BENZOTHIADIAZINES DERIVATIVES AS NOVEL ALLOSTERIC
MODULATORS OF KAINIC ACID RECEPTORS

L-Glutamate is the main excitatory neurotransmitter of the mammalian CNS and its signal transduction is mediated by metabotropic and ionotropic receptors located pre- and post-synaptically. Ionotropic glutamate receptors (iGluR) are composed of four major subtypes, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainic acid (KA), N-methyl-D-aspartate (NMDA) and delta receptors, with closely related structure but different pharmacological and signalling properties (1).

AMPA receptors (AMPARs) are assembled from 4 subunits, GluA1-4, and the diverse combinations confer different functional and pharmacological properties (1-3). KARs are composed of different combinations of subunits (GluK1-5) which may form homomeric or heteromeric receptors. GluK4 and GluK5 form functional receptors only when associated with GluK1-3 (1, 4-6).

AMPARs are mainly located post-synaptically, while KARs that play an important role pre-synaptically by modulating neurotransmitter release, are located at the post-synaptic membrane only in specific brain areas (7). By a physiological point of view, AMPAs and KARs mediate different forms of short and long-term plasticity (8).

Because of the staminal roles of iGluRs in synaptic plasticity, learning and memory processes (1, 9), alterations in their signalling result in several neurological, neurodegenerative and psychiatric diseases, such as epilepsy, Alzheimer’s disease, depression, and schizophrenia (1, 2, 5, 10). In the last decades, considerable effort has been devoted to the development of compounds, synthetic or of natural origin, able to modulate glutamatergic function and to be used in the treatment of neurological deseases (10, 11). The rapidly growing interest in positive allosteric modulators (PAMs) of AMPARs (AMPA-PAMs) stems from their therapeutic properties as enhancers of glutamatergic transmission, putatively devoid of the side effects associated with direct agonists use. Several chemical classes of AMPA-PAMs have been reported and can be grouped into five classes based on their binding mode (12). These molecules bind to an allosteric site and enhance the AMPA receptor mediated current by decreasing desensitization and/or deactivation of the receptor (13).

AMPAR-PAMs bind to the interface between the ligand binding domain (LBD) of two subunits, stabilize dimer interface and prevent the receptor desensitization.

It was demonstrated that PAMs induced conformational changes in the ligand-binding core reduce desensitization by stabilizing the intradimer interface through diverse mechanisms (13-15).

One class of AMPA-PAMs is represented by the benzothiadiazines, and within this class IDRA21 (Fig. 1) represents one of the most important and studied lead compounds. Since IDRA21 crosses the blood brain barrier (16), in vivo studies highlighted its ability in reducing cognitive impairment in patas monkeys (17) and in improving cognition in rats (18). Administration of IDRA21 in vitro potentiates glutamate evoked current (19, 20), promotes LTP induction (21), and modulates synaptic activity acting at both AMPA (22) and NMDA receptors (23).

Fig. 1. Chemical structure of IDRA21 and c2.

On the negative side, IDRA21 is not very potent and produces a small but significant neurotoxic effect when coapplied with AMPA in vitro (24), even though at concentrations that are one order of magnitude greater than those antagonizing the cognitive deficit elicited by benzodiazepines in rats (18) and monkeys (17).

With the aim of developing substances with a better pharmacological profile, our research group synthesized several benzothiadiazine derivatives (25-29). Among these, 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide (c2, Fig. 1) was the most interesting because of its capability of potently potentiate the currents mediated by AMPAR expressed in cortical neurons in culture and to pass blood brain barrier (26).

Because of the important contribution of KARs to synaptic glutamatergic transmission (1, 8) and of the paucity of PAMs for KAR (1, 30), we investigated the effects of c2 and IDRA21 on native KARs expressed in cerebellar neuronal culture.

Since numerous subtypes of AMPAR and KAR are present in the brain and can be selectively modulated by drugs, we rated as important to elucidate the modulation of IDRA21 and c2 also on recombinant AMPA and KAR assembled from distinct subunit combinations.

MATERIAL AND METHODS

Chemistry

IDRA21 and c2 were synthetized as previously reported (19, 26). All compounds were characterized by melting points and 1H-NMR, and spectra showed the expected chemical shifts.

