Structural Studies of the Taurine Transporter: A Potential Biological Target from the GABA Transporter Subfamily in Cancer Therapy.

The taurine transporter (TauT, SLC6A6) is a member of the solute carrier 6 (SLC6) family, which plays multiple physiological roles. The SLC6 family is divided into four subfamilies: GABA (γ-aminobutyric acid), monoamine, glycine and neutral amino acid transporters. Proteins from the GABA group, including the taurine transporter, are primarily considered therapeutic targets for treating central nervous system disorders. However, recent studies have suggested that inhibitors of SLC6A6 could also serve as anticancer agents. Overexpression of TauT has been associated with the progression of colon and gastric cancer. The pool of known ligands of this transporter is limited and the exact spatial structure of taurine transporter remains unsolved. Understanding its structure could aid in the development of novel inhibitors. Therefore, we utilized homology modelling techniques to create models of TauT. Docking studies and molecular dynamics simulations were conducted to describe protein-ligand interactions. We compared the obtained information for TauT with literature data on other members of the GABA transporter group. Our in silico analysis allowed us to characterize the transporter structure and point out amino acids crucial for ligand binding: Glu406, Gly62 and Tyr138. The significance of selected residues was confirmed through structural studies of mutants. These results will aid in the development of novel taurine transporter inhibitors, which can be explored as anticancer agents.

4-Oxypiperidine Ethers as Multiple Targeting Ligands at Histamine H3 Receptors and Cholinesterases.

This study examines the properties of a novel series of 4-oxypiperidines designed and synthesized as histamine H3R antagonists/inverse agonists based on the structural modification of two lead compounds, viz., ADS003 and ADS009. The products are intended to maintain a high affinity for H3R while simultaneously inhibiting AChE or/and BuChE enzymes. Selected compounds were subjected to hH3R radioligand displacement and gpH3R functional assays. Some of the compounds showed nanomolar affinity. The most promising compound in the naphthalene series was ADS031, which contained a benzyl moiety at position 1 of the piperidine ring and displayed 12.5 nM affinity at the hH3R and the highest inhibitory activity against AChE (IC50 = 1.537 µM). Eight compounds showed over 60% eqBuChE inhibition and hence were qualified for the determination of the IC50 value at eqBuChE; their values ranged from 0.559 to 2.655 µM. Therapy based on a multitarget-directed ligand combining H3R antagonism with additional AChE/BuChE inhibitory properties might improve cognitive functions in multifactorial Alzheimer's disease.

The SLC6A15-SLC6A20 Neutral Amino Acid Transporter Subfamily: Functions, Diseases, and Their Therapeutic Relevance.

The neutral amino acid transporter subfamily that consists of six members, consecutively SLC6A15-SLC620, also called orphan transporters, represents membrane, sodium-dependent symporter proteins that belong to the family of solute carrier 6 (SLC6). Primarily, they mediate the transport of neutral amino acids from the extracellular milieu toward cell or storage vesicles utilizing an electric membrane potential as the driving force. Orphan transporters are widely distributed throughout the body, covering many systems; for instance, the central nervous, renal, or intestinal system, supplying cells into molecules used in biochemical, signaling, and building pathways afterward. They are responsible for intestinal absorption and renal reabsorption of amino acids. In the central nervous system, orphan transporters constitute a significant medium for the provision of neurotransmitter precursors. Diseases related with aforementioned transporters highlight their significance; SLC6A19 mutations are associated with metabolic Hartnup disorder, whereas altered expression of SLC6A15 has been associated with a depression/stress-related disorders. Mutations of SLC6A18-SLCA20 cause iminoglycinuria and/or hyperglycinuria. SLC6A18-SLC6A20 to reach the cellular membrane require an ancillary unit ACE2 that is a molecular target for the spike protein of the SARS-CoV-2 virus. SLC6A19 has been proposed as a molecular target for the treatment of metabolic disorders resembling gastric surgery bypass. Inhibition of SLC6A15 appears to have a promising outcome in the treatment of psychiatric disorders. SLC6A19 and SLC6A20 have been suggested as potential targets in the treatment of COVID-19. In this review, we gathered recent advances on orphan transporters, their structure, functions, related disorders, and diseases, and in particular their relevance as therapeutic targets. SIGNIFICANCE STATEMENT: The following review systematizes current knowledge about the SLC6A15-SLCA20 neutral amino acid transporter subfamily and their therapeutic relevance in the treatment of different diseases.

