When Synthesis Gets It Wrong: Unexpected Epimerization Using PyBOP in the Synthesis of the Cyclic Peptide Thermoactinoamide A.

Chemical synthesis is commonly seen as the final proof of the structure of complex natural products, but even a seemingly easy and well-established synthetic procedure may lead to an unexpected result. This is what happened with the synthesis of thermoactinoamide A (1a), an antimicrobial and antitumor nonribosomal cyclic hexapeptide produced by the thermophilic bacterium Thermoactinomyces vulgaris. The synthetic thermoactinoamide A outsourced to a company and the one described in a synthetic paper showed spectroscopic data identical to each other but different from those of the natural product. After a detailed spectroscopic, degradative, and synthetic study, the synthetic compound was shown to be an epimer (1b) of the intended target compound, originating during the cyclization reaction by extensive epimerization at the activated C-terminal amino acid. This allowed confirmation of the structure of the natural product.

Molecular Networking Revealed Unique UV-Absorbing Phospholipids: Favilipids from the Marine Sponge Clathria faviformis.

Analysis of extracts of the marine sponge Clathria faviformis by high-resolution LC-MS2 and molecular networking resulted in the discovery of a new family of potentially UV-protecting phospholipids, the favilipids. One of them, favilipid A (1), was isolated and its structure determined by mass and tandem mass spectrometry, NMR, electronic circular dichroism (ECD), and computational studies. Favilipid A, which has no close analogues among natural products, possesses an unprecedented structure characterized by a 4-aminodihydropiridinium core, resulting in UV-absorbing properties that are very unusual for a phospholipid. Consequently, favilipid A could inspire the development of a new class of molecules to be used as sunscreen ingredients. In addition, favilipid A inhibited by 58-48% three kinases (JAK3, IKKß, and SYK) involved in the regulation of the immune system, suggesting a potential use for treatment of autoimmune diseases, hematologic cancers, and other inflammatory states.

Toll-like Receptor 4 in Acute Kidney Injury.

Acute kidney injury (AKI) is a common and devastating pathologic condition, associated with considerable high morbidity and mortality. Although significant breakthroughs have been made in recent years, to this day no effective pharmacological therapies for its treatment exist. AKI is known to be connected with intrarenal and systemic inflammation. The innate immune system plays an important role as the first defense response mechanism to tissue injury. Toll-like receptor 4 (TLR4) is a well-characterized pattern recognition receptor, and increasing evidence has shown that TLR4 mediated inflammatory response, plays a pivotal role in the pathogenesis of acute kidney injury. Pathogen-associated molecular patterns (PAMPS), which are the conserved microbial motifs, are sensed by these receptors. Endogenous molecules generated during tissue injury, and labeled as damage-associated molecular pattern molecules (DAMPs), also activate pattern recognition receptors, thereby offering an understanding of sterile types of inflammation. Excessive, uncontrolled and/or sustained activation of TLR4, may lead to a chronic inflammatory state. In this review we describe the role of TLR4, its endogenous ligands and activation in the inflammatory response to ischemic/reperfusion-induced AKI and sepsis-associated AKI. The potential regeneration signaling patterns of TLR4 in acute kidney injury, are also discussed.

Novel Insights in the Potential of Halogenated Polyketide-Peptide Molecules as Lead Compounds in Cancer Drug Discovery.

In this interdisciplinary study, we selected two compounds, namely, smenamide A, a peptide-polyketide, and smenolactone D, a polyketide, as models because they are representative of two different classes of molecules isolated from the marine sponge Smenospongia aurea. The organic extract of Smenospongia aurea was analyzed using a combination of high-resolution LC-MS/MS and molecular networking, a recently developed method for automated LC-MS data analysis. The analyses were targeted to highlight clusters made by chlorinated compounds present in the extracts. Then, the two model compounds were analyzed for their bioactivity. Data reported here show that smenamide A did not exhibit a cytotoxic effect, while smenolactone D was cytotoxic on different tumor cell lines and was able to induce different types of cell death, including ferroptosis and apoptosis.

A Glimpse at Siderophores Production by Anabaena flos-aquae UTEX 1444.

