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 identified two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors appear to target an allosteric site, which confers 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-specific detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely defined biochemical activities yet possess unanticipated structures and/or binding sites.

Allosteric Inhibition of PTP1B by a Nonpolar Terpenoid

“…The importance of a flexible α 7 helix in facilitating the binding of AD to its crystallographic site (loc1) and a close neighboring site (loc2) is consistent with previous kinetic measurements, which show that the removal of α 7 reduces the potency of AD. 35…”

“…The enzyme and inhibitor were incubated together for 5 min before adding the substrate. For each inhibitor, we used previously reported IC 50 s 13,35 to identify the concentrations of each inhibitor that inhibit the wild-type enzyme to a similar extent for both inhibitors (Figure S12). We report kinetic measurements in Table S4.…”

“…Motivated by the paucity of well-characterized allosteric inhibitors of PTPs, we used an engineered microbial system to search for terpenoids that inhibit PTP1B. 35 We reasoned that nonpolar terpenoids, if inhibitory, would bind outside of the positively charged active site. Indeed, kinetic analyses and X-ray crystallography showed that amorphadiene (AD) can inhibit PTP1B by binding to a hydrophobic declivity formed by a reorganization of its α 7 helix.…”

Recent research progress of biologically active peptides

“…Microorganisms can not only directly ferment to prepare BAPs, but also provide cheap commercial enzymes, creating good conditions for the preparation of BAPs (Figures 5 and 6). 62,[76][77][78][79][80][81] Wang et al used casein as raw material, using the method of combined fermentation of Lactobacillus helveticus and protease, acid protease was determined as the best protease. Under the optimal fermentation conditions, the yield of peptides was increased by 73.84% compared with the use of L. helveticus.…”

“…Compared with SPPS, liquid-phase peptide synthesis (LPPS) may be more inclined to synthesize high crude purity peptides, reduce reagent consumption, be easier to F I G U R E 5 Endogenous or microbial hydrolyzed protein 62 F I G U R E 6 Natural products using microbiological license 76 scale, and simpler detection and impurity separation. But it takes a long time and the physical properties of peptide intermediates are difficult to predict (Figure 9).…”