Damijan Knez, David Malinak, Anže Meden, Svit Ferjančič Benetik, Anja Pišlar, Zuzana Kohoutova, Iveta Vondrackova, Miroslav Psotka, Antonin Lycka, Lenka Pulkrabkova, Ondrej Soukup, Adam Skarka, Rudolf Andrys, Xavier Brazzolotto, Alexandre Igert, José Dias, Florian Nachon, Jan Detka, Filip Targosiński, Dziyana Hliabovich, Joseph S Brunzelle, Ludmilla Shuvalova, Kinga Sałat, Saktimayee Mitra Roy, Daniel Martin Watterson, Kamil Musílek, Stanislav Gobec
Chronic neuroinflammation and cholinergic dysfunction are major contributors to cognitive decline in Alzheimer's disease. Here, we report a series of pyridazine-based multitarget-directed ligands that simultaneously inhibit human butyrylcholinesterase (hBChE) and p38α mitogen-activated protein kinase (p38α MAPK). Pyridazines 8 and 21 were identified as potent dual inhibitors with submicromolar inhibitory potencies against target enzymes and high selectivity over acetylcholinesterase. X-ray crystallographic analyses revealed the experimental binding modes of 8 and 21 in hBChE and p38α MAPK, and identified conserved π-π, π-cation, and hinge region interactions that rationalize dual-target engagement. Both compounds were blood-brain barrier permeable, exhibited low cytotoxicity, and significantly attenuated lipopolysaccharide-induced proinflammatory responses and apoptosis in BV2 microglia. In vivo, compound 21 enhanced cognitive performance in mouse models of scopolamine-induced amnesia and LPS-stimulated neuroinflammatory cognitive impairment. Collectively, these results document the potential of structure-assisted design to achieve selective dual-target pharmacology within a single molecular entity.