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.

Guanidines: Synthesis of Novel Histamine H3R Antagonists with Additional Breast Anticancer Activity and Cholinesterases Inhibitory Effect.

This study examines the properties of novel guanidines, designed and synthesized as histamine H3R antagonists/inverse agonists with additional pharmacological targets. We evaluated their potential against two targets viz., inhibition of MDA-MB-231, and MCF-7 breast cancer cells viability and inhibition of AChE/BuChE. ADS10310 showed micromolar cytotoxicity against breast cancer cells, combined with nanomolar affinity at hH3R, and may represent a promising target for the development of an alternative method of cancer therapy. Some of the newly synthesized compounds showed moderate inhibition of BuChE in the single-digit micromolar concentration ranges. H3R antagonist with additional AChE/BuChE inhibitory effect might improve cognitive functions in Alzheimer's disease. For ADS10310, several in vitro ADME-Tox parameters were evaluated and indicated that it is a metabolically stable compound with weak hepatotoxic activity and can be accepted for further studies.

Taurine and Creatine Transporters as Potential Drug Targets in Cancer Therapy.

Cancer cells are characterized by uncontrolled growth, proliferation, and impaired apoptosis. Tumour progression could be related to poor prognosis and due to this fact, researchers have been working on novel therapeutic strategies and antineoplastic agents. It is known that altered expression and function of solute carrier proteins from the SLC6 family could be associated with severe diseases, including cancers. These proteins were noticed to play important physiological roles through transferring nutrient amino acids, osmolytes, neurotransmitters, and ions, and many of them are necessary for survival of the cells. Herein, we present the potential role of taurine (SLC6A6) and creatine (SLC6A8) transporters in cancer development as well as therapeutic potential of their inhibitors. Experimental data indicate that overexpression of analyzed proteins could be connected with colon or breast cancers, which are the most common types of cancers. The pool of known inhibitors of these transporters is limited; however, one ligand of SLC6A8 protein is currently tested in the first phase of clinical trials. Therefore, we also highlight structural aspects useful for ligand development. In this review, we discuss SLC6A6 and SLC6A8 transporters as potential biological targets for anticancer agents.

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.

Searching for new mTOR kinase inhibitors: Analysis of binding sites and validation of docking protocols.

The mammalian target of rapamycin (mTOR) is an important biological target for development of novel anticancer drugs and potential antiageing agents. Therefore, many scientific groups search for mTOR kinase inhibitors. Herein, we present structure-based approach which could be helpful in the studies on new bioactive compounds. Method validation was preceded by structural analysis of ATP catalytic cleft and FRB domain. In silico studies allowed us to point crucial amino acid residues for ligand binding and develop optimal docking protocols. The presented methodology could be applied for design and development of potential mTOR kinase inhibitors.

Discovery of small molecule mechanistic target of rapamycin inhibitors as anti-aging and anti-cancer therapeutics.

To date, the most studied drug in anti-aging research is the mTOR inhibitor - rapamycin. Despite its almost perfect anti-aging profile, rapamycin exerts one significant limitation - inappropriate physicochemical properties. Therefore, we have decided to utilize virtual high-throughput screening and fragment-based design in search of novel mTOR inhibiting scaffolds with suitable physicochemical parameters. Seven lead compounds were selected from the list of obtained hits that were commercially available (4, 5, and 7) or their synthesis was feasible (1, 2, 3, and 6) and evaluated in vitro and subsequently in vivo. Of all these substances, only compound 3 demonstrated a significant cytotoxic, senolytic, and senomorphic effect on normal and cancerous cells. Further, it has been confirmed that compound 3 is a direct mTORC1 inhibitor. Last but not least, compound 3 was found to exhibit anti-SASP activity concurrently being relatively safe within the test of in vivo tolerability. All these outstanding results highlight compound 3 as a scaffold worthy of further investigation.

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.

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.

Determination of Relative Density and Degree of Saturation in Mineral Soils Based on In Situ Tests.

