Docking Foundations: From Rigid to Flexible Docking.

Despite the development of methods for the experimental determination of protein structures, the dissonance between the number of known sequences and their solved structures is still enormous. This is particularly evident in protein-protein complexes. To fill this gap, diverse technologies have been developed to study protein-protein interactions (PPIs) in a cellular context including a range of biological and computational methods. The latter derive from techniques originally published and applied almost half a century ago and are based on interdisciplinary knowledge from the nexus of the fields of biology, chemistry, and physics about protein sequences, structures, and their folding. Protein-protein docking, the main protagonist of this chapter, is routinely treated as an integral part of protein research. Herein, we describe the basic foundations of the whole process in general terms, but step by step from protein representations through docking methods and evaluation of complexes to their final validation.

Machine Learning Methods in Protein-Protein Docking.

An exponential increase in the number of publications that address artificial intelligence (AI) usage in life sciences has been noticed in recent years, while new modeling techniques are constantly being reported. The potential of these methods is vast-from understanding fundamental cellular processes to discovering new drugs and breakthrough therapies. Computational studies of protein-protein interactions, crucial for understanding the operation of biological systems, are no exception in this field. However, despite the rapid development of technology and the progress in developing new approaches, many aspects remain challenging to solve, such as predicting conformational changes in proteins, or more "trivial" issues as high-quality data in huge quantities.Therefore, this chapter focuses on a short introduction to various AI approaches to study protein-protein interactions, followed by a description of the most up-to-date algorithms and programs used for this purpose. Yet, given the considerable pace of development in this hot area of computational science, at the time you read this chapter, the development of the algorithms described, or the emergence of new (and better) ones should come as no surprise.

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.

Metabolic and cardiovascular benefits and risks of 4-hydroxy guanabenz hydrochloride: α2-adrenoceptor and trace amine-associated receptor 1 ligand.

BACKGROUND: α2-adrenoceptor ligands have been investigated as potential therapeutic agents for the treatment of obesity. Our previous studies have shown that guanabenz reduces the body weight of obese rats, presumably through its anorectic action. This demonstrates an additional beneficial effect on selected metabolic parameters, including glucose levels. The purpose of this present research was to determine the activity of guanabenz's metabolite-4-hydroxy guanabenz hydrochloride (4-OH-Guanabenz). METHODS: We performed in silico analyses, involving molecular docking to targets of specific interest as well as other potential biological targets. In vitro investigations were conducted to assess the selectivity profile of 4-OH-Guanabenz binding to α-adrenoceptors, along with intrinsic activity studies involving α2-adrenoceptors and trace amine-associated receptor 1 (TAAR1). Additionally, the effects of 4-OH-Guanabenz on the body weight of rats and selected metabolic parameters were evaluated using the diet-induced obesity model. Basic safety and pharmacokinetic parameters were also examined. RESULTS: 4-OH-guanabenz is a partial agonist of α2A-adrenoceptor. The calculated EC50 value for it is 316.3 nM. It shows weak agonistic activity at TAAR1 too. The EC50 value for 4-OH-Guanabenz calculated after computer simulation is 330.6 µM. Its primary mode of action is peripheral. The penetration of 4-OH-Guanabenz into the brain is fast (tmax = 15 min), however, with a low maximum concentration of 64.5 ng/g. 4-OH-Guanabenz administered ip at a dose of 5 mg/kg b.w. to rats fed a high-fat diet causes a significant decrease in body weight (approximately 14.8% compared to the baseline weight before treatment), reduces the number of calories consumed by rats, and decreases plasma glucose and triglyceride levels. CONCLUSIONS: The precise sequence of molecular events within the organism, linking the impact of 4-OH-Guanabenz on α2A-adrenoceptor and TAAR1 with weight reduction and the amelioration of metabolic disturbances, remains an unresolved matter necessitating further investigation. Undoubtedly, the fact that 4-OH-Guanabenz is a metabolite of a well-known drug has considerable importance, which is beneficial from an economic point of view and towards its further development as a drug candidate.

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.

Benzophenone Derivatives with Histamine H3 Receptor Affinity and Cholinesterase Inhibitory Potency as Multitarget-Directed Ligands for Possible Therapy of Alzheimer's Disease.

