Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors.

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.

Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures.

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 IRAP 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.

Synthesis and preclinical evaluation of 68Ga-labeled collagelin analogs for imaging and quantification of fibrosis.

OBJECTIVES: Fibrosis affecting functionality of vital organs such as liver, lung, heart, and kidney, is involved in many chronic diseases. Positron emission tomography (PET) would not only provide precise localization and extent of the affected tissue but also allow the accurate quantification of the fibrotic process for the subsequent prognosis. METHODS: A cyclic peptide c[CPGRVMHGLHLGDDEGPC] conjugated either to 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (NOTA(tBu)2) or 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid (NODAGA(tBu)3) via polyethylene glycol link (PEG2) was synthesized and labeled with (68)Ga. Non-specific organ distribution, blood clearance, and excretion were investigated ex vivo in healthy rats. The binding specificity of the radioligands was assessed in vitro using autoradiography on cryosections of dog fibrotic heart tissue. RESULTS: The yield of NOTA-PEG2-c[CPGRVMHGLHLGDDEGPC] and NODAGA-PEG2-c[CPGRVMHGLHLGDDEGPC] was 56% and 41%, respectively. Non-decay-corrected radiochemical yield was 80 ± 5% with radiochemical purity of 95 ± 4%. Pharmacokinetic studies in healthy male Sprague-Dawley rats showed fast blood clearance and renal excretion. Lower uptake in liver, spleen, and kidney was found for [[(68)Ga]Ga-NOTA](+1)-PEG2-c[CPGRVMHGLHLGDDEGPC] as compared to [[(68)Ga]Ga-NODAGA](0)-PEG2-c[CPGRVMHGLHLGDDEGPC]. Histologic evaluation of the left ventricle (LV) myocardium from a dog with severe mitral regurgitation (MR), revealed mild to moderate perivascular and subendocardial, and mild diffuse interstitial fibrosis. The tracer binding to the cryosections of the tissue was specific with the equilibrium Kd of 2.3 ± 0.8 µM and 2.1 ± 0.9 µM, respectively for [(68)Ga]Ga-NO2A-Col and [(68)Ga]Ga-NODAGA-Col. CONCLUSIONS: Two novel peptide based agents for the imaging of fibrosis by PET were developed. Moderation of the biodistribution could be achieved by variation of the charge on the complex moiety of the agents. The combination of the fast clearance from non-target organs as well as organs of interest such as lung, heart, and liver and binding specificity to the target tissue suggests the potential of the analogs for the imaging of fibrosis.