Discovery and development of macrocyclic peptide modulators of the cannabinoid 2 receptor.

The cannabinoid-type 2 receptor (CB2R), a G protein-coupled receptor (GPCR), is an important regulator of immune cell function and a promising target to treat chronic inflammation and fibrosis. While CB2R is typically targeted by small molecules, including endo-, phyto- and synthetic cannabinoids, peptides - owing to their size - may offer a different interaction space to facilitate differential interactions with the receptor. Here we explore plant-derived cyclic cystine-knot peptides as ligands of the CB2R. Cyclotides are known for their exceptional biochemical stability. Recently they gained attention as GPCR modulators and as templates for designing peptide ligands with improved pharmacokinetic properties over linear peptides. Cyclotide-based ligands for CB2R were profiled based on a peptide-enriched extract library comprising nine plants. Employing pharmacology-guided fractionation and peptidomics we identified cyclotide vodo-C1 from sweet violet (Viola odorata) as a full agonist of CB2R with an affinity (Ki) of 1µM and a potency (EC50) of 8µM. Leveraging deep learning networks we verified the structural topology of vodo-C1 and modelled its molecular volume in comparison to the CB2R ligand binding pocket. In a fragment-based approach we designed and characterized vodo-C1-based bicyclic peptides (vBCL1-4), aiming to reduce size and improve potency. Opposite to vodo-C1, the vBCL peptides lacked the ability to activate the receptor but acted as negative allosteric modulators or neutral antagonists of CB2R. This study introduces a macrocyclic peptide phytocannabinoid, which served as template for the development of synthetic CB2R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors.

Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor.

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-ß-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-ß-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-ß-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.

Peptidomics.

Peptides are biopolymers, typically consisting of 2-50 amino acids. They are biologically produced by the cellular ribosomal machinery or by non-ribosomal enzymes and, sometimes, other dedicated ligases. Peptides are arranged as linear chains or cycles, and include post-translational modifications, unusual amino acids and stabilizing motifs. Their structure and molecular size render them a unique chemical space, between small molecules and larger proteins. Peptides have important physiological functions as intrinsic signalling molecules, such as neuropeptides and peptide hormones, for cellular or interspecies communication, as toxins to catch prey or as defence molecules to fend off enemies and microorganisms. Clinically, they are gaining popularity as biomarkers or innovative therapeutics; to date there are more than 60 peptide drugs approved and more than 150 in clinical development. The emerging field of peptidomics comprises the comprehensive qualitative and quantitative analysis of the suite of peptides in a biological sample (endogenously produced, or exogenously administered as drugs). Peptidomics employs techniques of genomics, modern proteomics, state-of-the-art analytical chemistry and innovative computational biology, with a specialized set of tools. The complex biological matrices and often low abundance of analytes typically examined in peptidomics experiments require optimized sample preparation and isolation, including in silico analysis. This Primer covers the combination of techniques and workflows needed for peptide discovery and characterization and provides an overview of various biological and clinical applications of peptidomics.

Multiplexed neuropeptide mapping in ant brains integrating microtomography and three-dimensional mass spectrometry imaging.

Neuropeptides are important regulators of animal physiology and behavior. Hitherto the gold standard for the localization of neuropeptides have been immunohistochemical methods that require the synthesis of antibody panels, while another limiting factor has been the brain's opacity for subsequent in situ light or fluorescence microscopy. To address these limitations, we explored the integration of high-resolution mass spectrometry imaging (MSI) with microtomography for a multiplexed mapping of neuropeptides in two evolutionary distant ant species, Atta sexdens and Lasius niger. For analyzing the spatial distribution of chemically diverse peptide molecules across the brain in each species, the acquisition of serial mass spectrometry images was essential. As a result, we have comparatively mapped the three-dimensional (3D) distributions of eight conserved neuropeptides throughout the brain microanatomy. We demonstrate that integrating the 3D MSI data into high-resolution anatomy models can be critical for studying organs with high plasticity such as brains of social insects. Several peptides, like the tachykinin-related peptides (TK) 1 and 4, were widely distributed in many brain areas of both ant species, whereas others, for instance myosuppressin, were restricted to specific regions only. Also, we detected differences at the species level; many peptides were identified in the optic lobe of L. niger, but only one peptide (ITG-like) was found in this region in A. sexdens. Building upon MS imaging studies on neuropeptides in invertebrate model systems, our approach leverages correlative MSI and computed microtomography for investigating fundamental neurobiological processes by visualizing the unbiased 3D neurochemistry in its complex anatomic environment.

