SARS-CoV-2 Spike Protein-Derived Cyclic Peptides as Modulators of Spike Interaction with GRP78.

The human glucose-regulated protein GRP78 is a human chaperone that translocactes to the cell surface when cells are under stress. Theoretical studies suggested it could be involved in SARS-CoV-2 virus entry to cells. In this work, we used in vitro surface plasmon resonance-based assays to show that human GRP78 indeed binds to SARS-CoV-2 spike protein. We have designed and synthesised cyclic peptides based on the loop structure of amino acids 480-488 of the SARS-CoV-2 spike protein S1 domain from the Wuhan and Omicron variants and showed that both peptides bind to GRP78. Consistent with the greater infectiousness of the Omicron variant, the Omicron-derived peptide displays slower dissociation from the target protein. Both peptides significantly inhibit the binding of wild-type S1 protein to the human protein GRP78 suggesting that further development of these cyclic peptide motifs may provide a viable route to novel anti-SARS-CoV-2 agents.

Exploring the Limits of Cyanobactin Macrocyclase PatGmac: Cyclization of PawS-Derived Peptide Sunflower Trypsin Inhibitor-1 and Cyclotide Kalata B1.

The subtilisin-like macrocyclase PatGmac is produced by the marine cyanobacterium Prochloron didemni. This enzyme is involved in the last step of the biosynthesis of patellamides, a cyanobactin type of ribosomally expressed and post-translationally modified cyclic peptides. PatGmac recognizes, cleaves, and cyclizes precursor peptides after a specific recognition motif comprised of a C-terminal tail with the sequence motif -AYDG. The result is the native macrocyclic patellamide, which has eight amino acid residues. Macrocyclase activity can be exploited by incorporating that motif in other short linear peptide precursors, which then are formed into head-to-tail cyclized peptides. Here, we explore the possibility of using PatGmac in the cyclization of peptides larger than the patellamides, namely, the PawS-derived peptide sunflower trypsin inhibitor-1 (SFTI-1) and the cyclotide kalata B1. These peptides fall under two distinct families of disulfide constrained macrocyclic plant peptides. They are both implicated as scaffolds for drug design due to their structures and unusual stability. We show that PatGmac can be used to efficiently cyclize the 14 amino acid residue long SFTI-1, but less so the 29 amino acid residue long kalata B1.

Chemoenzymatic Late-Stage Modifications Enable Downstream Click-Mediated Fluorescent Tagging of Peptides.

Aromatic prenyltransferases from cyanobactin biosynthetic pathways catalyse the chemoselective and regioselective intramolecular transfer of prenyl/geranyl groups from isoprene donors to an electron-rich position in these macrocyclic and linear peptides. These enzymes often demonstrate relaxed substrate specificity and are considered useful biocatalysts for structural diversification of peptides. Herein, we assess the isoprene donor specificity of the N1-tryptophan prenyltransferase AcyF from the anacyclamide A8P pathway using a library of 22 synthetic alkyl pyrophosphate analogues, of which many display reactive groups that are amenable to additional functionalization. We further used AcyF to introduce a reactive moiety into a tryptophan-containing cyclic peptide and subsequently used click chemistry to fluorescently label the enzymatically modified peptide. This chemoenzymatic strategy allows late-stage modification of peptides and is useful for many applications.

A cell-based assay system for activators of the environmental cell stress response.

Improved health span and lifespan extension in a wide phylogenetic range of species is associated with the induction of the environmental cell stress response through a signalling pathway regulated by the transcription factor Nrf2. Phytochemicals which stimulate this response may form part of therapeutic interventions which stimulate endogenous cytoprotective mechanisms, thereby delaying the onset of age-related diseases and promoting healthy ageing in humans. In order to identify compounds that activate the Nrf2 pathway, a cell-based reporter system was established in HepG2 cells using a luciferase reporter gene under the control of the Nqo1 promoter. Sulforaphane, an isothiocyanate derived from cruciferous vegetables and a known activator of the Nrf2 pathway, was used to validate the reporter system. The transfected cell line HepG2 C1 was subsequently used to screen natural product libraries. Five compounds were identified as activating the bioluminescent reporter by greater than 5-fold. The two most potent compounds, MBC20 and MBC37, were further characterised and shown to stimulate endogenous cytoprotective gene and protein expression. The bioluminescent reporter system will allow rapid, in vitro identification of novel compounds that have the potential to improve health span through activation of the environmental stress response.