Primary culture of cerebellar granule cells

Primary cultures were prepared from 7 days old Sprague-Dawley rats (20). Briefly, cells from cerebellum were dispersed with trypsin (0.24 mg/ml; Sigma Aldrich, Milan, Italy) and plated at a density of 106 cells/ml on glass coverslips in 35 mm Falcon dishes coated with poly-L-lysine (10 mg/ml, Sigma Aldrich, Milan, Italy). Cells were plated in basal Eagle’s Medium (BME; Celbio, Milan, Italy), supplemented with 10% fetal bovine serum (Celbio, Milan, Italy), 2 mM glutamine and 100 mg/ml gentamycin (Sigma Aldrich, Milan, Italy) and maintained at 37°C in 5% CO2. 24 hours after plating cytosine arabinofuranoside (10 µM, Sigma Aldrich) was added to prevent glia proliferation. The experiments were performed on cells after 7–10 days in culture.

All experiments were carried out in accordance with the Declaration of Helsinki and with the European Communities Council Directive of 24 November 1986 (86/609).

Transient transfection of HEK293 cells

Transformed human embryonic kidney cells 293 (HEK293) (ATCC CRL1573) were grown in minimal essential medium supplemented with 10% fetal bovine serum containing 100 units of streptomycin and 100 units of penicillin for ml. Exponentially growing cells were trypsinized and seeded at a concentration of 105 cells per 35-mm dish in 2 ml of growth medium. The calcium phosphate transfection method (31) was used to transfect HEK293 cells with plasmids containing the subunits of rat KARs: GluK1 (Q-edited), GluK2 (Q-edited), GluK4, GluK5, and of rat AMPARs: the edited and flop form of GluA1 and GluA2. The cells were used 1–3 days after transfection. Plasmids were a generous gift from the late Prof. P.H. Seeburg from Heidelberg, Germany.

Electrophysiological recordings

Patch-clamp recordings were performed at room temperature in the whole-cell configuration (32). During the experiments, the cells were continuously perfused at 5 ml/min with standard solution containing: 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 5 mM HEPES, 5 mM glucose, and 20 mM sucrose, and the pH adjusted to 7.4 with NaOH. Patch electrodes had a resistance of 3–5 MΩ and were filled with 140 mM KCl, 5 mM HEPES, 5 mM EGTA, 3 mM MgCl2, and 2 mM Na2ATP, and the pH adjusted to 7.2 with KOH. Cells were clamped at –60 mV and access resistance was monitored throughout the recordings. Currents were amplified with an Axopatch 1D amplifier, filtered at 5 kHz, digitized at 10 kHz. Drugs were applied directly by gravity through a Y-tube perfusion system (33). Drug application had a fast onset and achieved a complete local perfusion of the recorded cell. Off-line data analysis, curve fitting, and figure preparation were performed with pClamp 8 (Axon Instruments, Foster City, CA, USA), Microsoft Office and Origin (Microcal, Northampton, MA, USA) software.

Data analysis

Off-line data analysis, curve fitting, and figure preparation were performed with Clampfit 8 (Axon Instruments) and Origin 4.1 (Microcal, Northampton, MA). SigmaPlot 11 (SYSTAT) was used for data analysis. All data are expressed as the arithmetic mean ± standard error of the mean (SEM) of n experiments. A fitting of the dose–response relationship was performed using the logistic equation:

where y is the substance concentration, EC50 is the concentration of the agonist eliciting the half-maximal response and nH the Hill coefficient. Student’ t-test was used to compare the values of potency (EC50) and efficacy (MaxEff) of the two compounds in the different conditions. The parameters were considered significantly different at p<0.05.

RESULTS

Employing the patch clamp technique in the whole cell configuration, we tested the modulatory effects of IDRA21 and c2 on kainate (KA)-evoked currents in cultured cerebellar granule cells. In control condition long pulse of 100 µM KA evoked a non desensitizing current that was potentiated by IDRA21 (100 µM) and c2 (10 µM) and the effect was reversible and dose dependent (Fig. 2). The analysis of the concentration response curves of the two molecules effect highlighted that c2 was more potent than IDRA21 (EC50c2 = 8±4 µM vs. EC50IDRA21 = 133±37 µM) and also more efficacious (MaxEffc2 = 177±20% vs. MaxEffIDRA21 = 99±19%). The data obtained were in agreement with the results previously reported in cortical cultures (26). Application of KA activates both AMPAR and KAR, even though the contribution of KAR to the total current is very low.

Fig. 2. IDRA21 and c2 potentiate KA-evoked current in cerebellar granule cells. (A): Representative whole cell recording showing the current elicited in a cerebellar neuron by application of KA (100 µM) alone and together with 100 µM IDRA21 (empty circles) or 10 µM c2 (filled squares). Bars highlight the duration of drug application. Vh= –60 mV. (B): Dose-response curves of the potentiation (% of control) of KA current by IDRA21 and c2. Each data point is the mean ±SEM of 10 cells. The values of the EC50 are shown in the inset.

