Rational Search for Betaine/GABA Transporter 1 Inhibitors─In Vitro Evaluation of Selected Hit Compound.

Inhibitory neurotransmission mediated by γ-aminobutyric acid (GABA) plays an important role in maintaining body homeostasis. Disturbances in GABA signaling are implicated in a multitude of neurologic and psychiatric conditions, including epilepsy, ischemia, anxiety, depression, insomnia, and mood disorders. Clinically relevant increases in GABA neurotransmitter level can be achieved by inhibition of its uptake into presynaptic neurons and surrounding glial cells, driven by GABA transporters (GAT1, BGT1, GAT2, and GAT3). Herein, we focused on the search for inhibitors of the BGT1 transporter which is understudied and for which the therapeutic potential of its inhibition is partly unknown. We applied multilevel virtual screening to identify compounds with inhibitory properties. Among selected hits, compound 9 was shown to be a preferential inhibitor of BGT1 (IC50 13.9 µM). The compound also revealed some inhibitory activity against GAT3 (4x lower) while showing no or low activity (IC50 > 100 µM) toward GAT1 and GAT2, respectively. The predicted binding mode of compound 9 was confirmed by mutagenesis studies on E52A, E52Y, Q299L, and E52A+Q299L human BGT1 mutants. Subsequent evaluation showed that the selected hit displayed no affinity toward major GABAA receptor subtypes. Moreover, it was nontoxic when tested on normal human astrocytes and even showed some neuroprotective activity in SH-SY5Y cells. Compound 9 is considered a promising candidate for further evaluation of the therapeutic potential of BGT1 transporter inhibition and the development of novel inhibitors.

Discovery of novel multifunctional ligands targeting GABA transporters, butyrylcholinesterase, ß-secretase, and amyloid ß aggregation as potential treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a global health problem in the medical sector that will increase over time. The limited treatment of AD leads to the search for a new clinical candidate. Considering the multifactorial nature of AD, a strategy targeting number of regulatory proteins involved in the development of the disease is an effective approach. Here, we present a discovery of new multi-target-directed ligands (MTDLs), purposely designed as GABA transporter (GAT) inhibitors, that successfully provide the inhibitory activity against butyrylcholinesterase (BuChE), ß-secretase (BACE1), amyloid ß aggregation and calcium channel blockade activity. The selected GAT inhibitors, 19c and 22a - N-benzylamide derivatives of 4-aminobutyric acid, displayed the most prominent multifunctional profile. Compound 19c (mGAT1 IC50 = 10 µM, mGAT4 IC50 = 12 µM and BuChE IC50 = 559 nM) possessed the highest hBACE1 and Aß40 aggregation inhibitory activity (IC50 = 1.57 µM and 99 % at 10 µM, respectively). Additionally, it showed a decrease in both the elongation and nucleation constants of the amyloid aggregation process. In contrast compound 22a represented the highest activity and a mixed-type of eqBuChE inhibition (IC50 = 173 nM) with hBACE1 (IC50 = 9.42 µM), Aß aggregation (79 % at 10 µM) and mGATs (mGAT1 IC50 = 30 µM, mGAT4 IC50 = 25 µM) inhibitory activity. Performed molecular docking studies described the mode of interactions with GATs and enzymatic targets. In ADMET in vitro studies both compounds showed acceptable metabolic stability and low neurotoxicity. Successfully, compounds 19c and 22a at the dose of 30 mg/kg possessed statistically significant antiamnesic properties in a mouse model of amnesia caused by scopolamine and assessed in the novel object recognition (NOR) task or the passive avoidance (PA) task.

New cyclopentaquinoline and 3,5-dichlorobenzoic acid hybrids with neuroprotection against oxidative stress for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disease. Thus, drugs including donepezil, rivastigmine, and galantamine are not entirely effective in the treatment of this multifactorial disease. The present study evaluates eight derivatives (3a-3h) as candidates with stronger anti-AD potential but with less side effects. Reactive oxygen species (ROS) assays were used to assess oxidative stress which involve in the neurodegeneration. The neuroprotective properties of 3e against oxidative stress were done in three experiments using MTT test. The anti-AD potential was determined based on their anticholinesterase inhibition ability, determined using Ellman's method, Aß aggregation potential according to thioflavin (Th) fluorescence assay, and their antioxidative and anti-inflammatory activities. Compound 3e exhibited moderate cholinesterase inhibition activity (AChE, IC50 = 0.131 µM; BuChE, IC50 = 0.116 µM; SI = 1.13), significant inhibition of Aß(1-42) aggregation (55.7%, at 5 µM) and acceptable neuroprotective activity. Extensive analysis of in vitro and in vivo assays indicates that new cyclopentaquinoline derivatives offer promise as candidates for new anti-AD drugs.

