Marine natural products.

Motobamide (1), a new cyclic peptide containing a C-prenylated cyclotryptophan residue, was isolated from a marine Leptolyngbya sp. cyanobacterium. Its planar structure was established by spectroscopic and MS/MS analyses. The absolute configuration was elucidated based on a combination of chemical degradations, chiral-phase HPLC analyses, spectroscopic analyses, and computational chemistry. Motobamide (1) moderately inhibited the growth of bloodstream forms of Trypanosoma brucei rhodesiense (IC50 2.3 µM). However, it exhibited a weaker cytotoxicity against normal human cells (IC50 55 µM).

Motobamide, an Antitrypanosomal Cyclic Peptide from a Leptolyngbya sp. Marine Cyanobacterium.

ConspectusBiologically active peptides are a major growing class of drugs, but their therapeutic potential is constrained by several limitations including bioavailability and poor pharmacokinetics. The attachment of functional groups like lipids has proven to be a robust and effective strategy for improving their therapeutic potential. Biochemical and bioactivity-guided screening efforts have identified the cyanobactins as a large class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that are modified with lipids. These lipids are attached by the F superfamily of peptide prenyltransferase enzymes that utilize 5-carbon (prenylation) or 10-carbon (geranylation) donors. The chemical structures of various cyanobactins initially showed isoprenoid attachments on Ser, Thr, or Tyr. Biochemical characterization of the F prenyltransferases from the corresponding clusters shows that the different enzymes have different acceptor residue specificities but are otherwise remarkably sequence tolerant. Hence, these enzymes are well suited for biotechnological applications. The crystal structure of the Tyr O-prenyltransferase PagF reveals that the F enzyme shares a domain architecture reminiscent of a canonical ABBA prenyltransferase fold but lacks secondary structural elements necessary to form an enclosed active site. Binding of either cyclic or linear peptides is sufficient to close the active site to allow for productive catalysis, explaining why these enzymes cannot use isolated amino acids as substrates.Almost all characterized isoprenylated cyanobactins are modified with 5-carbon isoprenoids. However, chemical characterization demonstrates that the piricyclamides are modified with a 10-carbon geranyl moiety, and in vitro reconstitution of the corresponding PirF shows that the enzyme is a geranyltransferase. Structural analysis of PirF shows an active site nearly identical with that of the PagF prenyltransferase but with a single amino acid substitution. Of note, mutation at this residue in PagF or PirF can completely switch the isoprenoid donor specificity of these enzymes. Recent efforts have resulted in significant expansion of the F family with enzymes identified that can carry out C-prenylations of Trp, N-prenylations of Trp, and bis-N-prenylations of Arg. Additional genome-guided efforts based on the sequence of F enzymes identify linear cyanobactins that are α-N-prenylated and α-C-methylated by a bifunctional prenyltransferase/methyltransferase fusion and a bis-α-N- and α-C-prenylated linear peptide. The discovery of these different classes of prenyltransferases with diverse acceptor residue specificities expands the biosynthetic toolkit for enzymatic prenylation of peptide substrates.In this Account, we review the current knowledge scope of the F family of peptide prenyltransferases, focusing on the biochemical, structure-function, and chemical characterization studies that have been carried out in our laboratories. These enzymes are easily amenable for diversity-oriented synthetic efforts as they can accommodate substrate peptides of diverse sequences and are thus attractive catalysts for use in synthetic biology approaches to generate high-value peptidic therapeutics.

Catalysts for the Enzymatic Lipidation of Peptides.

Natural products derived from marine sponges have exhibited bioactivity and, in some cases, serve as potent quorum sensing inhibitory agents that prevent biofilm formation and attenuate virulence factor expression by pathogenic microorganisms. In this study, the inhibitory activity of the psammaplin-type compounds, psammaplin A (1) and bisaprasin (2), isolated from the marine sponge, Aplysinella rhax, are evaluated in quorum sensing inhibitory assays based on the Pseudomonas aeruginosa PAO1 lasB-gfp(ASV) and rhlA-gfp(ASV) biosensor strains. The results indicate that psammaplin A (1) showed moderate inhibition on lasB-gfp expression, but significantly inhibited the QS-gene promoter, rhlA-gfp, with IC50 values at 14.02 μM and 4.99 μM, respectively. In contrast, bisaprasin (2) displayed significant florescence inhibition in both biosensors, PAO1 lasB-gfp and rhlA-gfp, with IC50 values at 3.53 μM and 2.41 μM, respectively. Preliminary analysis suggested the importance of the bromotyrosine and oxime functionalities for QSI activity in these molecules. In addition, psammaplin A and bisaprasin downregulated elastase expression as determined by the standard enzymatic elastase assay, although greater reduction in elastase production was observed with 1 at 50 μM and 100 μM. Furthermore, the study revealed that bisaprasin (2) reduced biofilm formation in P. aeruginosa.

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Inhibition of the Quorum Sensing System, Elastase Production and Biofilm Formation in Pseudomonas aeruginosa by Psammaplin A and Bisaprasin

The tryptophan connection: cyclic peptide natural products linked <i>via</i> the tryptophan side chain

A new cyclic decapeptide, trikoramide A (1), has been isolated from samples of the marine cyanobacterium Symploca hydnoides, collected from Bintan Island, Indonesia. Trikoramide A (1) is a C-prenylated cyclotryptophan-containing cyanobactin. Its planar structure was deduced by 1D and 2D NMR spectroscopy as well as HR-MS/MS data. In addition, its absolute configuration was determined by Marfey's method and 2D NOESY NMR spectroscopic analysis. Compound 1 possessed cytotoxicity against the MOLT-4 and AML2 cancer cell lines with IC50 values of 4.8 and 8.2 μM, respectively.