In order to analyze the effect of IDRA21 and c2 only on AMPARs, HEK293 were transiently transfected with the GluA1 and GluA2 subunits of AMPARs. We focused our study only on these subunits because they are the main AMPAR subunits expressed in the CNS (34). Application of 100 µM glutamate (GLUT) to HEK293 cells expressing recombinant AMPAR elicited an inward current that was potentiated by c2 (100 µM) and IDRA21 (1 mM) as shown in Fig. 3. C2 was more potent and efficacious than IDRA21 on both homomeric receptors (GluA1: EC50IDRA21 585±130 µM and EC50c2 70±25 µM; GluA2: EC50IDRA21 532±80 µM and EC50c2 47±7 µM).

Fig. 3. IDRA21 and c2 are more efficacious at GluA1 than at GluA2 receptors. (A): Recordings displaying the effect of IDRA21 (1 mM) and c2 (100 µM) on GLUT (100 µM) - evoked current recorded from HEK293 cells transiently transfected with the GluA1 (upper panels) or GluA2 (lower panels) subunit of the AMPAR. Vh = –60mV. (B): Dose-response curve of the effect of IDRA21 (empty circles) and c2 (filled squares) on GLUT-evoked current (% of the control) at GluA1 (upper panel) and GluA2 (lower panel) expressing HEK293 cells. Each data point is the mean ±SEM of 7 cells. The values of the EC50 are shown in the inset.

The efficacy of c2 and IDRA21 was higher in GluA1 than in GluA2 (MaxEffc2 = 1600±200% for GluA1 vs. 529±290 % for GluA2; MaxEffIDRA21 = 300±62% for GluA1 vs. 105±11% for GluA2). Because, as previously mentioned, KA activates both KAR and AMPAR, we used GYKI 53655, a non-competitive antagonist of AMPA receptors, in order to dissect a “pure” KARs-mediated current. The co-application of KA and GYKI53655 elicited a response of low amplitude and fast desensitizing that was potentiated by IDRA21 (1 mM) and c2 (100 µM) (see Fig. 4A-4C).

Fig. 4. IDRA21 and c2 potentiate KARs-mediated current in cerebellar granule cells. Representative recording showing the current elicited in a cerebellar granule neuron by KA (100 µM) alone and in the presence of GYKI 53655 (100 µM) (A), the modulation of KA+GYKI evoked currents by 1 mM IDRA21 (B) and by 100 µM c2 (C), and (D): dose-response curves of IDRA21 (empty circles) and c2 (filled squares) effect on KA+GYKI plateau current. Each data point is the mean ±SEM of 10–14 cells.

C2 and IDRA21 potentiated native KAR-mediated currents in a dose-dependent manner with different potency (EC50c2 = 20±4 µM vs. EC50IDRA21 = 568±260 µM) and similar efficacy (MaxEffc2 = 500±70% vs. MaxEffIDRA21 = 375±110%). Because GYKI 53655 selectively blocks AMPAR, IDRA21 and c2 behave as positive allosteric modulators of KARs. To better investigate the activity of these compounds at the level of KAR, HEK293 cells were transiently transfected with diverse subunits of the receptor. The effect of IDRA21 and c2 was tested on GLUT-evoked current in HEK293 cells expressing homomeric receptors composed of GluK1 or GluK2 and heteromeric receptors assembled from GluK1 or GluK2 together with GluK4 or GluK5 subunits. For sake of simplicity, we are grouping the results based on the two “major” subunits GluK1 and GluK2 alone and in association with the “minor” subunits GluK4 or GluK5.

In Fig. 5 the effect of IDRA21 and c2 on GLUT-evoked current in cells expressing GluK1 receptors is shown. C2 was more potent on homomeric receptors but was significantly more efficacious in the heteromeric assemblies (MaxEffc2 = 275±10% in GluK1, 660±50% in GluK1-GluK4 and 710±85% in GluK1-GluK5). On the contrary, the potency and efficacy of IDRA21 was similar in all the subunit combinations containing GluK1 (see EC50 in the inset of Fig. 5).

Fig. 5. Modulation of Glut-evoked current by IDRA21 and c2 at recombinant KAR containing GluK1 subunit alone or with GluK4 and GluK5. (A): Representative traces showing the effect of IDRA21 (100 µM) (left panel) and of compound 2 (100 µM) (right panel) on GLUT (100 µ M)-evoked current recorded from HEK293 cells transiently transfected with GluK1 subunit. Vh= –60mV. (B): Dose-response curves of the effect of IDRA21 (left) and c2 (right) in HEK293 cells transfected with the GluK1alone or with GluK4 or GluK5 subunits. Each data point is the mean ±SEM of 6 cells.

We then tested the compounds effect in HEK293 transiently transfected with GluK2 alone or in combination with GluK4 or GluK5 (Fig. 6).

Fig. 6. Modulation of Glut-evoked current by IDRA21 and c2 on 293HEK cells expressing GluK2 subunit alone or with GluK4 and GluK5. (A): Representative whole cell recording showing the effect of IDRA21 (left) and compound 2 (right) on GLUT (100 µM)-evoked current in HEK293 cells transiently transfected with GluK2 and GluK5 subunits. Vh= –60mV. (B): Dose-response curve of the effect of IDRA21(left) and c2 (right) on GLUT-evoked current in HEK293 cells expressing GluK2 alone or in combination with GluK4 or GluK5. Each data point is the mean ±SEM of 5–9 cells.

C2 resulted more efficacious on homomeric receptors (MaxEffc2 = 800±120% in GluK2, 657±80% in GluK2-GluK4 and 448±85% in GluK2-GluK5). The efficacy of IDRA21 in cells expressing GluK2-GluK4 was lower compared to that of homomeric GluK2 and GluK2-GluK5 receptors (MaxEffIDRA21 = 400±100% in GluK2, 92±15% in GluK2-GluK4 and 293±18% in GluK2-GluK5). C2 potency was similar in all the subunit combinations containing K2 while IDRA21 was more potent in GluK2-GluK4.

The potency and efficacy of IDRA21 and c2 in native receptors and in the different subunit combinations of recombinant AMPA and KAR are summarized in Table 1.

Table 1. IDRA21 and c2 have different potency and efficacy depending on the subunit composition of AMPAR and KAR.

Potency (EC50) and efficacy (MaxEff) of IDRA21 and c2 (expressed as mean values ±SEM) are summarized in the different conditions. In parenthesis are the number of cells tested. *p<0.05, **p<0.001 Student’s t test. The comparison was made between IDRA21 and c2 effect.

DISCUSSION

The aim of this study was to analyze the activity of IDRA21 and of its new derivative on native and recombinant AMPAR and KAR responses. Here we show that these compounds, previously reported as AMPAR PAMs, exert also a strong modulatory effect on the currents mediated by KAR activation.

AMPA receptor modulation

In primary cultures of cerebellar neurons c2 is more potent than IDRA21 in potentiating KA-activated current and that its EC50 is in the low micromolar range, extending previous data obtained in cortical neurons (26).

The potency of both compounds was higher in native than in recombinant receptors. A similar behavior was highlighted also by Nagarajan et al. for some benzoylpiperidine derivatives belonging to the class of ampakines (22). The less efficient modulation detected in recombinant receptors can be due to different causes, for example to the expression, in granule cells, of receptors with different subunit isoforms, such as the GluA4c subunit (35) or to the activity of accessory proteins associated to native receptors (1, 36-38) that are not present in HEK293 cells.

In the mammalian brain, AMPAR are mainly formed by heteromeric assemblies between GluA2 and GluA1 or GluA3 subunits. Furthermore, they aggregate in macromolecular complexes comprising different constituents, such as the auxiliary proteins, which play important regulatory roles (39-41). For these reasons understanding the physiological functions and the pharmacological modulation of the different receptor assemblies is extremely complex.

C2 was more potent than IDRA21 in HEK293 cells transfected with homomeric AMPAR formed by GluA1 or GluA2 subunits. Interestingly, both compounds had low efficacy at GluA2 receptors, differently from ampakine that are more active at GluA2 than GluA1 receptors (22). It is difficult to forecast how this diverse selectivity can impact on the drugs modulation, we can surmise that the two classes of PAMs have distinct mechanisms of action. Our previous studies indicated that c2 binds, similarly to IDRA21, at the dimer interface of S1S2 GluA2 subunits (26). The increased activity of c2 with respect to IDRA21 can be explained by additional interactions with the ligand binding site due to heteroaryl substituent in the C5 position of the benzothiadiazine core.

Kainic acid receptor modulation

As for AMPAR, both compounds showed a weaker activity in recombinant compared to native KAR. Our study was performed in cerebellum granule cells that express GluK2 and K5 subunits together with the auxiliary subunit NETO2 (43). The auxiliary proteins have been shown to play a role in the receptor trafficking and also in determining KAR pharmacological and physiological properties (1, 7, 43-44). The low effect of IDRA21 and c2 at recombinant KAR could be ascribed, as previously mentioned for AMPAR, to a lack of accessory proteins or to KAR subunits with high affinity for the drugs expressed in native receptors.