Peptide modulators of cell migration: Overview, applications and future development.

Cell migration is a key physiological process in the development and homeostasis of multicellular organisms; errors in this complex system can trigger the development of cancer or inflammatory disorders. Therefore, modulating cell migration provides opportunities for drug discovery. Peptides are gaining importance on the global therapeutics market, given their unique properties compared with established small-molecule drugs or biologics. In this review, we identified over 470 peptides modulating cell migration and analyzed their characteristics. Over 95% of these peptides are in the discovery or preclinical stage, because the transition of peptide hits into drug leads often results in a bottleneck in the development process. We summarize chemical strategies in (pre-)clinical development to enhance drug-like properties of bioactive peptides.

The nature inspired peptide [T20K]-kalata B1 induces anti-tumor effects in anaplastic large cell lymphoma.

Ribosomally synthesized and post-translationally modified peptides, such as plant cyclotides, are a diverse group of natural products well known as templates in drug discovery and therapeutic lead development. The cyclotide kalata B1 (kB1) has previously been discovered as immunosuppressive agent on T-lymphocytes, and a synthetic version of this peptide, [T20K]kB1 (T20K), has been effective in reducing clinical symptoms, such as inflammation and demyelination, in a mouse model of multiple sclerosis. Based on its T-cell modulatory impact we studied the effects of T20K and several analogs on the proliferation of anaplastic large cell lymphoma (ALCL), a heterogeneous group of clinically aggressive diseases associated with poor prognosis. T20K, as a prototype drug candidate, induces apoptosis and a proliferation arrest in human lymphoma T-cell lines (SR786, Mac-2a and the Jurkat E6.1) in a concentration dependent fashion, at least partially via increased STAT5 and p53 signaling. In contrary to its effect on IL-2 signaling in lymphocytes, the cytokine levels are not altered in lymphoma cells. In vivo mouse experiments revealed a promising activity of T20K on these cancer cells including decreased tumor weight and increased apoptosis. This study opens novel avenues for developing cyclotide-based drug candidates for therapy of patients with ALCL.

Importance of the Cyclic Cystine Knot Structural Motif for Immunosuppressive Effects of Cyclotides.

The cyclotide T20K inhibits the proliferation of human immune cells and is currently in clinical trials for multiple sclerosis. Here, we provide novel functional data and mechanistic insights into structure-activity relationships of T20K. Analogs with partial or complete reduction of the cystine knot had loss of function in proliferation experiments. Similarly, an acyclic analog of T20K was inactive in lymphocyte bioassays. The lack of activity of non-native peptide analogs appears to be associated with the ability of cyclotides to interact with and penetrate cell membranes, since cellular uptake studies demonstrated fast fractional transfer only of the native peptide into the cytosol of human immune cells. Therefore, structural differences between cyclic and linear native folded peptides were investigated by NMR to elucidate structure-activity relationships. Acyclic T20K had a less rigid backbone and considerable structural changes in loops 1 and 6 compared to the native cyclic T20K, supporting the idea that the cyclic cystine knot motif is a unique bioactive scaffold. This study provides evidence that this structural motif in cyclotides governs bioactivity, interactions with and transport across biological membranes, and the structural integrity of these peptides. These observations could be useful to understand the structure-activity of other cystine knot proteins due to the structural conservation of the cystine knot motif across evolution and to provide guidance for the design of novel cyclic cysteine-stabilized molecules.