Structure-Based Design, Synthesis and Bioactivity of a New Anti-TNFα Cyclopeptide.

As opposed to small molecules, macrocyclic peptides possess a large surface area and are recognised as promising candidates to selectively treat diseases by disrupting specific protein-protein interactions (PPIs). Due to the difficulty in predicting cyclopeptide conformations in solution, the de novo design of bioactive cyclopeptides remains significantly challenging. In this study, we used the combination of conformational analyses and molecular docking studies to design a new cyclopeptide inhibitor of the interaction between the human tumour necrosis factor alpha (TNFα) and its receptor TNFR-1. This interaction is a key in mediating the inflammatory response to tissue injury and infection in humans, and it is also an important causative factor of rheumatoid arthritis, psoriasis and inflammatory bowel disease. The solution state NMR structure of the cyclopeptide was determined, which helped to deduce its mode of interaction with TNFα. TNFα sensor cells were used to evaluate the biological activity of the peptide.

N-Prenylation of Tryptophan by an Aromatic Prenyltransferase from the Cyanobactin Biosynthetic Pathway.

Aromatic prenylation is an important step in the biosynthesis of many natural products and leads to an astonishing diversity of chemical structures. Cyanobactin pathways frequently encode aromatic prenyltransferases that catalyze the prenylation of these macrocyclic and linear peptides. Here we characterized the anacyclamide ( acy) biosynthetic gene cluster from Anabaena sp. UHCC-0232. Partial reconstitution of the anacyclamide pathway, heterologous expression, and in vitro biochemical characterization demonstrate that the AcyF enzyme, encoded in the acy biosynthetic gene cluster, is a Trp N-prenyltransferase. Bioinformatic analysis suggests the monophyletic origin and rapid diversification of cyanobactin prenyltransferase enzymes and the multiple origins of N-1 Trp prenylation in prenylated natural products. The AcyF enzyme displayed high flexibility toward a range of Trp-containing substrates and represents an interesting new tool for biocatalytic applications.

Identification of Spongionella compounds as cyclosporine A mimics.

Marine sponges are found to be a wide source of bioactive compounds with different effects such as anti-inflammatory or anticancer actions among others. Cyclophilin A (Cyp A) is a target protein implicated in the mechanism of action of immunosuppressive compounds such as Cyclosporine A (CsA). In the present paper we studied the binding between 4 Spongionella compounds (Gracilins H, A, L and Tetrahydroaplysulphurin-1) and Cyp A immobilized over a CM5 sensor chip. Thus, we found that Spongionella compounds showed to have similar binding affinities than CsA with dissociation equilibrium constant in the range. Next, the effect of these Spongionella isolated compounds was tested over calcineurin phosphatase activity. The same than CsA, Gracilin H, A and Tetrahydroaplysulphurin-1 were able to inhibit phosphatase activity once the complex between Cyp A-CsA/Spongionella compounds was formed. The ability to avoid the dephosphorylation of NFATc1 was also checked in human T cells isolated from peripheral blood. First, cells were pre-treated with Spongionella compounds or CsA following by Concanavalin A (Con A) stimulation. In these conditions nuclear NFATc1 levels were diminished either by CsA or Gracilin A, L, and Tetrahydroaplysulphurin-1 treatment. Moreover, as happens with CsA due to the inhibition of NFATc1, Interleukine-2 (IL-2) released to the culture medium was significantly decreased with all Spongionella compounds. Results conclude that, Spongionella derivatives preserve T lymphocytes from activation modulating the same pathway than CsA. Thus, this mechanism of action suggests that these compounds could be interesting candidates in drug development as immunosuppressive or anti-inflammatory drugs.

