Covalent modification of a cysteine residue in the XPB subunit of the general transcription factor TFIIH through single epoxide cleavage of the transcription inhibitor triptolide

Abstract

Triptolide is a key component of the traditional Chinese medicinal plant Thunder God Vine and has potent anticancer and immunosuppressive activities. It is an irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit of the general transcription factor TFIIH. Cys342 of XPB was identified as the residue that undergoes covalent modification by the 12,13-epoxide group of triptolide. Mutation of Cys342 of XPB to threonine conferred resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB with the Cys342Thr mutant in a HEK293T cell line rendered it completely resistant to triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these results deepen our understanding of the interaction between triptolide and XPB and have implications for the future development of new analogues of triptolide as leads for anticancer and immunosuppressive drugs.

Keywords: inhibitors; medicinal chemistry; natural products; target validation; transcription factors.

Figure 1

Structures of triptolide and triptolide analogues under clinical development. Potential sites of attack by a nucleophile from a protein are marked with red arrows. Sections for which the analogues differ in structure from triptolide are highlighted in blue.

Figure 2

The C12,13-epoxide of triptolide (TPL) forms a covelant bond with XPB. A) The C12,13-epoxide of triptolide reacts with N-acetyl-L-cysteine methyl ester to form the adduct 4. B) Inhibition of cell proliferation and the ATPase activity of TFIIH by triptolide analogues. Mean values ± SEM from two independent experiments are shown. C) Covalent binding of triptolide analogues to XPB as determined by the lack of recovery of XPB ATPase activity upon dialysis of the analogue–XPB complex. Mean values ± SD from two independent experiments are shown. DMSO=dimethyl sulfoxide.

Figure 3

Triptolide binds covalently to C342 of XPB. A) MS/MS spectrum of a peptide from human XPB (TFIIH) treated with triptolide. Insets: MS/MS spectrum of m/z 730.8895, which led to the identification of a mass shift of +360.1573 Da at the Cys342 residue of ³³⁵SGVIVLPCGAGK³⁴⁶. The labels (b) and (y) designate N- and C-terminal fragments of the peptide, respectively. The label Δ designates (b) or (y) ions with water and/or ammonia loss. B) [³H]-triptolide does not bind covalently to recombinant C342A, C342S, or C342T XPB mutant proteins.

Figure 4

The C342T XPB mutant is resistant to triptolide in vitro and confers high-level triptolide resistance to HET293T cells. A) C342A, C342S, and C342T XPB mutant TFIIH complexes were resistant to triptolide at concentrations up to 100 μM and C342T-TFIIH has the highest ATPase activity. Mean values ± SD from two independent experiments are shown. B) A mutant knock-in cell line (T7115) expressing only the C342T XPB mutant is selectively resistant to triptolide in cell proliferation assay. Mean values ± SEM from four independent experiments are shown.