Analysis of Different Binding Modes for Tiagabine within the GAT-1 Transporter.

The recently obtained cryo-electron microscopy structure (PDB code: 7SK2) of the human γ-aminobutyric acid transporter type 1 (hGAT-1) in complex with the antiepileptic drug, tiagabine, revealed a rather unexpected binding mode for this inhibitor in an inward-open state of the transporter. The simultaneously released crystal structures of the modified dopamine transporter with mutations mimicking hGAT-1 indicated an alternative binding mode for the tiagabine analogues that were found to block the transporter in an outward-open state, which is more consistent with the results of previous biological and molecular modeling studies. In view of the above discrepancies, our study compares different hypothetical tiagabine binding modes using classical and accelerated molecular dynamics simulations, as well as MM-GBSA free binding energy (dG) calculations. The results indicate that the most stable and energetically favorable binding mode of tiagabine is the one where the nipecotic acid fragment is located in the main binding site (S1) and the aromatic rings are arranged within the S2 site of the hGAT-1 transporter in an outward-open state, confirming the previous molecular modelling findings. The position of tiagabine bound to hGAT-1 in an inward-open state, partially within the intracellular release pathway, was significantly less stable and the dG values calculated for this complex were higher. Furthermore, analysis of the cryo-electron map for the 7SK2 structure shows that the model does not appear to fit into the map optimally at the ligand binding site. These findings suggest that the position of tiagabine found in the 7SK2 structure is rather ambiguous and requires further experimental verification. The identification of the main, high-affinity binding site for tiagabine and its analogues is crucial for the future rational design of the GABA transporter inhibitors.

Discovery of the First Highly Selective Antagonist of the GluK3 Kainate Receptor Subtype.

Kainate receptors belong to the family of glutamate receptors ion channels, which are responsible for the majority of rapid excitatory synaptic transmission in the central nervous system. The therapeutic potential of kainate receptors is still poorly understood, which is also due to the lack of potent and subunit-selective pharmacological tools. In search of selective ligands for the GluK3 kainate receptor subtype, a series of quinoxaline-2,3-dione analogues was synthesized and pharmacologically characterized at selected recombinant ionotropic glutamate receptors. Among them, compound 28 was found to be a competitive GluK3 antagonist with submicromolar affinity and unprecedented high binding selectivity, showing a 400-fold preference for GluK3 over other homomeric receptors GluK1, GluK2, GluK5 and GluA2. Furthermore, in functional assays performed for selected metabotropic glutamate receptor subtypes, 28 did not show agonist or antagonist activity. The molecular determinants underlying the observed affinity profile of 28 were analyzed using molecular docking and molecular dynamics simulations performed for individual GluK1 and GluK3 ligand-binding domains.

Analysis of Binding Determinants for Different Classes of Competitive and Noncompetitive Inhibitors of Glycine Transporters.

Glycine transporters are interesting therapeutic targets as they play significant roles in glycinergic and glutamatergic systems. The search for new selective inhibitors of particular types of glycine transporters (GlyT-1 and GlyT-2) with beneficial kinetics is hampered by limited knowledge about the spatial structure of these proteins. In this study, a pool of homology models of GlyT-1 and GlyT-2 in different conformational states was constructed using the crystal structures of related transporters from the SLC6 family and the recently revealed structure of GlyT-1 in the inward-open state, in order to investigate their binding sites. The binding mode of the known GlyT-1 and GlyT-2 inhibitors was determined using molecular docking studies, molecular dynamics simulations, and MM-GBSA free energy calculations. The results of this study indicate that two amino acids, Gly373 and Leu476 in GlyT-1 and the corresponding Ser479 and Thr582 in GlyT-2, are mainly responsible for the selective binding of ligands within the S1 site. Apart from these, one pocket of the S2 site, which lies between TM3 and TM10, may also be important. Moreover, selective binding of noncompetitive GlyT-1 inhibitors in the intracellular release pathway is affected by hydrophobic interactions with Ile399, Met382, and Leu158. These results can be useful in the rational design of new glycine transporter inhibitors with desired selectivity and properties in the future.

Development of tricyclic N-benzyl-4-hydroxybutanamide derivatives as inhibitors of GABA transporters mGAT1-4 with anticonvulsant, antinociceptive, and antidepressant activity.

