Microbially Guided Discovery and Biosynthesis of Biologically Active Natural Products

Abstract

The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge-the inhibition of a human drug target-into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identi ed two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors target an allosteric site, which confers unusual selectivity, and can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4,464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded alternative PTP-speci c detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely de ned biochemical activities yet possess unanticipated structures and/or binding sites.

Evolution-Guided Biosynthesis of Terpenoid Inhibitors

“…coli. We used a bacterial two-hybrid (B2H) system that links the inhibition of PTP1B to the expression of a gene for antibiotic resistance (Figure C) . In this system, Src kinase phosphorylates a substrate domain, allowing it to bind to an Src homology 2 (SH2) domain; the substrate–SH2 complex activates the transcription of a resistance gene by localizing RNA polymerase to its promoter.…”

“…PTP1B dephosphorylates the substrate domain, preventing transcription, and the inactivation of PTP1B re-enables it. In prior work, we used this system to identify terpene synthases that can generate inhibitors of PTP1B . In a prior screen, both amorphadiene synthase and α-bisabolene synthase conferred a significant survival advantage (i.e., growth on solid media with ∼800 μg/mL spectinomycina concentration sufficient to kill strains with inactive variants of both terpene synthases); subsequent biophysical analyses indicated that amorphadiene inhibits PTP1B by binding to an allosteric site (Figure B) .…”

“…We used chemically competent NEB Turbo and electrocompetent One Shot Top10 (Invitrogen) cells for cloning and library preparation, BL2­(DE3) cells to express proteins for in vitro studies, s1030 for all B2H analyses, and DH5α for terpenoid isolation. When necessary, we generated chemically competent and electrocompetent cells with standard protocols (RbCl and washing, respectively) …”

“…Plasmids: pMBIS, the mevalonate-dependent isoprenoid pathway of S. cerevisiae; pTS, a terpene synthase; and pB2H, a two-hybrid system in which the inhibition of PTP1B permits a phosphorylation-mediated protein–protein interaction that activates the transcription of a gene for spectinomycin resistance . (D) A selection-based assay for identifying mutants of GHS that generate PTP1B inhibitors.…”

“…PTP1B is an influential regulatory enzyme, an important model system for biophysical studies, and an elusive drug target; new inhibitors could find broad use. GHS has a diverse, mutation-sensitive product profile and does not generate potent inhibitors of PTP1B in its wild-type form; it is a promising starting point for directed evolution. Using our synthetic objective as a guide, we uncovered mutants of GHS in which just one or two amino acid substitutions conferred a survival advantage by reducing GHS toxicity and/or by generating PTP1B inhibitors.…”