The main results of studies of these sites and their ligands obtained in recent years will be considered below.
3.1. The Orthosteric Binding Site for Agonists and Competitive AntagonistsAs explained above, the orthosteric site is located inside the “clamshell” of the ligand-binding domain and provides the binding of full and partial agonists as well as competitive antagonists of the receptor. Although extensive research on its ligands has already been performed in previous years, it still attracts attention as a target for the development of potential drugs and/or pharmacological tools.
It should be noted that AMPA receptor agonists can enhance desensitization and often cause a number of side effects, including seizures and neurotoxicity [55]. In this regard, the development of competitive receptor antagonists is more promising for clinical use.The development of the quinoxaline-2,3-dione scaffold, a classic one for competitive AMPA receptor antagonists, has made it possible to obtain a large series of analogs with different selectivity profiles for ionotropic glutamate receptors, including a selective AMPA receptor antagonist 5 [56]. A detailed analysis of the binding of quinoxaline-2,3-diones showed that these compounds (for example, dinitro-derivative DNQX 6) are easily deprotonated at pH close to physiological and interact with the receptor specifically in the anionic form [57]. By modifying this scaffold, reversible photoswitchable antagonists such as ShuBQX-3 (7) were developed (only the trans-form has antagonistic activity; it is formed upon irradiation by light with a wavelength of 600 nm and turns into an inactive cis-form at 400–500 nm, making it possible to accurately and non-invasively control the receptor operation in space and time) [58]. Based on the combination of quinoxaline-2,3-dione and kynurenic acid scaffolds, a series of hybrid quinazolinediones was constructed, including the potential antiepileptic drug selurampanel (8) [59].Recently, new chemotypes of AMPA receptor agonists and competitive antagonists were being actively developed. For example, conformationally restricted bicyclic analogs of glutamic acid CIP-AS (9) and LM-12b (10) [60] act as partial or full agonists of the AMPA and kainate receptors, with a strong preference for the kainate GluK3 subunits. Their selectivity profiles and binding modes were analyzed using X-ray data. Based on the scaffold of aryl- and hetaryl-substituted phenylalanines, a series of competitive AMPA receptor antagonists such as compound 11 was obtained, their possible binding modes were analyzed using molecular docking, and their anticonvulsant and antioxidant properties were confirmed [61,62,63].By modifying the structure of the natural compound (S)-willardiine 12, which is a partial agonist of the AMPA receptor, a series of bicyclic derivatives of pyrimidinedione 13 was created [64,65]. It has been demonstrated that the replacement of the heterocyclic fragment leads to a significant (up to 500-fold) change in affinity. Molecular modeling and analysis of structural data obtained by X-ray crystallography have shown that this effect is caused by a significant difference in interactions with the protein and, especially, the binding site water molecules.In the development of a series of glutamate and aspartate analogs 14–16, the 4-hydroxy-1,2,3-triazole fragment was used as a bioisostere for the distal carboxylic group [66]. Compounds 14a–c and 15a were shown to be selective AMPA receptor agonists; moreover, compound 14b exhibited selectivity to certain types of AMPA receptor subunits.The classical chemotypes of the positive allosteric modulators of AMPA receptors are benzamides, benzothiadiazines, biarylalkylsulfonamides, and trifluoromethylpyrazoles.
Among the first AMPAR positive allosteric modulators were benzamides based on aniracetam 17. The most promising molecules of this chemotype were CX516 (18), CX614 (21), CX691 (22), and CX717 (23) [80,81] (the only difference between the two latter structures is the replacement of the piperidine fragment in CX691 by morpholine). Later, the more distant racetam analogs were obtained such as S47445 (CX1632, tulrampator) 24 [82] and 25 [83]. The analysis of the AMPAR LBD complexes with aniracetam, CX516 (18), Me-CX516 (19), and CX614 (21) [80] has shown that all these molecules bind in the central subpocket in a very similar way, with a key role played by the hydrogen bonds with the network of water molecules that mediate the interaction of a ligand with amino acid residues in the binding site (Figure 6A).To date, the most significant therapeutic effects of CX516 have been shown in the animal models of intellectual disability (at 5 mg/kg dose for 5 days) [84] and hyperactivity commonly observed in schizophrenia and autism spectrum disorder (at 10–40 mg/kg) [85]. In recent studies, CX546 has shown reasonably good results in the autism models and was found to effectively mediate neurogenesis and dendritogenesis [86,87]. However, its low oral bioavailability limits the clinical development of this agent. In its turn, during preclinical studies, CX614 has shown itself as a promising brain-derived neurotrophic factor (BDNF) inductor [88] and enhanced the effects of antidepressants such as imipramine and reboxetine.CX691 was studied for its procognitive effects in an animal model of Alzheimer’s disease, and increased hippocampal BDNF expression and improved spatial learning and memory were found [89]. For CX717, it has been established that it acts as a PAM and has a strong antidepressant effect [90]; in addition, CX717 is considered as a potential agent for the treatment of attention deficit hyperactivity disorder (ADHD).In recent preclinical studies, it was found that S47445 (CX1632, tulrampator) significantly enhances synaptic plasticity and increases neurotrophin levels both in the hippocampus and the prefrontal cortex of aged mice [82,91]. Interestingly, the antidepressant and anxiolytic effects were also detected for this ligand in three animal models. These behavioral effects were accompanied by increased levels of hippocampal neurogenesis and BDNF [92,93]. S47445 has also shown procognitive effects in animal models [94,95,96]. Based on these results, Phase I clinical studies have been started for S47445 as a potential agent for the treatment of Alzheimer’s disease and dementia-associated depression (NCT02626572, NCT02805439). However, the results of the first double-blind placebo-controlled clinical study in patients with mild to moderate