Significance: The prevalence of obesity and type 2 diabetes (T2D) has increased worldwide. Obesity is correlated with unfavorable adipose tissue modifications that lead to metabolic dysregulation. Here, we show that P2Y ₆ R deficiency in adipocytes protects from diet-induced obesity due to enhanced energy expenditure, reduced inflammation, and white adipose tissue browning. Further, adipocytes lacking P2Y ₆ R show reduced JNK expression and activity, contributing to the improved phenotype. This proof-of-concept study strongly suggests that P2Y ₆ R antagonists may be beneficial for the treatment of obesity and T2D.

Allosteric antagonism by bitopic ligands, as reported for many receptors, is a distinct modulatory mechanism. Although several bitopic A_2A adenosine receptor (A_2AAR) ligand classes were reported as pharmacological tools, their receptor binding and functional antagonism patterns, i.e., allosteric or competitive, were not well characterized. Therefore, here we systematically characterized A_2AAR binding and functional antagonism of two distinct antagonist chemical classes. i.e., fluorescent conjugates of xanthine amine congener (XAC) and SCH442416. Bitopic ligands were potent, weak, competitive or allosteric, based on the combination of pharmacophore, linker and fluorophore. Among antagonists tested, XAC, XAC245, XAC488, SCH442416, MRS7352 showed K_i binding values consistent with K_B values from functional antagonism. Interestingly, MRS7396, XAC-X-BY630 (XAC630) and 5-(N,N-hexamethylene)amiloride (HMA) were 9–100 times weaker in displacing fluorescent MRS7416 binding than radioligand binding. XAC245, XAC630, MRS7396, MRS7416 and MRS7322 behaved as allosteric A_2AAR antagonists, whereas XAC488 and MRS7395 antagonized competitively. Schild analysis showed antagonism slopes of 0.42 and 0.47 for MRS7396 and XAC630, respectively. Allosteric antagonists HMA and MRS7396 were more potent in displacing [³H]ZM241385 binding than MRS7416 binding. Sodium site D52N mutation increased and decreased affinity of HMA and MRS7396, respectively, suggesting possible preference for different A_2AAR conformations. The allosteric binding properties of some bitopic ligands were rationalized and analyzed using the Hall two-state allosteric model. Thus, fluorophore tethering to an orthosteric ligand is not neutral pharmacologically and may confer unexpected properties to the conjugate.

5-Fluorouracil and 5-fluorouracil-based prodrugs have been used clinically for decades to treat cancer. Their anticancer effects are most prominently ascribed to inhibition of thymidylate synthase (TS) by metabolite 5-fluoro-2 '- deoxyuridine 5 '-monophosphate (FdUMP). However, 5-fluorouracil and FdUMP are subject to numerous unfavorable metabolic events that can drive undesired systemic toxicity. Our previous research on antiviral nucleotides suggested that substitution at the nucleoside 5 '-carbon imposes conformational restrictions on the corresponding nucleoside monophosphates, rendering them poor substrates for productive intracellular conversion to viral polymerase-inhibiting triphosphate metabolites. Accordingly, we hypothesized that 5 '-substituted analogs of FdUMP, which is uniquely active at the monophosphate stage, would inhibit TS while preventing undesirable metabolism. Free energy perturbation-derived relative binding energy calculations suggested that 5 '(R)-CH3 and 5 '(S)-CF3 FdUMP analogs would maintain TS potency. Herein, we report our computational design strategy, synthesis of 5 '-substituted FdUMP analogs, and pharmacological assessment of TS inhibitory activity.

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.