Discovery of α‐Substituted Imidazole‐4‐acetic Acid Analogues as a Novel Class of ρ1 γ‐Aminobutyric Acid Type A Receptor Antagonists with Effect on Retinal Vascular Tone

The ρ‐containing γ‐aminobutyric acid type A receptors (GABA_ARs) play an important role in controlling visual signaling. Therefore, ligands that selectively target these GABA_ARs are of interest. In this study, we demonstrate that the partial GABA_AR agonist imidazole‐4‐acetic acid (IAA) is able to penetrate the blood–brain barrier in vivo; we prepared a series of α‐ and N‐alkylated, as well as bicyclic analogues of IAA to explore the structure–activity relationship of this scaffold focusing on the acetic acid side chain of IAA. The compounds were prepared via IAA from l ‐histidine by an efficient minimal‐step synthesis, and their pharmacological properties were characterized at native rat GABA_ARs in a [³H]muscimol binding assay and at recombinant human α₁β₂γ_2S and ρ₁ GABA_ARs using the FLIPR? membrane potential assay. The (+)‐α‐methyl‐ and α‐cyclopropyl‐substituted IAA analogues ((+)‐ 6 a and 6 c , respectively) were identified as fairly potent antagonists of the ρ₁ GABA_AR that also displayed significant selectivity for this receptor over the α₁β₂γ_2S GABA_AR. Both 6 a and 6 c were shown to inhibit GABA‐induced relaxation of retinal arterioles from porcine eyes.     

Cinnamamide Derivatives for Central and Peripheral Nervous System Disorders—A Review of Structure–Activity Relationships

The cinnamamide scaffold has been incorporated in to the structure of numerous organic compounds with therapeutic potential. The scaffold enables multiple interactions, such as hydrophobic, dipolar, and hydrogen bonding, with important molecular targets. Additionally, the scaffold has multiple substitution options providing the opportunity to optimize and modify the pharmacological activity of the derivatives. In particular, cinnamamide derivatives have exhibited therapeutic potential in animal models of both central and peripheral nervous system disorders. Some have undergone clinical trials and were introduced on to the pharmaceutical market. The diverse activities observed in the nervous system included anticonvulsant, antidepressant, neuroprotective, analgesic, anti‐inflammatory, muscle relaxant, and sedative properties. Over the last decade, research has focused on the molecular mechanisms of action of these derivatives, and the data reported in the literature include targeting the γ‐aminobutyric acid type A (GABA_A) receptors, N‐methyl‐D ‐aspartate (NMDA) receptors, transient receptor potential (TRP) cation channels, voltage‐gated potassium channels, histone deacetylases (HDACs), prostanoid receptors, opioid receptors, and histamine H₃ receptors. Here, the literature data from reports evaluating cinnamic acid amide derivatives for activity in target‐based or phenotypic assays, both in vivo and in vitro, relevant to disorders of the central and peripheral nervous systems are analyzed and structure–activity relationships discussed.     

The Dual Role of the GABAA Receptor in Peripheral Inflammation and Neuroinflammation: A Study in Hyperammonemic Rats

Cognitive and motor impairment in minimal hepatic encephalopathy (MHE) are mediated by neuroinflammation, which is induced by hyperammonemia and peripheral inflammation. GABAergic neurotransmission in the cerebellum is altered in rats with chronic hyperammonemia. The mechanisms by which hyperammonemia induces neuroinflammation remain unknown. We hypothesized that GABA_A receptors can modulate cerebellar neuroinflammation. The GABA_A antagonist bicuculline was administrated daily (i.p.) for four weeks in control and hyperammonemic rats. Its effects on peripheral inflammation and on neuroinflammation as well as glutamate and GABA neurotransmission in the cerebellum were assessed. In hyperammonemic rats, bicuculline decreases IL-6 and TNFα and increases IL-10 in the plasma, reduces astrocyte activation, induces the microglia M2 phenotype, and reduces IL-1β and TNFα in the cerebellum. However, in control rats, bicuculline increases IL-6 and decreases IL-10 plasma levels and induces microglial activation. Bicuculline restores the membrane expression of some glutamate and GABA transporters restoring the extracellular levels of GABA in hyperammonemic rats. Blocking GABA_A receptors improves peripheral inflammation and cerebellar neuroinflammation, restoring neurotransmission in hyperammonemic rats, whereas it induces inflammation and neuroinflammation in controls. This suggests a complex interaction between GABAergic and immune systems. The modulation of GABA_A receptors could be a suitable target for improving neuroinflammation in MHE.     

