AR71, Histamine H3 Receptor Ligand-In Vitro and In Vivo Evaluation (Anti-Inflammatory Activity, Metabolic Stability, Toxicity, and Analgesic Action).

The future of therapy for neurodegenerative diseases (NDs) relies on new strategies targeting multiple pharmacological pathways. Our research led to obtaining the compound AR71 [(E)-3-(3,4,5-trimethoxyphenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one], which has high affinity for human H3R (Ki = 24 nM) and selectivity towards histamine H1 and H4 receptors (Ki > 2500 nM), and showed anti-inflammatory activity in a model of lipopolysaccharide-induced inflammation in BV-2 cells. The presented tests confirmed its antagonist/inverse agonist activity profile and good metabolic stability while docking studies showed the binding mode to histamine H1, H3, and H4 receptors. In in vitro tests, cytotoxicity was evaluated at three cell lines (neuroblastoma, astrocytes, and human peripheral blood mononuclear cells), and a neuroprotective effect was observed in rotenone-induced toxicity. In vivo experiments in a mouse neuropathic pain model demonstrated the highest analgesic effects of AR71 at the dose of 20 mg/kg body weight. Additionally, AR71 showed antiproliferative activity in higher concentrations. These findings suggest the need for further evaluation of AR71's therapeutic potential in treating ND and CNS cancer using animal experimental models.

Anti-Inflammatory Activities of 8-Benzylaminoxanthines Showing High Adenosine A2A and Dual A1/A2A Receptor Affinity.

Chronic inflammation plays an important role in the development of neurodegenerative diseases, such as Parkinson's disease (PD). In the present study, we synthesized 25 novel xanthine derivatives with variable substituents at the N1-, N3- and C8-position as adenosine receptor antagonists with potential anti-inflammatory activity. The compounds were investigated in radioligand binding studies at all four human adenosine receptor subtypes, A1, A2A, A2B and A3. Compounds showing nanomolar A2A and dual A1/A2A affinities were obtained. Three compounds, 19, 22 and 24, were selected for further studies. Docking and molecular dynamics simulation studies indicated binding poses and interactions within the orthosteric site of adenosine A1 and A2A receptors. In vitro studies confirmed the high metabolic stability of the compounds, and the absence of toxicity at concentrations of up to 12.5 µM in various cell lines (SH-SY5Y, HepG2 and BV2). Compounds 19 and 22 showed anti-inflammatory activity in vitro. In vivo studies in mice investigating carrageenan- and formalin-induced inflammation identified compound 24 as the most potent anti-inflammatory derivative. Future studies are warranted to further optimize the compounds and to explore their therapeutic potential in neurodegenerative diseases.

The Potent and Selective Histamine H3 Receptor Antagonist E169 Counteracts Cognitive Deficits and Mitigates Disturbances in the PI3K/AKT/GSK-3ß Signaling Pathway in MK801-Induced Amnesia in Mice.

The role of histamine H3 receptors (H3Rs) in memory and the prospective of H3R antagonists in pharmacological control of neurodegenerative disorders, e.g., Alzheimer's disease (AD), is well-accepted. Therefore, the procognitive effects of acute systemic administration of H3R antagonist E169 (2.5-10 mg/kg, i.p.) on MK801-induced amnesia in C57BL/6J mice using the novel object recognition test (NORT) were evaluated. E169 (5 mg) provided a significant memory-improving effect on MK801-induced short- and long-term memory impairments in NORT. The E169 (5 mg)-provided effects were comparable to those observed with the reference phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and were abrogated with the H3R agonist (R)-α-methylhistamine (RAMH). Additionally, our results demonstrate that E169 ameliorated MK801-induced memory deficits by antagonism of H3Rs and by modulation of the level of disturbance in the expression of PI3K, Akt, and GSK-3ß proteins, signifying that E169 mitigated the Akt-mTOR signaling pathway in the hippocampus of tested mice. Moreover, the results observed revealed that E169 (2.5-10 mg/kg, i.p.) did not alter anxiety levels and locomotor activity of animals in open field tests, demonstrating that performances improved following acute systemic administration with E169 in NORT are unrelated to changes in emotional response or in spontaneous locomotor activity. In summary, these obtained results suggest the potential of H3R antagonists such as E169, with good in silico physicochemical properties and stable retained key interactions in docking studies at H3R, in simultaneously modulating disturbed brain neurotransmitters and the imbalanced Akt-mTOR signaling pathway related to neurodegenerative disorders, e.g., AD.