Cyclotides Isolated From Violet Plants of Cameroon Are Inhibitors of Human Prolyl Oligopeptidase.

Traditional medicine and the use of herbal remedies are well established in the African health care system. For instance, Violaceae plants are used for antimicrobial or anti-inflammatory applications in folk medicine. This study describes the phytochemical analysis and bioactivity screening of four species of the violet tribe Allexis found in Cameroon. Allexis cauliflora, Allexis obanensis, Allexis batangae and Allexis zygomorpha were evaluated for the expression of circular peptides (cyclotides) by mass spectrometry. The unique cyclic cystine-rich motif was identified in several peptides of all four species. Knowing that members of this peptide family are protease inhibitors, the plant extracts were evaluated for the inhibition of human prolyl oligopeptidase (POP). Since all four species inhibited POP activity, a bioactivity-guided fractionation approach was performed to isolate peptide inhibitors. These novel cyclotides, alca 1 and alca 2 exhibited IC50 values of 8.5 and 4.4 µM, respectively. To obtain their amino acid sequence information, combinatorial enzymatic proteolysis was performed. The proteolytic fragments were evaluated in MS/MS fragmentation experiments and the full-length amino acid sequences were obtained by de novo annotation of fragment ions. In summary, this study identified inhibitors of the human protease POP, which is a drug target for inflammatory or neurodegenerative disorders.

Alterations of the Platelet Proteome in Lung Cancer: Accelerated F13A1 and ER Processing as New Actors in Hypercoagulability.

In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins calreticulin (CALR), endoplasmic reticulum chaperone BiP (HSPA5) and protein disulfide-isomerase (P4HB) were significantly elevated. Accelerated conversion of the fibrin stabilising factor XIII was detected in platelets of patients with lung cancer by elevated levels of a coagulation factor XIII (F13A1) 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma levels of the plasmin-α-2-antiplasmin complex and D-dimer suggests its enhanced degradation by the fibrinolytic system. Protein association network analysis showed that lung cancer-related proteins were involved in platelet degranulation and upregulated ER protein processing. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Nature-inspired dimerization as a strategy to modulate neuropeptide pharmacology exemplified with vasopressin and oxytocin.

Vasopressin (VP) and oxytocin (OT) are cyclic neuropeptides that regulate fundamental physiological functions via four G protein-coupled receptors, V1aR, V1bR, V2R, and OTR. Ligand development remains challenging for these receptors due to complex structure-activity relationships. Here, we investigated dimerization as a strategy for developing ligands with novel pharmacology. We regioselectively synthesised and systematically studied parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimer constructs of VP, OT and dVDAVP (1-deamino-4-valine-8-d-arginine-VP). All disulfide-linked dimers, except for the head-to-tail cyclized constructs, retained nanomolar potency despite the structural implications of dimerization. Our results support a single chain interaction for receptor activation. Dimer orientation had little impact on activity, except for the dVDAVP homodimers, where an antagonist to agonist switch was observed at the V1aR. This study provides novel insights into the structural requirements of VP/OT receptor activation and spotlights dimerization as a strategy to modulate pharmacology, a concept also frequently observed in nature.

Isolation and functional diversity of Bowman-Birk type serine proteinase inhibitors from Hyacinthus orientalis.