5‐(Piperidin‐4‐yl)‐3‐Hydroxypyrazole: A Novel Scaffold for Probing the Orthosteric γ‐Aminobutyric Acid Type A Receptor Binding Site

A series of bioisosteric N₁‐ and N₂‐substituted 5‐(piperidin‐4‐yl)‐3‐hydroxypyrazole analogues of the partial GABA_AR agonists 4‐PIOL and 4‐PHP have been designed, synthesized, and characterized pharmacologically. The unsubstituted 3‐hydroxypyrazole analogue of 4‐PIOL ( 2 a ; IC₅₀～300 μM ) is a weak antagonist at the α₁β₂γ₂ GABA_AR, whereas substituting the N₁‐ or N₂‐position with alkyl or aryl substituents resulted in antagonists with binding affinities in the high nanomolar to low micromolar range at native rat GABA_ARs. Docking studies using a α₁β₂γ₂ GABA_AR homology model along with the obtained SAR indicate that the N₁‐substituted analogues of 4‐PIOL and 4‐PHP, 2 a – k , and previously reported 3‐substituted 4‐PHP analogues share a common binding mode to the orthosteric binding site in the receptor. Interestingly, the core scaffold of the N₂‐substituted analogues of 4‐PIOL and 4‐PHP, 3 b – k , are suggested to flip 180° thereby adapting to the binding pocket and addressing a cavity situated above the core scaffold.     

Characterization of the Functional Cross-Talk between Surface GABAA and Dopamine D5 Receptors

The γ-aminobutyric acid type A receptor (GABA_AR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABA_AR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABA_ARs and D5Rs form dynamic surface clusters. Disrupting the GABA_AR–D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABA_ARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABA_ARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABA_AR and D5R, and uncover a non-canonical role in regulating synaptic transmission.     

Muscimol Directly Activates the TREK-2 Channel Expressed in GABAergic Neurons through Its N-Terminus

The two-pore domain K⁺ (K_2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABA_AR and GABA_BR) reduces cellular excitability through Cl^- influx and K⁺ efflux in neurons. Relatively little is known about the link between GABA_AR and the K⁺ channel. The present study was performed to identify the effect of GABAR agonists on K_2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABA_AR and GABA_BR agonists. In particular, muscimol, a GABA_AR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABA_AR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl^- influx in GABAergic neurons.     

Purinergic Signaling: Impact of GPCR Structures on Rational Drug Design

The purinergic signaling system includes membrane-bound receptors for extracellular purines and pyrimidines, and enzymes/transporters that regulate receptor activation by endogenous agonists. Receptors include: adenosine (A₁, A_2A, A_2B, and A₃) and P2Y (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) receptors (all GPCRs), as well as P2X receptors (ion channels). Receptor activation, especially accompanying physiological stress or damage, creates a temporal sequence of signaling to counteract this stress and either mobilize (P2Rs) or suppress (ARs) immune responses. Thus, modulation of this large signaling family has broad potential for treating chronic diseases. Experimentally determined structures represent each of the three receptor families. We focus on selective purinergic agonists (A₁, A₃), antagonists (A₃, P2Y₁₄), and allosteric modulators (P2Y₁, A₃). Examples of applying structure-based design, including the rational modification of known ligands, are presented for antithrombotic P2Y₁R antagonists and anti-inflammatory P2Y₁₄R antagonists and A₃AR agonists. A₃AR agonists are a potential, nonaddictive treatment for chronic neuropathic pain.     

Predicting the transmembrane secondary structure of ligand-gated ion channels

Recent mutational analyses of ligand-gated ion channels (LGICs)have demonstrated a plausible site of anesthetic action withintheir transmembrane domains. Although there is a consensus thatthe transmembrane domain is formed from four membrane-spanningsegments, the secondary structure of these segments is not known.We utilized 10 state-of-the-art bioinformatics techniques topredict the transmembrane topology of the tetrameric regionswithin six members of the LGIC family that are relevant to anestheticaction. They are the human forms of the GABA alpha 1 receptor,the glycine alpha 1 receptor, the 5HT3 serotonin receptor, thenicotinic AChR alpha 4 and alpha 7 receptors and the TorpedonAChR alpha 1 receptor. The algorithms utilized were HMMTOP,TMHMM, TMPred, PHDhtm, DAS, TMFinder, SOSUI, TMAP, MEMSAT andTOPPred2. The resulting predictions were superimposed on toa multiple sequence alignment of the six amino acid sequencescreated using the CLUSTAL W algorithm. There was a clear statisticalconsensus for the presence of four alpha helices in those regionsexperimentally thought to span the membrane. The consensus of10 topology prediction techniques supports the hypothesis thatthe transmembrane subunits of the LGICs are tetrameric bundlesof alpha helices.     