IDRA21 and c2 are less potent on KAR than on AMPAR; indeed, their EC50 is in the high micromolar range (Table1) suggesting that in vivo their modulatory action will be mainly on AMPAR function. However, c2 was always more potent than IDRA21 (t-test, p<0.05). The potency of c2 is similar within the diverse subunit combinations of KARs, except at homomeric GluK1 receptors that show a good affinity for the compound, even though the co-expression of this subunit with GluK4 or GluK5 caused a reduction in the compound’s potency associated though to a significant (t-test, p<0.05) increase in the efficacy (Table 1). The presence of GluK4 and GluK5 subunits in the receptor assemblies should cause a change in c2 affinity for its binding site explaining the drastic reduction in potency that we detected on GluK1-GluK4 and GluK1-GluK5 receptors.

IDRA21 modulation is less dependent on the KAR subunit combinations, except for the receptors containing GluK2 and GluK4 where it is more potent but less efficacious.

The selective activity of the two chemicals on the different subunit assemblies could be caused by a diverse binding modality in the receptor pocket. As we previously mentioned, c2, compared to IDRA21, interacts with an additional hydrophobic pocket within the AMPA receptor structure (26) and very likely it binds differently also in the binding pocket of KAR. Additional crystallographic and molecular modeling studies might help understanding the binding mode of c2 within the different KAR subunit assemblies.

Molecular mechanism of IDRA21 and c2 at AMPA and kainic acid receptors

It has been widely demonstrated that IDRA21 potentiates AMPAR-mediated current by reducing receptor desensitization and slowing down the rate of deactivation (22, 45) and c2, given its chemical similarity with IDRA21, could act in a similar fashion but with higher efficacy. It has been suggested that a group of AMPAR PAMs, including aniracetam, that potentiates glutamatergic current by slowing the deactivation, acts by stabilizing the GluA2-LB2 clam shell in its agonist bound conformation (13). On the other side, modulators that have a main effect on desensitization such as IDRA21 favor dimerization of the GluA2 - ligand binding domain (14).

In outside-out patches from recombinant GluA2 receptors and from native receptors IDRA21 slows down the time constant of desensitization producing an increase in the GLUT steady state current without affecting peak current (22). The same Authors suggested that IDRA21 binds preferentially to the non desensitized state of the receptor while ampakines bind only to the receptor when the agonist is bound and act by destabilizing the desensitized conformation of the receptors (22).

IDRA21 and c2 modulatory effects have different kinetics: the maximal potentiation induced by c2 takes time to develop and to be washed out, while the effect of IDRA21 reaches its maximal value almost immediately (Figs. 2-5). The slow kinetic of c2 effect could be determined by a difficulty in reaching the binding site within the receptor structure, as previously shown for other PAM, i.e. cyclothiazide (13, 14). Alternatively, this behavior suggests that the binding of c2 to the receptor, differently from that of IDRA21 (22), is promoted by the presence of the agonist. It is also possible that c2 binds better to the desensitized state, unlike IDRA21 that has higher affinity for the non desensitized state (22).

What is the mechanism of action of IDRA21 and c2 at kainate receptors?

It is possible that the potentiation of KAR - mediated currents by IDRA21 and c2 be obtained through a removal of receptor desensitization even though the desensitized structures of AMPA and KAR are different (46-48).

Indeed, in a very interesting paper Larsen et al. (30) reported that some N-4 alkylated benzothiadiazines, structurally related to IDRA21 and proved to be powerful AMPARs PAMs (49), were also active on recombinant KARs. They showed that these compounds, in outside-out patches from 293HEK cells expressing different homomeric KAR subunits, increase peak amplitude and slow down the decay of GLUT elicited current. The current potentiation was mainly due to a decrease in the KAR desensitization and to a lesser extent in the KAR deactivation kinetic. The same Authors, analyzing the compounds interaction at the level of the GluK1-LBD dimer, found that they bind at the dimer interface and by increasing the stability of this structure produce a strong reduction in receptor desensitization (30).

As shown in Table 1, IDRA21 and c2 have different efficacy depending on the subunit combination of the receptors; this diverse performance could be related to distinct desensitization properties of the receptor assemblies (50), for example receptors that are slowly desensitizing will be less sensitive to the drug while receptors that are highly desensitizing will be more impacted by the compounds modulation.

Importance of developing positive allosteric modulators for kainate receptors

The good activity of c2 as PAM of AMPAR and KAR in vitro suggests that it could exert important functions when administered in vivo, as a matter-of-fact mouse cerebral microdialysis studies indicate that it crosses the blood brain barrier after intraperitoneal injection (26).

