Targeting DYRK1A with rationally designed pyrazine-guanidine hybrids for Alzheimer's disease: design, synthesis, and biological evaluation.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) has been implicated in the pathogenesis of Alzheimer's disease (AD) by regulating tau hyperphosphorylation and neuroinflammatory pathways, suggesting DYRK1A as an important therapeutic target. Herein, we report the rational design, synthesis and evaluation of a new class of low-molecular-weight pyrazine-guanidine molecular hybrids as potential DYRK1A inhibitors via an integrated computational and experimental approach. Molecular docking studies predicted good protein-ligand interactions, with compounds F1, F5, and F7 exhibiting the highest binding affinity of -9.7 kcal mol-1, compared with the reference inhibitor Harmine (-8.8 kcal mol-1). Steady-state fluorescence further confirmed the formation of a protein-ligand complex with the highest binding affinity, K = 1.70 × 106 M-1 for compound F7. Enzyme inhibition assays showed concentration-dependent inhibition of DYRK1A, with IC50 values as low as 8.10, 12.46, and 24.05 µM for compounds F1, F7, and F11, respectively to the reference standard Harmine with 1.43 µM. The cytotoxicity of the tested compounds against N9 microglial cells indicated that they were mostly non-cytotoxic, with compound F7 having the best safety profile. Moreover, compounds F1 and F7 markedly reduced lipopolysaccharide-induced nitric oxide and reactive oxygen species production, indicating their potential to inhibit neuroinflammatory responses. The stability of the protein-ligand complex was further confirmed by molecular dynamics simulations, reporting sustained contacts within the active site of the kinase during the simulation. The compounds exhibit drug-like properties and good oral bioavailability as indicated by in silico physicochemical predictions. However, the poor predicted BBB permeability of the evaluated compounds represents an important limitation for CNS exposure and highlights the need for further structural optimization to improve brain penetration. Among the screened compounds, compound F7 was the most promising DYRK1A inhibitor with low cytotoxicity and significant anti-neuroinflammatory effects. The present findings identify the cationic heteroaryl pyrazine-guanidine scaffold as a useful framework for the development of DYRK1A inhibitors aimed at Alzheimer's disease.