Structure Prediction, Evaluation, and Validation of GPR18 Lipid Receptor Using Free Programs.

The GPR18 receptor, often referred to as the N-arachidonylglycine receptor, although assigned (along with GPR55 and GPR119) to the new class A GPCR subfamily-lipid receptors, officially still has the status of a class A GPCR orphan. While its signaling pathways and biological significance have not yet been fully elucidated, increasing evidence points to the therapeutic potential of GPR18 in relation to immune, neurodegenerative, and cancer processes to name a few. Therefore, it is necessary to understand the interactions of potential ligands with the receptor and the influence of particular structural elements on their activity. Thus, given the lack of an experimentally solved structure, the goal of the present study was to obtain a homology model of the GPR18 receptor in the inactive state, meeting all requirements in terms of protein structure quality and recognition of active ligands. To increase the reliability and precision of the predictions, different contemporary protein structure prediction methods and software were used and compared herein. To test the usability of the resulting models, we optimized and compared the selected structures followed by the assessment of the ability to recognize known, active ligands. The stability of the predicted poses was then evaluated by means of molecular dynamics simulations. On the other hand, most of the best-ranking contemporary CADD software/platforms for its full usability require rather expensive licenses. To overcome this down-to-earth obstacle, the overarching goal of these studies was to test whether it is possible to perform the thorough CADD experiments with high scientific confidence while using only license-free/academic software and online platforms. The obtained results indicate that a wide range of freely available software and/or academic licenses allow us to carry out meaningful molecular modelling/docking studies.

N-Substituted piperazine derivatives as potential multitarget agents acting on histamine H3 receptor and cancer resistance proteins.

Taking into account that multidrug resistance (MDR) is the main cause for chemotherapeutic failure in cancer treatment, the ability of novel histamine H3 receptor ligands to reverse the cancer MDR was evaluated, using the ABCB1 efflux pump inhibition assay in mouse MDR T-lymphoma cells. The most active compounds displayed significant cytotoxic and antiproliferative effects as well as a very potent MDR efflux pump inhibitory action, 3-5-fold stronger than that of reference inhibitor verapamil. Although these compounds possess weak antagonistic properties against histamine H3 receptors, they are valuable pharmacological tools in the search for novel anticancer molecules. Furthermore, for the most active compounds, an insight into mechanisms of action using either, the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp, was performed.

A Taxicab geometry quantification system to evaluate the performance of in silico methods: a case study on adenosine receptors ligands.

Among still comparatively few G protein-coupled receptors, the adenosine A2A receptor has been co-crystallized with several ligands, agonists as well as antagonists. It can thus serve as a template with a well-described orthosteric ligand binding region for adenosine receptors. As not all subtypes have been crystallized yet, and in order to investigate the usability of homology models in this context, multiple adenosine A1 receptor (A1AR) homology models had been previously obtained and a library of lead-like compounds had been docked. As a result, a number of potent and one selective ligand toward the intended target have been identified. However, in in vitro experimental verification studies, many ligands also bound to the A2AAR and the A3AR subtypes. In this work we asked the question whether a classification of the ligands according to their selectivity was possible based on docking scores. Therefore, we built an A3AR homology model and docked all previously found ligands to all three receptor subtypes. As a metric, we employed an in vitro/in silico selectivity ranking system based on taxicab geometry and obtained a classification model with reasonable separation. In the next step, the method was validated with an external library of, selective ligands with similarly good performance. This classification system might also be useful in further screens.

Novel, Dual Target-Directed Annelated Xanthine Derivatives Acting on Adenosine Receptors and Monoamine Oxidase B.

