Competitive inhibition of human carbonyl reductase 1 by the ketal-masked panaxfuraynes A and B: A computational-enzymatic study.

Abstract

Carbonyl reductase 1 (CBR1) is an NADPH-dependent enzyme that reduces doxorubicin to doxorubicinol and is therefore of pharmacological interest as a modulator of anthracycline metabolism. Panaxfuraynes A and B are tetrahydrofuran-type polyacetylene sophorosides from Panax ginseng C.A. Meyer whose biological activities have remained uncharacterised since their isolation in 2009. Here we identify both compounds as inhibitors of human CBR1 and examine the structural basis of their activity. The parent glycosides possess defined chemical structures with little bioactivity annotation, so ligand-based target prediction was uninformative; metabolic analysis instead suggested sequential deglycosylation and ring opening to expose a conjugated ynone, directing attention to carbonyl reduction and CBR1. Catalytic-site binding was evaluated by molecular docking and three independent 100-ns molecular-dynamics simulations, and potency and inhibition mode were then determined by enzyme kinetics against recombinant human CBR1 and the homologous CBR3. Velocity data measured across five substrate and four inhibitor concentrations were fitted globally and compared across four inhibition models by Akaike information criterion. At CBR1 the competitive model was preferred, giving inhibition constants of 1.06 ± 0.14 and 1.68 ± 0.24 μM for panaxfuraynes A and B against a Michaelis constant of 54.6 ± 6.6 μM. At CBR3 the mixed model was preferred and the competitive model was rejected, giving inhibition constants of 9.59 ± 2.61 and 18.37 ± 7.66 μM with α values of 9.2 ± 6.1 and 3.6 ± 2.5, so the preference for CBR1 is approximately ninefold. The apparent IC50 against CBR1 rose linearly from 3.2 to 14.5 μM as the substrate concentration rose from 50 to 800 μM, the relationship expected of competitive inhibition. Curcumin, assayed as a positive control (Table S7 and Fig. S9) under identical conditions, inhibited CBR1 with an apparent IC50 of 1.13 ± 0.02 μM. Detergent, thiol, extended-preincubation and enzyme-free controls supported reversible, non-aggregating inhibition. These findings establish the first reported biological activity of panaxfuraynes and identify a ketal at the carbon that carbonyl reductase would otherwise reduce as a structural feature associated with competitive inhibition of CBR1, providing chemical probes for carbonyl-reductase recognition.

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