An early GPCR positive allosteric modulator (PAM) was the 2-amino-3-benzoylthiophene derivative PD81,723, which enhances Gi-coupled A1 adenosine receptor (AR) agonist effects. Cryo-EM studies located its extrahelical binding site on TMs 5 and 6, and extensive empirical SAR is consistent with the experimental A1AR structure. At the anti-inflammatory A3AR, a series of 1H-imidazo[4,5-c]quinolin-4-amine PAMs, originally derived from an A1AR antagonist scaffold, was recently found, using mutagenesis and molecular modeling, to bind at an extrahelical, lipid-exposed site on the receptor. The N4 and C2 positions on the scaffold were modified with substituted phenyl and cycloalkyl/branched alkyl groups, respectively, as in the prototypical PAM LUF6000. Before the structural binding hypothesis, extensive synthetic optimization failed to introduce hydrophilic groups needed for aqueous compatibility. Eventually, secondary anchoring of these A3AR PAMs within the phospholipid bilayer (lipid trolling) succeeded in improving the pharmacological profile, based on the attraction of appended terminal cationic chains on the PAM with anionic phospholipid head groups. Molecular dynamics (MD) simulation has predicted a rearrangement of anionic lipids around H8 when long-amino chain PAMs bind, and an upward movement of H8 that is reported to be associated with Class A GPCR activation. N1 alkylation with benzyl groups removed residual A3AR antagonism and produced substantial enhancement of agonist potency as well as Emax. Both A1AR and A3AR PAMs can activate partially in the absence of orthosteric agonist. Thus, at both receptors, potent PAMs have been discovered and optimized for selective enhancement of endogenous adenosine and potentially for treatment of conditions such as chronic pain. Collectively, extrahelical, lipid-exposed pockets are druggable allosteric sites in Class A GPCRs and amenable to deliberate engagement of the membrane environment to optimize receptor modulation. The evolution from empirical screening to structure-guided and membrane-aware design provides a generalizable framework for targeting signaling across the GPCR superfamily beyond ARs.

Growing evidence is highlighting purinergic receptors as therapeutic targets in cardiac diseases. MRS2339, a charged (N)-methanocarba derivative of 2-Cl-adenosine monophosphate, is efficacious in multiple animal models of systolic heart failure with reduced ejection fraction following infarction, pressure overload, calsequestrin overexpression, rapid pacing, and other injuries. In this study, we explored the beneficial mechanisms of this nucleotide analog in dogs with rapid pacing-induced systolic heart failure and in mice with postinfarct ischemic heart failure. In dogs, sustained infusion of the nucleotide analog increased stroke volume, cardiac output, arterial blood pressure, and arterial oxygen level without any vascular effects, suggesting direct cardiac effects of the drug. In the murine model, MRS2339 caused an increase in myocardial cyclic GMP level, a known cardioprotective second messenger in wild type mice, but no effects in P2X4 receptor knockout mice. Previous data showed that MRS2339 was able to elicit a current in adult mouse cardiomyocytes. The present data showed that MRS2339 could not stimulate a current through rat homomeric P2X4 receptors expressed in HEK293 cells. While the composition of endogenous cardiomyocyte P2X receptors is not known, P2X4 receptors in the heart appear necessary for MRS2339-induced cyclic GMP increase. Our findings support a novel cardiac P2X 4 receptor-mediated cyclic GMP enhancement by MRS2339 as a cellular mechanism of action in heart failure. Stimulating cardiac P2X receptors with increased cyclic GMP level to yield hemodynamic improvement is a potential new therapeutic target in heart failure.

AST-004 is an adenosine A1R/A3R agonist and promising cerebroprotectant currently in Phase II clinical development. Clinical evaluations have been initiated in concussion, with trials in preparation for mild complicated traumatic brain injury (TBI) and acute ischemic stroke (AIS). AST-004 was discovered as the result of basic research on the P2Y1R-activated homeostatic roles of astrocytes in cerebroprotection, including control of edema, excitotoxicity and oxidative stress. AST-004 was identified as the active nucleoside metabolite of the P2Y1R agonist MRS2365. Subsequent research demonstrated that astrocyte mitochondrial ATP production could be activated by AST-004, resulting in significant efficacy in rodent models of permanent and transient occlusion AIS, porcine models of TBI and a non-human primate model of AIS as well as models of neurodegeneration and addiction. The purpose of this review is to highlight the research that led to the discovery of AST-004, summarize its widespread preclinical efficacy in a variety of disease models, and describe how this compound’s excellent safety and pharmaceutical profile is differentiated from prototypical A1R and A3R agonists formerly or currently in the clinic.