The multitarget-directed ligands demonstrating affinity to histamine H3 receptor and additional cholinesterase inhibitory potency represent a promising strategy for research into the effective treatment of Alzheimer's disease. In this study, a novel series of benzophenone derivatives was designed and synthesized. Among these derivatives, we identified compound 6 with a high affinity for H3R (Ki = 8 nM) and significant inhibitory activity toward BuChE (IC50 = 172 nM and 1.16 µM for eqBuChE and hBuChE, respectively). Further in vitro studies revealed that compound 6 (4-fluorophenyl) (4-((5-(piperidin-1-yl)pentyl)oxy)phenyl)methanone) displays moderate metabolic stability in mouse liver microsomes, good permeability with a permeability coefficient value (Pe) of 6.3 × 10-6 cm/s, and its safety was confirmed in terms of hepatotoxicity in the HepG2 cell line. Therefore, we investigated the in vivo activity of compound 6 in the Passive Avoidance Test and the Formalin Test. While compound 6 did not show a statistically significant influence on memory and learning, it showed analgesic properties in both acute (ED50 = 20.9 mg/kg) and inflammatory (ED50 = 17.5 mg/kg) pain.

Histamine H3 Receptor Ligands-KSK-59 and KSK-73-Reduce Body Weight Gain in a Rat Model of Excessive Eating.

Noting the worldwide rapid increase in the prevalence of overweight and obesity new effective drugs are now being sought to combat these diseases. Histamine H3 receptor antagonists may represent an effective therapy as they have been shown to modulate histamine synthesis and release and affect a number of other neurotransmitters (norepinephrine, acetylcholine, γ-aminobutyric acid, serotonin, substance P) thus influencing the food intake. Based on the preliminary studies determining affinity, intrinsic activity, and selected pharmacokinetic parameters, two histamine H3 receptor ligands were selected. Female rats were fed palatable food for 28 days and simultaneously administered the tested compounds intraperitoneally (i.p.) at a dose of 10 or 1 mg/kg b.w./day. Weight was evaluated daily and calorie intake was evaluated once per week. The plasma levels of cholesterol, triglycerides, leptin, adiponectin, ghrelin, corticosterone, CRP and IL-6 were determined at the end of experiment. The glucose tolerance test was also performed. To exclude false positives, the effect of tested compounds on spontaneous activity was monitored during the treatment, as well as the amount of consumed kaolin clay was studied as a reflection of possible gastrointestinal disturbances comparable to nausea. The histamine H3 receptor antagonists KSK-59 and KSK-73 administered i.p. at a dose of 10 mg/kg b.w. prevented weight gain in a rat model of excessive eating. They reduced adipose tissue deposits and improved glucose tolerance. Both compounds showed satisfying ability to penetrate through biological membranes determined in in vitro studies. Compound KSK-73 also reduced the caloric intake of the experimental animals what indicates its anorectic effect. These results show the pharmacological properties of histamine H3 receptor antagonists, (4-pyridyl)piperazine derivatives, as the compounds causing not only slower weight gain but also ameliorating some metabolic disorders in rats having the opportunity to overeat.

Metabolic benefits of novel histamine H3 receptor ligands in the model of excessive eating: The importance of intrinsic activity and pharmacokinetic properties.

AIMS: One of the therapeutic approaches in the treatment of obesity is the use of histamine H3 receptor ligands. Histamine plays a significant role in eating behavior because it causes a loss of appetite and is considered to be a satiety signal released during food intake. MATERIAL AND METHODS: Histamine ligands were selected based on the preliminary studies which included determination of intrinsic activity and selected pharmacokinetic parameters. Female Wistar rats were fed palatable feed for 28 days and simultaneously the tested compounds were administered intraperitoneally at a dose of 10 mg/kg b.w./day. Rats' weight was evaluated daily and calories intake was evaluated once per week. At the end of experiment insulin and glucose tolerance tests was performed. Plasma levels of cholesterol, triglycerides, leptin, insulin, glucose, C-peptide and CRP were also determined. In order to rule out false-positive results the influence of tested compounds on spontaneous activity of rats was monitored. RESULTS: Animals fed palatable feed and treated with KSK-61 or KSK-63 compounds showed the slowest weight gain which was comparable to the one observed in control animals. Both compounds with the highest pharmacological activity have also similar pharmacokinetic properties with quite long half-life and high volume of distribution indicating that they can freely cross most biological barriers. Some compounds, especially KSK-63, compensated for metabolic disorders. CONCLUSION: The presented study proves that search among the active histamine H3 receptor ligands for the new therapeutic agents to treat obesity is justified. Compounds KSK-61 and KSK-63 can be considered as the leading structures.

Discovery of Potential, Dual-Active Histamine H3 Receptor Ligands with Combined Antioxidant Properties.