Based on the results of dynamic probing (DP), time-domain reflectometry (TDR/MUX/MPTS), resistivity cone penetration tests (RCPT), Marchetti dilatometer tests (DMT), and seismic dilatometer tests (SDMT), it is possible to develop a relationship to calculate the relative density (Dr) and degree of saturation (Sr) of selected sandy soils. Compiled databases from documented research points for selected sandy soils were used to construct and develop direct correlations between the measured pressures p0 and p1 from DMT and shear wave velocity (Vs) from SDMT, along with pore water pressures (u0) and atmospheric pressure (Pa). The results allowed us to make a preliminary prediction when evaluating the parameters. Further, they allowed limiting the use of an additional device, especially in the case of multilayer heavy preconsolidated subsoils. Moreover, soil physical and mechanical characteristics (temperature, humidity, pressure, swelling, salinity) measured from TDR/MUX/MPTS (laboratory/field-operated meter for simultaneous measurements of soil moisture, matric potential, temperature, and salinity-bulk electrical conductivity) were assessed. The main achievement of this paper is the original proposal of using a new nomogram chart to determine the relative density and degree of saturation based on DMT and SDMT tests.

Novel Functionalized Amino Acids as Inhibitors of GABA Transporters with Analgesic Activity.

Neuropathic pain resistance to pharmacotherapy has encouraged researchers to develop effective therapies for its treatment. γ-Aminobutyric acid (GABA) transporters 1 and 4 (mGAT1 and mGAT4) have been increasingly recognized as promising drug targets for neuropathic pain (NP) associated with imbalances in inhibitory neurotransmission. In this context, we designed and synthesized new functionalized amino acids as inhibitors of GABA uptake and assessed their activities toward all four mouse GAT subtypes (mGAT1-4). According to the obtained results, compounds 2RS,4RS-39c (pIC50 (mGAT4) = 5.36), 50a (pIC50 (mGAT2) = 5.43), and 56a (with moderate subtype selectivity that favored mGAT4, pIC50 (mGAT4) = 5.04) were of particular interest and were therefore evaluated for their cytotoxic and hepatotoxic effects. In a set of in vivo experiments, both compounds 50a and 56a showed antinociceptive properties in three rodent models of NP, namely, chemotherapy-induced neuropathic pain models (the oxaliplatin model and the paclitaxel model) and the diabetic neuropathic pain model induced by streptozotocin; however compound 56a demonstrated predominant activity. Since impaired motor coordination is also observed in neuropathic pain conditions, we have pointed out that none of the test compounds induced motor deficits in the rotarod test.

Design, Synthesis, and In Vitro Antiproliferative Activity of Hydantoin and Purine Derivatives with the 4-Acetylphenylpiperazinylalkyl Moiety.

Cancer represents one of the most serious health problems and the second leading cause of death around the world. Heterocycles, due to their prevalence in nature as well as their structural and chemical diversity, play an immensely important role in anti-cancer drug discovery. In this paper, a series of hydantoin and purine derivatives containing a 4-acetylphenylpiperazinylalkyl moiety were designed, synthesized, and biologically evaluated for their anticancer activity on selected cancer cell lines (PC3, SW480, SW620). Compound 4, a derivative of 3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione, was the most effective against SW480, SW620, and PC3 cancer cell lines. Moreover, 4 has high tumor-targeting selectivity. Based on docking studies, it was concluded that R isomers of 3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione could be further studied as promising scaffolds for the development of thymidine phosphorylase inhibitors.