Alzheimer’s disease and depressive symptoms suggested that S47445, although well tolerated, did not show significant improvement in cognitive functions [97].For compound 25, extensive in vitro and in vivo preclinical studies have been conducted, confirming that it acts as a neuroprotective agent and can significantly reduce neurological deficits and restore cognitive functions after ischemic brain injury [83].Although the derivatives and analogs of benzothiadiazine dioxide, starting with cyclothiazide (CTZ 26), represent one of the most thoroughly researched classes of the AMPAR positive allosteric modulators, their studies continue to attract considerable interest. During the search for new chemotypes of modulators and the investigation of their structure–activity relationships, a number of highly potent structures were found, for example, 27 [98], 28a and its unsaturated metabolite with comparable activity 28b [99]. In some cases, the “unexpected” structure–activity relationships discovered during the study could be explained using thermodynamic analysis, experimental data on the binding modes, and molecular modeling [100,101]. Unlike most of the studied benzothiadiazine dioxides, which admit independent binding of two modulator molecules in different regions of the binding site at the LBD dimer interface, the larger molecules of the phenoxy derivative 29 [102] and the specially designed dimeric compounds 30 [76] occupy several regions of the site by binding of one molecule (Figure 6B) and exhibit activity in the nanomolar range.Recently, an attempt was made to develop new positive allosteric modulators by switching from the benzothiadiazine dioxide scaffold to the thiochromane dioxide scaffold using a classical bioisosteric replacement [103]. However, the resulting series of compounds 31 exhibits lower activity toward the AMPA receptor compared to the original benzothiadiazine dioxides.Many of the studied negative allosteric modulators (non-competitive antagonists, non-competitive inhibitors) of the AMPA receptor bind in the linker region between the ligand-binding and transmembrane domains, preventing the displacement of the M3 helix and hindering the ion channel opening. Although for a number of antagonists with a confirmed non-competitive nature of action there is still no information on the binding site or the action mechanism, they will also be considered in this section.
A preeminent example of negative allosteric modulators (noncompetitive antagonists) of the AMPA receptor is perampanel 52 (PMP), approved by the FDA in 2012 as an antiepileptic drug [133]. Since then, research and clinical experience indicate that it is an effective antiepileptic agent with a broad spectrum and a novel mechanism of action [134,135], and may also be useful in alleviating functional and cognitive impairment after stroke [136,137]. In addition, its antitumor activity against brain tumors [138,139] and the possible effectiveness of negative allosteric modulators with a similar mechanism of action against tumors of other organs [140] have been demonstrated.Classical chemotypes of negative allosteric modulators of the AMPA receptor are 2,3-benzodiazepines and quinozalin-4-one derivatives. The binding mode and the mechanism of action of such modulators, using CP465022 (53), GYKI53655 (54), and perampanel as an example, were analyzed in detail by means of the X-ray diffraction analysis, site-directed mutagenesis, molecular and quantum mechanics, molecular docking, and molecular dynamics [141,142,143,144]. All these modulators bind in the interface region of the transmembrane domain (Figure 10). The large size and flexibility of the binding site “pocket”, which is able to adapt to different structures and orientations of the ligands, leads to the binding of modulators with significantly different structures in the same site. Modulator molecules play the role of “wedges” between the transmembrane segments, preventing the M3 helix from shifting when the channel opens. The binding occurs due to multiple weak interactions including hydrophobic contacts, π-stacking, and hydrogen bonds. At the same time, the interaction with certain amino acid residues depends on the state of the receptor, and the significance of some interactions remains debatable.Despite previous extensive studies of the 2,3-benzodiazepine scaffold, it continues to attract some interest in the search for new negative AMPA receptor modulators. Particularly, in recent years, its isoxazoline derivatives such as 55 [145] and m-chlorophenyl analog 56 (the o-chloro derivative is inactive) [146] have been obtained.In addition, new chemotypes of negative allosteric modulators of the AMPA receptor are currently being actively developed. The most interesting among them are the derivatives of phthalazine-1,4-dione (e.g., 57) [147], pyridothiazinone (e.g., 58) [148], benzodioxole (e.g., 59) [149], and 5-chloro-2-oxo-3H-benzoxazole (e.g., 60) [150]. A non-competitive inhibitory effect and influence on the kinetics of desensitization and deactivation for various subtypes of the AMPA receptor were also found for a number of curcumin derivatives 61 and 62 [151,152,153]. Several promising non-competitive AMPAR antagonists 63 were obtained in the study of arctigenin analogs, with molecular docking results suggesting a binding mode in the transmembrane domain similar to that of the known AMPA receptor non-competitive antagonists such as perampanel [154].For some new scaffolds of the allosteric AMPA receptor modulators, the activity cliffs are observed, that is, significant changes in the magnitude and even nature of activity occur with minor changes in the structure of the compound. Possible reasons for this phenomenon are currently under investigation.
For instance, among bis(tetrahydroquinazoline) derivatives [158,159,160], compounds 65a, 65b, 65d, 65e, 65f, 65h, and 65i are positive modulators of the AMPA receptor in a broad range of concentrations (maximum potentiation by 70%, 55%, 66%, 53%, 77%, 51%, and 61% at 1 nM, respectively), while compounds 65c, 65g, and 66 are negative modulators (maximum current reduction by 30% at 0.1 nM; by 50% at 1 μM or 30% at 0.1 nM; EC50 = 14 µM, respectively). In the bis(amide) series, compound 67a is a positive modulator in a broad range of concentrations (maximum potentiation by 40% at 1 nM), while compounds 67b–67d are negative modulators (maximum current decrease at 1 nM by 20%, 50%, and 40%, respectively) [161,162].
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