In an attempt to find new dual acting histamine H3 receptor (H3R) ligands, we designed a series of compounds, structurally based on previously described in our group, a highly active and selective human histamine H3 receptor (hH3R) ligand KSK63. As a result, 15 obtained compounds show moderate hH3R affinity, the best being the compound 17 (hH3R Ki = 518 nM). Docking to the histamine H3R homology model revealed two possible binding modes, with key interactions retained in both cases. In an attempt to find possible dual acting ligands, selected compounds were tested for antioxidant properties. Compound 16 (hH3R Ki = 592 nM) showed the strongest antioxidant properties at the concentration of 10-4 mol/L. It significantly reduced the amount of free radicals presenting 50-60% of ascorbic acid activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, as well as showed antioxidative properties in the ferric reducing antioxidant power (FRAP) assay. Despite the yet unknown antioxidation mechanism and moderate hH3R affinity, 16 (QD13) constitutes a starting point for the search of potential dual acting H3R ligands-promising tools for the treatment of neurological disorders associated with increased neuronal oxidative stress.

Structural modifications in the distal, regulatory region of histamine H3 receptor antagonists leading to the identification of a potent anti-obesity agent.

A series of 4-pyridylpiperazine derivatives with varying regulatory region substituents proved to be potent histamine H3 receptor (H3R) ligands in the nanomolar concentration range. The most influential modification that affected the affinity toward the H3R appeared by introducing electron-withdrawing moieties into the distal aromatic ring. In order to finally discuss the influence of the characteristic 4-pyridylpiperazine moiety on H3R affinity, two Ciproxifan analogues 2 and 3 with a slight modification in their basic part were obtained. The replacement of piperazine in 3 with piperidine in compound 2, led to slightly reduced affinity towards the H3R (Ki = 3.17 and 7.70 nM, respectively). In fact, 3 showed the highest antagonistic properties among all compounds in this series, hence affirming our previous assumptions, that the 4-pyridylpiperazine moiety is the key element for suitable interaction with the human histamine H3 receptor. While its structural replacement to piperidine is also tolerated for H3R binding, the heteroaromatic 4-pyridyl moiety seems to be essential for proper ligand-receptor interaction. The putative protein-ligand interactions responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at the H3R, as well as drug-like properties of ligands were evaluated using in vitro methods. Moreover, pharmacological in vivo test results of compound 9 (structural analogue of Abbott's A-331440) clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound.

Dual-targeting Approach on Histamine H3 and Sigma-1 Receptor Ligands as Promising Pharmacological Tools in the Treatment of CNS-linked Disorders.

With the recent market approval of Pitolisant (Wakix®), the interest in clinical application for novel multifunctional histamine H3 receptor antagonists has clearly increased. Several combinations of different H3R pharmacophores with pharmacophoric elements of other G-protein coupled receptors, transporters, or enzymes have been synthesized by numerous pharmaceutical companies and academic institutions. Since central nervous system disorders are characterized by diverse physiological dysfunctions and deregulations of a complex network of signaling pathways, optimal multipotent drugs should simultaneously and peculiarly modulate selected groups of biological targets. Interestingly, very recent studies have shown that some clinically evaluated histamine H3 receptor antagonists possess a nanomolar affinity for sigma-1 receptor binding sites, suggesting that this property might play a role in their overall efficacy. The sigma-1 receptor, unusual and yet obscure protein, is supposed to be involved in numerous CNS pathologies through neuroprotection and neuroplasticity. These two different biological structures, histamine H3 and sigma-1 receptors, combined, can represent a potential fruitful target for therapeutic developments in tackling numerous human diseases.

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.

Rational design of new multitarget histamine H3 receptor ligands as potential candidates for treatment of Alzheimer's disease.

Design and development of multitarget-directed ligands (MTDLs) has become a very important approach in the search of new therapies for Alzheimer's disease (AD). In our present research, a number of xanthone derivatives were first designed using a pharmacophore model for histamine H3 receptor (H3R) antagonists/inverse agonists, and virtual docking was then performed for the enzyme acetylcholinesterase. Next, 23 compounds were synthesised and evaluated in vitro for human H3R (hH3R) affinity and inhibitory activity on cholinesterases. Most of the target compounds showed hH3R affinities in nanomolar range and exhibited cholinesterase inhibitory activity with IC50 values in submicromolar range. Furthermore, the inhibitory effects of monoamine oxidases (MAO) A and B were investigated. The results showed low micromolar and selective human MAO B (hMAO B) inhibition. Two azepane derivatives, namely 23 (2-(5-(azepan-1-yl)pentyloxy)-9H-xanthen-9-one) and 25 (2-(5-(azepan-1-yl)pentyloxy)-7-chloro-9H-xanthen-9-one), were especially very promising and showed high affinity for hH3R (Ki = 170 nM and 100 nM respectively) and high inhibitory activity for acetylcholinesterase (IC50 = 180 nM and 136 nM respectively). Moreover, these compounds showed moderate inhibitory activity for butyrylcholinesterase (IC50 = 880 nM and 394 nM respectively) and hMAO B (IC50 = 775 nM and 897 nM respectively). Furthermore, molecular docking studies were performed for hH3R, human cholinesterases and hMAO B to describe the mode of interactions with these biological targets. Next, the two most promising compounds 23 and 25 were selected for in vivo studies. The results showed significant memory-enhancing effect of compound 23 in dizocilpine-induced amnesia in rats in two tests: step-through inhibitory avoidance paradigm (SIAP) and transfer latency paradigm time (TLPT). In addition, favourable analgesic effects of compound 23 were observed in neuropathic pain models. Therefore, compound 23 is a particularly promising structure for further design of new MTDLs for AD.

