Unveiling an indole alkaloid diketopiperazine biosynthetic pathway that features a unique stereoisomerase and multifunctional methyltransferase.

The 2,5-diketopiperazines are a prominent class of bioactive molecules. The nocardioazines are actinomycete natural products that feature a pyrroloindoline diketopiperazine scaffold composed of two D-tryptophan residues functionalized by N- and C-methylation, prenylation, and diannulation. Here we identify and characterize the nocardioazine B biosynthetic pathway from marine Nocardiopsis sp. CMB-M0232 by using heterologous biotransformations, in vitro biochemical assays, and macromolecular modeling. Assembly of the cyclo-L-Trp-L-Trp diketopiperazine precursor is catalyzed by a cyclodipeptide synthase. A separate genomic locus encodes tailoring of this precursor and includes an aspartate/glutamate racemase homolog as an unusual D/L isomerase acting upon diketopiperazine substrates, a phytoene synthase-like prenyltransferase as the catalyst of indole alkaloid diketopiperazine prenylation, and a rare dual function methyltransferase as the catalyst of both N- and C-methylation as the final steps of nocardioazine B biosynthesis. The biosynthetic paradigms revealed herein showcase Nature's molecular ingenuity and lay the foundation for diketopiperazine diversification via biocatalytic approaches.

Biomimetic Total Synthesis of (+)-Nocardioazine B and Analogs.

Nocardioazines A and B are prenylated, bioactive pyrroloindoline natural products, isolated from Nocardiopsis, with a desymmetrized cyclo-d-Trp-d-Trp DKP core. Based on our deeper biosynthetic understanding, a biomimetic total synthesis of (+)-nocardioazine B is accomplished in merely seven steps and 23.2% overall yield. This pathway accesses regio- and stereoselectively C3-isoprenylated analogs of (+)-nocardioazine B, using the same number of steps and in similar efficiency. The successful strategy mandated that the biomimetic C3-prenylation step be executed early. The use of an unprotected carboxylic acid of Trp led to high diastereoselectivity toward formation of key intermediates exo-12a, exo-12b, and exo-12c (>19:1). Evidence shows that N1-methylation causes the prenylation reaction to bifurcate away to result in a C2-normal-prenylated isomer. Nocardioazine A, possessing an isoprenoidal-epoxide bridge, inhibits P-glycoprotein (P-gp)-mediated membrane efflux, in multidrug-resistant mammalian colon cancer cells. As several P-gp inhibitors have failed due to their toxicity effects, endogenous amino-acid-derived noncytotoxic inhibitors (from the nocardioazine core) are worthy leads toward a rejuvenated strategy against resistant carcinomas. This total synthesis provides direct access to Trp-derived isoprenylated DKP natural products and their derivatives.

Bioinspired Brønsted Acid-Promoted Regioselective Tryptophan Isoprenylations.

Tryptophan-containing isoprenoid indole alkaloid natural products are well known for their intricate structural architectures and significant biological activities. Nature employs dimethylallyl tryptophan synthases (DMATSs) or aromatic indole prenyltransferases (iPTs) to catalyze regio- and stereoselective prenylation of l-Trp. Regioselective synthetic routes that isoprenylate cyclo-Trp-Trp in a 2,5-diketopiperazine (DKP) core, in a desymmetrizing manner, are nonexistent and are highly desirable. Herein, we present an elaborate report on Brønsted acid-promoted regioselective tryptophan isoprenylation strategy, applicable to both the monomeric amino acid and its dimeric l-Trp DKP. This report outlines a method that regio- and stereoselectively increases sp3 centers of a privileged bioactive core. We report on conditions involving screening of Brønsted acids, their conjugate base as salt, solvent, temperature, and various substrates with diverse side chains. Furthermore, we extensively delineate effects on regio- and stereoselection of isoprenylation and their stereochemical confirmation via NMR experiments. Regioselectively, the C3-position undergoes normal-isoprenylation or benzylation and forms exo-ring-fused pyrroloindolines selectively. Through appropriate prenyl group migrations, we report access to the bioactive tryprostatin alkaloids, and by C3-normal-farnesylation, we access anticancer drimentines as direct targets of this method. The optimized strategy affords iso-tryprostatin B-type products and predrimentine C with 58 and 55% yields, respectively. The current work has several similarities to biosynthesis, such as-reactions can be performed on unprotected substrates, conditions that enable Brønsted acid promotion, and they are easy to perform under ambient conditions, without the need for stoichiometric levels of any transition metal or expensive ligands.