Knockout of the UDP-glucose-activated P2Y 14 receptor (P2Y 14 R) in adipocytes or whole-body has been reported to provide metabolic benefits in obese mice. We hypothesized that selective P2Y 14 R activation would lead to metabolic impairments, whereas pharmacological antagonism would improve metabolic deficits in diet-induced obese (DIO) mice. Here, we investigated the metabolic effects of a synthetic P2Y 14 R agonist, UDP-like methylene-bridged MRS2905. Acute P2Y 14 R activation with MRS2905 triggered a robust and prolonged hyperglycemic effect in lean and obese mice with impaired glucose homeostasis. Moreover, MRS2905 treatment of obese mice lowered fasting plasma insulin and increased glucagon levels, along with upregulation of liver JNK phosphorylation and the expression of rate-limiting gluconeogenic genes. The MRS2905-induced hyperglycemic effect was blunted in whole-body P2Y 14 R knockout mice compared with wild-type control. In contrast, a potent P2Y 14 R antagonist mono-ester prodrug (MRS4779) partially reversed the agonist-induced hyperglycemia and restored proper glucose homeostasis after acute treatment. Chronic MRS4779 administration in DIO mice reduced fat mass and improved various metabolic parameters including liver steatosis. Additionally, we examined the roles of P2Y 14 R in hepatocytes of DIO mice. Here, we report that P2Y 14 R was upregulated in liver and hepatocytes from obese mice compared to lean mice. Overnight fasting also upregulated hepatic P2Y 14 R expression. P2Y 14 R deletion from hepatocytes in DIO mice improved fasting blood glucose level and lipid metabolism without improving glucose homeostasis. These results suggest a novel P2Y 14 R function in hepatic lipid metabolism, and P2Y 14 R antagonists may prove useful for the treatment of obesity and obesity-related metabolic disorders.

Following previous observations that N 6-elongated (N)-methanocarba adenosine derivatives modulate efflux pump activity associated with multidrug resistance (MDR) in tumors, we synthesized and characterized 34 nucleoside analogues. Effects on ATPase activity of both ABCG2 and P-glycoprotein identified m-substituted N 6-benzyl derivative 16 (MRS8288) as a dual inhibitor, with IC50 1.0 and 1.4 μM, respectively. Compound 30 (MRS8431) inhibited ABCG2-ATPase activity with IC50 160 ± 9 nM, while compound 40 (MRS8432) stimulated it potently with EC50 7.5 ± 2 nM. Selected (N)-methanocarba nucleosides, notably 16, 30, and 40, inhibited ABCG2-mediated substrate transport, with marginal effects on the activity of P-gp and were not transported by ABCG2. Compound 30, but not 16 or 40, sensitized ABCG2-expressing HEK-293-R5 cells to mitoxantrone. Distinct docking modes of compounds 30 and 40 to ABCG2 predict the structural determinants for the inhibition of ATPase. These results reveal novel rigid, extended nucleoside inhibitors of ABC transporters with varied activities that attenuate MDR in cells.

Adenosine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Adenosine Receptors [114], and updated [155]) are activated by the endogenous ligand adenosine (potentially inosine also at A3 receptors). Crystal and cryo-EM structures for all four adenosine receptors have been solved, occupied by either agonists (sometimes in the presence of an allosteric modulator) or antagonists. Many of these structures were incorporated in a recent review [155]. More recently, structures for the A2B receptor [58, 48] and the A3 receptor [279, 47] were elucidated. The A2A receptor is used as a workhorse in GPCR structure elucidation: almost 100 structures are available in the Protein Data Bank (www.rcsb.org). istradefylline, a selective A2A receptor antagonist, is on the market for the treatment of Parkinson's disease, while caffeine's mechanism of action is largely due to its antagonism of at least three of the four adenosine receptor subtypes. Allosteric modulators, particular PAMs of A1 and A3 receptors, have been explored chemically and structurally [88, 293].