8-Benzylaminoxanthine scaffold variations for selective ligands acting on adenosine A2A receptors. Design, synthesis and biological evaluation.

A library of 34 novel compounds based on a xanthine scaffold was explored in biological studies for interaction with adenosine receptors (ARs). Structural modifications of the xanthine core were introduced in the 8-position (benzylamino and benzyloxy substitution) as well as at N1, N3, and N7 (small alkyl residues), thereby improving affinity and selectivity for the A2A AR. The compounds were characterized by radioligand binding assays, and our study resulted in the development of the potent A2A AR ligands including 8-((6-chloro-2-fluoro-3-methoxybenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12d; Ki human A2AAR: 68.5 nM) and 8-((2-chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12h; Ki human A2AAR: 71.1 nM). Moreover, dual A1/A2AAR ligands were identified in the group of 1,3-diethyl-7-methylxanthine derivatives. Compound 14b displayed Ki values of 52.2 nM for the A1AR and 167 nM for the A2AAR. Selected A2AAR ligands were further evaluated as inactive for inhibition of monoamine oxidase A, B and isoforms of phosphodiesterase-4B1, -10A, which represent classical targets for xanthine derivatives. Therefore, the developed 8-benzylaminoxanthine scaffold seems to be highly selective for AR activity and relevant for potent and selective A2A ligands. Compound 12d with high selectivity for ARs, especially for the A2AAR subtype, evaluated in animal models of inflammation has shown anti-inflammatory activity. Investigated compounds were found to display high selectivity and may therefore be of high interest for further development as drugs for treating cancer or neurodegenerative diseases.

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.

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.

Search for new multi-target compounds against Alzheimer's disease among histamine H3 receptor ligands.

Multi-target-directed ligands seem to be an interesting approach to the treatment of complex disorders such as Alzheimer's disease. The aim of the present study was to find novel multifunctional compounds in a non-imidazole histamine H3 receptor ligand library. Docking-based virtual screening was applied for selection of twenty-six hits which were subsequently evaluated in Ellman's assay for the inhibitory potency toward acetyl- (AChE) and butyrylcholinesterase (BuChE). The virtual screening with high success ratio enabled to choose multi-target-directed ligands. Based on docking results, all selected ligands were able to bind both catalytic and peripheral sites of AChE and BuChE. The most promising derivatives combined the flavone moiety via a six carbon atom linker with a heterocyclic moiety, such as azepane, piperidine or 3-methylpiperidine. They showed the highest inhibitory activities toward cholinesterases as well as well-balanced potencies against H3R and both enzymes. Two derivatives were chosen - 5 (IC50 = 0.46 µM (AChE); 0.44 µM (BuChE); Ki = 159.8 nM (H3R)) and 17 (IC50 = 0.50 µM (AChE); 0.76 µM (BuChE); Ki = 228.2 nM (H3R)), and their inhibition mechanism was evaluated in kinetic studies. Both compounds displayed non-competitive mode of AChE and BuChE inhibition. Compounds 5 and 17 might serve as good lead structures for further optimization and development of novel multi-target anti-Alzheimer's agents.

Structural modifications and in vitro pharmacological evaluation of 4-pyridyl-piperazine derivatives as an active and selective histamine H3 receptor ligands.

A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part substituents proved to be potent histamine H3 receptor (hH3R) ligands in the nanomolar concentration range. While paying attention to their alkyl linker length, derivatives with a six methylene linker tend to be more potent than their five methylene homologues. Moreover, in the case of both phenoxyacetyl- and phenoxypropionyl- derivatives, an eight methylene linkers possess lower activity than their seven methylene homologues. However, in global analysis of collected data on the influence of alkyl linker length, a three methylene homologues appeared to be of highest hH3R affinity among all described 4-pyridylpiperazine derivatives from our group up to date. In the case of biphenyl and benzophenone derivatives, compounds with para- substituted second aromatic ring were of higher affinity than their meta analogues. Interestingly, benzophenone derivative 18 showed the highest affinity among all tested compounds (hH3R Ki = 3.12 nM). The likely protein-ligand interactions, responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at H3R, as well as drug-like properties of selected ligands were evaluated using in vitro methods.