γ-Aminobutyric acid (GABA) neurotransmission has a significant impact on the proper functioning of the central nervous system. Numerous studies have indicated that inhibitors of the GABA transporters mGAT1-4 offer a promising strategy for the treatment of several neurological disorders, including epilepsy, neuropathic pain, and depression. Following our previous results, herein, we report the synthesis, biological evaluation, and structure-activity relationship studies supported by molecular docking and molecular dynamics of a new series of N-benzyl-4-hydroxybutanamide derivatives regarding their inhibitory potency toward mGAT1-4. This study allowed us to identify compound 23a (N-benzyl-4-hydroxybutanamide bearing a dibenzocycloheptatriene moiety), a nonselective GAT inhibitor with a slight preference toward mGAT4 (pIC50 = 5.02 ± 0.11), and compound 24e (4-hydroxy-N-[(4-methylphenyl)-methyl]butanamide bearing a dibenzocycloheptadiene moiety) with relatively high inhibitory activity toward mGAT2 (pIC50 = 5.34 ± 0.09). In a set of in vivo experiments, compound 24e successively showed predominant anticonvulsant activity and antinociception in the formalin model of tonic pain. In contrast, compound 23a showed significant antidepressant-like properties in mice. These results were consistent with the available literature data, which indicates that, apart from seizure control, GABAergic neurotransmission is also involved in the pathophysiology of several psychiatric diseases, however alternative mechanisms underlying this action cannot be excluded. Finally, it is worth noting that the selected compounds showed unimpaired locomotor skills that have been indicated to give reliable results in behavioral assays.

Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates.

The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.

Molecular Modeling Studies on the Multistep Reactivation Process of Organophosphate-Inhibited Acetylcholinesterase and Butyrylcholinesterase.

Poisoning with organophosphorus compounds used as pesticides or misused as chemical weapons remains a serious threat to human health and life. Their toxic effects result from irreversible blockade of the enzymes acetylcholinesterase and butyrylcholinesterase, which causes overstimulation of the cholinergic system and often leads to serious injury or death. Treatment of organophosphorus poisoning involves, among other strategies, the administration of oxime compounds. Oximes reactivate cholinesterases by breaking the covalent bond between the serine residue from the enzyme active site and the phosphorus atom of the organophosphorus compound. Although the general mechanism of reactivation has been known for years, the exact molecular aspects determining the efficiency and selectivity of individual oximes are still not clear. This hinders the development of new active compounds. In our research, using relatively simple and widely available molecular docking methods, we investigated the reactivation of acetyl- and butyrylcholinesterase blocked by sarin and tabun. For the selected oximes, their binding modes at each step of the reactivation process were identified. Amino acids essential for effective reactivation and those responsible for the selectivity of individual oximes against inhibited acetyl- and butyrylcholinesterase were identified. This research broadens the knowledge about cholinesterase reactivation and demonstrates the usefulness of molecular docking in the study of this process. The presented observations and methods can be used in the future to support the search for new effective reactivators.

Structure modeling of γ-aminobutyric acid transporters - Molecular basics of ligand selectivity.

γ-Aminobutyric acid transporters are responsible for regulating the GABA level in the synaptic cleft. In this way, they affect GABA-ergic transmission which is important for the proper functioning of the central nervous system. The exact structure of GABA transporters is still unknown, which hinders the design of new, potent and selective inhibitors. For these reasons, we decided to create models of all types of human gamma-aminobutyric acid transporters. They were built based on crystal structures of related proteins from the SLC6 family using homology modeling methods. The reliability of the received models has been confirmed by a number of tools assessing the quality of protein models. To determine the ligand binding mode and indicate the amino acids responsible for selectivity, docking studies and molecular dynamics simulations were performed. The amino acids lining the bottom of the main binding site have a major impact on the selective ligand binding. In addition, an important element is the non-helical fragment of the transmembrane domain 10, and several amino acids within the vestibule of the transporters, which affect its volume. To check whether obtained models are suitable to distinguish active compounds from inactive ones, enrichment plots were prepared. Results suggest that our models may be useful in the search for new inhibitors of GABA transporters of the desired selectivity.

γ-Aminobutyric acid transporters as relevant biological target: Their function, structure, inhibitors and role in the therapy of different diseases.

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the nervous system. It plays a crucial role in many physiological processes. Upon release from the presynaptic element, it is removed from the synaptic cleft by reuptake due to the action of GABA transporters (GATs). GATs belong to a large SLC6 protein family whose characteristic feature is sodium-dependent relocation of neurotransmitters through the cell membrane. GABA transporters are characterized in many contexts, but their spatial structure is not fully known. They are divided into four types, which differ in occurrence and role. Herein, the special attention was paid to these transporting proteins. This comprehensive review presents the current knowledge about GABA transporters. Their distribution in the body, physiological functions and possible utilization in the therapy of different diseases were fully discussed. The important structural features were described based on published data, including sequence analysis, mutagenesis studies, and comparison with known SLC6 transporters for leucine (LeuT), dopamine (DAT) and serotonin (SERT). Moreover, the most important inhibitors of GABA transporters of various basic scaffolds, diverse selectivity and potency were presented.

New Tetrahydroacridine Hybrids with Dichlorobenzoic Acid Moiety Demonstrating Multifunctional Potential for the Treatment of Alzheimer's Disease.

