Allosterism in the adenosine A2A and cannabinoid CB2 heteromer.

BACKGROUND AND PURPOSE: Allosterism is a regulatory mechanism for GPCRs that can be attained by ligand-binding or protein-protein interactions with another GPCR. We have studied the influence of the dimer interface on the allosteric properties of the A2A receptor and CB2 receptor heteromer. EXPERIMENTAL APPROACH: We have evaluated cAMP production, phosphorylation of signal-regulated kinases (pERK1/2), label-free dynamic mass redistribution, ß-arrestin 2 recruitment and bimolecular fluorescence complementation assays in the absence and presence of synthetic peptides that disrupt the formation of the heteromer. Molecular dynamic simulations provided converging evidence that the heteromeric interface influences the allosteric properties of the A2AR-CB2R heteromer. KEY RESULTS: Apo A2AR blocks agonist-induced signalling of CB2R. The disruptive peptides, with the amino acid sequence of transmembrane (TM) 6 of A2AR or CB2R, facilitate CB2R activation, suggesting that A2AR allosterically prevents the outward movement of TM 6 of CB2R for G protein binding. Significantly, binding of the selective antagonist SCH 58261 to A2AR also facilitated agonist-induced activation of CB2R. CONCLUSIONS AND IMPLICATIONS: It is proposed that the A2AR-CB2R heteromer contains distinct dimerization interfaces that govern its functional properties. The molecular interface between protomers of the A2AR-CB2R heteromer interconverted from TM 6 for apo or agonist-bound A2AR, blocking CB2R activation, to mainly the TM 1/7 interface for antagonist-bound A2AR, facilitating the independent opening of intracellular cavities for G protein binding. These novel results shed light on a different type of allosteric mechanism and extend the repertoire of GPCR heteromer signalling.

Molecular glues as potential GPCR therapeutics.

"Molecular Glues" are defined as small molecules that can either be endogenous or synthetic which promote interactions between proteins at their interface. Allosteric modulators, specifically GPCR allosteric modulators, can promote both the association and the dissociation of a given receptor's transducer but accomplishes this "at a distance" from the interface. However, recent structures of GPCR G protein complexes in the presence of allosteric modulators indicate that some GPCR allosteric modulators can act as "molecular glues" interacting with both the receptor and the transducer at the interface biasing transducer signaling in both a positive and negative manner depending on the transducer. Given these phenomena we discuss the implications for this class of allosteric modulators to be used as molecular tools and for future drug development.

Amplification sensing manipulated by a sumanene-based supramolecular polymer as a dynamic allosteric effector.

The synthesis of signal-amplifying chemosensors induced by various triggers is a major challenge for multidisciplinary sciences. In this study, a signal-amplification system that was flexibly manipulated by a dynamic allosteric effector (trigger) was developed. Herein, the focus was on using the behavior of supramolecular polymerization to control the degree of polymerization by changing the concentration of a functional monomer. It was assumed that this control was facilitated by a gradually changing/dynamic allosteric effector. A curved-π buckybowl sumanene and a sumanene-based chemosensor (SC) were employed as the allosteric effector and the molecular binder, respectively. The hetero-supramolecular polymer, (SC·(sumanene)n), facilitated the manipulation of the degree of signal-amplification; this was accomplished by changing the sumanene monomer concentration, which resulted in up to a 62.5-fold amplification of a steroid. The current results and the concept proposed herein provide an alternate method to conventional chemosensors and signal-amplification systems.

High Pressure Promotes Binding of the Allosteric Inhibitor Zn2+-Cyclen in Crystals of Activated H-Ras.

In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP-bound and inactive GDP-bound states. Various states of GTP-bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+-cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H-Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+-cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+-cyclen binding is observed at 500 MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+-cyclen to H-Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x-ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low-populated conformations at ambient conditions, enabling the design of specific inhibitors.

Antinociceptive effects of α2/α3-subtype selective GABAA receptor positive allosteric modulators KRM-II-81 and NS16085 in rats: behavioral specificity.

