First Potent Macrocyclic A3 Adenosine Receptor Agonists Reveal G-Protein and ß-Arrestin2 Signaling Preferences.

(N)-Methanocarba adenosine derivatives (A3 adenosine receptor (AR) agonists containing bicyclo[3.1.0]hexane replacing furanose) were chain-extended at N6 and C2 positions with terminal alkenes for ring closure. The resulting macrocycles of 17-20 atoms retained affinity, indicating a spatially proximal orientation of these receptor-bound chains, consistent with molecular modeling of 12. C2-Arylethynyl-linked macrocycle 19 was more A3AR-selective than 2-ether-linked macrocycle 12 (both 5'-methylamides, human (h) A3AR affinities (Ki): 22.1 and 25.8 nM, respectively), with lower mouse A3AR affinities. Functional hA3AR comparison of two sets of open/closed analogues in ß-arrestin2 and Gi/o protein assays showed certain signaling preferences divergent from reference agonist Cl-IB-MECA 1. The potencies of 1 at all three Gαi isoforms were slightly less than its hA3AR binding affinity (Ki: 1.4 nM), while the Gαi1 and Gαi2 potencies of macrocycle 12 were roughly an order of magnitude higher than its radioligand binding affinity. Gαi2-coupling was enhanced in macrocycle 12 (EC50 2.56 nM, ∼40% greater maximal efficacy than 1). Di-O-allyl precursor 18 cyclized to form 19, increasing the Gαi1 potency by 7.5-fold. The macrocycles 12 and 19 and their open precursors 11 and 18 potently stimulated ß-arrestin2 recruitment, with EC50 values (nM) of 5.17, 4.36, 1.30, and 4.35, respectively, and with nearly 50% greater efficacy compared to 1. This example of macrocyclization altering the coupling pathways of small-molecule (nonpeptide) GPCR agonists is the first for potent and selective macrocyclic AR agonists. These initial macrocyclic derivatives can serve as a guide for the future design of macrocyclic AR agonists displaying unanticipated pharmacology.

Structure activity relationships of 5-HT2B and 5-HT2C serotonin receptor antagonists: N6, C2 and 5'-Modified (N)-methanocarba-adenosine derivatives.

(N)-Methanocarba adenosine derivatives were structurally modified to target 5-HT2B serotonin receptors as antagonists, predominantly containing branched N6-alkyl groups. N6-Dicycloalkyl-methyl groups, including their asymmetric variations, as well as 2-iodo, were found to generally favor 5-HT2Rs, while only N6-dicyclohexyl-methyl derivative 35 showed weak 5-HT2AR affinity (Ki 3.6 µM). The highest 5-HT2BR affinities were Ki 11-23 nM (N6-dicyclopropyl-methyl-2-iodo 11, 2-chloro-5'-deoxy-5'-methylthio 15 and N6-((R)-cyclobuty-cyclopropyl-methyl)-2-iodo 43), and Ki 73 nM at 5-HT2CR for 36. Direct comparison of adenine ribosides and their corresponding rigid (N)-methanocarba derivatives (cf. 51 and MRS8099 45) indicated a multifold affinity enhancement with the bicyclic ring system. Compounds 43, 45 and 48 were functional 5-HT2BR (KB 2-3 nM) and 5-HT2CR (KB 79-328 nM) antagonists in a Gq-mediated calcium flux assay, with 5-HT2BR functional selectivity ranging from 45- (48) to 113-fold (43). Substantial adenosine receptor (AR) affinity (Ki, A1AR < Ki, A3AR < Ki, A2AAR) was still present in this series, suggestive of dual acting compounds: 5-HT2B antagonist and A1AR agonist, potentially useful for treating chronic conditions (fibrosis; pain). Given its affinity (17 nM) and moderate 5-HT2BR binding selectivity (32-fold vs. 5-HT2CR, 4-fold vs. A1AR), 43 (MRS7925) could potentially be useful for anti-fibrotic therapy.

Fluorescent A2A and A3 adenosine receptor antagonists as flow cytometry probes.