Annelated purinedione derivatives have been shown to act as possible multiple-target ligands, addressing adenosine receptors and monoaminooxidases. In this study, based on our previous results, novel annelated pyrimido- and diazepino[2,1-f]purinedione derivatives were designed as dual-target-directed ligands combining A2A adenosine receptor (AR) antagonistic activity with blocking monoamine oxidase B. A library of 19 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. This allowed 9-(2-chloro-6-fluorobenzyl)-3-ethyl-1-methyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione (13 e; Ki human A2A AR: 264 nM and IC50 human MAO-B: 243 nM) to be identified as the most potent dual-acting ligand from this series. ADMET parameters were estimated in vitro, and analysis of the structure-activity relationships was complemented by molecular-docking studies based on previously published X-ray structures of the protein targets. Such dual-acting ligands, by selectively blocking A2A AR, accompanied by the inhibition of dopamine metabolizing enzyme MAO-B, might provide symptomatic and neuroprotective effects in, among others, the treatment of Parkinson disease.

KSK19 - Novel histamine H3 receptor ligand reduces body weight in diet induced obese mice.

AIMS: Histamine H3 receptors ligands act anorectic by blocking the H3 autoreceptors in the CNS, that results in increased synthesis and disinhibition of histamine release. Histamine further influencing H1 receptors participates in the leptin-dependent inhibition of food intake. It also affects the peripheral metabolism by increasing white adipose tissue lipolysis. Thus, ligands such as KSK19 with significant antagonistic properties at the H3 receptor might serve as an useful treatment for obesity. MATERIALS AND METHODS: To induce obesity, female CD-1 mice were fed a high-fat diet for 14 weeks. The test compound at the doses of 10 or 15 mg/kg, i.p. was administrated for 21 days. Glucose and insulin tolerance tests was performed at the beginning of week 15. At the end of study, amount of intraperitoneal fat pads, AlAT, IL-6 and TNF-α plasma levels were determined. RESULTS: Animals fed with high-fat diet and treated with test compound at the dose of 15 mg/kg showed significantly less weight gain, than mice from the control group. The use of KSK19 for 21 days in obese mice also significantly improved glucose tolerance and insulin resistance. In the tested doses KSK19 did not affect locomotor activity neither in lean nor in obese mice after single i.p. administration, but spontaneous activity increased during three hour after twentieth administrations. CONCLUSION: KSK19 is a strong, selective histamine H3 receptor antagonist with a favorable influence on body weight after multiple administration at the dose of 15 mg/kg. Moreover it significantly improves glucose tolerance.

Optimization and preclinical evaluation of novel histamine H3receptor ligands: Acetyl and propionyl phenoxyalkyl piperazine derivatives.

As a continuation of our search for novel histamine H3 receptor ligands, a series of new acetyl and propionyl phenoxyalkylamine derivatives (2-25) was synthesized. Compounds with three to four carbon atoms alkyl chain spacer, composed of six various 4N-substituted piperazine moieties were evaluated for their binding properties at human histamine H3 receptors (hH3R). In vitro test results proved the 4-pyridylpiperazine moiety as crucial element for high hH3R affinity (hH3R Ki = 5.2-115 nM). Moreover introduction of carbonyl group containing residues in the lipophilic part of molecules instead of branched alkyl substituents resulted in increased affinity in correlation to previously described series, whereas propionyl derivatives showed slightly higher affinities than those of acetyl (16 and 22vs.4 and 10; hH3R Ki = 5.2 and 15.4 nM vs. 10.2 and 115 nM, respectively). These findings were confirmed by molecular modelling studies, demonstrating multiple ligand-receptor interactions. Furthermore, pharmacological in vivo test results of compound 4 clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound. Likewise, its protective action against hyperglycemia and the development of overweight has been shown. In order to estimate drug-likeness of compound 4, in silico and experimental evaluation of metabolic stability in human liver microsomes was performed.

Discovery of novel lead in the group of N-substituted piperazine ether derivatives with potential histamine H3 receptor activity.

The search for novel lead from the group of various substituted N-piperazine ether derivatives was performed. Acyl- and pyridylpiperazine ethyl/propyl ethers were obtained via three different synthetic pathways. Affinity to histamine H3 receptor was established, as well as, for selected compounds, selectivity towards histamine H4R. Docking studies to the histamine H3R homology model strengthened the position of (4-(3-(4-(3-chlorobenzoyl)piperazin-1- yl)propoxy)phenyl)(cyclopropyl)methanone (compound 26) as a novel lead for further studies on histamine H3 receptor antagonist/inverse agonist.