KAR are involved in synaptic modulation at pre-, post- and/or extrasynaptic sites (1, 8). KAR exert their functions at both excitatory and inhibitory synapses, by inducing postsynaptic depolarization, modulating neurotransmitter release, and, through a metabotropic signaling, regulating ion channel function and intrinsic excitability. A biphasic activity of KAR in modulating glutamate release (i.e. facilitating at low concentrations and depressing at higher doses) has been reported in several CNS areas, including hippocampus, cerebellum and cortex (51). In the spinal cord, a functional interaction between KAR and other presynaptic receptors, such as GABAA, has also been observed (52).

Furthermore, several forms of short- and long -term synaptic plasticity require the activation of KAR, leading to the modulation of KAR function itself or to the regulation of other synaptic mechanisms, such as AMPAR expression or neurotransmitter release (53).

The role of KAR desensitization in synaptic modulation and plasticity has been scarcely investigated. However, a decrease of KAR desensitization could importantly affect several aspects of neural activity, including summation of postsynaptic responses, frequency-dependence of action potential firing, and presynaptic modulation of transmitter release. PAMs acting on KAR, such as c2, could be of great use as pharmacological tools to understand the role of KAR desensitization in synaptic modulation and plasticity. KAR have been shown to play important role in several pathological states of the CNS such as epilepsy, ischemic brain injury, schizophrenia, and depression (5).

Since few modulators of KAR are available so far (30), the 3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide core could represent an interesting scaffold to obtain more potent and selective PAMs on both AMPARs and KARs.

In conclusion, we show here that small molecules such as benzothiadizines are able to modulate KA receptors expressed in neurons. We demonstrated for the first time that two positive allosteric modulators of AMPAR, i.e., IDRA21 and 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide potentiate the currents mediated by native and recombinant AMPA and KAR with different sensitivities.

Additional studies might be required to extrapolate the structural features necessary to retain a good affinity and efficacy only at native KARs since selective PAM for KAR will be a very important tool to study in vivo KAR function but also important regulators of neuronal network activity.

Abbreviations: AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CNS, central nervous system; IDRA21, 7-chloro-5-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide; c2, 7-chloro-5-3-furanyl-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide; iGluR, ionotropic glutamate receptors; KA, kainic acid; KAR, kainate receptor; NMDA, N-methyl-D-aspartate; PAM, positive allosteric modulators;

Author contributions: G. Puja was responsible for conceiving and supervise the project. U.M. Battisti and C. Citti carried out the synthesis of the compounds, and G. Cannazza was responsible for the supervision. F. Ravazzini and G. Losi conducted the electrophysiological experiments and F. Ravazzini and G. Puja were responsible for the analysis of the data and figures preparation. G. Puja, F. Ravazzini, U.M. Battisti, R. Bardoni wrote the manuscript.

Conflict of interests: None declared.