Trikoramide A, a Prenylated Cyanobactin from the Marine Cyanobacterium Symploca hydnoides.

Trikoveramides A-C, cyclic depsipeptides from the marine cyanobacterium Symploca hydnoides

A new cyclic depsipeptide, triproamide (1), containing the rare 4-phenylvaline (dolaphenvaline, Dpv) and a β-amino acid, dolamethylleucine (Dml), originally found in dolastatin 16, was isolated from the polar VLC-derived fraction of the extracts prepared from the marine cyanobacterium Symploca hydnoides. Triproamide (1) was isolated along with the known molecule kulokainalide-1 (2), as well as its two new analogues, pemukainalides A (3) and B (4). Their planar structures were elucidated based on extensive NMR and mass spectrometric data. The absolute and relative configurations of the compounds were determined utilizing a combination of Marfey's method, J-based configuration, and chiral-phase HPLC analyses. Kulokainalide-1 (2) and pemukainalide A (3) exhibited cytotoxicity against the MOLT-4 leukemia cell line with IC50 values of 5.9 and 5.6 μM, respectively.

Triproamide and Pemukainalides, Cyclic Depsipeptides from the Marine Cyanobacterium Symploca hydnoides.

Three new cyanobactins, trikoramides B (1)–D (3), have been isolated from the marine cyanobacterium, Symploca hydnoides, following a preliminary bioassay-guided isolation of the two most active polar fractions based on the brine shrimp toxicity assay. These new cyanobactins are new analogues of the previously reported cytotoxic trikoramide A (4) with differences mainly in the C-prenylated cyclotryptophan unit. Their planar structures were elucidated from their 1D and 2D NMR spectral data in combination with the HRMS/MS data. Marfey’s method, 2D NOESY NMR spectroscopic and ECD spectra analyses were used to determine the absolute stereochemistry of trikoramides B (1)–D (3). Trikoramides B (1) and D (3) exhibited cytotoxicity against MOLT-4 acute lymphoblastic leukemia cell line with IC50 values of 5.2 µM and 4.7 µM, respectively. Compounds 1 and 3 were also evaluated for their quorum-sensing inhibitory assay based on the Pseudomonas aeruginosa PAO1 lasB-gfp and rhlA-gfp bioreporter strains. Although trikoramide B (1) exhibited weak quorum-sensing inhibitory activity, the Br-containing trikoramide D (3) exhibited moderate to significant dose-dependent quorum-sensing inhibitory activities against PAO1 lasB-gpf and rhlA-gfp bioreporter strains with IC50 values of 19.6 µM and 7.3 µM, respectively.

Trikoramides B-D, Bioactive Cyanobactins from the Marine Cyanobacterium Symploca hydnoides.

Marine sponges are known to host a complex microbial consortium that is essential to the health and resilience of these benthic invertebrates. These sponge-associated microbes are also an important source of therapeutic agents. The Neptune’s Cup sponge, Cliona patera, once believed to be extinct, was rediscovered off the southern coast of Singapore in 2011. The chance discovery of this sponge presented an opportunity to characterize the prokaryotic community of C. patera. Sponge tissue samples were collected from the inner cup, outer cup and stem of C. patera for 16S rRNA amplicon sequencing. C. patera hosted 5,222 distinct OTUs, spanning 26 bacterial phyla, and 74 bacterial classes. The bacterial phylum Proteobacteria, particularly classes Gammaproteobacteria and Alphaproteobacteria, dominated the sponge microbiome. Interestingly, the prokaryotic community structure differed significantly between the cup and stem of C. patera, suggesting that within C. patera there are distinct microenvironments. Moreover, the cup of C. patera had lower diversity and evenness as compared to the stem. Quorum sensing inhibitory (QSI) activities of selected sponge-associated marine bacteria were evaluated and their organic extracts profiled using the MS-based molecular networking platform. Of the 110 distinct marine bacterial strains isolated from sponge samples using culture-dependent methods, about 30% showed quorum sensing inhibitory activity. Preliminary identification of selected QSI active bacterial strains revealed that they belong mostly to classes Alphaproteobacteria and Bacilli. Annotation of the MS/MS molecular networkings of these QSI active organic extracts revealed diverse classes of natural products, including aromatic polyketides, siderophores, pyrrolidine derivatives, indole alkaloids, diketopiperazines, and pyrone derivatives. Moreover, potential novel compounds were detected in several strains as revealed by unique molecular families present in the molecular networks. Further research is required to determine the temporal stability of the microbiome of the host sponge, as well as mining of associated bacteria for novel QS inhibitors.

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Jun Xian Goh

Papers

Trikoramide A, a Prenylated Cyanobactin from the Marine Cyanobacterium Symploca hydnoides.

Trikoveramides A-C, cyclic depsipeptides from the marine cyanobacterium Symploca hydnoides

Triproamide and Pemukainalides, Cyclic Depsipeptides from the Marine Cyanobacterium Symploca hydnoides.

Trikoramides B-D, Bioactive Cyanobactins from the Marine Cyanobacterium Symploca hydnoides.

Assessing the Diversity and Biomedical Potential of Microbes Associated With the Neptune's Cup Sponge, Cliona patera.