Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq‐Signaling Inhibitor FR900359

Abstract

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC-1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.

Cell‐permeable high‐affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

“…MD simulation studies of Gα q protein in complex with FR and YM, respectively, indicated that the kinetic differences are largely contributed by the isopropyl (R 2 in Figure b) interaction with Phe75 in the binding pocket. The new tracers will allow to study binding kinetics of other compounds that bind to the same site, for example, novel analogues and derivatives of YM and FR, that are accessible by chemical synthesis (Rensing, Uppal, Blumer, & Moeller, ; Xiong et al, ; Zhang et al, ), and by biotechnological approaches (Crüsemann et al, ; Taniguchi et al, ). The new tracers will help to design and develop G q inhibitors with specific, desired kinetic profiles.…”

“…YM is produced by Chromobacterium sp. (Taniguchi et al, ), while FR was isolated from the plant Ardisia crenata Sims and is produced by the bacterial endophyte Candidatus Burkholderia crenata that is present as a symbiont in the leaves of the plant (Crüsemann et al, ; Fujioka, Koda, & Morimoto, ). A few analogues of FR have also been isolated, however, in tiny amounts (Crüsemann et al, ; Reher et al, ).…”

“…(Taniguchi et al, ), while FR was isolated from the plant Ardisia crenata Sims and is produced by the bacterial endophyte Candidatus Burkholderia crenata that is present as a symbiont in the leaves of the plant (Crüsemann et al, ; Fujioka, Koda, & Morimoto, ). A few analogues of FR have also been isolated, however, in tiny amounts (Crüsemann et al, ; Reher et al, ). Recently, the total syntheses of 1 and 2 and some analogues were described, but they represent labour‐intensive procedures providing only small amounts of the products; all of the synthesized analogues showed moderate potency or were inactive (Xiong et al, ; Zhang et al, ).…”

Recent achievements in developing selective Gq inhibitors

“…Synthetic FR900359 has been pharmacologically characterized on G q ‐, G s ‐, and G i ‐mediated signaling and compared to natural (isolated) FR900359, showing identical biological activity (Figure ). Recently, Crüsemann et al reported the first heterologous biosynthesis of FR900359 in a cultivable, bacterial ( Escherichia coli ) host by expression of the FR nonribosomal peptide synthetase (frs) genes, which were revealed by sequencing the uncultured endosymbiont of A. crenata …”

Early steps in the evolution of vertical transmission revealed by a plant-bacterium symbiosis

“…Burkholderia leaf nodule symbionts show extensive signs of reductive genome evolution, with coding capacities ranging from 41.7 % to 67.3 % and an accumulation of pseudogenes and insertion sequences [10–12]. Despite extensive genome erosion, some symbionts have been shown to produce secondary metabolites, likely involved in the protection of the host from herbivory, such as the insecticidal kirkamide and the depsipeptide FR900359, as well as the herbicidal streptol-glucoside possibly involved in allelopathic interactions [12–14]. Because of genomic instability and evolved co-dependence, it is unclear whether secondary metabolism was present in the ancestor of leaf nodule Burkholderia or acquired as a secondary trait.…”