Identification of new PTP1B-inhibiting decipiene diterpenoid esters from Eremophila clarkei by high-resolution PTP1B inhibition profiling, enzyme kinetics analysis, and molecular docking

Abstract

In this study, an extract of the leaves of Eremophila clarkei Oldfield & F.Muell. showed protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with an IC50 value of 33.0 μg/mL. The extract was therefore investigated by high-resolution PTP1B inhibition profiling to pinpoint the constituents responsible for the activity. Subsequent isolation and purification using analytical-scale HPLC led to identification of eight previously undescribed decipiene diterpenoids, eremoclarkanes A-H, as well as eremoclarkic acid, a biogenetically related new phenolic acid. In addition, one known decipiene diterpenoid and ten known O-methylated flavonoids were isolated. The structures of the isolated compounds were elucidated by extensive analysis of their HRMS and 1D and 2D NMR spectra. The absolute configuration of decipiene diterpenoids was determined by comparison of experimental and calculated ECD spectra. The flavonoid hispidulin (2b) and the four decipiene diterpenoids 13a, 13b, 13f, and 14b exhibited PTP1B inhibitory activity with IC50 values ranging from 22.8 to 33.6 μM. This is the first report of PTP1B inhibitory activity of decipienes, and enzyme kinetics revealed that 13a and 13b are competitive inhibitors of PTP1B, whereas 13f and 14b displayed mixed-type-mode inhibition of PTP1B. Finally, molecular docking indicated that 13a, 13b, 13f, and 14b showed comparable binding affinity towards the active and/or allosteric site of PTP1B enzyme. Structure-activity relationship (SAR) of the identified O-methylated flavonoids and decipiene diterpenoids towards PTP1B is discussed. Plausible enzymatic and photochemically driven routes for the formation of the decipienes and conversion products thereof are presented and discussed.