Structure,Dynamics, and Ligand Recognition of Human-Specific CHRFAM7A (Dupα7) Nicotinic Receptor Linked to Neuropsychiatric Disorders

Cholinergic α7 nicotinic receptors encoded by the CHRNA7 gene are ligand-gated ion channels directly related to memory and immunomodulation. Exons 5–7 in CHRNA7 can be duplicated and fused to exons A-E of FAR7a, resulting in a hybrid gene known as CHRFAM7A, unique to humans. Its product, denoted herein as Dupα7, is a truncated subunit where the N-terminal 146 residues of the ligand binding domain of the α7 receptor have been replaced by 27 residues from FAM7. Dupα7 negatively affects the functioning of α7 receptors associated with neurological disorders, including Alzheimer’s diseases and schizophrenia. However, the stoichiometry for the α7 nicotinic receptor containing dupα7 monomers remains unknown. In this work, we developed computational models of all possible combinations of wild-type α7 and dupα7 pentamers and evaluated their stability via atomistic molecular dynamics and coarse-grain simulations. We assessed the effect of dupα7 subunits on the Ca²⁺ conductance using free energy calculations. We showed that receptors comprising of four or more dupα7 subunits are not stable enough to constitute a functional ion channel. We also showed that models with dupα7/α7 interfaces are more stable and are less detrimental for the ion conductance in comparison to dupα7/dupα7 interfaces. Based on these models, we used protein–protein docking to evaluate how such interfaces would interact with an antagonist, α-bungarotoxin, and amyloid Aβ₄₂. Our findings show that the optimal stoichiometry of dupα7/α7 functional pentamers should be no more than three dupα7 monomers, in favour of a dupα7/α7 interface in comparison to a homodimer dupα7/dupα7 interface. We also showed that receptors bearing dupα7 subunits are less sensitive to Aβ₄₂ effects, which may shed light on the translational gap reported for strategies focused on nicotinic receptors in ‘Alzheimer’s disease research.     

In Silico Screening of Novel α1-GABAA Receptor PAMs towards Schizophrenia Based on Combined Modeling Studies of Imidazo [1,2-a]-Pyridines

The ionotropic GABA_A receptor (GABA_AR) has been proven to be an important target of atypical antipsychotics. A novel series of imidazo [1,2-a]-pyridine derivatives, as selective positive allosteric modulators (PAMs) of α1-containing GABA_ARs with potent antipsychotic activities, have been reported recently. To better clarify the pharmacological essentiality of these PAMs and explore novel antipsychotics hits, three-dimensional quantitative structure–activity relationships (3D-QSAR), molecular docking, pharmacophore modeling, and molecular dynamics (MD) were performed on 33 imidazo [1,2-a]-pyridines. The constructed 3D-QSAR models exhibited good predictive abilities. The dockings results and MD simulations demonstrated that hydrogen bonds, π–π stackings, and hydrophobic interactions play essential roles in the binding of these novel PAMs in the GABA_AR binding pocket. Four hit compounds (DS01–04) were then screened out by the combination of the constructed models and computations, including the pharmacophore model, Topomer Search, molecular dockings, ADME/T predictions, and MD simulations. The compounds DS03 and DS04, with higher docking scores and better predicted activities, were also found to be relatively stable in the binding pocket by MD simulations. These results might provide a significant theoretical direction or information for the rational design and development of novel α1-GABA_AR PAMs with antipsychotic activities.     

Expanding the Genetic Code in Xenopus laevis Oocytes

Heterologous expression of ligand‐gated ion channels (LGICs) in Xenopus laevis oocytes combined with site‐directed mutagenesis has been demonstrated to be a powerful approach to study structure–function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl‐tRNA synthetase (aaRS)/suppressor tRNA_CUA pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N‐terminal domain of N‐methyl‐D ‐aspartate receptors (NMDARs) serve as photo‐crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes.     

Application of the guanidine-acylguanidine bioisosteric approach to argininamide-type NPY Y₂ receptor antagonists