Adenosine receptor (AR) ligands are being developed for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A2A and A3ARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled to eight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at A2AAR were Ki 144-316 nM for 10, 12, and 19, and at A3AR affinity of Ki 21.6 nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface A2AAR was imaged using confocal microscopy. Compound 19 MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for A3AR characterization in whole cells by flow cytometry (Kd 11.8 nM), and its bitopic interaction mode with an A3AR homology model was predicted. Given its affinity and selectivity (11-fold vs. A2AAR, ~ 50-fold vs. A1AR and A2BAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the A3AR.

Effects of Purinergic Receptor Deletion or Pharmacologic Modulation on Pulmonary Inflammation in Mice.

COVID-19 disease is associated with progressive accumulation of SARS-CoV-2-specific mRNA, which is recognized by innate immune receptors, such as TLR3. This in turn leads to dysregulated production of multiple cytokines, including IL-6, IFN-γ, CXCL1, and TNF-α. Excessive production of these cytokines leads to acute lung injury (ALI), which consequently compromises alveolar exchange of O2 and CO2. It is therefore of considerable interest to develop novel therapies that reduce pulmonary inflammation and stem production of pro-inflammatory cytokines, potentially for COVID-19 patients that are at high risk of developing severe disease. Purinergic signaling has a central role in fine-tuning the innate immune system, with P2 (nucleotide) receptor antagonists and adenosine receptor agonists having anti-inflammatory effects. Accordingly, we focused here on the potential role of purinergic receptors in driving neutrophilic inflammation and cytokine production in a mouse model of pulmonary inflammation. To mimic the effects of SARS-CoV-2-specific RNA accumulation in mice, we administered progressively increasing daily doses of a viral mimetic, polyinosinic:polycytidylic acid [poly(I:C)] into the airways of mice over the course of 1 week. Some mice also received increasing daily doses of ovalbumin to mimic virus-encoded protein accumulation. Animals receiving both poly(I:C) and ovalbumin displayed particularly high cytokine levels and neutrophilia, suggestive of both innate and antigen-specific, adaptive immune responses. The extent of these responses was diminished by genetic deletion (P2Y14R, P2X7R) or pharmacologic modulation (P2Y14R antagonists, A3AR agonists) of purinergic receptors. These results suggest that pharmacologic modulation of select purinergic receptors might be therapeutically useful in treating COVID-19 and other pulmonary infections.

Selective A3 Adenosine Receptor Antagonist Radioligand for Human and Rodent Species.

The A3 adenosine receptor (A3AR) is a target for pain, ischemia, and inflammatory disease therapy. Among the ligand tools available are selective agonists and antagonists, including radioligands, but most high-affinity non-nucleoside antagonists are limited in selectivity to primate species. We have explored the structure-activity relationship of a previously reported A3AR antagonist DPTN 9 (N-[4-(3,5-dimethylphenyl)-5-(4-pyridyl)-1,3-thiazol-2-yl]nicotinamide) for radiolabeling, including 3-halo derivatives (3-iodo, MRS7907), and characterized 9 as a high -affinity radioligand [3H]MRS7799. A3AR K d values were (nM): 0.55 (human), 3.74 (mouse), and 2.80 (rat). An extended methyl acrylate (MRS8074, 19) maintained higher affinity (18.9 nM) than a 3-((5-chlorothiophen-2-yl)ethynyl) derivative 20. Compound 9 had an excellent brain distribution in rats (brain/plasma ratio ∼1). Receptor docking predicted its orthosteric site binding by engaging residues that were previously found to be essential for AR binding. Thus the new radioligand promises to be a useful species-general antagonist tracer for receptor characterization and drug discovery.

Interaction of A3 adenosine receptor ligands with the human multidrug transporter ABCG2.

Various adenosine receptor nucleoside-like ligands were found to modulate ATP hydrolysis by the multidrug transporter ABCG2. Both ribose-containing and rigidified (N)-methanocarba nucleosides (C2-, N6- and 5'-modified), as well as adenines (C2-, N6-, and deaza modified), were included. 57 compounds out of 63 tested either stimulated (50) or inhibited (7) basal ATPase activity. Structure-activity analysis showed a separation of adenosine receptor and ABCG2 activities. The 7-deaza modification had favorable effects in both (N)-methanocarba nucleosides and adenines. Adenine 37c (MRS7608) and (N)-methanocarba 7-deaza-5'-ethyl ester 60 (MRS7343) were found to be potent stimulators of ABCG2 ATPase activity with EC50 values of 13.2 ± 1.7 and 13.2 ± 2.2 nM, respectively. Both had affinity in the micromolar range for A3 adenosine receptor and lacked the 5'-amide agonist-enabling group (37c was reported as a weak A3 antagonist, Ki 6.82 µM). Compound 60 significantly inhibited ABCG2 substrate transport (IC50 0.44 µM). Docking simulations predicted the interaction of 60 with 21 residues in the drug-binding pocket of ABCG2.