Recent studies suggest that amongst the GABAA receptor subtype heterogeneity, α2/α3 subunits of GABAA receptors mediate pain processing. Therefore, α2/α3-subtype selective GABAA receptor positive allosteric modulators (PAMs) may be candidate analgesics. Antinociceptive effects of α2/α3-subtype selective GABAA receptor PAMs have been reported, but the behavioral effects of these compounds have not been systematically evaluated. This study examined the behavioral effects of two α2/α3 subtype-selective GABAA receptor PAMs, KRM-II-81 and NS16085, in male rats. The antinociceptive effects of KRM-II-81 and NS16085 were examined using rat models of inflammatory (complete Freund's adjuvant) and neuropathic pain (chronic constriction injury). The effect of KRM-II-81 on affective pain was measured using the place escape/avoidance paradigm (PEAP). Rate-response of food-maintained operant responding, horizontal wire test, and the spontaneous alternation T-maze, were assessed to study the side-effect profiles of KRM-II-81 and NS16085. The benzodiazepine midazolam was used as a comparator in these studies. KRM-II-81 and NS16085 attenuated mechanical allodynia but not thermal hyperalgesia in both pain states, and their effects were attenuated by the benzodiazepine receptor antagonist flumazenil. KRM-II-81 attenuated affective pain-related behavior in the PEAP test. In the operant responding procedure and horizontal wire test, only midazolam produced significant effects at the dose that produced maximal antinociception. In the T-maze assay, only midazolam significantly decreased the percentage of alternation at an antinociceptive dose. Thus, KRM-II-81 and NS16085 but not midazolam selectively produced antinociceptive effects. Collectively, these data suggest that α2/α3-subtype selective GABAA PAMs could be a novel class of analgesics and warrant further investigation. Significance Statement This study demonstrates that α2/α3-subtype selective GABAA PAMs KRM-II-81 and NS16085 produce selective antinociceptive effects devoid of sedation, myorelaxation, cognitive impairment in two rat models of persistent pain. Unlikely classical benzodiazepines, this study supports the development of α2/α3-subtype selective GABAA PAMs as safe and novel analgesics for pain management.

Why classical receptor theory, which ignores allostery, can effectively measure the strength of an allosteric effect as agonist's efficacy.

BACKGROUND AND PURPOSE: The classical theory of receptor action has been used for decades as a powerful tool to estimate molecular determinants of ligand-induced receptor activation (i.e., affinity and efficacy) from experimentally observable biological responses. However, it is also a well-recognized fact that the receptor-binding and activation mechanisms, and the parameters thereof, described in the classical theory contradict with the modern view of receptor activation based on allosteric principles. EXPERIMENTAL APPROACH: We used mathematical analysis, along with some numerical simulations, to answer the key question as to what extent the classical theory is compatible-if at all-with the modern understanding of receptor activation. KEY RESULTS: Here, we showed conclusively that (1) receptor activation equations based on allosteric principles contain the logic of the classical theory in disguise, and therefore, (2) estimates of "intrinsic efficacy" (ε) obtained by means of classical techniques (i.e., null methods or fitting the operational model to concentration-response data) are equivalent to the allosteric coupling factors that represent the molecular efficacy of ligands. CONCLUSION AND IMPLICATIONS: Thus, we conclude that despite the justified criticisms it has received so far, the classical theory may continue to be useful in estimating ligand efficacy from experimental data, if used properly. Here, we also provide rigorous criteria for the proper use of the theory. These findings not only have implications for ligand classification but also resolve some long lasting discussions in the field of bias agonism in GPCR, which requires reasonable estimates of relative ligand efficacies at different signalling pathways.

Novel 1-(1-Arylimiazolin-2-Yl)-3-Arylalkilurea Derivatives with Modulatory Activity on Opioid MOP Receptors.

µ-opioid receptor ligands such as morphine and fentanyl are the most known and potent painkillers. However, the severe side effects seen with their use significantly limit their widespread use. The continuous broadening of knowledge about the properties of the interactions of the MOP receptor (human mu opioid receptor, OP3) with ligands and specific intracellular signaling pathways allows for the designation of new directions of research with respect to compounds with analgesic effects in a mechanism different from classical ligands. Allosteric modulation is an extremely promising line of research. Compounds with modulator properties may provide a safer alternative to the currently used opioids. The aim of our research was to obtain a series of urea derivatives of 1-aryl-2-aminoimidazoline and to determine their activity, mechanism of biological action and selectivity toward the MOP receptor. The obtained compounds were subjected to functional tests (cAMP accumulation and ß-arrestin recruitment) in vitro. One of the obtained compounds, when administered alone, did not show any biological activity, while when co-administered with DAMGO, it inhibited ß-arrestin recruitment. These results indicate that this compound is a negative allosteric modulator (NAM) of the human MOP receptor.