A Virtual Screening Platform Identifies Chloroethylagelastatin A as a Potential Ribosomal Inhibitor.

Chloroethylagelastatin A (CEAA) is an analogue of agelastatin A (AA), a natural alkaloid derived from a marine sponge. It is under development for therapeutic use against brain tumors as it has excellent central nervous system (CNS) penetration and pre-clinical therapeutic activity against brain tumors. Recently, AA was shown to inhibit protein synthesis by binding to the ribosomal A-site. In this study, we developed a novel virtual screening platform to perform a comprehensive screening of various AA analogues showing that AA analogues with proven therapeutic activity including CEAA have significant ribosomal binding capacity whereas therapeutically inactive analogues show poor ribosomal binding and revealing structural fingerprint features essential for drug-ribosome interactions. In particular, CEAA was found to have greater ribosomal binding capacity than AA. Biological tests showed that CEAA binds the ribosome and contributes to protein synthesis inhibition. Our findings suggest that CEAA may possess ribosomal inhibitor activity and that our virtual screening platform may be a useful tool in discovery and development of novel ribosomal inhibitors.

An Isotopic Ratio Outlier Analysis Approach for Global Metabolomics of Biosynthetically Talented Actinomycetes.

Actinomycetes are powerhouses of natural product biosynthesis. Full realization of this biosynthetic potential requires approaches for recognizing novel metabolites and determining mediators of metabolite production. Herein, we develop an isotopic ratio outlier analysis (IROA) ultra-high performance liquid chromatography-mass spectrometry (UHPLC/MS) global metabolomics strategy for actinomycetes that facilitates recognition of novel metabolites and evaluation of production mediators. We demonstrate this approach by determining impacts of the iron chelator 2,2'-bipyridyl on the Nocardiopsis dassonvillei metabolome. Experimental and control cultures produced metabolites with isotopic carbon signatures that were distinct from corresponding "standard" culture metabolites, which were used as internal standards for LC/MS. This provided an isotopic MS peak pair for each metabolite, which revealed the number of carbon atoms and relative concentrations of metabolites and distinguished biosynthetic products from artifacts. Principal component analysis (PCA) and random forest (RF) differentiated bipyridyl-treated samples from controls. RF mean decrease accuracy (MDA) values supported perturbation of metabolites from multiple amino acid pathways and novel natural products. Evaluation of bipyridyl impacts on the nocazine/XR334 diketopiperazine (DKP) pathway revealed upregulation of amino acid precursors and downregulation of late stage intermediates and products. These results establish IROA as a tool in the actinomycete natural product chemistry arsenal and support broad metabolic consequences of bipyridyl.

Correction: The expanding spectrum of diketopiperazine natural product biosynthetic pathways containing cyclodipeptide synthases.

Correction for 'The expanding spectrum of diketopiperazine natural product biosynthetic pathways containing cyclodipeptide synthases' by Paul Borgman et al., Org. Biomol. Chem., 2019, DOI: 10.1039/c8ob03063d.

The expanding spectrum of diketopiperazine natural product biosynthetic pathways containing cyclodipeptide synthases.

Microorganisms are remarkable chemists, with enzymes as their tools for executing multi-step syntheses to yield myriad natural products. Microbial synthetic aptitudes are illustrated by the structurally diverse 2,5-diketopiperazine (DKP) family of bioactive nonribosomal peptide natural products. Nonribosomal peptide synthetases (NRPSs) have long been recognized as catalysts for formation of DKP scaffolds from two amino acid substrates. Cyclodipeptide synthases (CDPSs) are more recently recognized catalysts of DKP assembly, employing two aminoacyl-tRNAs (aa-tRNAs) as substrates. CDPS-encoding genes are typically found in genomic neighbourhoods with genes encoding additional biosynthetic enzymes. These include oxidoreductases, cytochrome P450s, prenyltransferases, methyltransferases, and cyclases, which equip the DKP scaffold with groups that diversify chemical structures and confer biological activity. These tailoring enzymes have been characterized from nine CDPS-containing biosynthetic pathways to date, including four during the last year. In this review, we highlight these nine DKP pathways, emphasizing recently characterized tailoring reactions and connecting new developments to earlier findings. Featured pathways encompass a broad spectrum of chemistry, including the formation of challenging C-C and C-O bonds, regioselective methylation, a unique indole alkaloid DKP prenylation strategy, and unprecedented peptide-nucleobase bond formation. These CDPS-containing pathways also provide intriguing models of metabolic pathway evolution across related and divergent microorganisms, and open doors to synthetic biology approaches for generation of DKP combinatorial libraries. Further, bioinformatics analyses support that much unique genetically encoded DKP tailoring potential remains unexplored, suggesting opportunities for further expansion of Nature's biosynthetic spectrum. Together, recent studies of DKP pathways demonstrate the chemical ingenuity of microorganisms, highlight the wealth of unique enzymology provided by bacterial biosynthetic pathways, and suggest an abundance of untapped biosynthetic potential for future exploration.

Accelerated bottom-up drug design platform enables the discovery of novel stearoyl-CoA desaturase 1 inhibitors for cancer therapy.

Here we present an innovative computational-based drug discovery strategy, coupled with machine-based learning and functional assessment, for the rational design of novel small molecule inhibitors of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1). Our methods resulted in the discovery of several unique molecules, of which our lead compound SSI-4 demonstrates potent anti-tumor activity, with an excellent pharmacokinetic and toxicology profile. We improve upon key characteristics, including chemoinformatics and absorption/distribution/metabolism/excretion (ADME) toxicity, while driving the IC50 to 0.6 nM in some instances. This approach to drug design can be executed in smaller research settings, applied to a wealth of other targets, and paves a path forward for bringing small-batch based drug programs into the Clinic.

Two Distinct Cyclodipeptide Synthases from a Marine Actinomycete Catalyze Biosynthesis of the Same Diketopiperazine Natural Product.

Diketopiperazine natural products are structurally diverse and offer many biological activities. Cyclodipeptide synthases (CDPSs) were recently unveiled as a novel enzyme family that employs aminoacyl-tRNAs as substrates for 2,5-diketopiperazine assembly. Here, the Nocardiopsis sp. CMB-M0232 genome is predicted to encode two CDPSs, NozA and NcdA. Metabolite profiles from E. coli expressing these genes and assays with purified recombinant enzymes revealed that NozA and NcdA catalyze cyclo(l-Trp-l-Trp) (1) biosynthesis from tryptophanyl-tRNA and do not accept other aromatic aminoacyl-tRNA substrates. Fidelity is uncommon among characterized CDPSs, making NozA and NcdA important CDPS family additions. Further, 1 was previously supported as a biosynthetic precursor of the nocardioazines; the current study suggests that Nocardiopsis sp. may derive this precursor from both NozA and NcdA. This study offers a rare example of a single bacterium encoding multiple phylogenetically distinct enzymes that yield the same secondary metabolite and provides tools for chemoenzymatic syntheses of indole alkaloid diketopiperazines.

Correction: Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis.

Correction for 'Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis' by Norah Alqahtani et al., Org. Biomol. Chem., 2015, 13, 7177-7192.

Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis.

Marine actinomycete-derived natural products continue to inspire chemical and biological investigations. Nocardioazines A and B (3 and 4), from Nocardiopsis sp. CMB-M0232, are structurally unique alkaloids featuring a 2,5-diketopiperazine (DKP) core functionalized with indole C3-prenyl as well as indole C3- and N-methyl groups. The logic of their assembly remains cryptic. Bioinformatics analyses of the Nocardiopsis sp. CMB-M0232 draft genome afforded the noz cluster, split across two regions of the genome, and encoding putative open reading frames with roles in nocardioazine biosynthesis, including cyclodipeptide synthase (CDPS), prenyltransferase, methyltransferase, and cytochrome P450 homologs. Heterologous expression of a twelve gene contig from the noz cluster in Streptomyces coelicolor resulted in accumulation of cyclo-l-Trp-l-Trp DKP (5). This experimentally connected the noz cluster to indole alkaloid natural product biosynthesis. Results from bioinformatics analyses of the noz pathway along with challenges in actinomycete genetics prompted us to use asymmetric synthesis and mass spectrometry to determine biosynthetic intermediates in the noz pathway. The structures of hypothesized biosynthetic intermediates 5 and 12-17 were firmly established through chemical synthesis. LC-MS and MS-MS comparison of these synthetic compounds with metabolites present in chemical extracts from Nocardiopsis sp. CMB-M0232 revealed which of these hypothesized intermediates were relevant in the nocardioazine biosynthetic pathway. This established the early and mid-stages of the biosynthetic pathway, demonstrating that Nocardiopsis performs indole C3-methylation prior to indole C3-normal prenylation and indole N1'-methylation in nocardioazine B assembly. These results highlight the utility of merging bioinformatics analyses, asymmetric synthetic approaches, and mass spectrometric metabolite profiling in probing natural product biosynthesis.

Characterization of the nocardiopsin biosynthetic gene cluster reveals similarities to and differences from the rapamycin and FK-506 pathways.

Macrolide-pipecolate natural products, such as rapamycin (1) and FK-506 (2), are renowned modulators of FK506-binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB-M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A-D (4-7) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L-lysine into the L-pipecolic acid incorporated into 4 and 5. Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP-binding metabolites and for probing new enzymology in nature's polyketide tailoring arsenal.

Analysis of glycylsarcosine transport by lobster intestine using gas chromatography.

Gas chromatography was used to measure transepithelial transport of glycylsarcosine (Gly-Sar) by perfused lobster (Homarus americanus) intestine. Unidirectional and net fluxes of dipeptide across the tissue and luminal factors affecting their magnitude and direction were characterized by perfusing the lumen with the dipeptide and measuring its appearance in saline on the serosal side of the organ. Transmural transport of 10 mM Gly-Sar resulted in serosal accumulation of only the dipeptide; no appearance of corresponding monomeric amino acids glycine or sarcosine was observed. Carrier-mediated and diffusional transmural intestinal transport of Gly-Sar was estimated at 1-15 mM luminal concentrations and followed a curvilinear equation providing a K m = 0.44 ± 0.17 mM, a J max = 1.27 ± 0.12 nmol cm(-2) min(-1), and a diffusional coefficient = 0.026 ± 0.008 nmol cm(-2) min(-1) mM(-1). Unidirectional mucosal to serosal and serosal to mucosal fluxes of 10 mM Gly-Sar provided a significant (p < 0.05) net absorptive flux toward the serosa of 3.54 ± 0.77 nmol cm(-2) min(-1), further supporting carrier-mediated dipeptide transport across the gut. Alkaline (pH 8.5) luminal pH more than doubled transmural Gly-Sar transport as compared to acidic (pH 5.5) luminal pH, while luminal amino acid-metal chelates (e.g., Leu-Zn-Leu), and high concentrations of amino acids alone significantly (p < 0.001) reduced intestinal Gly-Sar transfer by inhibiting carrier transport of the dipeptide. Proposed mechanisms accounting for intestinal dipeptide transport and luminal factors affecting this process are discussed.

Characterization of an orphan diterpenoid biosynthetic operon from Salinispora arenicola.

While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe-microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms.

Bioactivity-guided genome mining reveals the lomaiviticin biosynthetic gene cluster in Salinispora tropica.

The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico-chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo- and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora tropica CNB-440 by a DNA interference bioassay to isolate DNA-targeting enediyne polyketides. An organic extract of S. tropica showed DNA-interference activity that surprisingly was not abolished in genetic mutants of the targeted enediyne pathways, ST_pks1 and spo. Instead we showed that the product of the orphan type II polyketide synthase pathway, ST_pks2, is solely responsible for the DNA-interfering activity of the parent strain. Subsequent comparative metabolic profiling revealed the lomaiviticins, glycosylated diazofluorene polyketides, as the ST_pks2 products. This study marks the first report of the 59 open reading frame lomaiviticin gene cluster (lom) and supports the biochemical logic of their dimeric construction through a pathway related to the kinamycin monomer.

Structures and comparative characterization of biosynthetic gene clusters for cyanosporasides, enediyne-derived natural products from marine actinomycetes.

Cyanosporasides are marine bacterial natural products containing a chlorinated cyclopenta[a]indene core of suspected enediyne polyketide biosynthetic origin. Herein, we report the isolation and characterization of novel cyanosporasides C-F (3-6) from the marine actinomycetes Salinispora pacifica CNS-143 and Streptomyces sp. CNT-179, highlighted by the unprecedented C-2' N-acetylcysteamine functionalized hexose group of 6. Cloning, sequencing, and mutagenesis of homologous ~50 kb cyanosporaside biosynthetic gene clusters from both bacteria afforded the first genetic evidence supporting cyanosporaside's enediyne, and thereby p-benzyne biradical, biosynthetic origin and revealed the molecular basis for nitrile and glycosyl functionalization. This study provides new opportunities for bioengineering of enediyne derivatives and expands the structural diversity afforded by enediyne gene clusters.

Optimization of peptide hydroxamate inhibitors of insulin-degrading enzyme reveals marked substrate-selectivity.

Insulin-degrading enzyme (IDE) is an atypical zinc-metallopeptidase that degrades insulin and the amyloid ß-protein and is strongly implicated in the pathogenesis of diabetes and Alzheimer's disease. We recently developed the first effective inhibitors of IDE, peptide hydroxamates that, while highly potent and selective, are relatively large (MW > 740) and difficult to synthesize. We present here a facile synthetic route that yields enantiomerically pure derivatives comparable in potency to the parent compounds. Through the generation of truncated variants, we identified a compound with significantly reduced size (MW = 455.5) that nonetheless retains good potency (ki = 78 ± 11 nM) and selectivity for IDE. Notably, the potency of these inhibitors was found to vary as much as 60-fold in a substrate-specific manner, an unexpected finding for active site-directed inhibitors. Collectively, our findings demonstrate that potent, small-molecule IDE inhibitors can be developed that, in certain instances, can be highly substrate selective.

Ecological leads for natural product discovery: Novel sesquiterpene hydroquinones from the red macroalga Peyssonnelia sp.

Pharmacologically-motivated marine natural product investigations have yielded a large variety of structurally unique compounds with interesting biomedical properties, but the natural roles of these molecules often remain unknown. While secondary metabolites may function as antimicrobial chemical defenses, few studies have examined this hypothesis. In the present investigation, chromatographic fractions from 69 collections of Fijian red macroalgae representing at least 43 species were evaluated for growth inhibition of three microbial pathogens and saprophytes of marine macrophytes. At least one microbe was suppressed by fraction(s) of all evaluated algae, suggesting that antimicrobial defenses are common among tropical seaweeds. From these leads, peyssonoic acids A-B (1-2), novel sesquiterpene hydroquinones, were isolated from the crustose red alga Peyssonnelia sp. At ecologically realistic concentrations, both compounds inhibited growth of Pseudoalteromonas bacteriolytica, a bacterial pathogen of marine algae, and Lindra thalassiae, a fungal pathogen of marine algae, and exhibited modest antineoplastic activity against ovarian cancer cells. The peyssonoic acids included one novel carbon skeleton and illustrated the utility of ecological studies in natural product discovery.

Biochemical warfare on the reef: the role of glutathione transferases in consumer tolerance of dietary prostaglandins.

BACKGROUND: Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. PRINCIPAL FINDINGS: Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A(2) (PGA(2)) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA(2)) were also the most potent inhibitors. In vivo estimates of PGA(2) concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA(2) and operating at or near physiological capacity. SIGNIFICANCE: The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as 'all-purpose' detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.