The temperate marine sponge, Tsitsikamma favus, produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of T. favus sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as T. favus and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo-ortho-quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a Tsitsikamma sponge species, and the first description of distinct chemotypes within a species of the Latrunculiidae family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponges.

Sponges of the Latrunculiidae family produce bioactive pyrroloiminoquinone alkaloids including makaluvamines, discorhabdins, and tsitsikammamines. The aim of this study was to use LC-ESI-MS/MS-driven molecular networking to characterize the pyrroloiminoquinone secondary metabolites produced by six latrunculid species. These are Tsitsikamma favus, Tsitsikamma pedunculata, Cyclacanthia bellae, and Latrunculia apicalis as well as the recently discovered species, Tsitsikamma nguni and Tsitsikamma michaeli. Organic extracts of 43 sponges were analyzed, revealing distinct species-specific chemical profiles. More than 200 known and unknown putative pyrroloiminoquinones and related compounds were detected, including unprecedented makaluvamine-discorhabdin adducts and hydroxylated discorhabdin I derivatives. The chemical profiles of the new species T. nguni closely resembled those of the known T. favus (chemotype I), but with a higher abundance of tsitsikammamines vs. discorhabdins. T. michaeli sponges displayed two distinct chemical profiles, either producing mostly the same discorhabdins as T. favus (chemotype I) or non- or monobrominated, hydroxylated discorhabdins. C. bellae and L. apicalis produced similar pyrroloiminoquinone chemistry to one another, characterized by sulfur-containing discorhabdins and related adducts and oligomers. This study highlights the variability of pyrroloiminoquinone production by latrunculid species, identifies novel isolation targets, and offers fundamental insights into the collision-induced dissociation of pyrroloiminoquinones.

Pyrroloiminoquinones are a group of cytotoxic alkaloids most commonly isolated from marine sponges. Structurally, they are based on a tricyclic pyrrolo[4,3,2-de]quinoline core and encompass marine natural products such as makaluvamines, tsitsikammamines and discorhabdins. These diverse compounds are known to exhibit a broad spectrum of biological activities including anticancer, antiplasmodial, antimicrobial, antifungal and antiviral activities as well as the inhibition of several key cellular enzymes. The resurgence of interest in pyrroloiminoquinones and the convoluted understanding regarding their biological activities have prompted this review. Herein, we provided a concise summary of key findings and recent developments pertaining to their structural diversity, distribution, biogenesis, and their potential as chemical probes for drug development, including a discussion of promising synthetic analogs.

Sponges are important sources of bioactive secondary metabolites. These compounds are frequently synthesized by bacterial symbionts, which may be recruited from the surrounding seawater or transferred to the sponge progeny by the parent. In this study, we investigated the bacterial communities associated with the sponge Tethya rubra Samaai and Gibbons 2005. Sponge specimens were collected from Evans Peak and RIY Banks reefs in Algoa Bay, South Africa and taxonomically identified by spicule analysis and molecular barcoding. Crude chemical extracts generated from individual sponges were profiled by ultraviolet high performance liquid chromatography (UV-HPLC) and subjected to bioactivity assays in mammalian cells. Next-generation sequencing analysis of 16S rRNA gene sequences was used to characterize sponge-associated bacterial communities. T. rubra sponges collected from the two locations were morphologically and genetically indistinguishable. Chemical extracts from sponges collected at RIY banks showed mild inhibition of the metabolic activity of mammalian cells and their UV-HPLC profiles were distinct from those of sponges collected at Evans Peak. Similarly, the bacterial communities associated with sponges from the two locations were distinct with evidence of vertical transmission of symbionts from the sponge parent to its embryos. We conclude that these distinct bacterial communities may be responsible for the differences observed in the chemical profiles of the two Algoa Bay T. rubra Samaai and Gibbons 2005 populations.

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