Structure-Based Design of AG-946, a Pyruvate Kinase Activator.

Pyruvate kinase (PK) is the enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate to pyruvate and adenosine triphosphate in glycolysis and plays a crucial role in regulating cell metabolism. We describe the structure-based design of AG-946, an activator of PK isoforms, including red blood cell-specific forms of PK (PKR). This was designed to have a pseudo-C2-symmetry matching its allosteric binding site on the PK enzyme, which increased its potency toward PKR while reducing activity against off-targets observed from the original scaffold. AG-946 (1) demonstrated activation of human wild-type PK (half-maximal activation concentration [AC50 ]=0.005 µM) and a panel of mutated PK proteins (K410E [AC50 =0.0043 µM] and R510Q [AC50 =0.0069 µM]), (2) displayed a significantly longer half-time of activation (>150-fold) compared with 6-(3-methoxybenzyl)-4-methyl-2-(methylsulfinyl)-4,6-dihydro-5H-thieno[2',3':4,5]pyrrolo[2,3-d]pyridazin-5-one, and (3) stabilized PKR R510Q, an unstable mutant PKR enzyme, and preserved its catalytic activity under increasingly denaturing conditions. As a potent, oral, small-molecule allosteric activator of wild-type and mutant PKR, AG-946 was advanced to human clinical trials.

Mechanistic Insight into how ß-Ketoacyl ACP Synthase I (KasA) Recognizes the Fatty Acid Chain Length of its Substrate.

ß-ketoacyl ACP synthase I (KasA) has been considered as a promising drug target against Tuberculosis because it is known to play a pivotal role in the survival of Mycobacterium Tuberculosis, a causative agent of Tuberculosis. KasA catalyzes the reaction elongating only the acyl chain that is 16 carbon atoms in length or longer, but the molecular details of how KasA selectively recognizes only the substrate longer than a certain length still remain unknown. In the present study, this challenging subject is addressed, and to this end, molecular dynamics (MD) simulations and free energy calculations for actual substrate binding process are carried out. The results illustrate that the substrate specificity of KasA is highly linked to its cooperativity and this cooperativity is realized through the activation of catalytic residues. Through these results, the mechanistic details of how KasA can be selectively activated only by the substrate with a proper length are suggested.

The molecular basis of dapsone activation of CYP2C9-catalyzed nonsteroidal anti-inflammatory drug oxidation.

Positive heterotropic cooperativity, or "activation," results in an instantaneous increase in enzyme activity in the absence of an increase in protein expression. Thus, cytochrome P450 (CYP) enzyme activation presents as a potential drug-drug interaction mechanism. It has been demonstrated previously that dapsone activates the CYP2C9-catalyzed oxidation of a number of nonsteroidal anti-inflammatory drugs in vitro. Here, we conducted molecular dynamics simulations (MDS) together with enzyme kinetic investigations and site-directed mutagenesis to elucidate the molecular basis of the activation of CYP2C9-catalyzed S-flurbiprofen 4'-hydroxylation and S-naproxen O-demethylation by dapsone. Supplementation of incubations of recombinant CYP2C9 with dapsone increased the catalytic efficiency of flurbiprofen and naproxen oxidation by 2.3- and 16.5-fold, respectively. MDS demonstrated that activation arises predominantly from aromatic interactions between the substrate, dapsone, and the phenyl rings of Phe114 and Phe476 within a common binding domain of the CYP2C9 active site, rather than involvement of a distinct effector site. Mutagenesis of Phe114 and Phe476 abrogated flurbiprofen and naproxen oxidation, and MDS and kinetic studies with the CYP2C9 mutants further identified a pivotal role of Phe476 in dapsone activation. MDS additionally showed that aromatic stacking interactions between two molecules of naproxen are necessary for binding in a catalytically favorable orientation. In contrast to flurbiprofen and naproxen, dapsone did not activate the 4'-hydroxylation of diclofenac, suggesting that the CYP2C9 active site favors cooperative binding of nonsteroidal anti-inflammatory drugs with a planar or near-planar geometry. More generally, the work confirms the utility of MDS for investigating ligand binding in CYP enzymes.

