Case Studies in Molecular Network-Guided Marine Biodiscovery.

In reviewing a selection of recent case studies from our laboratory, we revealed some lessons learned and benefits accrued from the application of mass spectrometry (MS/MS) molecular networking in the field of marine sponge natural products. Molecular networking proved pivotal to our discovery of many new natural products and even new classes of natural product, some of which were opaque to alternate dereplication and prioritization strategies. Case studies included the discovery of: (i) trachycladindoles, an exceptionally rare class of bioactive indole alkaloid previously only known from a single southern Australia sample of Trachycladus laevispirulifer; (ii) dysidealactams, an unprecedented class of sesquiterpene glycinyl-lactam and glycinyl-imide from a Dysidea sp., a sponge genera often discounted as having been exhaustively studied; (iii) cacolides, an unprecedented family of sesterterpene α-methyl-γ-hydroxybutenolides from a Cacospongia sp., all too easily mischaracterized and deprioritized during dereplication as a well-known class of sponge sesterterpene tetronic acids; and (iv) thorectandrins, a new class of indole alkaloid which revealed unexpected insights into the chemical and biological properties of the aplysinopsins, one of the earliest and more extensively reported class of sponge natural products.

Secreted NF-κB suppressive microbial metabolites modulate gut inflammation.

Emerging evidence suggests that microbiome-host crosstalk regulates intestinal immune activity and predisposition to inflammatory bowel disease (IBD). NF-κB is a master regulator of immune function and a validated target for the treatment of IBD. Here, we identify five Clostridium strains that suppress immune-mediated NF-κB activation in epithelial cell lines, PBMCs, and gut epithelial organoids from healthy human subjects and patients with IBD. Cell-free culture supernatant from Clostridium bolteae AHG0001 strain, but not the reference C. bolteae BAA-613 strain, suppresses inflammatory responses and endoplasmic reticulum stress in gut epithelial organoids derived from Winnie mice. The in vivo responses to Clostridium bolteae AHG0001 and BAA-613 mirror the in vitro activity. Thus, using our in vitro screening of bacteria capable of suppressing NF-κB in the context of IBD and using an ex vivo organoid-based approach, we identify a strain capable of alleviating colitis in a relevant pre-clinical animal model of IBD.

New from Old: Thorectandrin Alkaloids in a Southern Australian Marine Sponge, Thorectandra choanoides (CMB-01889).

Thorectandra choanoides (CMB-01889) was prioritized as a source of promising new chemistry from a library of 960 southern Australian marine sponge extracts, using a global natural products social (GNPS) molecular networking approach. The sponge was collected at a depth of 45 m. Chemical fractionation followed by detailed spectroscopic analysis led to the discovery of a new tryptophan-derived alkaloid, thorectandrin A (1), with the GNPS cluster revealing a halo of related alkaloids 1a-1n. In considering biosynthetic origins, we propose that Thorectandrachoanoides (CMB-01889) produces four well-known alkaloids, 6-bromo-1',8-dihydroaplysinopsin (2), 6-bromoaplysinopsin (3), aplysinopsin (4), and 1',8-dihydroaplysinopsin (10), all of which are susceptible to processing by a putative indoleamine 2,3-dioxygenase-like (IDO) enzyme to 1a-1n. Where the 1',8-dihydroalkaloids 2 and 10 are fully transformed to stable ring-opened thorectandrins 1 and 1a-1b, and 1h-1j, respectively, the conjugated precursors 3 and 4 are transformed to highly reactive Michael acceptors that during extraction and handling undergo complete transformation to artifacts 1c-1g, and 1k-1n, respectively. Knowledge of the susceptibility of aplysinopsins as substrates for IDOs, and the relative reactivity of Michael acceptor transformation products, informs our understanding of the pharmaceutical potential of this vintage marine pharmacophore. For example, the cancer tissue specificity of IDOs could be exploited for an immunotherapeutic response, with aplysinopsins transforming in situ to Michael acceptor thorectandrins, which covalently bind and inhibit the enzyme.

