The Kavaratamides: Discovery of Linear Lipodepsipeptides from the Marine Cyanobacterium Moorena bouillonii Using a Comparative Chemogeographic Analysis

Kavaratamide A (1), a new linear lipodepsipeptide possessing an unusual isopropyl-O-methylpyrrolinone moiety, was discovered from the tropical marine filamentous cyanobacterium Moorena bouillonii collected from Kavaratti, India. A comparative chemogeographic analysis of M. bouillonii collected from six different geographical regions led to the prioritized isolation of this metabolite from India as distinctive among our data sets. AI-based structure annotation tools, including SMART 2.1 and DeepSAT, accelerated the structure elucidation by providing useful structural clues, and the full planar structure was elucidated based on comprehensive HRMS, MS/MS fragmentation, and NMR data interpretation. Subsequently, the absolute configuration of 1 was determined using advanced Marfey’s analysis, modified Mosher’s ester derivatization, and chiral-phase HPLC. The structures of kavaratamides B (2) and C (3) are proposed based on a detailed analysis of their MS/MS fragmentations. The biological activity of kavaratamide A was also investigated and found to show moderate cytotoxicity to the D283-medullablastoma cell line.

Fresh HMI-9 medium (99 µL/well) was added to the assay plate.Parasites in the exponential phase were suspended at 2 × 10 5 parasites/mL in HMI-9 medium and added to each well (100 µL) to a total density of 2 × 10 4 trypanosomes/well.Assay plates were incubated at 37 °C and 5% CO 2 for 70 h, followed by the addition of 20 µL/well of resazurin 0.5 mM (Faria et al., 2015).The plates were incubated for an additional 2h and fluorescence was measured at 535 nm and 590 nm excitation and emission wavelengths, respectively, using a 2104 EnVision® multilabel plate reader (PerkinElmer, Waltham, MA).The viability of the parasites was normalized to positive and negative controls in each assay plate.The screening was performed in technical quadruplicate, and pentamidine at a fixed concentration of 4 µM was employed as a positive drug control.

Methods S2. Enzymatic assays of T. brucei cathepsin L (TbrCATL) and T. cruzi cruzain (CRZ)
The recombinant forms of TbrCATL and cruzain were expressed and purified as previously described by Caffrey et al. (2001) and Silva et al. (2019), respectively.Proteolytic activity was measured by monitoring the cleavage of the fluorogenic substrate Z-Phe-Argaminomethylcoumarin (Z-FRAMC), in a Synergy HTX (Biotek) fluorimeter.All assays were performed in a 384-well black plate format, at a final volume of 30 µL, in a buffer solution of 0.1 M sodium acetate, pH 5.5, containing 1 mM dithiothreitol, 0.01% Triton X-100, 0.5 nM enzyme, and 2.5 µM substrate.The assay was performed after a 10 min pre-incubation of 10 µM kavaratamide A (1) with the enzymes.Initial rates of substrate hydrolysis were calculated relative to a DMSO control.For each assay, two independent experiments were performed, each in triplicate.Trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), at 1 µM, was used as an inhibitor control.

Figure S1 .
Figure S1.Map of collection sites of M. bouillonii

Figure S20 .
Figure S20.Kavaratamides MS/MS-based cluster with the sums of the intensities of the precursor ions of each compound used to determine their relative abundance.These sums were used to depict the node sizes.

Table S2 .
Antitrypanosomal activity screening results of kavaratamide A (1) Positive control for T. b. brucei assay; ** Positive control for enzymatic assay.