Cyanobiphenyls: Novel H3 receptor ligands with cholinesterase and MAO B inhibitory activity as multitarget compounds for potential treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a complex and incurable illness that requires the urgent approval of new effective drugs. However, since 2003, no new molecules have shown successful results in clinical trials, thereby making the common "one compound - one target" paradigm questionable. Recently, the multitarget-directed ligand (MTDL) approach has gained popularity, as compounds targeting at least two biological targets may be potentially more effective in treating AD. On the basis of these findings, we designed, synthesized, and evaluated through biological assays a series of derivatives of alicyclic amines linked by an alkoxy bridge to an aromatic lipophilic moiety of [1,1'-biphenyl]-4-carbonitrile. The research results revealed promising biological activity of the obtained compounds toward the chosen targets involved in AD pathophysiology; the compounds showed high affinity (mostly low nanomolar range of Ki values) for human histamine H3 receptors (hH3R) and good nonselective inhibitory potency (micromolar range of IC50 values) against acetylcholinesterase from electric eel (eeAChE) and equine serum butyrylcholinesterase (eqBuChE). Moreover, micromolar/submicromolar potency against human monoamine oxidase B (hMAO B) was detected for some compounds. The study identified compound 5 as a multiple hH3R/eeAChE/eqBuChE/hMAO B ligand (5: hH3R Ki = 9.2 nM; eeAChE IC50 = 2.63 µM; eqBuChE IC50 = 1.30 µM; hMAO B IC50 = 0.60 µM). Further in vitro studies revealed that compound 5 exhibits a mixed type of eeAChE and eqBuChE inhibition, good metabolic stability, and moderate hepatotoxicity effect on HepG2 cells. Finally, compound 5 showed a beneficial effect on scopolamine-induced memory impairments, as assessed by the passive avoidance test, thus revealing the potential of this compound as a promising agent for further optimization for AD treatment.

Biphenylalkoxyamine Derivatives-Histamine H3 Receptor Ligands with Butyrylcholinesterase Inhibitory Activity.

Neurodegenerative diseases, e.g., Alzheimer's disease (AD), are a key health problem in the aging population. The lack of effective therapy and diagnostics does not help to improve this situation. It is thought that ligands influencing multiple but interconnected targets can contribute to a desired pharmacological effect in these complex illnesses. Histamine H3 receptors (H3Rs) play an important role in the brain, influencing the release of important neurotransmitters, such as acetylcholine. Compounds blocking their activity can increase the level of these neurotransmitters. Cholinesterases (acetyl- and butyrylcholinesterase) are responsible for the hydrolysis of acetylcholine and inactivation of the neurotransmitter. Increased activity of these enzymes, especially butyrylcholinesterase (BuChE), is observed in neurodegenerative diseases. Currently, cholinesterase inhibitors: donepezil, rivastigmine and galantamine are used in the symptomatic treatment of AD. Thus, compounds simultaneously blocking H3R and inhibiting cholinesterases could be a promising treatment for AD. Herein, we describe the BuChE inhibitory activity of H3R ligands. Most of these compounds show high affinity for human H3R (Ki < 150 nM) and submicromolar inhibition of BuChE (IC50 < 1 µM). Among all the tested compounds, 19 (E153, 1-(5-([1,1'-biphenyl]-4-yloxy)pentyl)azepane) exhibited the most promising in vitro affinity for human H3R, with a Ki value of 33.9 nM, and for equine serum BuChE, with an IC50 of 590 nM. Moreover, 19 (E153) showed inhibitory activity towards human MAO B with an IC50 of 243 nM. Furthermore, in vivo studies using the Passive Avoidance Task showed that compound 19 (E153) effectively alleviated memory deficits caused by scopolamine. Taken together, these findings suggest that compound 19 can be a lead structure for developing new anti-AD agents.

Guanidine Derivatives: How Simple Structural Modification of Histamine H3R Antagonists Has Led to the Discovery of Potent Muscarinic M2R/M4R Antagonists.

This article describes the discovery of novel potent muscarinic receptor antagonists identified during a search for more active histamine H3 receptor (H3R) ligands. The idea was to replace the flexible seven methylene linker with a semirigid 1,4-cyclohexylene or p-phenylene substituted group of the previously described histamine H3R antagonists ADS1017 and ADS1020. These simple structural modifications of the histamine H3R antagonist led to the emergence of additional pharmacological effects, some of which unexpectedly showed strong antagonist potency at muscarinic receptors. This paper reports the routes of synthesis and pharmacological characterization of guanidine derivatives, a novel chemotype of muscarinic receptor antagonists binding to the human muscarinic M2 and M4 receptors (hM2R and hM4R, respectively) in nanomolar concentration ranges. The affinities of the newly synthesized ADS10227 (1-{4-{4-{[4-(phenoxymethyl)cyclohexyl]methyl}piperazin-1-yl}but-1-yl}-1-(benzyl)guanidine) at hM2R and hM4R were 2.8 nM and 5.1 nM, respectively.

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.