Dinucleoside polyphosphates, or dinucleotides (Np(n)N'; N, N' = A, U, G, C; n = 2-7), are naturally occurring ubiquitous physiologically active compounds. Despite the interest in dinucleotides, and the relevance of their conformation to their biological function, the conformation of dinucleotides has been insufficiently studied. Therefore, here we performed conformational analysis of a series of Np(n)N' Na(+) salts (N = A, G, U, C; N' = A, G, U, C; n = 2-5) by various NMR techniques. All studied dinucleotides, except for Up(4/5)U, formed intramolecular base stacking interactions in aqueous solutions as indicated by NMR. The conformation around the glycosidic angle in Np(n)N's was found to be anti/high anti and the preferred conformation around the C4'-C5', C5'-O5' bonds was found to be gauche-gauche (gg). The ribose moiety in Np(n)N's showed a small preference for the S conformation, but when attached to cytosine the ribose ring preferred the N conformation. However, no predominant conformation was observed for the ribose moiety in any of the dinucleotides. Molecular dynamics simulations of Ap(2)A and Ap(4)A Na(+) salts supported the experimental results. In addition, three modes of base-stacking were found for Ap(2/4)A: α-α, β-β and α-β, which exist in equilibrium, while none is dominant. We conclude that natural, free Np(n)N's (n = 2-5) at physiological pH exist mostly in a folded (stacked), rather than extended conformation, in several interconverting stacking modes. Intramolecular base stacking of Np(n)N's does not alter the conformation of each of the nucleotide moieties, which remains the same as that of the mononucleotides in solution.

P2Y nucleotide receptors (P2Y-Rs) play important physiological roles. However, most of the P2Y-R subtypes are still lacking potent and selective agonists and antagonists. Based on data mining analysis of binding interactions in 44 protein-uridine nucleos(t)ides complexes, we designed uracil nucleotides, substituted at the C5/C6 position. All C6-substituted derivatives were inactive at the P2Y(2,4,6)-Rs, while out of the C5-substituted analogues, only 5-OMe-UD(T)P showed activity. To rationalize the data, the ionization and conformation of these analogues were evaluated. The pK(a) values of most analogues substituted at the C5/C6 positions were unaltered compared to UTP (pK(a) 9.42), except for 5-F-UTP nucleotide (pK(a) 7.85). C6-substituted analogues adopt the syn or high-syn conformations, which are disfavored by the receptors, while 5-OMe-UD(T)P adopt the favored anti conformation. Furthermore, 5-OMe-UDP adopts the S sugar puckering, which is the conformation preferred by the P2Y(6)-R, but not the P2Y(2)- or P2Y(4)-Rs. 5-OMe-UDP fulfills the conformational and H-bonding requirements of P2Y(6)-R, thus, making a potent P2Y(6)-R agonist (EC(50) 0.08 microM), more than UDP (EC(50) 0.14 microM).

On the basis of the high affinity of Zn(2+) to sulfur and imidazole, we targeted nucleotides such as GDP-β-S, ADP-β-S, and AP3(β-S)A, as potential biocompatible Zn(2+)-chelators. The thiophosphate moiety enhanced the stability of the Zn(2+)-nucleotide complex by about 0.7 log units. ATP-α,β-CH2-γ-S formed the most stable Zn(2+)-complex studied here, log K 6.50, being ~0.8 and ~1.1 log units more stable than ATP-γ-S-Zn(2+) and ATP-Zn(2+) complexes, and was the major species, 84%, under physiological pH. Guanine nucleotides Zn(2+) complexes were more stable by 0.3-0.4 log units than the corresponding adenine nucleotide complexes. Likewise, AP3(β-S)A-zinc complex was ~0.5 log units more stable than AP3A complex. (1)H- and (31)P NMR monitored Zn(2+) titration showed that Zn(2+) coordinates with the purine nucleotide N7-nitrogen atom, the terminal phosphate, and the adjacent phosphate. In conclusion, replacement of a terminal phosphate by a thiophosphate group resulted in decrease of the acidity of the phosphate moiety by approximately one log unit, and increase of stability of Zn(2+)-complexes of the latter analogues by up to 0.7 log units. A terminal phosphorothioate contributed more to the stability of nucleotide-Zn(2+) complexes than a bridging phosphorothioate.

Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.

BackgroundAlmost 90% of patients with dementia suffer from some type of neurobehavioral symptom, and there are no approved medications to address these symptoms.ObjectiveTo evaluate the safety and efficacy of the medical cannabis oil “Avidekel” for the reduction of behavioral disturbances among patients with dementia.Materials and methodsIn this randomized, double-blind, single-cite, placebo-controlled trial conducted in Israel (ClinicalTrials.gov: NCT03328676), patients aged at least 60, with a diagnosis of major neurocognitive disorder and associated behavioral disturbances were randomized 2:1 to receive either “Avidekel,” a broad-spectrum cannabis oil (30% cannabidiol and 1% tetrahydrocannabinol: 295 mg and 12.5 mg per ml, respectively; n = 40) or a placebo oil (n = 20) three times a day for 16 weeks. The primary outcome was a decrease, as compared to baseline, of four or more points on the Cohen-Mansfield Agitation Inventory score by week 16.ResultsFrom 60 randomized patients [mean age, 79.4 years; 36 women (60.0%)], 52 (86.7%) completed the trial (all eight patients who discontinued treatment were from the investigational group). There was a statistically significant difference in the proportion of subjects who had a Cohen-Mansfield Agitation Inventory score reduction of ≥ 4 points at week 16: 24/40 (60.0%) and 6/20 (30.0%) for investigational and control groups, respectively (χ2 = 4.80, P = 0.03). There was a statistically significant difference in the proportion of subjects who had a Cohen-Mansfield Agitation Inventory score reduction of ≥ 8 points at week 16: 20/40 (50%) and 3/20 (15%), respectively (χ2 = 6.42, P = 0.011). The ANOVA repeated measures analysis demonstrated significantly more improvement in the investigational group compared to the control group at weeks 14 and 16 (F = 3.18, P = 0.02). Treatment was mostly safe, with no significant differences in the occurrence of adverse events between the two groups.ConclusionIn this randomized controlled trial, ‘Avidekel’ oil significantly reduced agitation over placebo in patients suffering from behavioral disturbances related to dementia, with non-serious side-effects. Further research is required with a larger sample size.