In this study, a strain of Anabaena flos-aquae UTEX 1444 was cultivated in six different concentrations of iron (III). Cultures were extracted with organic solvents and analyzed using our dereplication strategy, based on the combined use of high-resolution tandem mass spectrometry and molecular networking. The analysis showed the presence of the siderophores' family, named synechobactins, only in the zero iron (III) treatment culture. Seven unknown synechobactin variants were present in the extract, and their structures have been determined by a careful HRMS/MS analysis. This study unveils the capability of Anabaena flos-aquae UTEX 1444 to produce a large array of siderophores and may be a suitable model organism for a sustainable scale-up exploitation of such bioactive molecules, for the bioremediation of contaminated ecosystems, as well as in drug discovery.

Computational Metabolomics Tools Reveal Subarmigerides, Unprecedented Linear Peptides from the Marine Sponge Holobiont Callyspongia subarmigera.

A detailed examination of a unique molecular family, restricted to the Callyspongia genus, in a molecular network obtained from an in-house Haplosclerida marine sponge collection (including Haliclona, Callyspongia, Xestospongia, and Petrosia species) led to the discovery of subarmigerides, a series of rare linear peptides from Callyspongia subarmigera, a genus mainly known for polyacetylenes and lipids. The structure of the sole isolated peptide, subarmigeride A (1) was elucidated through extensive 1D and 2D NMR spectroscopy, HRMS/MS, and Marfey's method to assign its absolute configuration. The putative structures of seven additional linear peptides were proposed by an analysis of their respective MS/MS spectra and a comparison of their fragmentation patterns with the heptapeptide 1. Surprisingly, several structurally related analogues of subarmigeride A (1) occurred in one distinct cluster from the molecular network of the cyanobacteria strains of the Guadeloupe mangroves, suggesting that the true producer of this peptide family might be the microbial sponge-associated community, i.e., the sponge-associated cyanobacteria.

Dittrichia graveolens (L.) Greuter, a Rapidly Spreading Invasive Plant: Chemistry and Bioactivity.

Dittrichia graveolens L. Greuter belonging to the Asteraceae family, is an aromatic herbaceous plant native to the Mediterranean region. This plant species has been extensively studied for its biological activities, including antioxidant, antitumor, antimicrobial, antifungal, anti-inflammatory, anticholinesterase, and antityrosinase, and for its peculiar metabolic profile. In particular, bioactivities are related to terpenes and flavonoids metabolites, such as borneol (40), tomentosin (189), inuviscolide (204). However, D. graveolens is also well known for causing health problems both in animals and humans. Moreover, the species is currently undergoing a dramatic northward expansion of its native range related to climate change, now including North Europe, California, and Australia. This review represents an updated overview of the 52 literature papers published in Scopus and PubMed dealing with expansion, chemistry (262 different compounds), pharmacological effects, and toxicology of D. graveolens up to October 2021. The review is intended to boost further studies to determine the molecular pathways involved in the observed activities, bioavailability, and clinical studies to explore new potential applications.

Cyanochelins, an Overlooked Class of Widely Distributed Cyanobacterial Siderophores, Discovered by Silent Gene Cluster Awakening.

Cyanobacteria require iron for growth and often inhabit iron-limited habitats, yet only a few siderophores are known to be produced by them. We report that cyanobacterial genomes frequently encode polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) biosynthetic pathways for synthesis of lipopeptides featuring ß-hydroxyaspartate (ß-OH-Asp), a residue known to be involved in iron chelation. Iron starvation triggered the synthesis of ß-OH-Asp lipopeptides in the cyanobacteria Rivularia sp. strain PCC 7116, Leptolyngbya sp. strain NIES-3755, and Rubidibacter lacunae strain KORDI 51-2. The induced compounds were confirmed to bind iron by mass spectrometry (MS) and were capable of Fe3+ to Fe2+ photoreduction, accompanied by their cleavage, when exposed to sunlight. The siderophore from Rivularia, named cyanochelin A, was structurally characterized by MS and nuclear magnetic resonance (NMR) and found to contain a hydrophobic tail bound to phenolate and oxazole moieties followed by five amino acids, including two modified aspartate residues for iron chelation. Phylogenomic analysis revealed 26 additional cyanochelin-like gene clusters across a broad range of cyanobacterial lineages. Our data suggest that cyanochelins and related compounds are widespread ß-OH-Asp-featuring cyanobacterial siderophores produced by phylogenetically distant species upon iron starvation. Production of photolabile siderophores by phototrophic cyanobacteria raises questions about whether the compounds facilitate iron monopolization by the producer or, rather, provide Fe2+ for the whole microbial community via photoreduction. IMPORTANCE All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited. To face this problem, microorganisms synthesize low-molecular-weight metabolites capable of iron scavenging, i.e., the siderophores. Although cyanobacteria inhabit the majority of the Earth's ecosystems, their repertoire of known siderophores is remarkably poor. Their genomes are known to harbor a rich variety of gene clusters with unknown function. Here, we report the awakening of a widely distributed class of silent gene clusters by iron starvation to yield cyanochelins, ß-hydroxy aspartate lipopeptides involved in iron acquisition. Our results expand the limited arsenal of known cyanobacterial siderophores and propose products with ecological function for a number of previously orphan gene clusters.

Exploring Chemical Diversity of Phorbas Sponges as a Source of Novel Lead Compounds in Drug Discovery.

Porifera, commonly referred to as marine sponges, are acknowledged as major producers of marine natural products (MNPs). Sponges of the genus Phorbas have attracted much attention over the years. They are widespread in all continents, and several structurally unique compounds have been identified from this species. Terpenes, mainly sesterterpenoids, are the major secondary metabolites isolated from Phorbas species, even though several alkaloids and steroids have also been reported. Many of these compounds have presented interesting biological activities. Particularly, Phorbas sponges have been demonstrated to be a source of cytotoxic metabolites. In addition, MNPs exhibiting cytostatic, antimicrobial, and anti-inflammatory activities have been isolated and structurally characterized. This review provides an overview of almost 130 secondary metabolites from Phorbas sponges and their biological activities, and it covers the literature since the first study published in 1993 until November 2021, including approximately 60 records. The synthetic routes to the most interesting compounds are briefly outlined.

Molecular Docking and Biophysical Studies for Antiproliferative Assessment of Synthetic Pyrazolo-Pyrimidinones Tethered with Hydrazide-Hydrazones.

Chemotherapy represents the most applied approach to cancer treatment. Owing to the frequent onset of chemoresistance and tumor relapses, there is an urgent need to discover novel and more effective anticancer drugs. In the search for therapeutic alternatives to treat the cancer disease, a series of hybrid pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered with hydrazide-hydrazones, 5a-h, was synthesized from condensation reaction of pyrazolopyrimidinone-hydrazide 4 with a series of arylaldehydes in ethanol, in acid catalysis. In vitro assessment of antiproliferative effects against MCF-7 breast cancer cells, unveiled that 5a, 5e, 5g, and 5h were the most effective compounds of the series and exerted their cytotoxic activity through apoptosis induction and G0/G1 phase cell-cycle arrest. To explore their mechanism at a molecular level, 5a, 5e, 5g, and 5h were evaluated for their binding interactions with two well-known anticancer targets, namely the epidermal growth factor receptor (EGFR) and the G-quadruplex DNA structures. Molecular docking simulations highlighted high binding affinity of 5a, 5e, 5g, and 5h towards EGFR. Circular dichroism (CD) experiments suggested 5a as a stabilizer agent of the G-quadruplex from the Kirsten ras (KRAS) oncogene promoter. In the light of these findings, we propose the pyrazolo-pyrimidinone scaffold bearing a hydrazide-hydrazone moiety as a lead skeleton for designing novel anticancer compounds.

New Tricks with an Old Sponge: Feature-Based Molecular Networking Led to Fast Identification of New Stylissamide L from Stylissa caribica.

Feature-based molecular networking was used to re-examine the secondary metabolites in extracts of a very well studied marine sponge, Stylissa caribica, known to contain a large array of cyclic peptides and brominated alkaloids. The analysis revealed the presence of 13 cyclic peptides in the sponge that had never been detected in previous work and appeared to be new compounds. The most abundant one was isolated and shown to be a new proline-rich cyclic heptapetide that was called stylissamide L (1). Structure of compound 1, including the cis/trans geometry of the three proline residues, was determined by extensive NMR studies; the l configuration of the seven amino acid residues was determined using Marfey's method. Stylissamide L was tested for activity as a cell growth inhibitor and cell migration inhibitor on two cancer cell lines but, unlike other members of the stylissamide family, it showed no significant activity. This approach showed that even a thoroughly studied species such as S. caribica may contain new chemistry that can be revealed if studied with the right tools.

Identification of Quorum Sensing Activators and Inhibitors in The Marine Sponge Sarcotragus spinosulus.

Marine sponges, a well-documented prolific source of natural products, harbor highly diverse microbial communities. Their extracts were previously shown to contain quorum sensing (QS) signal molecules of the N-acyl homoserine lactone (AHL) type, known to orchestrate bacterial gene regulation. Some bacteria and eukaryotic organisms are known to produce molecules that can interfere with QS signaling, thus affecting microbial genetic regulation and function. In the present study, we established the production of both QS signal molecules as well as QS inhibitory (QSI) molecules in the sponge species Sarcotragus spinosulus. A total of eighteen saturated acyl chain AHLs were identified along with six unsaturated acyl chain AHLs. Bioassay-guided purification led to the isolation of two brominated metabolites with QSI activity. The structures of these compounds were elucidated by comparative spectral analysis of 1HNMR and HR-MS data and were identified as 3-bromo-4-methoxyphenethylamine (1) and 5,6-dibromo-N,N-dimethyltryptamine (2). The QSI activity of compounds 1 and 2 was evaluated using reporter gene assays for long- and short-chain AHL signals (Escherichia coli pSB1075 and E. coli pSB401, respectively). QSI activity was further confirmed by measuring dose-dependent inhibition of proteolytic activity and pyocyanin production in Pseudomonas aeruginosa PAO1. The obtained results show the coexistence of QS and QSI in S. spinosulus, a complex signal network that may mediate the orchestrated function of the microbiome within the sponge holobiont.

Discovery of Unusual Cyanobacterial Tryptophan-Containing Anabaenopeptins by MS/MS-Based Molecular Networking.

Heterocytous cyanobacteria are among the most prolific sources of bioactive secondary metabolites, including anabaenopeptins (APTs). A terrestrial filamentous Brasilonema sp. CT11 collected in Costa Rica bamboo forest as a black mat, was studied using a multidisciplinary approach: genome mining and HPLC-HRMS/MS coupled with bioinformatic analyses. Herein, we report the nearly complete genome consisting of 8.79 Mbp with a GC content of 42.4%. Moreover, we report on three novel tryptophan-containing APTs; anabaenopeptin 788 (1), anabaenopeptin 802 (2), and anabaenopeptin 816 (3). Furthermore, the structure of two homologues, i.e., anabaenopeptin 802 (2a) and anabaenopeptin 802 (2b), was determined by spectroscopic analysis (NMR and MS). Both compounds were shown to exert weak to moderate antiproliferative activity against HeLa cell lines. This study also provides the unique and diverse potential of biosynthetic gene clusters and an assessment of the predicted chemical space yet to be discovered from this genus.

Losartan through Hsp70 Avoids Angiotensin II Induced Mesenchymal Epithelial Transition in Proximal Tubule Cells from Spontaneously Hypertensive Rats.

BACKGROUND/AIMS: Renal injury related to hypertension is characterized by glomerular and tubulointerstitial damage. The overactivation of the renin-angiotensin system mainly by angiotensin II (AII) seems to be a main contributor to progressive renal fibrosis. Epithelial to mesenchymal transition (EMT) is a mechanism that promotes renal fibrosis. Owing to heat shock protein 70 (Hsp70) cytoprotective properties, the chaperone exhibits an important potential as a therapeutic target. We investigate the role of Hsp70 on Angiotensin II induced epithelial mesenchymal transition within the Losartan effect in proximal tubule cells (PTCs) from a genetic model of hypertension in rats (SHR). METHODS: Primary cell culture of PTCs from SHR and Wistar Kyoto (WKY) rats were stimulated with AII, treated with Losartan (L), (L+AII) or untreated (Cc). The functional Hsp70 role in Losartan effect, after silencing its expression by cell transfection, was determined by Immunofluorescence; Western blotting; Gelatin Zymography assays; Scratch wound assays; flow cytometry; and Live Cell Time-lapse microscopy. RESULTS: (L) and (L+AII) treatments induced highly organized actin filaments and increased cortical actin in SHR PTCs. However, SHR PTCs (Cc) and (AII) treated cells showed disorganized actin. After Hsp72 knockdown in SHR PTCs, (L) was unable to stabilize the actin cytoskeleton. We demonstrated that (L) and (L+AII) increased E-cadherin levels and decreased vinculin, α-SMA, vimentin, pERK, p38 and Smad2-3 activation compared to (AII) and (Cc) SHR PTCs. Moreover, (L) inhibited MMP-2 and MMP-9 secretion, reduced migration and cellular displacement, stabilizing intercellular junctions. Notably, (L) treatment in shHsp72 knockdown SHR PTCs showed results similar to SHR PTCs (Cc). CONCLUSION: Our results demonstrate that Losartan through Hsp70 inhibits the EMT induced by AII in proximal tubule cells derived from SHR.

Rab7b participation on the TLR4 (Toll-like receptor) endocytic pathway in Shiga toxin-associated Hemolytic Uremic Syndrome (HUS).

BACKGROUND: The inflammatory response of the host to Shiga toxin and/or lipopolysaccharide (LPS) of Escherichia coli (E. coli) is included in (HUS). The TLR4-LPS complex is internalized and TLR4 induced inflammatory signaling is stopped by targeting the complex for degradation. Rab7b, a small guanosine triphosphatase (GTPase) expressed in monocytes, regulates the later stages of the endocytic pathway. OBJECTIVE: we studied the Rab7b participation on the TLR4 endocytic pathway and its effect on monocyte cytokine production along the acute course of pediatric Shiga toxin-associated HUS. METHODS AND RESULTS: Monocytes were identified according to their positivity in CD14 expression. Surface TLR4 expression in monocytes from 18 HUS patients significantly increased by day 1 to 6, showing the highest increase on day 4 compared to monocytes of 10 healthy children. Significant higher surface TLR4 expression was accompanied by increased proinflammatory intracellular cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In contrast, after these time points, surface TLR4 expression and intracellular TNF-α levels, returned to near control levels after 10 days. Furthermore, confocal immunofluorescence microscopy proved colocalization of increased intracellular TLR4/Rab7b determined by Pearson's coefficient in monocytes from HUS patients from day 1 on the highest colocalization of both proteins by day 4. Decreased TLR4/Rab7b colocalization was shown 10 days after HUS onset. CONCLUSION: The colocalization of TLR4 and Rab7b allows us to suggest Rab7b participation in the control of the TLR4 endocytic pathway in HUS patient monocytes. A consequential fall in cytokine production throughout the early follow up of HUS is demonstrated.

Fast Detection of Two Smenamide Family Members Using Molecular Networking.

Caribbean sponges of the genus Smenospongia are a prolific source of chlorinated secondary metabolites. The use of molecular networking as a powerful dereplication tool revealed in the metabolome of S. aurea two new members of the smenamide family, namely smenamide F (1) and G (2). The structure of smenamide F (1) and G (2) was determined by spectroscopic analysis (NMR, MS, ECD). The relative and the absolute configuration at C-13, C-15, and C-16 was determined on the basis of the conformational rigidity of a 1,3-disubstituted alkyl chain system (i.e., the C-12/C-18 segment of compound (1). Smenamide F (1) and G (2) were shown to exert a selective moderate antiproliferative activity against cancer cell lines MCF-7 and MDA-MB-231, while being inactive against MG-63.

A Collection of Bioactive Nitrogen-Containing Molecules from the Marine Sponge Acanthostrongylophora ingens.

Thirteen nitrogen-containing molecules (1a/1b and 2-12) were isolated from the Indonesian sponge Acanthostrongylophora ingens, highlighting the richness of this organism as a source of alkaloids. Their structures were elucidated using one- and two-dimensional NMR spectroscopy and HR-ESI-MS, while the stereochemistry of the diketopiperazines was established using Marfey's method. All compounds were screened in our standard bioactivity assays, including antibacterial, antikinases, and amyloid ß-42 assays. The most interesting bioactivity result was obtained with the known acanthocyclamine A (3), which revealed for the first time a specific Escherichia coli antimicrobial activity and an inhibitory effect on amyloid ß-42 production induced by aftin-5 and no cytotoxicity at the dose of 26 µM. These results highlight the potentiality of a bipiperidine scaffold as a promising skeleton for preventing or reducing the production of amyloid ß-42, a key player in the initiation of Alzheimer's disease.

Clogging the Ubiquitin-Proteasome Machinery with Marine Natural Products: Last Decade Update.

The ubiquitin-proteasome pathway (UPP) is the central protein degradation system in eukaryotic cells, playing a key role in homeostasis maintenance, through proteolysis of regulatory and misfolded (potentially harmful) proteins. As cancer cells produce proteins inducing cell proliferation and inhibiting cell death pathways, UPP inhibition has been exploited as an anticancer strategy to shift the balance between protein synthesis and degradation towards cell death. Over the last few years, marine invertebrates and microorganisms have shown to be an unexhaustive factory of secondary metabolites targeting the UPP. These chemically intriguing compounds can inspire clinical development of novel antitumor drugs to cope with the incessant outbreak of side effects and resistance mechanisms induced by currently approved proteasome inhibitors (e.g., bortezomib). In this review, we report about (a) the role of the UPP in anticancer therapy, (b) chemical and biological properties of UPP inhibitors from marine sources discovered in the last decade, (c) high-throughput screening techniques for mining natural UPP inhibitors in organic extracts. Moreover, we will tell about the fascinating story of salinosporamide A, the first marine natural product to access clinical trials as a proteasome inhibitor for cancer treatment.

Isolation of Smenopyrone, a Bis-γ-Pyrone Polypropionate from the Caribbean Sponge Smenospongia aurea.

The organic extract of the Caribbean sponge Smenospongia aurea has been shown to contain an array of novel chlorinated secondary metabolites derived from a mixed PKS-NRPS biogenetic route such as the smenamides. In this paper, we report the presence of a biogenetically different compound known as smenopyrone, which is a polypropionate containing two γ-pyrone rings. The structure of smenopyrone including its relative and absolute stereochemistry was determined by spectroscopic analysis (NMR, MS, ECD) and supported by a comparison with model compounds from research studies. Pyrone polypropionates are unprecedented in marine sponges but are commonly found in marine mollusks where their biosynthesis by symbiotic bacteria has been hypothesized and at least in one case demonstrated. Since pyrones have recently been recognized as bacterial signaling molecules, we speculate that smenopyrone could mediate inter-kingdom chemical communication between S. aurea and its symbiotic bacteria.

Smenamide A Analogues. Synthesis and Biological Activity on Multiple Myeloma Cells.

Smenamides are an intriguing class of peptide/polyketide molecules of marine origin showing antiproliferative activity against lung cancer Calu-1 cells at nanomolar concentrations through a clear pro-apoptotic mechanism. To probe the role of the activity-determining structural features, the 16-epi-analogue of smenamide A and eight simplified analogues in the 16-epi series were prepared using a flexible synthetic route. The synthetic analogues were tested on multiple myeloma (MM) cell lines showing that the configuration at C-16 slightly affects the activity, since the 16-epi-derivative is still active at nanomolar concentrations. Interestingly, it was found that the truncated compound 8, mainly composed of the pyrrolinone terminus, was not active, while compound 13, essentially lacking the pyrrolinone moiety, was 1000-fold less active than the intact substance and was the most active among all the synthesized compounds.