Strongly basic groups such as guanidine moieties are crucial structural elements, but they compromise the drug‐likeness of numerous biologically active compounds, including ligands of G‐protein‐coupled receptors (GPCRs). As part of a project focused on the search for guanidine bioisosteres, argininamide‐type neuropeptide Y (NPY) Y₂ receptor (Y₂R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, N^G‐acylated argininamides were obtained by guanidinylation with tailor‐made mono‐Boc‐protected N‐acyl‐S‐methylisothioureas. The compounds were investigated for Y₂R antagonism (calcium assays), Y₂R affinity, and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the N^G‐substituted (S)‐argininamides showed Y₂R antagonistic activities and binding affinities similar to those of the parent compound, whereas N^G‐acylated or ‐carbamoylated analogues with a terminal amine were superior (Y₂R: K_i and K_B values in the low nanomolar range). This demonstrates that the basicity of the compounds, although 4–5 orders of magnitude lower than that of guanidines, is sufficient to form key interactions with acidic amino acids of the Y₂R. The acylguanidines bind with high affinity and selectivity to Y₂R over the Y₁, Y₄, and Y₅ receptors. As derivatization of the amino group is tolerated, these compounds can be considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for in vitro studies of the Y₂R. The results support the concept of bioisosteric guanidine–acylguanidine exchange as a broadly applicable approach to retain pharmacological activity despite decreased basicity.     

Design,Synthesis, and Characterization of a Novel Fluoroprobe for Live Human Islet Cell Imaging of Serotonin 5-HT1A Receptor

Mounting evidence suggests that the serotonin system serves in signal transmission to regulate insulin secretion in pancreatic islets of Langerhans. Among the 5-HT receptor subtype found in pancreatic islets, serotonin receptor 1A (5-HT_1A) demonstrates a unique ability to inhibit β-cell insulin secretion. We report the design, synthesis, and characterization of two novel fluorescent probes for the 5-HT_1A receptor. The compounds were prepared by conjugating the scaffold of the 5-HT_1A receptor agonist 8-OH-DPAT with two fluorophores suitable for live-cell imaging. Compound 5a {5-(dimethylamino)-N-[5-[(8-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino]pentyl]naphtalen-1-sulfonammide} showed high affinity for the 5-HT_1A receptor (K_i=1.8 nM). Fluoroprobe 5a was able to label the 5-HT_1A receptor in pancreatic islet cell cultures in a selective manner, as the fluorescence emission was significantly attenuated by co-administration of the 5-HT_1A receptor antagonist WAY-100635. Thus, fluoroprobe 5a showed useful properties to further characterize this unique receptor‘s role.     

Effect of Linker Length and Composition on Heterobivalent Ligand‐Mediated Receptor Cross‐Talk between the A1 Adenosine and β2 Adrenergic Receptors

Heterobivalent ligands that possess pharmacophores designed to interact with both the A₁ adenosine receptor (A₁AR) and the β₂ adrenergic receptor (β₂AR) were prepared. More specifically, these ligands contain an adenosine moiety that is linked via its N⁶‐position to the amino group of the saligenin‐substituted ethanolamine moiety present in the well‐known β₂AR agonist, salbutamol. The affinities of these ligands were determined at both receptors and found to vary with linker length and composition. With all compounds, affinity and functional potencies were found to have selectivity for the A₁AR over the β₂AR. In all cases, cAMP accumulation (a β₂AR‐mediated response) was mainly observed when the A₁AR was blocked or its function decreased by pertussis toxin or chronic agonist treatment. This suggests that heterobivalent compounds for receptors that mediate opposite responses might be useful for elucidating the mechanisms of receptor cross‐talk and how this interaction, in terms of responsiveness, may change under pathophysiological conditions.     

The inhibitory glycine receptor-simple views of a complicated channel

The strychnine-sensitive glycine receptor is the principal mediator of fast inhibitory synaptic transmission in the mammalian spinal cord and brain stem. As a member of the ligand-gated ion-channel family, it shares structural homology with the nicotinic acetylcholine, GABA(A/C) and serotonin 5-HT(3) receptors. Ion-channel activation and desensitisation are controlled by a variety of factors such as subunit composition, posttranslational modification, absence or presence of modulatory ions or other agents and possibly protein-protein interactions. Glycine-receptor mutations, either associated with the human motor disorder hyperekplexia or artificially introduced, have helped to define the regulatory domains of the receptor protein. In addition to their effects on glycine-receptor function, allelic variants of glycine-receptor genes may also affect biogenesis, assembly and degradation of the receptor.     

Binding Kinetics of ZM241385 Derivatives at the Human Adenosine A2A Receptor

Classical drug design and development rely mostly on affinity‐ or potency‐driven structure–activity relationships (SAR). Thus far, a given compound’s binding kinetics have been largely ignored, the importance of which is now being increasingly recognized. In the present study, we performed an extensive structure–kinetics relationship (SKR) study in addition to a traditional SAR analysis at the adenosine A_2A receptor (A_2AR). The ensemble of 24 A_2AR compounds, all triazolotriazine derivatives resembling the prototypic antagonist ZM241385 (4‐(2‐((7‐amino‐2‐(furan‐2‐yl)‐[1,2,4]triazolo[1,5‐a][1,3,5]triazin‐5‐yl)amino)ethyl)phenol), displayed only minor differences in affinity, although they varied substantially in their dissociation rates from the receptor. We believe that such a combination of SKR and SAR analyses, as we have done with the A_2AR, will have general importance for the superfamily of G protein‐coupled receptors, as it can serve as a new strategy to tailor the interaction between ligand and receptor.     

