Discovery of Caralluma-derived pregnane glycosides as potent and selective cholinesterase inhibitors: integrated in silico and in vitro evaluation

Abstract

Alzheimer's disease (AD) is the fourth leading cause of death among elderly people worldwide. It has a complex pathogenesis, making multitarget-directed ligands (MTDLs) a key therapeutic strategy. This study evaluated pregnane glycosides isolated from Caralluma species (Apocynaceae) as potential cholinesterase inhibitors targeting acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes for AD treatment. In silico molecular docking against AChE (PDB: 4EY7) and BuChE (PDB: 8CGO) identified caratuberside E and awdelioside B as top AChE binders (−11.09 and −11.49 kcal mol⁻¹, outperforming the cocrystal inhibitor at −9.52 kcal mol⁻¹). For BuChE, caratuberside G and penicilloside C showed superior scores (−10.94 and −11.55 kcal mol⁻¹ vs. −8.89 kcal mol⁻¹ for the cocrystal). These results were validated by 200 ns molecular dynamics simulations (stable RMSD values) and MM-GBSA binding free-energy calculations, confirming strong interactions and favourable energetics. In vitro assays (using donepezil as reference) demonstrated potent inhibition: caratuberside E was most active against AChE (IC₅₀ = 0.69 ± 0.07 µM), followed by awdelioside B (IC₅₀ = 18.99 ± 0.06 µM); caratuberside G (IC₅₀ = 1.59 ± 0.16 µM) and penicilloside C (IC₅₀ = 12.38 ± 0.51 µM) excelled against BuChE. Collectively, these pregnane glycosides from Caralluma show promise as selective cholinesterase inhibitors and potential MTDLs for AD therapy.