Nucleoside-2’,3’/3’,5’-Bis(thio)phosphate Analogues are Anti-oxidants and Promising Agents for Dissolution of Aβ42-Zn(II)/Cu(II) Aggregates

Bosmat Levi Hevroni, Chemistry, Bar Ilan University, Ramat Gan, Israel
Bilha Fischer, Chemistry, Bar Ilan University, Ramat Gan, Israel

Amyloid beta (Aβ) peptide forms aggregates with zinc and copper ions in brains of Alzheimer’s disease (AD) patients. Metal-ion-chelation was suggested as therapy for the metal-ion-induced Aβ aggregation, metal-ion overload, and oxidative stress. In a quest for biocompatible metal-ion chelators potentially useful for AD therapy, we synthesized a series of adenine/guanine 2′,3′- or 3′,5′-bisphosphate and -bisphosphorothioate analogues, 1−6, as agents for re-solubilization of Cu(II)/Zn(II)-Aβ-aggregates, and inhibitors of Fenton reaction in Cu(I) or Fe(II)/H₂O₂ systems. We found that inhibition Fe(II)-induced production of OH radicals from H₂O₂, by bisphosphate derivatives 1, 3, and 5 (IC₅₀ = 36, 24, and 40 μM, respectively), was more effective than it was by EDTA, by a factor of 1.5, 2, and 1.4, respectively as monitored by EPR. Moreover, 2′-dA-3’5’PO, 1, was 1.8- and 4.7-times more potent than 5′-AMP and 5′-ADP, respectively.[1] Next, we evaluated analogues 1-6 for their ability to dissolve Aβ₄₂–M(II) aggregates, EDTA was highly effective in dissolving Aβ₄₂–Zn(II) aggregates, 51% and 52% disaggregation, at 3 and 6 eq, respectively, as observed by turbidity assay. A2’3’PS, 6, was found to be almost as effective as EDTA with 42% and 45% disaggregation at 3 and 6 eq, respectively. Nucleotides 1, 2, 4 and 5 presented a moderate re-solubilization ability with 10-20% disaggregation of Aβ₄₂–Zn(II). Most of the tested nucleotide analogues were equi-efficacious or even more effective than EDTA in dissolution of Aβ₄₂–Cu(II) aggregates. Compounds 1 and 2 presented 26% and 33% disaggregation at 6 eqiuv, respectively. Bisthiophosphates 4 and 6 dissolved 56% and 50% of the aggregates at 6 eq, which was respectively 2.5- and 2.2-fold better than EDTA (23% at 6 eq). Analogues 4 and 6 were found superior to 5’-AMP, 3’-AMP and ADP. For instance, ADP, at 6 eq dissolved only 15-20% of Zn(II)- and Cu(II)-Aβ₄₂ aggregation. Using circular dichroism (CD) we found that Aβ₄₂ adopted a mixture of b-sheet, a-helix and random coil structures. Upon addition of an equimolar amount of Zn(II)/Cu(II) Aβ₄₂ adopted mainly b-sheet structure. Next, 6 eq of compounds 1, 2, 4, 6 and EDTA as a control, were added to Aβ₄₂–M(II) mixtures. After 1 h compounds 4, 6, and EDTA reverted the Aβ₄₂–M(II) morphology, back to that of free Aβ₄₂. Therefore, we propose 1, 4, and 6 as potential lead candidates for AD therapy capable of both reducing OH radical production and re-solubilizing of Aβ₄₂–M(II) aggregates.