P2-receptors (P2-Rs) represent important targets for novel drug development. Most ATP analogues proposed as potential drug candidates have shortcomings such as limited receptor-selectivity and limited stability that justify the search for new P2-R agonists. Therefore, a novel series of nucleotides based on the adenosine 5'-O-(1-boranotriphosphate) (ATP-alpha-B) scaffold was developed and tested as P2Y(1)-R agonists. An efficient four-step one-pot synthesis of several ATP-alpha-B analogues from the corresponding nucleosides was developed, as well as a facile method for the separation of the diastereoisomers (A and B isomers) of the chiral products. The potency of the new analogues as P2Y(1)-R agonists was evaluated by the agonist-induced Ca2+ release of HEK 293 cells stably transfected with rat-brain P2Y(1)-R. ATP-alpha-B A isomer was equipotent with ATP (EC50 = 2 x 10(-7) M). However, 2-MeS- and 2-Cl- substitutions on ATP-alpha-B (A isomer) increased the potency of the agonist up to 100-fold, with EC50 values of 4.5 x 10(-9) and 3.6 x 10(-9) M, compared to that of the ATP-alpha-B (A isomer). Diastereoisomers A of all ATP-alpha-B analogues were more potent in inducing Ca2+ release than the corresponding B counterparts, with a 20-fold difference for 2-MeS-ATP-alpha-B analogues. The chemical stability of the new P2Y(1)-R agonists was evaluated by 31P NMR under physiological and gastric-juice pH values at 37 degrees C, with rates of hydrolysis of 2-MeS-ATP-alpha-B of 1.38 x 10(-7) s-1 (t1/2 of 1395 h) and 3.24 x 10(-5) s-1 (t1/2 = 5.9 h), respectively. The enzymatic stability of the new analogues toward spleen NTPDase was evaluated. Most of the new analogues were poor substrates for the NTPDase, with ATP-alpha-B (A isomer) hydrolysis being 5% of the hydrolysis rate of ATP. Diastereoisomers A and B exhibited different stability, with A isomers being significantly more stable, up to 9-fold. Furthermore, A isomers that are potent P2Y(1)-R agonists barely interact with NTPDase, thus exhibiting protein selectivity. Therefore, on the basis of our findings, the new, highly water-soluble, P2Y(1)-R agonists may be considered as potentially promising drug candidates.

Dinucleoside polyphosphates, NpnN', exert their physiological effects via P2 receptors. They are attractive drug targets as they offer better stability and specificity compared to nucleotides, the most common P2-receptor ligands. To further improve the properties of NpnN', which are still pharmacologically unsatisfactory, we developed novel boranophosphate isosteres of dinucleoside polyphosphates, denoted as Npn(B)N. These analogues were obtained in a facile and efficient synthesis as the exclusive products in a concerted reaction of two nucleoside phosphorimidazolides and inorganic boranophosphate. This unusual reaction is due to the preorganization of three reactant molecules by the Mg2+ ion. We found that Ap3/5(beta/gamma-B)A analogues were potent P2Y1-R agonists. Ap5(gamma-B)A was equipotent to 2-MeS-ADP (EC50 6.3x10(-8) M), thus making it one of the most potent P2Y1-R agonists currently known. Moreover, Ap5(gamma-B)A did not activate P2Y2-R. In contrast, Up3/5(beta/gamma-B)U analogues were extremely poor agonists of both P2Y1-R and P2Y2-R. Npn(B)N analogues exhibited remarkable chemical stability under physiological conditions. Under conditions mimicking gastric juice, Np3(beta-B)N analogues exhibited a half-life (t1/2) of 1.3 h, whereas Np5(gamma-B)N degraded at a much faster rate (t1/2 18 min). The hydrolysis of Ap3(beta-B)A by human nucleotide pyrophosphatase phosphodiesterases (NPP1 and NPP3) was slowed by 40% and 59%, respectively, as compared to Ap3A. However, this effect of the boranophosphate was position-dependent, as Np5(gamma-B)N was degraded at a rate comparable to that of Np5N. In summary, Ap5(gamma-B)A appears to be a highly potent and selective P2Y1-R agonist, as compared to the parent compound. This promising scaffold will be applied in the design of future metabolically stable analogues.

In the companion paper, part 1, we described the construction of an improved molecular model for the h-P2Y1 receptor (h-P2Y1-R) and proposed a rational for the stereoelectronic selectivity of the receptor. Here, we extend our studies on the molecular recognition of the h-P2Y1-R to the exploration of the diastereoselectivity of this receptor. For this purpose, we implemented an integrative approach combining synthesis, spectral analysis, biochemical assays, and computational analysis. Specifically, we selected and synthesized novel ATP analogues bearing a chiral center on the phosphate chain. We analyzed the conformation of the chiral ATP analogues in solution by 1H/13C NMR and assigned the absolute configuration of the diastereoisomers. The coordination mode of these analogues with a Mg2+ ion was evaluated by 31P NMR. These chiral analogues were biochemically evaluated and found to be potent h-P2Y1-R ligands. An EC50 difference of ca. 20-fold was observed between the diastereoisomers. Their spectral absolute configuration assignment was confirmed by comparison of the biochemical results to those of ATP-alpha-S diastereoisomers whose chirality is known. Finally, a computational analysis was performed for the elucidation of molecular recognition employing molecular mechanics (docking) studies on the receptor:ligands complexes. On the basis of the current results, we hypothesize that h-P2Y1-R's chiral discrimination originates from the requirement that the nucleotide analogue interacts with a Mg2+ ion within the receptor binding site. This Mg2+ ion is possibly coordinated with both Asp204 and the ATP's alpha, beta, gamma-phosphates in a Lambda configuration.

We previously developed orthosteric M1 muscarinic agonists (e.g. AF102B, AF267B and AF292), which act as cognitive enhancers and potential disease modifiers. We now report on a novel compound, AF710B, a highly potent and selective allosteric M1 muscarinic and σ1 receptor agonist. AF710B exhibits an allosteric agonistic profile on the M1 muscarinic receptor; very low concentrations of AF710B significantly potentiated the binding and efficacy of carbachol on M1 receptors and their downstream effects (p-ERK1/2, p-CREB). AF710B (1-30 µg/kg, p.o.) was a potent and safe cognitive enhancer in rats treated with the M1 antagonist trihexyphenidyl (passive avoidance impairment). These effects of AF710B involve σ1 receptor activation. In agreement with its antiamnesic properties, AF710B (at 30 nM), via activation of M1 and a possible involvement of σ1 receptors, rescued mushroom synapse loss in PS1-KI and APP-KI neuronal cultures, while AF267B (1 µM) was less potent in PS1-KI and ineffective in APP-KI models, respectively. In female 3xTg-AD mice, AF710B (10 µg/kg, i.p./daily/2 months) (i) mitigated cognitive impairments in the Morris water maze; (ii) decreased BACE1, GSK3β activity, p25/CDK5, neuroinflammation, soluble and insoluble Aβ₄₀, Aβ₄₂, plaques and tau pathologies. AF710B differs from conventional σ1 and M1 muscarinic (orthosteric, allosteric or bitopic) agonists. These results highlight AF710B as a potential treatment for Alzheimer's disease (e.g. improving cognitive deficits, synaptic loss, amyloid and tau pathologies, and neuroinflammation) with a superior profile over a plethora of other therapeutic strategies.