Chlorinated Enyne Fatty Acid Amides from a Marine Cyanobacterium: Discovery of Taveuniamides L-M and Pharmacological Characterization of Taveuniamide F as a GPCR Antagonist with CNR1 Selectivity

Abstract

NMR and MS/MS-based metabolomics of a cyanobacterial extract from Piti Bomb Holes, Guam, indicated the presence of unique enyne-containing halogenated fatty acid amides. We isolated three new compounds of this class, taveuniamides L-N (1-3), along with the previously reported taveuniamide F (4), which was the most abundant analog. The planar structures of the new compounds were established using 1D and 2D NMR as well as mass spectrometry. We established the configuration of this chemical class to be R at C-8 via Mosher's analysis of 4 after reduction of the carboxamide group. Our biological investigations with 4 revealed that the compound binds to the cannabinoid receptor CNR1, acting as an antagonist/inverse agonist in the canonical G-protein signaling pathways. In selectivity profiling against 168 GPCR targets using the β-arrestin functional assay, we found that 4 antagonizes GPR119, NPSR1b, CCR9, CHRM4, GPR120, HTR2A, and GPR103, in addition to CNR1. Interestingly, 4 showed a 6.8-fold selectivity for CNR1 over CNR2. The binding mode of 4 to CNR1 was investigated using docking and molecular dynamics simulations with both natural and unnatural stereoisomers, revealing important CNR1 residues for the interaction and also providing a possible reasoning for the observed CNR1/CNR2 selectivity.

Keywords: CNR1; GPCR; chlorinated enyne; cyanobacteria; fatty acid amides.

Figure 1

Metabolomics discovery of new and known compounds from marine cyanobacterium VPG14-26. (a) Chemical structures of isolated new taveuniamides L-N (1–3) and known taveunimade F (4) [15]. (b) GNPS generated network analysis using MS/MS data of the organic fraction from which 1–4 were purified.

Figure 2

Key 2D NMR correlations for taveuniamide L (1).

Figure 3

Mosher’s (tertiary amide) analysis of the reduced form of taveuniamide F (4). (a) The deshielding effect of the phenyl group of the MTPA on the corresponding side chains of the stereogenic center. The Z-amide configuration is depicted since it is determined to be the more stable isomer based on DFT calculations. (b) ∆δ^S-R defining the configuration of taveuniamide F (4) as R.

Figure 4

Taveuniamide F (4) binding and functional response characterization against CNR1. (a) Competitive radioligand binding assay of 4 to CNR1. Membrane homogenate of rat hematopoietic Chem-1 cells expressing human recombinant CNR1 receptors is incubated with 2 nM [3H]SR141716A for 60 min at 37 °C. Non-specific binding is estimated in the presence of 10 μM CP 55,940 (K_i = 0.025 μM; historical value at Eurofins Panlabs = 0.03 μM). (b) Effect of 4 in CNR1 cAMP functional assay in agonist and antagonist mode. Experiments were carried out as technical duplicates. Data are presented as mean ± SD (n = 2).

Figure 5

GPCR profiling of taveuniamide F (4) using cell-based functional screen (β-arrestin pathway). (a) Heatmap showing the screening of 4 (20 μM) against 168 GPCR targets in agonist (% activation) and antagonist mode (% inhibition). (b) Scatter plot of targets screened at 20 μM of 4. Hits identified in the screen with ≥75% activity or inhibitions are labeled. (c) Dose–response curve of 4 against GPCR hits identified in the PathHunter β-arrestin primary screen. (d) Dose–response curve of 4 against CNR1 and CNR2. The experiments were carried out as technical duplicates. Data are presented as mean ± SD (n = 2).

Figure 6

Molecular modeling of taveuniamide F enantiomers into CNR1. (a) Docking poses obtained for (R)-(left) and (S)-taveuniamide F (right) stereoisomers using Induced Fit Docking with GlideSP precision. Yellow lines are hydrogen bonds, orange are halogen bonds, and purple are salt bridges. (b) Protein–ligand interactions during 100 ns MD, for (R)-(left) and (S)-taveuniamide F (right). In the frequency plots, the bar height represents the fraction of the simulated time in which the specific interaction was present. Green bars are hydrogen bonds, purple are hydrophobic contacts, and blue are water-bridged interactions.