RESUMEN
Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 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 IC50 value of 1.0 µM. 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.
Asunto(s)
Aminopeptidasas , Insulina , Animales , Insulina Regular Humana , Antígenos CD13 , Leucil Aminopeptidasa , PiridinasRESUMEN
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 (1â 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.
Asunto(s)
Cistinil Aminopeptidasa/antagonistas & inhibidores , Oxindoles/síntesis química , Inhibidores de Proteasas/síntesis química , Quinazolinonas/síntesis química , Animales , Sitios de Unión , Evaluación Preclínica de Medicamentos , Humanos , Modelos Moleculares , Oxindoles/metabolismo , Inhibidores de Proteasas/metabolismo , Unión Proteica , Conformación Proteica , Quinazolinonas/metabolismo , Ratas , Receptores de Angiotensina/metabolismo , Solubilidad , Relación Estructura-ActividadRESUMEN
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 10â¯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.
Asunto(s)
Cistinil Aminopeptidasa/antagonistas & inhibidores , Espinas Dendríticas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipocampo/citología , Sulfonamidas/farmacología , Animales , Antígenos CD13/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Cistinil Aminopeptidasa/metabolismo , Espinas Dendríticas/enzimología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/metabolismo , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Sulfonamidas/síntesis químicaRESUMEN
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 10 µM. 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 µM 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.
Asunto(s)
Cistinil Aminopeptidasa/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Inhibidores de Proteasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células CHO , Células Cultivadas , Cricetulus , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Inhibidores de Proteasas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Angiotensin IV (Ang IV) and related peptide analogs, as well as nonpeptide 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 macrocyclic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N terminus 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 terminus to human IRAP were predicted by docking and molecular dynamics simulations. The binding free energies calculated with the linear interaction energy 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.
Asunto(s)
Cistinil Aminopeptidasa/antagonistas & inhibidores , Cistinil Aminopeptidasa/metabolismo , Espinas Dendríticas/metabolismo , Disulfuros/metabolismo , Compuestos Macrocíclicos/metabolismo , Animales , Células Cultivadas , Cristalografía , Cistinil Aminopeptidasa/análisis , Espinas Dendríticas/química , Disulfuros/farmacología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Células HEK293 , Humanos , Compuestos Macrocíclicos/farmacología , Embarazo , Unión Proteica/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Transition state and high energy intermediate mimetics have the potential to be very potent enzyme inhibitors. In this study, a model of peptide hydrolysis in the active site of insulin-regulated aminopeptidase (IRAP) was developed using density functional theory calculations and the cluster approach. The 3D structure models of the reaction coordinates were used for virtual screening to obtain new chemical starting points for IRAP inhibitors. This mechanism-based virtual screening process managed to identify several known peptidase inhibitors from a library of over 5 million compounds, and biological testing identified one compound not previously reported as an IRAP inhibitor. This novel methodology for virtual screening is a promising approach to identify new inhibitors mimicking key transition states or intermediates of an enzymatic reaction.
Asunto(s)
Cistinil Aminopeptidasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Teoría Cuántica , Termodinámica , Animales , Sitios de Unión , Células CHO , Cricetulus , Cistinil Aminopeptidasa/química , Evaluación Preclínica de Medicamentos , Modelos Moleculares , Estructura MolecularRESUMEN
Sirtuins (SIRTs) catalyze the NAD(+)-dependent deacetylation of N(ε)-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.