REFERENCES

1. Hansen KB, Wollmuth LP, Bowie D, et al. Structure, function, and pharmacology of glutamate receptor ion channels. Pharmacol Rev 2021; 73: 298-487.
2. Greger IH, Mayer ML. Structural biology of glutamate receptor ion channels: towards an understanding of mechanism. Curr Opin Struct Biol 2019; 57: 185-195.
3. Hansen KB, Yuan H, Traynelis SF. Glutamate receptor ion channel: structure, regulation and Structural aspects of AMPA receptor activation, desensitization and deactivation. Curr Opin Neurobiol 2007; 17: 281-288.
4. Jane DE, Lodge D, Collingridge GL. Kainate receptors: pharmacology, function and therapeutic potential. Neuropharmacology 2009; 56: 90-113.
5. Lerma J, Marques JM. Kainate receptors in health and disease. Neuron 2013; 80: 292-311.
6. Mayer ML. Structural biology of Kainate receptors. Neuropharmacology 2021; 190: 108511 doi: 10.1016/j.neuropharm.2021.108511
7. Valbuena S, Lerma J. Kainate receptors, homeostatic gatekeepers of synaptic plasticity. Neuroscience 2021; 456: 17-26.
8. Negrete-Diaz JV, Falcon-Moya R, Rodriguez-Moreno A. Kainate receptors: from synaptic activity to disease. FEBS J 2021; Jun 18: doi: 10.1111/febs.16081.
9. Peng S, Zhang Y, Zhang J, Wang H, Ren B. Glutamate receptors and signal transduction in learning and memory. Mol Biol Rep 2011; 38: 453-460.
10. Chang PK, Verbich D, McKinney RA. AMPA receptors drug targeting neurological disease: advantage, caveats, and future outlook. Eur J Neurosci 2012; 35: 1908-1916.
11. Rahman HM, Javaid S, Ashraf W, et al. Neuropharmacological investigation, ultra-high performance liquid chromatography analysis, and in silico studies of Phyla nodiflora. J Physiol Pharmacol 2021; 72: 637-654.
12. Frydenvang K, Pickering DS, Kastrup JS. Structural basis for positive allosteric modulators of AMPA and kainate receptors. J Physiol 2022; 600: 181-200.
13. Jin R, Clark S, Weeks AM, Dudman JT, Gouaux E, Partin KM. Mechanism of positive allosteric modulators acting on AMPA receptors. J Neurosci 2005; 25: 9027-9036.
14. Sun Y, Olson R, Horning M, Armstrong N, Mayer ML, Gouaux E. Mechanism of glutamate receptor desensitization. Nature 2002; 417: 245-253.
15. Veran J, Kumar J, Pinheiro PS, et al. Zinc potentiates GLUK3 glutamate receptor function by stabilizing the ligand binding domain interface. Neuron 2012; 76: 565-578.
16. Buccafusco JJ, Weiser T, Winter K, Klinder K, Terry AV. The effects of IDRA 21, a positive modulator of the AMPA receptor, on delayed matching performance by young and aged rhesus monkeys. Neuropharmacology 2004; 46: 10-22.
17. Thompson DM, Guidotti A, DiBella M, Costa E. 7-Chloro-3-methyl-3,4-dihydr o-2H-1,2,4-bezothiadiazine-S, S-dioxide (IDRA-21), a congener of aniracetam, potently abates pharmacologically induced cognitive impairments in patas monkeys. Proc Natl Acad Sci USA 1995; 92: 7667-7671.
18. Zivkovic I, Thompson DM, Bertolino M et al. 7-Chloro-3-methyl-3-4-dihydro-2H-1,2,4 benzothiadiazine S, S-dioxide (IDRA 21): a benzothiadiazine derivative that enhances cognition by attenuating DL-alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor desensitization. J Pharmacol Exp Ther 1995; 272: 300-309.
19. Bertolino M, Baraldi M, Parenti C, et al. Modulation f AMPA/kainate receptors by analogues of diazoxide and cyclothiazide in thin slices of rat hippocampus. Receptor Channels 1993; 1: 267-278.
20. Puia G, Losi G, Razzini G, Braghiroli D, DiBella M, Baraldi M. Modulation of kainate - activated currents by diazoxide and cyclothiazide analogues (IDRA) in cerebellar granule neurons. Prog Neuro Psychopharmacol Biol Psych 2000; 24: 1007-1015.
21. Arai A, Guidotti A, Costa E, Lynch G. Effect of the AMPA modulator IDRA-21 on LTP in hippocampal slices. NeuroReport 1996; 7: 2211-2215.
22. Nagarajan N, Quast C, Boxall AR, Shahid M, Rosenmund C. Mechanism and impact of allosteric AMPA receptor modulation by the ampakine CX546. Neuropharmacology 2001; 41: 650-663.
23. Losi G, Puia G, Braghiroli D, Baraldi M. IDRA 21, a positive AMPA receptor modulator, inhibits synaptic and extrasynaptic NMDA receptor mediated events in cultured cerebellar granule cell. Neuropharmacology 2004; 46: 1105-1113.
24. Impagnatiello F, Oberto A, Longone P, Costa E, Guidotti A. 7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S, S-dioxide: a partial modulator of AMPA receptor desensitization devoid of neurotoxicity. Proc Natl Acad Sci USA 1997; 94: 7053-7058.
25. Braghiroli D, Puia G, Cannazza G, et al. Synthesis of 3,4-dihydro-2H-1,2,4-benzo-thiadiazine 1,1-dioxide derivatives as potential allosteric modulators of AMPA/kainate receptors. J Med Chem 2002; 45: 2355-2357.
26. Battisti UM, Jozwiak K, Cannazza G, et al. 5-Arylbenzothiadiazine type compounds as positive allosteric modulators of AMPA/kainate receptors. ACS Med Chem Lett 2011; 3: 25-29.
