Chaxines from the edible mushroom Agrocybe chaxingu are electronically competent Michael acceptors but do not engage the cathepsin K active site: An in silico target-deconvolution study
9th International Eurasian Conference on Biological and Chemical Sciences (EurasianBioChem 2026), Ankara, Türkiye, 19 - 21 Ekim 2026, ss.15-23, (Tam Metin Bildiri)
- Yayın Türü: Bildiri / Tam Metin Bildiri
- Basıldığı Şehir: Ankara
- Basıldığı Ülke: Türkiye
- Sayfa Sayıları: ss.15-23
- Trakya Üniversitesi Adresli: Evet
Özet
The edible mushroom Agrocybe chaxingu produces chaxines, degraded ergostane-type metabolites that suppress osteoclast formation without cytotoxicity, yet their molecular target has never been identified. Because the chaxines carry a cross-conjugated α,β-unsaturated carbonyl and because osteoclast function depends on the cysteine protease cathepsin K, covalent engagement of the catalytic Cys25 is a plausible but untested hypothesis. Chaxines B–E were evaluated against a nine-receptor panel comprising eight osteoclast effectors (cathepsins K, L, S and B, carbonic anhydrase II, MMP-9, c-Src and CSF1R) and RORγt, using AutoDock Vina with three seeds. Every receptor was validated by redocking its own co-crystallised ligand; seven of nine reproduced the crystallographic pose, cathepsin K most accurately at 0.29 Å. The chaxines bound the cathepsin K site at −6.6 to −7.2 kcal/mol and outscored no cognate reference at any interpretable validated target: only MMP-9, whose zinc-chelating hydroxamate reference is systematically underestimated, and cathepsin L, whose reference is a covalent adduct, gave negative values. Electronic-structure calculations placed them in the electrophilicity range of established Michael acceptors and far from the non-electrophilic sterol ergosterol. However, a geometric criterion calibrated against a crystallographic pre-covalent nitrile complex showed the electrophilic β-carbon approaching Cys25 Sγ edge-on. Of 144 poses across four cysteine proteases, twelve came within 4.5 Å, yet none qualified at 30° or 40° from the perpendicular and a single pose appeared only at 50°, beyond the range compatible with conjugate addition. Ensemble docking over nine crystal forms, whole-surface blind docking and orthogonal rescoring reproduced the result. Retrospective validation against 60 known actives and 582 decoys gave a ROC-AUC of 0.602 with no early enrichment: scores carry little information in this pocket, and the conclusion rests on pose geometry rather than affinity. Direct active-site inhibition of cathepsin K is therefore an unlikely explanation for the osteoclast-suppressing phenotype of these mushroom metabolites.