A series of new tetrahydroacridine and 3,5-dichlorobenzoic acid hybrids with different spacers were designed, synthesized, and evaluated for their ability to inhibit both cholinesterase enzymes. Compounds 3a, 3b, 3f, and 3g exhibited selective butyrylcholinesterase (EqBuChE) inhibition with IC50 values ranging from 24 to 607 nM. Among them, compound 3b was the most active (IC50 = 24 nM). Additionally, 3c (IC50 for EeAChE = 25 nM and IC50 for EqBuChE = 123 nM) displayed dual cholinesterase inhibitory activity and was the most active compound against acetylcholinesterase (AChE). Active compound 3c was also tested for the ability to inhibit Aß aggregation. Theoretical physicochemical properties of the compounds were calculated using ACD Labs Percepta and Chemaxon. A Lineweaver-Burk plot and docking study showed that 3c targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Moreover, 3c appears to possess neuroprotective activity and could be considered a free-radical scavenger. In addition, 3c did not cause DNA damage and was found to be less toxic than tacrine after oral administration; it also demonstrated little inhibitory activity towards hyaluronidase (HYAL), which may indicate that it possesses anti-inflammatory properties. The screening for new in vivo interactions between 3c and known receptors was realized by yeast three-hybrid technology (Y3H).

Structure Modeling of the Norepinephrine Transporter.

The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on Drosophila melanogaster dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds.

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.

Discovery of New Cyclopentaquinoline Analogues as Multifunctional Agents for the Treatment of Alzheimer's Disease.

Here we report the two-step synthesis of 8 new cyclopentaquinoline derivatives as modifications of the tetrahydroacridine structure. Next, the biological assessment of each of them was performed. Based on the obtained results we identified 6-chloro-N-[2-(2,3-dihydro-1H-cyclopenta[b]quinolin-9-ylamino)-hexyl]]-nicotinamide hydrochloride (3e) as the most promising compound with inhibitory potencies against EeAChE and EqBuChE in the low nanomolar level 67 and 153 nM, respectively. Moreover, 3e compound is non-hepatotoxic, able to inhibit amyloid beta aggregation, and shows a mix-type of cholinesterase's inhibition. The mixed type of inhibition of the compound was confirmed by molecular modeling. Then, yeast three-hybrid (Y3H) technology was used to confirm the known ligand-receptor interactions. New derivatives do not show antioxidant activity (confirmed by the use of two different tests). A pKa assay method was developed to identify the basic physicochemical properties of 3e compound. A LogP assay confirmed that 3e compound fulfills Lipinsky's rule of five.

Determination of ligand efficiency indices in a group of 7H-purine-2,6-dione derivatives with psychotropic activity using micellar electrokinetic chromatography.

Discovering hit compounds and optimization processes in medicinal chemistry nowadays could be improved by predictive tools, based on the relationship between structure of molecules and lipophilic properties. Lipophilicity of drug candidate can affect both the pharmacokinetic and pharmacodynamics properties, in particular, the ability of a molecule to cross the cell membrane. Among the new methods for determination of the lipophilicity of compounds, micellar electrokinetic chromatography (MEKC) is considered to be an appropriate one for bioactive molecules, as it closely mimics the physiological conditions. In this paper MEKC was used for the estimation of the lipophilicity of 24 derivatives of 8-alkoxy-7H-purine-2,6-dione, designed and synthesized as potential antidepressant/anxiolytic and antipsychotic agents. The results of experimental method were compared with calculated in silico parameters (AlogPs and milogP by Virtual Computational Laboratory website, log PPallas by Pallas 3.1, Mlog P by Marvin, log PChemS by ChemSketch, log PChemDraw by ChemBioUltra) using Principal Component Analysis (PCA) method. Finally, using estimated log P values for selected compounds ligand - lipophilicity efficiency (LLE), per cent efficiency index (PEI), and binding efficiency index (BEI) parameters were calculated. Applied MEKC procedure could be used for selection of potential lead structure in a group of 7H-purine-2,6-dione derivatives.

Novel carbamate derivatives as selective butyrylcholinesterase inhibitors.

Selective butyrylcholinesterase inhibitors could be the promising drug candidates, used in treatment of Alzheimer's disease. The study describes the synthesis and biological activity of novel carbamate derivatives with N-phenylpiperazine, N-benzylpiperazine and 4-benzylpiperidine moieties. Biological studies revealed that most of these compounds displayed significant activity against BuChE. Compound 16 (3-(4-phenyl-piperazin-1-ylmethyl)-phenyl phenylcarbamate) turned out to be the most active (IC50 = 2.00 µM for BuChE). For all synthesized compounds lipophilicity and other physicochemical properties were calculated using computer programs. Relationship between these properties and activity was also checked. Binding mode with enzyme and the ensuing differences in activity were explained by the molecular modeling studies.