27. Citti C, Battisti UM, Cannazza G, et al. 7-Chloro-5-(furan-3-yl)-3-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide as positive allosteric modulator of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. The end of the unsaturated-inactive paradigm? ACS Chem Neurosci 2016; 7: 149-160.
28. Carrozzo M, Battisti UM, Cannazza G, at al. Design, stereoselective synthesis, configurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e] pyrrol[2,1- c][1,2,4]thiadiazine 5,5-dioxide. Bioorg Med Chem 2014; 22: 4667-4676.
29. Cannazza G, Battisti UM, Carrozzo M, et al. Development of an in vitro liquid chromatography-mass spectrometry method to evaluate stereo and chemical stability of new drug candidates employing immobilized artificial membrane column. J Chromatogr A 2014; 1363: 216-225.
30. Larsen AP, Fievre S, Frydenvang K, et al. Identification and structure-function study of positive allosteric modulators of kainate receptors. Mol Pharmacol 2017; 91: 576-585.
31. Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 1987; 7: 2745-2752.
32. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth B, Hamill FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflug Arch 1981; 391: 85-100.
33. Murase K, Ryu, PD, Randic M. Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons. Neurosci Lett 1989; 103: 56-63.
34. Lu W, Shi Y, Alexander C, et al. Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach. Neuron 2009; 62: 254-268.
35. Kamalova A, Nakagawa T. AMPA receptor structure and auxiliary subunits. J Physiol 2021; 599: 453-469.
36. Greger H, Watson JF, Cull-Candy SG. Structural and functional architecture of AMPA-type glutamate receptors and their auxiliary proteins. Neuron 2017; 94: 713-730.
37. Chen S, Gouaux E. Structure and mechanism of AMPA receptor auxiliary protein complexes. Curr Opin Struct Biol 2019; 54: 104-111.
38. Kawahara Y, Ito K, Sun H, Ito M, Kanazawa I, Kwak S. GluR4c, an alternative splicing isoform of GluR4, is abundantly expressed in the adult human brain. Mol Br Res 2004; 127: 150-115.
39. Jackson AC, Milstein AD, Soto D, Farrant M, Cull-Candy SG, Nicoll RA. Probing TARP modulation of AMPA receptor conductance with polyamine toxins. J Neurosci 2011; 31: 7511-7520.
40. Miguez-Cabello F, Sanchez-Fernandez N, Yefimenko N, et al. AMPAR/TARP stoichiometry differentially modulates channel properties. 2020 Elife May 26; 9: e53946. doi: 10.7554/eLife.53946
41. Shi Y, Lu W, Milstein AD, Nicoll RA. The stoichiometry of AMPA receptors and TARPs varies by neuronal cell type. Neuron 2009; 62: 633-640.
42. Tang M, Ivakine E, Mahadevan V, Salter MW, McInnes RR. Neto2 interacts with the scaffolding protein GRIP and regulates synaptic abundance of kainate receptors PLoS One 2012; 7: e51433. doi: 10.1371/journal.pone.0051433
43. Vinnakota R, Dhingra S, Kumari J, et al. Role of Neto1 extracellular domain in modulation of kainate receptors. Int J Biol Macromol 2021; 192: 525-536.
44. Fisher JL. The auxiliary subunits Neto1 and Neto2 have distinct, subunit-dependent effects at recombinant GluK1- and GluK2-containing kainate receptors. Neuropharmacology 2015; 99: 471-480.
45. Yamada KA, Hill MW, Hu Y, Covey DF. The diazoxide derivative 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-S, S-dioxide augments AMPA- and GABA-mediated synaptic responses in cultured hippocampal neurons. Neurobiol Dis 1998; 5: 196-205.
46. Meyerson JR, Chittori S, Merk A, et al. Structural basis of kainate subtype glutamate receptor desensitization. Nature 2016; 537: 567-571.
47. Chaudhry C, Weston MC, Schuck P, Rosenmund C, Mayer ML. Stability of ligand-binding domain dimer assembly controls Kainate receptor desensitization. EMBO J 2009; 28: 1518-1530.
48. Klykov O, Gangwar SP, Yelshanskaya MV, Yen L, Sobolevsky AI. Structure and desensitization of AMPA receptor complexes with type II TARP γ5 and GSG1L. Mol Cell 2021; 81: 4771-4783.e7.
49. Norholm A-B, Francotte P, Olsen L, et al. Synthesis, pharmacological and structural characterization, and thermodynamic aspects of GluA2-positive allosteric modulators with a 3,4-dihydro-2H-1,2,4-benzothiadiazine1,1-dioxide scaffold. J Med Chem 2013; 56: 8736-8745.
50. Mott DD, Rojas A, Fisher JL, Dingledine RJ, Benveniste M. Subunit-specific desensitization of heteromeric kainate receptors. J Physiol 2010; 588: 683-700.
51. Falcon-Moya R, Rodriguez-Moreno A. Metabotropic actions of kainate receptors modulating glutamate release. Neuropharmacology 2021; 197: 108696. doi: 10.1016/j.neuropharm.2021.108696
52. Betelli C, MacDermott AB, Bardoni R. Transient, activity dependent inhibition of transmitter release from low threshold afferents mediated by GABAA receptors in spinal cord lamina III/IV. Mol Pain 2015; 11: 64. doi: 10.1186/s12990-015-0067-5
53. Nair JD, Wilkinson KA, Henley JM, Mellor JR. Kainate receptors and synaptic plasticity. Neuropharmacology 2021; 196: 108540. doi: 10.1016/j.neuropharm.2021.108540