A3 adenosine receptor agonists containing dopamine moieties for enhanced interspecies affinity.

Following our study of 4'-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A3AR affinity, we incorporated dopamine-related N6 substituents in the full agonist 5'-methylamide series. N6-(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed Ki (nM) 0.563 at hA3AR (∼20,000-fold selective) and 1.54 at mA3AR. 2-Alkyl ethers maintained A3 affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and ß-arrestin 2 (ßarr2)-dependent pathways indicate these are full agonists but not biased. Through use of computational modeling, we hypothesized that phenyl OH/OMe groups interact with polar residues, particularly Gln261, on the mA3AR extracellular loops as the basis for the affinity enhancement. Although the pharmacokinetics indicated facile clearance of parent O-methyl catechol nucleosides 21 and 31, prolonged mA3AR activation in vivo was observed in a hypothermia model, suggested potential formation of active metabolites through demethylation. Selected analogues induced mouse hypothermia following i.p. injection, indicative of peripheral A3AR agonism in vivo.

Convergent synthesis of 2-thioether-substituted (N)-methanocarba-adenosines as purine receptor agonists.

A linear route has been used to prepare (N)-methanocarba-nucleoside derivatives, which serve as purine receptor ligands having a pre-established, receptor-preferred conformation. To introduce this rigid ribose substitute, a Mitsunobu reaction of a [3.1.0]bicyclohexane 5'-trityl intermediate 3 with a nucleobase is typically followed by functional group modifications. We herein report an efficient scalable convergent synthesis for 2-substituted (N)-methanocarba-adenosines, which were demonstrated to bind to the A3 adenosine receptor. The adenine moiety was pre-functionalized with 2-thioethers and other groups before coupling to the bicyclic precursor (3) as a key step to facilitate a high yield Mitsunobu product. This new approach provided the (N)-methanocarba-adenosines in moderate to good yield, which effectively increased the overall yield compared to a linear synthesis and conserved a key intermediate 3 (a product of nine sequential steps). The generality of this convergent synthesis, which is suitable as an optimized preclinical synthetic route, was demonstrated with various 2-thioether and 2-methoxy substituents.

Mechanism of 6-Hydroxynicotinate 3-Monooxygenase, a Flavin-Dependent Decarboxylative Hydroxylase Involved in Bacterial Nicotinic Acid Degradation.

6-Hydroxynicotinate 3-monooxygenase (NicC) is a Group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria. Two mechanisms for the decarboxylative hydroxylation half-reaction have been proposed [Hicks, K., et al. (2016) Biochemistry 55, 3432-3446]. Results with Bordetella bronchiseptica RB50 NicC here show that a homocyclic analogue of 6-HNA, 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420-fold lower catalytic efficiency than is 6-HNA. The 13( V/ K), measured with wild-type NicC by isotope ratio mass spectrometry following the natural abundance of 13C in the CO2 product, is inverse for both 6-HNA (0.9989 ± 0.0002) and 4-HBA (0.9942 ± 0.0004) and becomes negligible (0.9999 ± 0.0004) for 5-chloro-6-HNA, an analogue that is 10-fold more catalytically efficient than 6-HNA. Covalently bound 6-HNA complexes of NicC are not observed by mass spectrometry. Comparative steady-state kinetic and Kd6HNA analyses of active site NicC variants (C202A, H211A, H302A, H47E, Y215F, and Y225F) identify Tyr215 and His47 as critical determinants both of 6-HNA binding ( KdY215F/ KdWT > 240; KdH47E/ KdWT > 350) and in coupling rates of 2,5-DHP and NAD+ product formation ([2,5-DHP]/[NAD+] = 1.00 (WT), 0.005 (Y215F), and 0.07 (H47E)]. Results of these functional analyses are in accord with an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC.