Substrate Promiscuity of the Triceptide Maturase XncB Leads to Incorporation of Various Amino Acids and Detection of Oxygenated Products.

Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides. The characteristic cross-links are formed between an aromatic ring to Cß on three-residue Ω1X2X3 motifs (Ω1 = aromatic). Here, we explored the promiscuity of the XYE family triceptide maturase, XncB from Xenorhabdus nematophila DSM 3370. Single amino acid variants were coexpressed with XncB in vivo in Escherichia coli, and we show that a variety of amino acids can be incorporated into the Phe-Gly-Asn cyclophane. Aromatic amino acids at the X3 position were accepted by the enzyme but yielded hydroxylated, rather than the typical cyclophane, products. These studies show that oxygen can be inserted but diverges in the final product formed relative to daropeptide maturases. Finally, truncations of the leader peptide showed that it is necessary for complete modification by XncB.

Marine-Derived Anticancer Agents Targeting Apoptotic Pathways: Exploring the Depths for Novel Cancer Therapies.

Extensive research has been conducted on the isolation and study of bioactive compounds derived from marine sources. Several natural products have demonstrated potential as inducers of apoptosis and are currently under investigation in clinical trials. These marine-derived compounds selectively interact with extrinsic and intrinsic apoptotic pathways using a variety of molecular mechanisms, resulting in cell shrinkage, chromatin condensation, cytoplasmic blebs, apoptotic bodies, and phagocytosis by adjacent parenchymal cells, neoplastic cells, or macrophages. Numerous marine-derived compounds are currently undergoing rigorous examination for their potential application in cancer therapy. This review examines a total of 21 marine-derived compounds, along with their synthetic derivatives, sourced from marine organisms such as sponges, corals, tunicates, mollusks, ascidians, algae, cyanobacteria, fungi, and actinobacteria. These compounds are currently undergoing preclinical and clinical trials to evaluate their potential as apoptosis inducers for the treatment of different types of cancer. This review further examined the compound's properties and mode of action, preclinical investigations, clinical trial studies on single or combination therapy, and the prospective development of marine-derived anticancer therapies.

Functional and Promiscuity Studies of Three-Residue Cyclophane Forming Enzymes Show Nonnative C-C Cross-Linked Products and Leader-Dependent Cyclization.

Enzymes catalyzing peptide macrocyclization are important biochemical tools in drug discovery. The three-residue cyclophane-forming enzymes (3-CyFEs) are an emerging family of post-translational modifying enzymes that catalyze the formation of three-residue peptide cyclophanes. In this report, we introduce three additional 3-CyFEs, including ChlB, WnsB, and FnnB, that catalyze cyclophane formation on Tyr, Trp, and Phe, respectively. To understand the promiscuity of these enzymes and those previously reported (MscB, HaaB, and YxdB), we tested single amino acid substitutions at the three-residue motif of modification (Ω1X2X3, Ω1 = aromatic). Collectively, we observe that substrate promiscuity is observed at the Ω1 and X2 positions, but a greater specificity is observed for the X3 residue. Two nonnative cyclophane products were characterized showing a Phe-C3 to Arg-Cß and His-C2 to Pro-Cß cross-links, respectively. We also tested the leader dependence of selected 3-CyFEs and show that a predicted helix region is important for cyclophane formation. These results demonstrate the biocatalytic potential of these maturases and allow rational design of substrates to obtain a diverse array of genetically encoded 3-residue cyclophanes.

The Biosynthetic Landscape of Triceptides Reveals Radical SAM Enzymes That Catalyze Cyclophane Formation on Tyr- and His-Containing Motifs.