Avidin cooperative allosterism upon binding biotin observed by differential changes in intrinsic fluorescence.

Similar to streptavidin, the binding of biotin by avidin does not appear to be cooperative in the traditional sense of altered binding strength, though it appears to be cooperative in terms of ligand induced structural communication across subunits in the protein as previously shown for streptavidin. In this work we provide data from intrinsic tryptophan fluorescence as evidence of a cooperative structural change. The technique involves examination of the changes in fluorescence emission corresponding to the various tryptophan populations accompanying avidin-biotin binding. We note that the 335 nm emission population (i.e. more hydrophobic local environment) saturates prior to full ligation and the saturation of the 350 nm emission population commonly used in standard binding activity assays. We also note that total integrated fluorescence emission and peak height during the titration of ligand into streptavidin also reach saturation prior to the 4:1 stoichiometric end point. Unique to avidin and distinct from the behavior of streptavidin described in our prior work, the wavelength of maximum emission and full width at half maximum (FWHM) data do not saturate prior to the 4:1 stoichiometric end point. Avidin also exhibited larger FWHM for both apo and holo forms suggesting greater heterogeneity in local tryptophan environments, as compared to streptavidin. Taken together, the data suggests that the binding of the first 3 biotins effect greater structural changes in the protein than the final ligand in a similar way for avidin and streptavidin.

The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation.

Human liver pyruvate kinase (hlPYK) catalyzes the final step in glycolysis, the formation of pyruvate (PYR) and ATP from phosphoenolpyruvate (PEP) and ADP. Fructose 1,6-bisphosphate (FBP), a pathway intermediate of glycolysis, serves as an allosteric activator of hlPYK. Zymomonas mobilis pyruvate kinase (ZmPYK) performs the final step of the Entner-Doudoroff pathway, which is similar to glycolysis in that energy is harvested from glucose and pyruvate is generated. The Entner-Doudoroff pathway does not have FBP as a pathway intermediate, and ZmPYK is not allosterically activated. In this work, we solved the 2.4 Å X-ray crystallographic structure of ZmPYK. The protein is dimeric in solution as determined by gel filtration chromatography, but crystallizes as a tetramer. The buried surface area of the ZmPYK tetramerization interface is significantly smaller than that of hlPYK, and yet tetramerization using the standard interfaces from higher organisms provides an accessible low energy crystallization pathway. Interestingly, the ZmPYK structure showed a phosphate ion in the analogous location to the 6-phosphate binding site of FBP in hlPYK. Circular Dichroism (CD) was used to measure melting temperatures of hlPYK and ZmPYK in the absence and presence of substrates and effectors. The only significant difference was an additional phase of small amplitude for the ZmPYK melting curves. We conclude that the phosphate ion plays neither a structural or allosteric role in ZmPYK under the conditions tested. We hypothesize that ZmPYK does not have sufficient protein stability for activity to be tuned by allosteric effectors as described for rheostat positions in the allosteric homologues.

Kinetic analysis of fluorescent ligand binding to cell surface receptors: Insights into conformational changes and allosterism in living cells.

Equilibrium binding assays are one of the mainstays of current drug discovery efforts to evaluate the interaction of drugs with receptors in membranes and intact cells. However, in recent years, there has been increased focus on the kinetics of the drug-receptor interaction to gain insight into the lifetime of drug-receptor complexes and the rate of association of a ligand with its receptor. Furthermore, drugs that act on topically distinct sites (allosteric) from those occupied by the endogenous ligand (orthosteric site) can induce conformational changes in the orthosteric binding site leading to changes in the association and/or dissociation rate constants of orthosteric ligands. Conformational changes in the orthosteric ligand binding site can also be induced through interaction with neighbouring accessory proteins and receptor homodimerisation and heterodimerisation. In this review, we provide an overview of the use of fluorescent ligand technologies to interrogate ligand-receptor kinetics in living cells and the novel insights that they can provide into the conformational changes induced by drugs acting on a variety of cell surface receptors including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and cytokine receptors.

