Analogues of this hit were synthesized, and their inhibitory capacities were determined. A small structure-activity relationship study revealed that the sulfonamide function and the tetrazole ring are crucial for IRAP inhibition. The inhibitors exhibited a moderate inhibitory potency with an IC50=1.1±0.5 μm for the best inhibitor in the series. Further optimization of this new class of IRAP inhibitors is required to make them attractive as research tools and as potential cognitive enhancers.
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| 4. |
- Çevik Aras, Hülya, 1975, et al.
(author)
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A non-peptide receptor inhibitor with selectivity for one of the neutrophil formyl peptide receptors, FPR 1.
- 2012
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In: Biochemical pharmacology. - : Elsevier BV. - 1873-2968 .- 0006-2952. ; 83:12, s. 1655-1662
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Journal article (peer-reviewed)abstract
- The neutrophil formyl peptide receptors (FPR1 and FPR2) are members of the G-protein coupled receptor family. The signals generated by occupied FPRs are both pro-inflammatory and anti-inflammatory. Accordingly, these receptors have become a therapeutic target for the development of novel drugs that may be used to reduce injuries in inflammatory diseases including asthma, rheumatoid arthritis, Alzheimer's disease and cardiovascular diseases. To support the basis for a future pharmacological characterization, we have identified a small molecular non-peptide inhibitor with selectivity for FPR1. We used the FPR1 and FPR2 specific ligands fMLF and WKYMVM, respectively, and an earlier described ratio technique, to determine inhibitory activity combined with selectivity. We show that the compound 3,5-dichloro-N-(2-chloro-5-methyl-phenyl)-2-hydroxy-benzamide (BVT173187) fulfills the criteria for an FPR1 inhibitor selective for FPR1 over FPR2, and it inhibits the same functional repertoire in neutrophils as earlier described peptide antagonists. Accordingly, the new inhibitor reduced neutrophil activation with FPR1 agonists, leading to mobilization of adhesion molecules (CR3) and the generation of superoxide anion from the neutrophil NADPH-oxidase. The effects of a number of structural analogs were determined but these were either without activity or less active/specific than BVT173187. The potency of the new inhibitor for reduction of FPR1 activity was the same as that of the earlier described FPR1 antagonist cyclosporine H, but signaling through the C5aR and CXCR (recognizing IL8) was also affected by BVT173187.
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| 5. |
- Diwakarla, Shanti, et al.
(author)
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Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures
- 2016
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In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 7:10, s. 1383-1392
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Journal article (peer-reviewed)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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| 6. |
- Diwakarla, Shanti, et al.
(author)
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Binding to and Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Macrocyclic Disulfides Enhances Spine Density
- 2016
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In: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 89:4, s. 413-424
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Journal article (peer-reviewed)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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| 7. |
- Engen, Karin, et al.
(author)
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Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening
- 2016
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In: Assay and drug development technologies. - : Mary Ann Liebert Inc. - 1540-658X .- 1557-8127. ; 14:3, s. 180-193
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Journal article (peer-reviewed)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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| 8. |
- Engen, Karin, et al.
(author)
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Inhibition of Insulin-Regulated Aminopeptidase by Imidazo[1,5-α]pyridines; Synthesis and Evaluation
- 2024
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067.
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Journal article (peer-reviewed)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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| 9. |
- Engen, Karin, et al.
(author)
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Microwave Heated Flow Synthesis of Spiro-oxindole Dihydroquinazolinone Based IRAP Inhibitors
- 2014
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In: Organic Process Research & Development. - : American Chemical Society (ACS). - 1083-6160 .- 1520-586X. ; 18:11, s. 1582-1588
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Journal article (peer-reviewed)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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| 10. |
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