Isoflurane Potentiation of GABAA Receptors Is Reduced but Not Eliminated by the β3(N265M) Mutation

Background: Mice carrying the GABA_A receptor β3(N265M) point mutation, which renders receptors incorporating β3-subunits insensitive to many general anesthetics, have been used experimentally to link modulation of different receptor subtypes to distinct behavioral endpoints. Remarkably, however, the effect of the mutation on the susceptibility to modulation by isoflurane (a standard reference agent for inhalational vapors) has never been tested directly. Therefore, we compared the modulation by isoflurane of expressed α5β3(N265M)γ2L receptors with their wild type counterparts. Methods: Using whole-cell electrophysiological recording and rapid solution exchange techniques, we tested the effects of isoflurane at concentrations ranging from 80 μM to 320 μM on currents activated by 1 μM GABA. We measured drug modulation of wild-type α5β3γ2L GABA_A receptors and their counterparts harboring the β3(N265M) mutation. Results: Currents elicited by GABA were enhanced two- to four-fold by isoflurane, in a concentration-dependent manner. Under the same conditions, receptors incorporating the β3(N265M) mutation were enhanced by approximately 1.5- to two-fold; i.e., modulation by isoflurane was attenuated by approximately one-half. Direct activation by isoflurane was also present in mutant receptors but also attenuated. Conclusions: In contrast to the complete insensitivity of β3(N265M) mutant receptors to etomidate and propofol, the mutation has only a partial effect on receptor modulation by isoflurane. Therefore, the persistence of isoflurane effects in mutant mice does not exclude a possible contribution of β3-GABA_A receptors.     

Synthesis and in vivo Evaluation of Fluorine‐18 and Iodine‐123 Pyrazolo[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine Derivatives as PET and SPECT Radiotracers for Mapping A2A Receptors

Imaging agents that target adenosine type 2A (A_2A) receptors play an important role in evaluating new pharmaceuticals targeting these receptors, such as those currently being developed for the treatment of movement disorders like Parkinson′s disease. They are also useful for monitoring progression and treatment efficacy by providing a noninvasive tool to map changes in A_2A receptor density and function in neurodegenerative diseases. We previously described the successful evaluation of two A_2A‐specific radiotracers in both nonhuman primates and in subsequent human clinical trials: [¹²³I]MNI‐420 and [¹⁸F]MNI‐444. Herein we describe the development of both of these radiotracers by selection from a series of A_2A ligands, based on the pyrazolo[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine core of preladenant. Each of this series of 16 ligands was found to bind to recombinant human A_2A receptor in the low nanomolar range, and of these 16, six were radiolabeled with either fluorine‐18 or iodine‐123 and evaluated in nonhuman primates. These initial in vivo results resulted in the identification of 7‐(2‐(4‐(4‐(2‐[¹⁸F]fluoroethoxy)phenyl)piperazin‐1‐yl)ethyl)‐2‐(furan‐2‐yl)‐7H‐pyrazolo[4,3‐e][1,2,4]triazolo[1,5‐c]pyrimidin‐5‐amine ([¹⁸F]MNI‐444) and 7‐(2‐(4‐(2‐fluoro‐4‐[¹²³I]iodophenyl)piperazin‐1‐yl)ethyl)‐2‐(furan‐2‐yl)‐7H‐imidazo[1,2‐c]pyrazolo[4,3‐e]pyrimidin‐5‐amine ([¹²³I]MNI‐420) as PET and SPECT radiopharmaceuticals for mapping A_2A receptors in brain.     

Methamphetamine Blocks Adenosine A2A Receptor Activation via Sigma 1 and Cannabinoid CB1 Receptors

Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A_2A receptor (A_2AR). First, we noticed that methamphetamine does not affect A_2A functionality if the receptor is expressed in a heterologous system. However, A_2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB₁ receptor (CB₁R) and the sigma 1 receptor (σ₁R). Signaling via both adenosine A_2AR and cannabinoid CB₁R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A_2AR–CB₁R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A_2AR- and the CB₁R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusion, methamphetamine, with the participation of σ₁R, alters the expression and function of two interacting receptors, A_2AR, which is a therapeutic target for neuroprotection, and CB₁R, which is the most abundant G protein-coupled receptor (GPCR) in the brain.