Lapcin, a potent dual topoisomerase I/II inhibitor discovered by soil metagenome guided total chemical synthesis

Abstract

In natural product discovery programs, the power of synthetic chemistry is often leveraged for the total synthesis and diversification of characterized metabolites. The synthesis of structures that are bioinformatically predicted to arise from uncharacterized biosynthetic gene clusters (BGCs) provides a means for synthetic chemistry to enter this process at an early stage. The recent identification of non-ribosomal peptides (NRPs) containing multiple ρ-aminobenzoic acids (PABAs) led us to search soil metagenomes for BGCs that polymerize PABA. Here, we use PABA-specific adenylation-domain sequences to guide the cloning of the lap BGC directly from soil. This BGC was predicted to encode a unique N-acylated PABA and thiazole containing structure. Chemical synthesis of this structure gave lapcin, a dual topoisomerase I/II inhibitor with nM to pM IC50s against diverse cancer cell lines. The discovery of lapcin highlights the power of coupling metagenomics, bioinformatics and total chemical synthesis to unlock the biosynthetic potential contained in even complex uncharacterized BGCs.

Fig. 1. Discovery of the lap gene cluster.

a Overview of the PABA-specific A-domain guided discovery of the lapcin (lap) BGC from the soil metagenome. i DNA extracted from soil was used to construct metagenomic libraries. ii NPSTs generated from arrayed metagenomic libraries and iii the resulting NPSTs were searched for PABA-specific A-domains based on a signature sequence derived from known PABA A-domains. iv Phylogenetic analysis of predicted PABA NPSTs was used to identify sequences that arose from a new family of BGCs. v Clones containing NRPS BGCs of interest were recovered from the arrayed library subpools and fully sequenced to reveal BGCs that encode PABA-based natural products. b The lap BGC, which is shown here, was recovered from an archived soil metagenome library using this process. olive, resistance gene; red, NRPS biosynthesis; green, PABA tailoring; blue, thiazole formation; pink, PABA core.

Fig. 2. Bioinformatic prediction of lapcin from the lap gene cluster.

a The substrate of each lapcin A-domain was inferred by comparing the 10 residues that make up the substrate binding pocket of each lapcin A-domain with characterized NRPS A-domains (red, difference between lap and known substrate binding pockets). b The structure of lapcin was predicted bioinformatically based on the NRPS modules and accessory enzymes found in the lap gene cluster. c The predicted structure of lapcin.

Fig. 3. Overview of the synthesis of lapcin.

Lapcin was synthesized in three fragments A–C which were coupled using a series of amid bond forming reactions. i Coupling fragment AB (S18): isobutyl chloroformate, TEA, THF. ii Coupling AB (S18) to fragment C (16): HBTU, DIPEA, DMF.

Fig. 4. Topoisomerase inhibition by lapcin.

a Type I topoisomerase DNA relaxation assay (SC, supercoil plasmid) b Type II topoisomerase DNA decatenation assay (KDNA, Kineotoplast DNA). c Type I and d II topoisomerase inhibition plots. n = 3 independent assays. Mean value and SD are shown.