The indole alkaloid meleagrin, from the olive tree endophytic fungus Penicillium chrysogenum, as a novel lead for the control of c-Met-dependent breast cancer proliferation, migration and invasion.

Fungi of the genus Penicillium produce unique and chemically diverse biologically active secondary metabolites, including indole alkaloids. The role of dysregulated hepatocyte growth factor (HGF) and its receptor, c-Met, in the development and progression of breast carcinoma is documented. The goal of this work is to explore the chemistry and bioactivity of the secondary metabolites of the endophytic Penicillium chrysogenum cultured from the leaf of the olive tree Olea europea, collected in its natural habitat in Egypt. This fungal extract showed good inhibitory activities against the proliferation and migration of several human breast cancer lines. The CH2Cl2 extract of P. chrysogenum mycelia was subjected to bioguided chromatographic separation to afford three known indole alkaloids; meleagrin (1), roquefortine C (2) and DHTD (3). Meleagrin inhibited the growth of the human breast cancer cell lines MDA-MB-231, MDA-468, BT-474, SK BR-3, MCF7 and MCF7-dox, while similar treatment doses were found to have no effect on the growth and viability of the non-tumorigenic human mammary epithelial cells MCF10A. Meleagrin also showed excellent ATP competitive c-Met inhibitory activity in Z-Lyte assay, which was further confirmed via molecular docking studies and Western blot analysis. In addition, meleagrin treatment caused a dose-dependent inhibition of HGF-induced cell migration, and invasion of breast cancer cell lines. Meleagrin treatment potently suppressed the invasive triple negative breast tumor cell growth in an orthotopic athymic nude mice model, promoting this unique natural product from hit to a lead rank. The indole alkaloid meleagrin is a novel lead c-Met inhibitory entity useful for the control of c-Met-dependent metastatic and invasive breast malignancies.

Accurate quantification of modified cyclic peptides without the need for authentic standards.

There is a growing interest in the use of cyclic peptides as therapeutics, but their efficient production is often the bottleneck in taking them forward in the development pipeline. We have recently developed a method to synthesise azole-containing cyclic peptides using enzymes derived from different cyanobactin biosynthetic pathways. Accurate quantification is crucial for calculation of the reaction yield and for the downstream biological testing of the products. In this study, we demonstrate the development and validation of two methods to accurately quantify these compounds in the reaction mixture and after purification. The first method involves the use of a HPLC coupled in parallel to an ESMS and an ICPMS, hence correlating the calculated sulfur content to the amount of cyclic peptide. The second method is an NMR ERETIC method for quantifying the solution concentration of cyclic peptides. These methods make the quantification of new compounds much easier as there is no need for the use of authentic standards when they are not available.

Spongionella Secondary Metabolites Regulate Store Operated Calcium Entry Modulating Mitochondrial Functioning in SH-SY5Y Neuroblastoma Cells.

BACKGROUND/AIMS: The effect of four secondary metabolites isolated from sponge Spongionella, gracilins H, A, L and tetrahydroaplysulphurin-1 on Calcium ion (Ca2+) fluxes were studied in SH-SY5Y neuroblastoma cells. METHODS AND RESULTS: These compounds did not modify cytosolic baseline Ca2+-levels. Nevertheless, when cytosolic Ca2+-influx through store operated calcium channels (SOC channels) was stimulated with Thapsigargin (Tg), a strong inhibition was observed in the presence of gracilin A, gracilin L and tetrahydroaplysulphurin-1. Since these compounds were able to protect mitochondria from oxidative stress, the role of this organelle in the Ca2+-influx inhibition was tested. In this sense, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and Cyclosporine A (CsA) were used. Surprisingly, both the inhibitory effect over Tg-sensitive stores and Ca2+ influx through SOC channels produced by FCCP were abolished with different potencies by Spongionella compounds in a similar way than CsA. CsA is able to avoid Mitochondrial Permeability Transition Pore (mPTP) opening. As well as CsA, Spongionella compounds reverted mPTP opening induced by FCCP. In the case of CsA the mPTP blockade is due to the direct binding to Cyclophilin D (Cyp D), a mitochondrial matrix protein. This association was also observed between gracilin L and tetrahydroaplysulphurin-1 and Cyp D. Therefore, Spongionella compounds modulate mitochondrial activity by preventing mPTP opening by binding to Cyp D. CONCLUSIONS: These effects make Spongionella compounds as new family of compounds with promising activity in human diseases where mitochondrial alterations are implicated.

Spongionella secondary metabolites protect mitochondrial function in cortical neurons against oxidative stress.

The marine habitat provides a large number of structurally-diverse bioactive compounds for drug development. Marine sponges have been studied over many years and are found to be a rich source of these bioactive chemicals. This study is focused on the evaluation of the activity of six diterpene derivatives isolated from Spongionella sp. on mitochondrial function using an oxidative in vitro stress model. The test compounds include the Gracilins (A, H, K, J and L) and tetrahydroaplysulphurin-1. Compounds were co-incubated with hydrogen peroxide for 12 hours to determine their protective capacities and their effect on markers of apoptosis and Nrf2/ARE pathways was evaluated. Results conclude that Gracilins preserve neurons against oxidative damage, and that in particular, tetrahydroaplysulphurin-1 shows a complete neuroprotective activity. Oxidative stress is linked to mitochondrial dysfunction and consequently to neurodegenerative disorders like Parkinson and Alzheimer diseases, Friedreich ataxia or Amyotrophic lateral sclerosis. This neuroprotection against oxidation conditions suggest that these metabolites could be interesting lead candidates in drug development for neurodegenerative diseases.

An efficient method for the in vitro production of azol(in)e-based cyclic peptides.

Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine-derived enzymes and substrates obtained from a family of ribosomally produced and post-translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non-native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6-9 residues representing 11 out of the 20 canonical amino acids.

Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS-CoV-2 activity.

The recent COVID-19 pandemic shows the critical need for novel broad spectrum antiviral agents. Scorpion venoms are known to contain highly bioactive peptides, several of which have demonstrated strong antiviral activity against a range of viruses. We have generated the first annotated reference transcriptome for the Androctonus amoreuxi venom gland and used high performance liquid chromatography, transcriptome mining, circular dichroism and mass spectrometric analysis to purify and characterize twelve previously undescribed venom peptides. Selected peptides were tested for binding to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and inhibition of the spike RBD - human angiotensin-converting enzyme 2 (hACE2) interaction using surface plasmon resonance-based assays. Seven peptides showed dose-dependent inhibitory effects, albeit with IC50 in the high micromolar range (117-1202 µM). The most active peptide was synthesized using solid phase peptide synthesis and tested for its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7). On exposure to the synthetic peptide of a human lung cell line infected with replication-competent SARS-CoV-2, we observed an IC50 of 200 nM, which was nearly 600-fold lower than that observed in the RBD - hACE2 binding inhibition assay. Our results show that scorpion venom peptides can inhibit the SARS-CoV-2 replication although unlikely through inhibition of spike RBD - hACE2 interaction as the primary mode of action. Scorpion venom peptides represent excellent scaffolds for design of novel anti-SARS-CoV-2 constrained peptides. Future studies should fully explore their antiviral mode of action as well as the structural dynamics of inhibition of target virus-host interactions.

An enzymatic route to selenazolines.

Ringing the changes: Selenazolines have applications in medicinal chemistry, but their synthesis is challenging. We report a new convenient and less toxic route to these heterocycles that starts from commercially available selenocysteine. The new route depends on a heterocyclase enzyme that creates oxazolines and thiazolines from serines/threonines and cysteines.

Structure of PatF from Prochloron didemni.

Patellamides are macrocyclic peptides with potent biological effects and are a subset of the cyanobactins. Cyanobactins are natural products that are produced by a series of enzymatic transformations and a common modification is the addition of a prenyl group. Puzzlingly, the pathway for patellamides in Prochloron didemni contains a gene, patF, with homology to prenylases, but patellamides are not themselves prenylated. The structure of the protein PatF was cloned, expressed, purified and determined. Prenylase activity could not be demonstrated for the protein, and examination of the structure revealed changes in side-chain identity at the active site. It is suggested that these changes have inactivated the protein. Attempts to mutate these residues led to unfolded protein.

A Synthetic Cyclized Antimicrobial Peptide with Potent Effects against Drug-Resistant Skin Pathogens.

Dermal infections requiring treatment are usually treated with conventional antibiotics, but the rise of bacterial resistance to first-line antibiotics warrants alternative therapeutics. Here, we report that a backbone-cyclized antimicrobial peptide, CD4-PP, designed from the human host defense peptide LL-37, has strong direct antibacterial effects on antibiotic sensitive as well as resistant-type strains and clinical isolates of common skin pathogens in the low (<2) µM range. In addition, it influences innate immunity in keratinocytes, and treatment with CD4-PP is able to clear bacterial infections in infected keratinocytes. Additionally, CD4-PP treatment significantly reduces the wound area in a lawn of keratinocytes infected with MRSA. In conclusion, CD4-PP has the potential to serve as a future drug treating wounds infected with antibiotic-resistant bacteria.

The discovery of new cyanobactins from Cyanothece PCC 7425 defines a new signature for processing of patellamides.

Cyanobactins, including patellamides, are a group of cyanobacterial post-translationally modified ribosomal cyclic peptides. The final product should in theory be predictable from the sequence of the precursor peptide and the associated tailoring enzymes. Understanding the mechanism and recognition requirements of these enzymes will allow their rational engineering. We have identified three new cyanobactins from a Cyanothece PCC 7425 culture subjected to a heat shock. One of these compounds revealed a novel signature signal for ThcA, the subtilisin-like serine protease that is homologous to the patellamide protease PatA. The crystal structure of the latter and modelling studies allow rationalisation of the recognition determinants for both enzymes, consistent with the ribosomal biosynthetic origin of the new compounds.

Biochemical characterization of a cyanobactin arginine-N-prenylase from the autumnalamide biosynthetic pathway.

Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl-D-Arg-containing autumnalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications.

Diverse metabolic profiles of a Streptomyces strain isolated from a hyper-arid environment.

The metabolic profile of Streptomyces sp. strain C34, isolated from the Chilean hyper-arid Atacama Desert soil, is dependent on the culture media used for its growth. The application of an OSMAC approach on this strain using a range of cultivation media resulted in the isolation and identification of three new compounds from the rare class of 22-membered macrolactone polyketides, named chaxalactins A-C (1-3). In addition, the known compounds deferroxamine E (4), hygromycin A (5), and 5″-dihydrohygromycin A (6) were detected. The isolated compounds were characterized by NMR spectroscopy and accurate mass spectrometric analysis. Compounds 1-3 displayed strong activity against Gram-positive but weak activity Gram-negative strains tested.

Chaxamycins A-D, bioactive ansamycins from a hyper-arid desert Streptomyces sp.

Streptomyces sp. strain C34, isolated from soil collected in the Chilean hyper-arid Atacama Desert, was cultured on different media, resulting in the isolation and identification of four new ansamycin-type polyketides. The organism was selected for chemical investigation on the basis of a genome-mining PCR-based experiment targeting the gene encoding rifamycin-specific 3-amino-5-hydroxybenzoic acid synthetase (AHBA). The isolated compounds were structurally characterized using NMR and MS techniques and named chaxamycins A-D (1-4). Compounds 1-4 were tested for their antibacterial activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 and for their ability to inhibit the intrinsic ATPase activity of the heat shock protein 90 (Hsp90). Chaxamycin D (4), which showed a selective antibacterial activity against S. aureus ATCC 25923, was tested further against a panel of MRSA clinical isolates. In a virtual screening experiment, chaxamycins A-D (1-4) have also been docked into the ATP-binding pocket in the N-terminal domain of the Hsp90, and the observed interactions are discussed.