Structural insights into the half-of-sites reactivity in homodimeric and homotetrameric metalloenzymes.

Half-of-sites reactivity in many homodimeric and homotetrameric metalloenzymes has been known for half a century, yet its benefit remains poorly understood. A recently reported cryo-electron microscopy structure has given some clues on the less optimized reactivity of Escherichia coli ribonucleotide reductase with an asymmetric association of α2ß2 subunits during catalysis. Moreover, nonequivalence of enzyme active sites has been reported in many other enzymes, possibly as a means of regulation. They are often induced by substrate binding or caused by a critical component introduced from a neighboring subunit in response to substrate loadings, such as in prostaglandin endoperoxide H synthase, cytidine triphosphate synthase, glyoxalase, tryptophan dioxygenase, and several decarboxylases or dehydrogenases. Overall, half-of-sites reactivity is likely not an act of wasting resources but rather a method devised in nature to accommodate catalytic or regulatory needs.

Homogeneous, Synthetic, Non-Saccharide Glycosaminoglycan Mimetics as Potent Inhibitors of Human Cathepsin G.

Cathepsin G (CatG) is a pro-inflammatory neutrophil serine protease that is important for host defense, and has been implicated in several inflammatory disorders. Hence, inhibition of CatG holds much therapeutic potential; however, only a few inhibitors have been identified to date, and none have reached clinical trials. Of these, heparin is a well-known inhibitor of CatG, but its heterogeneity and bleeding risk reduce its clinical potential. We reasoned that synthetic small mimetics of heparin, labeled as non-saccharide glycosaminoglycan mimetics (NSGMs), would exhibit potent CatG inhibition while being devoid of bleeding risks associated with heparin. Hence, we screened a focused library of 30 NSGMs for CatG inhibition using a chromogenic substrate hydrolysis assay and identified nano- to micro-molar inhibitors with varying levels of efficacy. Of these, a structurally-defined, octasulfated di-quercetin NSGM 25 inhibited CatG with a potency of ~50 nM. NSGM 25 binds to CatG in an allosteric site through an approximately equal contribution of ionic and nonionic forces. Octasulfated 25 exhibits no impact on human plasma clotting, suggesting minimal bleeding risk. Considering that octasulfated 25 also potently inhibits two other pro-inflammatory proteases, human neutrophil elastase and human plasmin, the current results imply the possibility of a multi-pronged anti-inflammatory approach in which these proteases are likely to simultaneously likely combat important conditions, e.g., rheumatoid arthritis, emphysema, or cystic fibrosis, with minimal bleeding risk.

On the Rational Design of Cooperative Receptors.

Cooperativity (homotropic allostery) is the primary mechanism by which evolution steepens the binding curves of biomolecular receptors to produce more responsive input-output behavior in biomolecular systems. Motivated by the ubiquity with which nature employs this effect, over the past 15 years we, together with other groups, have engineered this mechanism into several otherwise noncooperative receptors. These efforts largely aimed to improve the utility of such receptors in artificial biotechnologies, such as synthetic biology and biosensors, but they have also provided the first quantitative, experimental tests of longstanding ideas about the mechanisms underlying cooperativity. In this article, we review the literature on the design of this effect, paying particular attention to the design strategies involved, the extent to which each can be rationally applied to (and optimized for) new receptors, and what each teaches us about the origins and optimization of this important phenomenon.

Allosteric Regulation of Aptamer Affinity through Mechano-Chemical Coupling.

The capacity to precisely modulate aptamer affinity is important for a wide variety of applications. However, most such engineering strategies entail laborious trial-and-error testing or require prior knowledge of an aptamer's structure and ligand-binding domain. We describe here a simple and generalizable strategy for allosteric modulation of aptamer affinity by employing a double-stranded molecular clamp that destabilizes aptamer secondary structure through mechanical tension. We demonstrate the effectiveness of the approach with a thrombin-binding aptamer and show that we can alter its affinity by as much as 65-fold. We also show that this modulation can be rendered reversible by introducing a restriction enzyme cleavage site into the molecular clamp domain and describe a design strategy for achieving even more finely-tuned affinity modulation. This strategy requires no prior knowledge of the aptamer's structure and binding mechanism and should thus be generalizable across aptamers.

