Genetically Encoded Detection of Biosynthetic Protease Inhibitors.

Proteases are an important class of drug targets that continue to drive inhibitor discovery. These enzymes are prone to resistance mutations, yet their promise for treating viral diseases and other disorders continues to grow. This study develops a general approach for detecting microbially synthesized protease inhibitors and uses it to screen terpenoid pathways for inhibitory compounds. The detection scheme relies on a bacterial two-hybrid (B2H) system that links protease inactivation to the transcription of a swappable reporter gene. This system, which can accomodate multiple biochemical outputs (i.e., luminescence and antibiotic resistance), permitted the facile incorporation of four disease-relevant proteases. A B2H designed to detect the inactivation of the main protease of severe acute respiratory syndrome coronavirus 2 enabled the identification of a terpenoid inhibitor of modest potency. An analysis of multiple pathways that make this terpenoid, however, suggested that its production was necessary but not sufficient to confer a survival advantage in growth-coupled assays. This finding highlights an important challenge associated with the use of genetic selection to search for inhibitors─notably, the influence of pathway toxicity─and underlines the value of including multiple pathways with overlapping product profiles in pathway screens. This study provides a detailed experimental framework for using microbes to screen libraries of biosynthetic pathways for targeted protease inhibitors.

Allosteric Inhibition of PTP1B by a Nonpolar Terpenoid.

Protein tyrosine phosphatases (PTPs) are promising drug targets for treating a wide range of diseases such as diabetes, cancer, and neurological disorders, but their conserved active sites have complicated the design of selective therapeutics. This study examines the allosteric inhibition of PTP1B by amorphadiene (AD), a terpenoid hydrocarbon that is an unusually selective inhibitor. Molecular dynamics (MD) simulations carried out in this study suggest that AD can stably sample multiple neighboring sites on the allosterically influential C-terminus of the catalytic domain. Binding to these sites requires a disordered α7 helix, which stabilizes the PTP1B-AD complex and may contribute to the selectivity of AD for PTP1B over TCPTP. Intriguingly, the binding mode of AD differs from that of the most well-studied allosteric inhibitor of PTP1B. Indeed, biophysical measurements and MD simulations indicate that the two molecules can bind simultaneously. Upon binding, both inhibitors destabilize the α7 helix by disrupting interactions at the α3-α7 interface and prevent the formation of hydrogen bonds that facilitate closure of the catalytically essential WPD loop. These findings indicate that AD is a promising scaffold for building allosteric inhibitors of PTP1B and illustrate, more broadly, how unfunctionalized terpenoids can engage in specific interactions with protein surfaces.

Evolution-Guided Biosynthesis of Terpenoid Inhibitors.

Terpenoids, the largest and most structurally diverse group of natural products, include a striking variety of biologically active compounds, from flavors to medicines. Despite their well-documented biochemical versatility, the evolutionary processes that generate new functional terpenoids are poorly understood and difficult to recapitulate in engineered systems. This study uses a synthetic biochemical objective─a transcriptional system that links the inhibition of protein tyrosine phosphatase 1B (PTP1B), a human drug target, to the expression of a gene for antibiotic resistance in Escherichia coli (E. coli)─to evolve a terpene synthase to produce enzyme inhibitors. Site saturation mutagenesis of poorly conserved residues on γ-humulene synthase (GHS), a promicuous enzyme, yielded mutants that improved fitness (i.e., the antibiotic resistance of E. coli) by reducing GHS toxicity and/or by increasing inhibitor production. Intriguingly, a combination of two mutations enhanced the titer of a minority product─a terpene alcohol that inhibits PTP1B─by over 50-fold, and a comparison of similar mutants enabled the identification of a site where mutations permit efficient hydroxylation. Findings suggest that the plasticity of terpene synthases enables an efficient sampling of structurally distinct starting points for building new functional molecules and provide an experimental framework for exploiting this plasticity in activity-guided screens.

Microbially Guided Discovery and Biosynthesis of Biologically Active Natural Products.

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 4464 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.