Dysidealactams and Dysidealactones: Sesquiterpene Glycinyl-Lactams, Imides, and Lactones from a Dysidea sp. Marine Sponge Collected in Southern Australia.

A GNPS molecular networking approach mapped a library of 960 southern Australian marine sponges and prioritized Dysidea sp. (CMB-01171) for chemical investigation. Although the published natural products literature on Australian Dysidea sponges extends back over half a century and suffers from the perception of being near exhausted, fractionation of Dysidea sp. (CMB-01171) led to the discovery of a family of 10 new biosynthetically and chemically related sesquiterpenes. Detailed spectroscopic analysis guided structure elucidation identified dysidealactams A-F (1-6), dysidealactones A and B (7 and 8), and two solvolysis artifacts, 9 and 10. The dysidealactams A-D (1-4) incorporate a rare glycinyl-lactam functionality, while dysidealactam E (5) is particularly noteworthy in incorporating an unprecedented glycinyl-imide moiety. In addition to expanding knowledge of Dysidea natural products, this study demonstrates the value of applying GNPS molecular networking to map chemical diversity and prioritize the selection of marine sponge extracts for more detailed chemical analysis.

Solvolysis Artifacts: Leucettazoles as Cryptic Macrocyclic Alkaloid Dimers from a Southern Australian Marine Sponge, Leucetta sp.

Keywords: ethanolysis; solvolysis; artifact; leucettazoles; leucettazines; macrocyclic alkaloids; Leucetta; Australian sponge; GNPS.

Cacolides: Sesterterpene Butenolides from a Southern Australian Marine Sponge, Cacospongia sp.

Chemical analysis of a marine sponge, Cacospongia sp. (CMB-03404), obtained during deep sea commercial fishing activities off the southern coast of Australia, yielded an unprecedented family of sesterterpene α-methyl-γ-hydroxybutenolides, cacolides A⁻L (1⁻12), together with biosynthetically related norsesterterpene carboxylic acids, cacolic acids A⁻C (13⁻15). Structures were assigned on the basis of detailed spectroscopic analysis with comparisons to known natural products and biosynthetic considerations. In addition to revealing new chemical diversity, this study provided a valuable platform for comparing and contrasting the capabilities of the traditional dereplication technologies of HPLC-DAD, HPLC-MS and NMR, with those of the emerging HPLC-MS/MS approach known as global natural products social molecular networking (GNPS), as applied to marine sponge sesterterpene tetronic acids.

Screening of Natural Bioactive Metabolites and Investigation of Antioxidant, Antimicrobial, Antihyperglycemic, Neuropharmacological, and Cytotoxicity Potentials of Litsea polyantha Juss. Ethanolic Root Extract.

This study was designed to identify some bioactive phytochemicals from ethanolic extract of roots of Litsea polyantha and to evaluate some of its pharmacological activities. Phytochemical tests indicated the presence of reducing sugar, combined reducing sugar, tannins, flavonoids, alkaloids, terpenoids, and phenol. In the antioxidant assay using 2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging method, the IC50 value was found to be 82.31 µg/mL. Total content of phenolic compounds, flavonoid, and tannin was found to be 152.69 mg GAE/gm, 85.60 mg QE/gm, and 77.22 mg GAE/gm of dry extract, respectively. In disc diffusion antibacterial assay, the extract exhibited highest zone of inhibition up to 12.25 mm against Escherichia coli at the concentration of 500 µg/disc. For brine shrimp lethality bioassay, the extract exhibited LC50 56.082 µg/mL. In in vivo antihyperglycemic activity test by oral glucose tolerance test using Swiss Albino mice at the oral dose of 250 and 500 mg/kg, the extract showed statistically significant antihyperglycemic effect. Finally, in vivo, the extract exhibited the dose dependent CNS depressant effects by reducing the locomotors of Swiss Albino mice which was confirmed through three different neuropharmacological activity tests such as open field, hole cross, and hole board test.