A side-by-side pharmacological comparison of ribose and (N)-methanocarba (bicyclo[3.1.0]hexane) nucleosides as A 3 AR agonists indicated that the bicyclic pseudoribose ring constraint provided higher affinity/selectivity at human and mouse A 3 AR. The mean affinity enhancement for 5 pairs of 5′methylamides was 11-fold at hA 3 AR and 42-fold at mA 3 AR. Novel C2-(5-fluorothien-2-ylethynyl) substitution enhanced affinity in the methanocarba but not ribose series, with highly hA 3 ARselective 16 (MRS7334) displaying K i 280 pM and favorable pharmacokinetics and off-target activity profile. Molecular dynamics comparison of 16 and its corresponding riboside 8 suggested a qualitative entropic advantage of 16 in hA 3 AR binding. The 5-F substitution tended to increase hA 3 AR affinity (cf. 5-Cl) for methanocarba but not ribose derivatives. A representative methanocarba agonist 4 was shown to interact potently exclusively with A 3 AR, among 240 GPCRs and 466 kinases. Thus, despite added synthetic difficulty, the (N)-methanocarba modification has distinct advantages for A 3 AR agonists, which have translational potential for chronic disease treatment.

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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 13 C in the CO 2 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 K d 6HNA 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 (K d Y215F /K d WT > 240; K d H47E /K d WT > 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.

The A 3 adenosine receptor (A 3 AR) 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 A 3 AR 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 [ 3 H]MRS7799. A 3 AR 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.