P2X receptors (P2XR) are a family of seven cation channels gated by extracellular ATP (eATP). Activation of P2XRs results in diverse cellular responses, including cell signalling, proliferation, differentiation, and death-all critically important in multiple physiological and pathophysiological states. These receptors, therefore, represent therapeutic targets of considerable interest. However, P2XRs, while structurally related, exhibit highly divergent and context-dependent functions. Their spatiotemporal and functional complexity is evident by overlapping expression across multiple cell types that can shift dynamically during physiological processes or disease progression. Furthermore, P2XRs can assemble as homo- or hetero-trimers, with distinct functional properties. These factors complicate definitive identification of a given P2XR responsible for a specific pathophysiological effect. Receptor activity in vivo is transient because of receptor-specific mechanisms and follows eATP breakdown by ectonucleotidases. Any correlation of ATP release with receptor engagement, as assessed in vitro, often does not correspond with the in vivo dynamics. Translation from animal models to humans is complicated by the species-specific pharmacology of some P2XRs, confounded by many animal models in use not fully replicating human P2XR function and regulation in pathology. Furthermore, there are no clinical biomarkers to distinguish incomplete receptor blockade from lack of therapeutic effect. Thus, translation has been very limited. To identify and validate specific P2XR functionalities, future experimental designs should use approaches and assays that can reliably assess receptor involvement, while reducing methodologically flawed findings. We propose guidelines developed in consultation with the purinergic community for consistent and reliable research practices in P2XR studies.

Ecto-5'-nucleotidase (CD73) is a potential new drug target for cancer immunotherapy. Its overexpression is associated with various aggressive cancers, including triple-negative breast cancer (TNBC) and pancreatic cancer, making it a promising target for diagnostic imaging. Besides antibodies, small-molecule CD73 inhibitors have been developed and are currently in clinical trials. This study aimed to develop and evaluate fluorine-18 labeled high-affinity CD73 inhibitors as tracers for the non-invasive positron emission tomography (PET) imaging of CD73 expression in cancer. Two CD73 inhibitors were selected for radiolabeling based on their high potency (Ki values of ca. 1 nM) and favorable pharmacokinetic properties, yielding [18F]PSB-19427 ([18F]1) and [18F]MRS-4648 ([18F]2). Ex vivo imaging studies on human breast cancer tissues indicated specific binding of both radiotracers. Subsequent in vivo studies proved [18F]1 to be superior due to its long elimination half-life and its accumulation in TNBC and pancreatic cancer tissues, suggesting its potential as a versatile PET tracer for imaging of various solid tumors. Compared to [18F]FDG, [18F]1 was superior in visualizing TNBC, offering potential advantages over [18F]FDG in terms of specificity and diagnostic accuracy. Thus, [18F]1 is a PET tracer with outstanding properties suitable for broad application in cancer diagnosis and personalized medicine.

Galectin-3 (Gal-3) is a β-galactoside-binding lectin implicated in metabolic inflammation, cardiovascular and renal dysfunction, neurodegenerative disorders, and obesity-related pathologies. Although Gal-3 is recognized as a clinically relevant biomarker, the mechanisms controlling its tissue expression and circulating abundance remain poorly defined. O-GlcNAcase (Oga; encoded by Mgea5), the enzyme that removes O-linked β-N-acetylglucosamine (O-GlcNAc) from proteins, regulates nutrient-sensitive signaling and transcriptional processes that overlap with Gal-3 associated disease pathways. To investigate the relationship between metabolic status and Gal-3 expression, male mice were fed a high-fat diet (HFD) for eight weeks to induce obesity. HFD-fed mice exhibited significant increases in body weight and fasting and fed blood glucose levels compared with lean controls, confirming metabolic dysregulation. ELISA revealed approximately threefold higher serum and plasma Gal-3 concentrations in obese mice, indicating enhanced Gal-3 production in diet-induced obesity. To determine whether Oga regulates Gal-3 expression, Oga wild-type (WT), heterozygous (HET), and knockout (KO) mice were analyzed. Circulating Gal-3 protein levels were significantly reduced in Oga KO mice, with intermediate levels in Oga HET animals. RT-qPCR revealed genotype-dependent modulation of Gal-3 (Lgals3) mRNA expression across multiple tissues, demonstrating tissue-specific regulation by Oga. These findings establish Oga as a critical regulator of Gal-3 expression and systemic abundance. The data reveal a mechanistic link between O-GlcNAc signaling enzyme Oga, and lectin-mediated metabolic inflammation, suggesting that Oga activity influences Gal-3 homeostasis and may affect its interpretation as a biomarker in metabolic disease.

Stress is a common trigger of headache and widespread somatic pain. Repeated exposure to stress establishes latent sensitization that is tonically suppressed by endogenous inhibitory systems. Using brain-penetrating and peripherally restricted receptor antagonists in a mouse model of repetitive restraint stress, we systematically identified these protective pathways. Peripheral kappa opioid receptors as well as GABA type A and type B (GABAA and GABAB) receptors, alongside central opioid and cannabinoid signaling, actively suppress headache-related facial mechanical hypersensitivity in stressed mice. Blocking any pathway quickly reinstated cephalic allodynia. Peripheral opioid and GABAA receptor signaling also inhibited stress-induced latent sensitization on hindpaw. In contrast, depletion of anti-inflammatory regulatory T (Treg) cells prolonged facial but not hindpaw sensitization, suggesting that Tregs preferentially limit stress-induced chronic headache. Next, we administered low-dose interleukin-2 (LD-IL-2) to stressed mice to preferentially expand and activate Treg cells. Following LD-IL-2 treatment, neither subthreshold pain triggers nor blockade of endogenous inhibitory pathways reinstated cephalic or hindpaw allodynia in either sex, indicating elimination of stress-induced sensitization. Mechanistically, LD-IL-2 acted through Treg cells to recruit multiple peripheral cytokine pathways without engaging endogenous opioid, GABA, or cannabinoid receptor signaling. Notably, LD-IL-2 was more effective than anti-CGRP (calcitonin gene-related peptide) therapy in preventing headache-related chronic sensitization in stressed mice. Collectively, these findings reveal multiple central and peripheral pathways that act concertedly to mask stress-induced latent sensitization and strongly support further evaluation of LD-IL-2 as a novel treatment for stress-induced headache and widespread pain with a distinct mechanism of action.

Ischemic injury triggers extracellular ATP release, activating P2X4 receptors (P2 × 4R) on immune and cardiac cells, which exacerbates inflammation and tissue damage. We evaluated MRS4719, a selective P2 × 4R antagonist, in aged mice subjected to transient middle cerebral artery occlusion (tMCAo) and cardiac ischemia/reperfusion (CI/R) injury. MRS4719 exhibited a nonlinear dose response, with an intermediate dose (2.25 mg/kg/day) and short-term treatment (2 days) optimally improving sensorimotor and cognitive recovery while reducing brain tissue atrophy. Treatment initiated up to 12 h post-stroke significantly decreased infarct volume. Additionally, MRS4719 preserved cardiac contractile function following ischemia/reperfusion injury. These findings suggest that targeted P2X4R inhibition mitigates inflammatory injury across multiple organs and supports functional recovery, highlighting MRS4719’s therapeutic potential for cerebral and cardiac ischemic disorders.

Several adenosine A1 receptor (A1R) radiotracers for positron emission tomography (PET) have been developed to study their neuromodulatory functions and role in brain disorders. While two xanthine-based radiotracers ([11C]-MPDX and [18F]-CPFPX) have been used in humans, we aimed to improve the metabolic stability and specific binding. Guided by structure-activity relationship (SAR) studies, 10 derivatives were synthesized with binding affinities up to 0.12 nM. Three subnanomolar candidates (3, 8, 9) were radiolabeled with C-11 (t 1/2 = 20.4 min) for evaluation using in vivo PET imaging and ex vivo rodent brain biodistribution. Although [11C]8 demonstrated a higher blood-brain barrier (BBB) permeability, negligible in vivo specific binding was observed. Ex vivo studies indicated that all three compounds are substrates for brain efflux pumps. Despite optimized affinity, BBB permeability and in vivo binding specificity remain challenges. These findings inform development of nonxanthine A1R radiotracers and highly potent CNS A1R drugs.

P2Y14 receptors respond to uracil-diphosphate-hexose conjugates, yet how this receptor selectively recognizes both uracil-nucleotides and hexose moieties of diverse agonists remains unclear. Here we report the active agonist-bound G protein-bound states of the P2Y14 G protein-coupled receptor (GPCR) bound to endogenous agonist uridine diphosphate glucose (UDP-glucose) and 2-thiouridine-5′-O-(α,β-methylene)diphosphate (MRS2905). The cryo-EM structures of the heterotrimeric complexes with 3.19 Å and 3.05 Å global resolution, respectively, with local refinements reaching 2.87 Å and 3.22 Å for the masked receptor region. Our structures reveal a pronounced extracellular facing electronegative vestibule connecting to a smaller nucleotide binding subpocket (~300Å3 volume) that is shielded by extracellular loop 2 (ECL2). A glucose-binding subpocket is spatially delimited by residue V93; mutation to Trp selectively blocks UDP-glucose while permitting MRS2905 and antagonist binding. These findings provide atomic insights into uracil recognition, reveal how the receptor accommodates diverse flexible UDP-sugars, and promise to enable rational drug discovery of therapeutic P2Y14R modulators. The authors cracked P2Y14’s “sweet tooth”: structures bound to UDP-glucose and MRS2905 decode hexose and uracil nucleotide selectivity. A bipartite binding pocket with distinct nucleotide and hexose subpockets divided by V93 enables rational therapeutic design.

The author presents his personal story from early contributions in purinergic receptor research to present-day structure-guided medicinal chemistry. Modulating purinergic signaling (encompassing pyrimidine nucleotides as well) and other nucleoside targets with small molecules is fruitful for identifying new directions for therapeutic intervention. Purinergic signaling encompasses four adenosine receptors, eight P2Y receptors that respond to various extracellular nucleotides, and trimeric P2X receptors that respond mainly to ATP. Each organ and tissue in the body expresses some combination of this family of cell-surface receptors, along with the enzymes and transporters that form, degrade, and process the native nucleoside and nucleotide agonists. The purinergic signaling system responds to physiological stress to an organ, for example by increasing the energy supply or decreasing the energy demand. The receptors are widespread on immune cells, such that P2Y and P2X receptor activation boosts the immune response when and where it is needed, for example to repel infection. In contrast, the adenosine receptors, which are activated later in the process─as stress-elevated ATP is hydrolyzed locally to adenosine by ectonucleotidases─tend to put the brakes on inflammation and can be used to correct an imbalance in pro- versus anti-inflammatory signals, such as in chronic pain. Hypoxia activates the immunosuppressive extracellular adenosine-A2A adenosine receptor axis, as originally formulated by Sitkovsky, which suppresses the immune response in the tumor microenvironment to make a cancer more aggressive. Conversely, the anti-inflammatory effects of adenosine receptor agonists have numerous therapeutic applications. Modulators of P2Y receptors, which respond to extracellular nucleotides, also show promise for treating chronic pain, metabolic disorders, and inflammation. Thus, control of this signaling system can be harnessed for treating a wide range of conditions, from cancer and neurodegeneration to autoimmune inflammatory diseases to ischemia of the brain or heart. The author's receiving the American Chemical Society's top award for medicinal chemistry in 2023 provides an opportunity to summarize these developments from their origins in empirical probing of receptor-ligand structure-activity relationship (SAR) to the current structure-based approaches, including conformational control of selectivity toward purinergic signaling. The work on each target receptor began either before or soon after it was cloned, and the initial focus was an academic exercise to use organic chemistry to develop a SAR for each target. The Jacobson lab has introduced chemical probes for 17 of the purinergic receptors as well as for associated regulators. Furthermore, surprisingly, some of the conformationally constrained nucleoside analogues can be designed to inhibit non-purinergic targets selectively, such as opioid and serotonin receptors and monoamine transporters. Only later did therapeutic applications of these pharmacological probes become apparent. Thus, the medicinal chemistry has largely enabled biological research on purinergic signaling by making definitive tool compounds available. Five compounds from the Jacobson laboratory (four adenosine derivatives) are currently in clinical trials for various chronic (autoimmune inflammatory and liver conditions) and acute (stroke, traumatic brain injury) conditions.

A human dopamine transporter (DAT) cryo-EM structure was recently reported, as stabilized by an allosteric inhibitor, i.e. rigid nucleoside MRS7292 1, a tropane orthosteric inhibitor and Zn2+. We have synthesized multiple North (N)-methanocarba-adenosine analogues of 1, with N6, C2 and 4' modifications to examine their effects on DAT radioligand binding (either enhancement or inhibition) and at the norepinephrine (NET) and serotonin (SERT) transporters (generally inhibition). Small N6 groups provided the most pronounced DAT enhancement (≥500 % at 10 μM for N6-methyl 2 and N6-cyclopropyl 8 5'-ethyl esters, and N6-methyl-4'-cyanomethyl 44 analogues). Various N6-(ω-phenyl-alkyl) groups caused binding inhibition and compensated for the removal of stabilizing interactions shown in the 1-DAT structure, e.g. an N7 H-bond, by accessing a previously uncharacterized DAT distal binding region. The optimal chain length was five methylenes for bitopic N6-(ω-phenyl-alkyl) derivatives, e.g. 16 and 31, having favored 5'-ethyl ester (but not 4'-cyanomethyl) and 2-(arylethynyl) groups. Surprisingly, the previously noted reduced DAT activity with a 2-iodo group, could be compensated by N6-(ω-phenyl-alkyl) groups. N6-(6-Phenylhexyl)-2-iodo compounds, 53 (5'-hydroxy, Ki 0.89 μM, pan-inhibitor) and 54 (4'-cyanomethyl, Ki 0.40 μM), inhibited DAT binding. Chiral, branched N6 groups displayed binding stereoselectivity. Key nucleosides were docked to outward-facing hDAT cryo-EM structures. Molecular dynamics simulation predicted π-π interactions of the N6-(ω-phenyl-alkyl) substituent and aromatic side chains of F208EL2 and H375EL4 (aromatic conserved at NET) to define a preferred binding mode for the extended chain. Thus, we characterized the transporter SAR of this series, either enhancement or inhibition of orthosteric radioligand binding, and transport inhibition, and with DAT structural predictions.

Alterations in mitochondrial function are the linchpin in numerous disease states including in the development of chemotherapy-induced neuropathic pain (CIPN), a major dose-limiting toxicity of widely used chemotherapeutic cytotoxins. In CIPN, mitochondrial dysfunction is characterized by deficits in mitochondrial bioenergetics (e.g., decreased ATP production) that are thought to drive the degeneration of the peripheral nerve sensory axon terminal sensory arbors in the skin (the intraepidermal nerve fibers; IENFs) and induce abnormal spontaneous discharge in peripheral nerve sensory axons. Preserving mitochondrial function is anticipated to prevent CIPN. We have now discovered that the G-protein-coupled receptor, A3adenosine receptor subtype (A3AR), is expressed on the mitochondrial outer membrane. Ex vivo application of a highly selective A3AR agonist, MRS5980, to saphenous nerve microfilaments harvested from male oxaliplatin-treated rats reversed the loss in ATP production underscoring mitoprotective effects resulting from A3AR activation on mitochondria. Moreover, in vivo administration of A3AR agonists to rats during oxaliplatin treatment was associated with reduced IENF loss and a lower incidence of spontaneous discharge in peripheral afferent axons. These effects are accompanied by improved mitochondrial ATP production in primary afferent sensory axons and overall inhibition of the development of neuropathic pain. These data identify for the first time mitochondrial A3AR and indicate that activation of A3AR protects mitochondrial function in primary afferent sensory axons against chemotherapy-induced neurotoxicity. Repurposing A3AR agonists that are already in clinical trials as anticancer agents as adjunct to chemotherapeutics will address a major unmet medical need for which there are no FDA-approved drugs.