Kinetics of Drug Molecule Interactions with a Newly Developed Nano-Gold-Modified Spike Protein Electrochemical Receptor Sensor.

In March 2020, the World Health Organization (WHO) declared COVID-19 a pandemic, and the spike protein has been reported to be an important drug target for anti-COVID-19 treatment. As such, in this study, we successfully developed a novel electrochemical receptor biosensor by immobilizing the SARS-CoV-2 spike protein and using AuNPs-HRP as an electrochemical signal amplification system. Moreover, the time-current method was used to quantify seven antiviral drug compounds, such as arbidol and chloroquine diphosphate. The results show that the spike protein and the drugs are linearly correlated within a certain concentration range and that the detection sensitivity of the sensor is extremely high. In the low concentration range of linear response, the kinetics of receptor-ligand interactions are similar to that of an enzymatic reaction. Among the investigated drug molecules, bromhexine exhibits the smallest Ka value, and thus, is most sensitively detected by the sensor. Hydroxychloroquine exhibits the largest Ka value. Molecular docking simulations of the spike protein with six small-molecule drugs show that residues of this protein, such as Asp, Trp, Asn, and Gln, form hydrogen bonds with the -OH or -NH2 groups on the branched chains of small-molecule drugs. The electrochemical receptor biosensor can directly quantify the interaction between the spike protein and drugs such as abidor and hydroxychloroquine and perform kinetic studies with a limit of detection 3.3 × 10-20 mol/L, which provides a new research method and idea for receptor-ligand interactions and pharmacodynamic evaluation.

Three Binding Conformations of BIO124 in the Pocket of the PICK1 PDZ Domain.

The PDZ family has drawn attention as possible drug targets because of the domains' wide ranges of function and highly conserved binding pockets. The PICK1 PDZ domain has been proposed as a possible drug target because the interactions between the PICK1 PDZ domain and the GluA2 subunit of the AMPA receptor have been shown to progress neurodegenerative diseases. BIO124 has been identified as a sub µM inhibitor of the PICK1-GluA2 interaction. Here, we use all-atom molecular dynamics simulations to reveal the atomic-level interaction pattern between the PICK1 PDZ domain and BIO124. Our simulations reveal three unique binding conformations of BIO124 in the PICK1 PDZ binding pocket, referred to here as state 0, state 1, and state 2. Each conformation is defined by a unique hydrogen bonding network and a unique pattern of hydrophobic interactions between BIO124 and the PICK1 PDZ domain. Interestingly, each conformation of BIO124 results in different dynamic changes to the PICK1 PDZ domain. Unlike states 1 and 2, state 0 induces dynamic coupling between BIO124 and the αA helix. Notably, this dynamic coupling with the αA helix is similar to what has been observed in other PDZ-ligand complexes. Our analysis indicates that the interactions formed between BIO124 and I35 may be the key to inducing dynamic coupling with the αA helix. Lastly, we suspect that the conformational shifts observed in our simulations may affect the stability and thus the overall effectiveness of BIO124. We propose that a physically larger inhibitor may be necessary to ensure sufficient interactions that permit stable binding between a drug and the PICK1 PDZ domain.

Adenosine receptor signalling in Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia in the elderly and its increasing prevalence presents treatment challenges. Despite a better understanding of the disease, the current mainstay of treatment cannot modify pathogenesis or effectively address the associated cognitive and memory deficits. Emerging evidence suggests adenosine G protein-coupled receptors (GPCRs) are promising therapeutic targets for Alzheimer's disease. The adenosine A1 and A2A receptors are expressed in the human brain and have a proposed involvement in the pathogenesis of dementia. Targeting these receptors preclinically can mitigate pathogenic ß-amyloid and tau neurotoxicity whilst improving cognition and memory. In this review, we provide an accessible summary of the literature on Alzheimer's disease and the therapeutic potential of A1 and A2A receptors. Although there are no available medicines targeting these receptors approved for treating dementia, we provide insights into some novel strategies, including allosterism and the targeting of oligomers, which may increase drug discovery success and enhance the therapeutic response.