Computationally-Assisted Discovery and Assignment of a New Class of 6/6/5/5 Fused-Ring Diterpene Acting as Pregnane X Receptor Ligands from Isodon serra

Abstract

We report here the orchestration of molecular ion networking (MoIN) and a set of computationally assisted structural elucidation approaches in the discovery and assignment of a new class of rearranged 4,5-seco-abietane diterpenoids including serra A (1), which possesses an unusual 6/6/5/5 fused-ring skeleton system, together with two previously unreported diterpenoids serras B-C (2-3) and five known compounds were isolated from Isodon serra (I. serra). The structures were elucidated by spectroscopic analysis in conjunction with computationally assisted structure elucidation tools. In silico, serras A-C (1-3) bind well to PXR, suggesting their potential role in reducing inflammation. The results of serra A (1) with hPXR demonstrated agonist activity with an EC₅₀ value of 15 μM. Serra A (1), graciliflorin F (4), gerardianin C (5), 11,12,15-trihydroxy-8,11,13-abietatrien-7-one (6), rabdosin D (7), and 15-hydroxysalprionin (8) exhibited promising anti-inflammatory activities in lipopolysaccharide (LPS)-induced RAW 267.4 cells, and their inhibition rates on NO production were more than 65% at 10 μM.

Figure 1.

Whole MoIN map for the ethanol extract of I. serra. The zoomed out part is for the diterpenoids of the whole MoIN map.

Figure 2.

Structure and key HMBC (H → C) and COSY (H → H) correlations of serras A–C (1–3).

Figure 3.

DFT, TDDFT and DP4+ analysis of compounds 2 and 3. Correlations that do not meet this criterion are highlighted in red.

Figure 4.

Experimental and calculated ECD spectra of serra A (1S) and ¹³C NMR calculations of serra A (1S).

Figure 5.

Experimental and calculated ECD spectra of serra B (1R,2R), and serra C (1R,2S) in MeOH.

Figure 6.

(A) PXR protein structure and binding domain. (B) Binding domain subdivided into subpockets 1–4 with respective ligands. (C) Binding positions and affinities of serra A (1) (pink; −9.4 kcal/mol), oxadiazon (orange; −9.8 kcal/mol), serra B (2) (green; −8.7 kcal/mol), serra C (3) (yellow; −9.5 kcal/mol), α-zearalanol (blue; −10.7 kcal/mol), pretilachlor (red; −7.0 kcal/mol), and estradiol (purple; −9.7 kcal/mol).

Figure 7.

Interaction of serra A (1) (pink) and oxadizon (orange) with the amino acid residues within the PXR binding domain.

Figure 8.

Transcriptional responses of PXR in GAL4-hPXR-LBD-transfected Hek293 cells exposed to different concentrations of serra A (1). Assays were performed in triplicate in at least three independent experiments, and data are expressed as the mean (±sd).