Peptide-derived cyclophanes inhabit a unique niche in the chemical space of macrocyclic peptides with several examples of pharmaceutical importance. Although both synthetic and biocatalytic methods are available for constructing these macrocycles, versatile (bio)catalysts able to incorporate a variety of amino acids that compose the macrocycle would be useful for the creation of diverse peptide cyclophanes. In this report, we synergized the use of bioinformatic tools to map the biosynthetic landscape of radical SAM enzymes (3-CyFEs) that catalyze three-residue cyclophane formation in the biosynthesis of a new family of RiPP natural products, the triceptides. This analysis revealed 3940 (3113 unique) putative precursor sequences predicted to be modified by 3-CyFEs. Several uncharacterized maturase systems were identified that encode unique precursor types. Functional studies were carried out in vivo in Escherichia coli to identify modified precursors containing His and Tyr residues. NMR analysis of the products revealed that Tyr and His can also be incorporated into cyclophane macrocycles by 3-CyFEs. Collectively, all aromatic amino acids can be incorporated by 3-CyFEs, and the cyclophane formation strictly occurs via a C(sp2)-C(sp3) cross-link between the (hetero)aromatic ring to Cß. In addition to 3-CyFEs, we functionally validated an Fe(II)/α-ketoglutarate-dependent hydroxylase, resulting in ß-hydroxylated residues within the cyclophane rings. This study reveals the potential breadth of triceptide precursors and a systematic approach for studying these enzymes to broaden the diversity of peptide macrocycles.

The Isorhamnetin-Containing Fraction of Philippine Honey Produced by the Stingless Bee Tetragonula biroi Is an Antibiotic against Multidrug-Resistant Staphylococcus aureus.

Honey exhibits antibacterial and antioxidant activities that are ascribed to its diverse secondary metabolites. In the Philippines, the antibacterial and antioxidant activities, as well as the bioactive metabolite contents of the honey, have not been thoroughly described. In this report, we investigated the in vitro antibacterial and antioxidant activities of honey from Apis mellifera and Tetragonula biroi, identified the compound responsible for the antibacterial activity, and compared the observed bioactivities and metabolite profiles to that of Manuka honey, which is recognized for its antibacterial and antioxidant properties. The secondary metabolite contents of honey were extracted using a nonionic polymeric resin followed by antibacterial and antioxidant assays, and then spectroscopic analyses of the phenolic and flavonoid contents. Results showed that honey extracts produced by T. biroi exhibits antibiotic activity against Staphylococcal pathogens as well as high antioxidant activity, which are correlated to its high flavonoid and phenolic content as compared to honey produced by A. mellifera. The bioassay-guided fractionation paired with Liquid Chromatography Mass Spectrometry (LCMS) and tandem MS analyses found the presence of the flavonoid isorhamnetin (3-methylquercetin) in T. biroi honey extract, which was demonstrated as one of the compounds with inhibitory activity against multidrug-resistant Staphylococcus aureus ATCC BAA-44. Our findings suggest that Philippine honey produced by T. biroi is a potential nutraceutical that possesses antibiotic and antioxidant activities.

Anthracycline Shunt Metabolites From Philippine Marine Sediment-Derived Streptomyces Destroy Cell Membrane Integrity of Multidrug-Resistant Staphylococcus aureus.

The rise of antibiotic resistance (ABR) and the drying up of the pipeline for the development of new antibiotics demands an urgent search for new antibiotic leads. While the majority of clinically available antibiotics were discovered from terrestrial Streptomyces, related species from marine sediments as a source of antibiotics remain underexplored. Here, we utilized culture-dependent isolation of thirty-five marine sediment-derived actinobacterial isolates followed by a screening of their antibacterial activity against multidrug-resistant S. aureus ATCC BAA-44. Our results revealed that the crude extract of Streptomyces griseorubens strain DSD069 isolated from marine sediments collected in Romblon, Philippines displays the highest antibacterial activity, with 96.4% growth inhibition. The S. aureus ATCC BAA-44 cells treated with crude extract of Streptomyces griseorubens strain DSD069 showed cell membrane damage as demonstrated by (a) leakage and loss of vital cell constituents, including DNA and proteins, (b) irregular shrinkage of cells, and (c) increase membrane permeability. The antibiotic compounds were identified as Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone with MIC value of 6.25 µg/mL and 50.00 µg/mL, respectively. Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone are shunt metabolites in the biosynthesis of anticancer anthracycline derivatives namely doxorubicin, daunorubicin, and cinerubins. It is rare, however, that shunt metabolites are accumulated during fermentation of marine sediment-derived Streptomyces strain without genetic modification. Thus, our study provides evidence that natural bacterial strain can produce Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone as antibiotic leads to combat ABR.