The pyrazolo4,3-cpyrazole core as a novel and versatile scaffold for developing dual DYRK1A-CLK1 inhibitors targeting key processes of Alzheimer's disease pathology

In the current study, we designed, synthesized, and characterized a series of substituted pyrazolo4,3-cpyrazoles. These novel compounds were evaluated in vitro for their inhibitory activity over a panel of protein kinases to determine their potential therapeutic applications against Alzheimer's disease. To gain deeper insight into the binding interactions between the most potent analogues and their respective kinase targets, advanced molecular simulations were performed. In parallel, the ability of pyrazolo4,3-cpyrazoles to inhibit Aβ40 aggregation was assessed using biophysical techniques such as circular dichroism and Thioflavin T assays. Our results highlight the specific heterocycle as a highly promising and synthetically versatile scaffold for developing inhibitors of both AD-relevant kinases and amyloid-β aggregation. Although more effort is needed to assess the possibility of developing multi-target inhibitors, pyrazolo4,3-cpyrazole analogues demonstrated significant activities against their individual targets, indicating substantial capacity of the heterocyclic scaffold for further optimization toward both directions. Overall, our findings emphasize the potential of properly substituted pyrazolo4,3-cpyrazoles as multifunctional agents targeting key processes in Alzheimer's disease pathology.