Feature-Based Molecular Networking for the Targeted Identification of G_q-Inhibiting FR900359 Derivatives

Abstract

Both the soil bacterium Chromobacterium vaccinii and the bacterial endosymbiont Candidatus Burkholderia crenata of the plant Ardisia crenata are producers of FR900359 (FR). This cyclic depsipeptide is a potent and selective Gq protein inhibitor used extensively to investigate the intracellular signaling of G protein coupled receptors (GPCRs). In this study, the metabolomes of both FR producers were investigated and compared using feature-based molecular networking (FBMN). As a result, 30 previously unknown FR derivatives were identified, one-third being unique to C. vaccinii. Guided by MS, a novel FR derivative, FR-6 (compound 1), was isolated, and its structure unambiguously established. In a whole-cell biosensing assay based on detection of dynamic mass redistribution (DMR) as readout for Gq inhibition, FR-6 suppressed Gq signaling with micromolar potency (pIC50 = 5.56). This functional activity was confirmed in radioligand binding assays (pK i = 7.50). This work demonstrates the power of molecular networking, guiding the way to a novel Gq-inhibiting FR derivative and underlining the potency of FR as a Gq inhibitor.

Promoter‐Driven Overexpression in Chromobacterium vaccinii Facilitates Access to FR900359 and Yields Novel Low Abundance Analogs

“…Analysis of the culture extracts of the C. vaccinii vioP mutant additionally revealed a second peak with an exact mass identical to FR, eluting at a shorter retention time in HPLC (5, Figure 1C). This finding has also been recently reported by Hanke et al., [14] but the authors did not succeed so far in isolation and structure elucidation of this potential FR isomer. As a consequence of higher titers of FR produced by the C. vaccinii vioP mutant, the production of compound 5 is also increased, thereby enabling its isolation and subsequent structure elucidation, along with its analog 6.…”

Targeting Oncogenic Gαq/11 in Uveal Melanoma

“…However, having a method of chemical synthesis provides an avenue to rationally modify these compounds, determine crucial moieties for G q/11 inhibition, and possibly design compounds that may be able to more specifically target the oncogenic mutant Gα q/11 . Some have taken to using biosynthesis methods of creating new YM/FR analogs [ 136 ], while others are utilizing feature-based molecular networking techniques to identify new FR analogs produced by other bacterial strains [ 135 , 137 ]. A recent study using tritium-labelled YM and FR compounds in a high throughput competition binding assay discovered novel G q inhibitors, which inhibited G q signaling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation, albeit with significantly less potency than FR [ 117 ].…”

Heterotrimeric G Protein α-Subunits - Structures, Peptide-Derived Inhibitors, and Mechanisms

“…Additionally, another β-HyLeu residue is attached to the free OH-group of a β-HyLeu residue of the cyclic core structure, which contributes greatly to the binding affinity of FR, and presumably also of YM [127]. In addition to YM and FR, several other derivatives and analogs have been described, which were either obtained by total synthesis [131,[153][154][155], extraction from A. crenata [156], or from recombinant bacterial cultures [127,157]. These derivatives mostly contain an unaltered macrocyclic core structure, but harbor different modifications of the core or the branched β-HyLeu side chain.…”