Genomics-guided discovery of thailanstatins A, B, and C As pre-mRNA splicing inhibitors and antiproliferative agents from Burkholderia thailandensis MSMB43

Abstract

Mining the genome sequence of Burkholderia thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonas sp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (1), B (2), and C (3), were isolated from the fermentation broth of B. thailandensis MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase pathway. They differ from 4 by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4 as tested in phosphate buffer at pH 7.4. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as 4, with half-maximal inhibitory concentrations in the single to sub-μM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development and new structure-activity information for chemical optimization of related spliceosome inhibitors.

Figure 1

Genomics-guided discovery of thailanstatins (1–3). (A) A graphical comparison of the FR901464 (4) biosynthetic gene cluster (fr9) and the thailanstatin biosynthetic gene cluster (tst). (B) Identification with RT-PCR analysis of three growth media in which the regulatory tstA gene is expressed. (C) HPLC profiling of a crude extract of B. thailandensis MSMB43 fermentation broth and identification of three compound peaks with a conjugated diene-like characteristic UV absorbance (UV_max at 235 nm).

Figure 2

Proposed biosynthetic pathway of thailanstatin A (1) in B. thailandensis MSMB43. Crossed domains are inactive or nonfunctional, star-labeled domains are atypical. See text for explanations. A, adenylation; ACP, acyl carrier protein; AT, acyltransferase; C, condensation; DH, dehydratase; ECH, enoyl-CoA hydratase; ER, enoyl reductase; GAT, glyceryl transferase/phosphatase; KR, ketoreductase; KS, ketosynthetase; MT, methyltransferase; OX, FAD-dependent monooxygenase; PCP, peptidyl carrier protein; TE, thioesterase.

Figure 3

Thailanstatins (1–3) are more stable than FR901464 (4). Compounds were placed in phosphate buffer (pH 7.4) at 37 °C and examined by HPLC at multiple time points.

Figure 4

Thailanstatins (1–3) inhibit pre-mRNA splicing in vitro. FR901464 (4) and 1–3 were added to splicing reactions containing ³²P-labeled CDC14–15 pre-mRNA at the final concentrations indicated. AMP-PNP (5 mM) and DMSO were used as positive and negative controls, respectively. RNA was purified and resolved on denaturing PAGE and gels were autoradiographed at −80 °C. Identities of splicing intermediates are depicted to the right of gels.