Bowman-Birk inhibitors (BBIs) are plant-derived serine proteinase inhibitors. Endogenously, they function as defense molecules against pathogens and insects, but they also have been explored for applications in cancer treatment and inflammatory disorders. Here, we isolated 15 novel BBIs from the bulb of Hyacinthus orientalis (termed HOSPIs). These isoinhibitors consisted of two or three chains, respectively, that are linked by disulfides bonds based on proposed cleavage sites in the canonical BBI reactive site loop. They strongly inhibited trypsin (Ki = 0.22-167 nM) and α-chymotrypsin (Ki = 19-1200 nM). Notably, HOSPI-B4 contains a six-residue reactive loop, which appears to be the smallest such motif discovered in BBIs to date. HOSPI-A6 and -A7 contain an unusual reactive site, i.e. Leu-Met at the P1-P1' position and have strong inhibitory activity against trypsin, α-chymotrypsin, and elastase. Analysis of the cDNA encoding HOSPIs revealed that the precursors have HOSPI-like domains repeated at least twice with a defined linker sequence connecting individual domains. Lastly, mutational analysis of HOSPIs suggested that the linker sequence does not affect the inhibitory activity, and a Thr residue at the P2 site and a Pro at the P3' site are crucial for elastase inhibition. Using mammalian proteases as representative model system, we gain novel insight into the sequence diversity and proteolytic activity of plant BBI. These results may aid the rational design of BBI peptides with potent and distinct inhibitory activity against human, pathogen, or insect serine proteinases.

Discovery of a Beetroot Protease Inhibitor to Identify and Classify Plant-Derived Cystine Knot Peptides.

Plant peptide protease inhibitors are important molecules in seed storage metabolism and to fight insect pests. Commonly they contain multiple disulfide bonds and are exceptionally stable molecules. In this study, a novel peptide protease inhibitor from beetroot (Beta vulgaris) termed bevuTI-I was isolated, and its primary structure was determined via mass spectrometry-based amino acid sequencing. By sequence homology analysis a few peptides with high similarity to bevuTI-I, also known as the Mirabilis jalapa trypsin inhibitor subfamily of knottin-type protease inhibitors, were discovered. Hence, we assessed bevuTI-I for inhibitory activity toward trypsin (IC50 = 471 nM) and human prolyl oligopeptidase (IC50 = 11 µM), which is an emerging drug target for neurodegenerative and inflammatory disorders. Interestingly, using a customized bioinformatics approach, bevuTI-I was found to be the missing link to annotate 243 novel sequences of M. jalapa trypsin inhibitor-like peptides. According to their phylogenetic distribution they appear to be common in several plant families. Therefore, the presented approach and our results may help to discover and classify other plant-derived cystine knot peptides, a class of plant molecules that play important functions in plant physiology and are currently being explored as lead molecules and scaffolds in drug development.

Isolation of Cysteine-Rich Peptides from Citrullus colocynthis.

The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

Critical Role of Neprilysin in Kidney Angiotensin Metabolism.

RATIONALE: Kidney homeostasis is critically determined by the coordinated activity of the renin-angiotensin system (RAS), including the balanced synthesis of its main effector peptides Ang (angiotensin) II and Ang (1-7). The condition of enzymatic overproduction of Ang II relative to Ang (1-7) is termed RAS dysregulation and leads to cellular signals, which promote hypertension and organ damage, and ultimately progressive kidney failure. ACE2 (angiotensin-converting enzyme 2) and NEP (neprilysin) induce the alternative, and potentially reno-protective axis by enhancing Ang (1-7) production. However, their individual contribution to baseline RAS balance and whether their activities change in chronic kidney disease (CKD) has not yet been elucidated. OBJECTIVE: To examine whether NEP-mediated Ang (1-7) generation exceeds Ang II formation in the healthy kidney compared with diseased kidney. METHODS AND RESULTS: In this exploratory study, we used liquid chromatography-tandem mass spectrometry to measure Ang II and Ang (1-7) synthesis rates of ACE, chymase and NEP, ACE2, PEP (prolyl-endopeptidase), PCP (prolyl-carboxypeptidase) in kidney biopsy homogenates in 11 healthy living kidney donors, and 12 patients with CKD. The spatial expression of RAS enzymes was determined by immunohistochemistry. Healthy kidneys showed higher NEP-mediated Ang (1-7) synthesis than Ang II formation, thus displaying a strong preference towards the reno-protective alternative RAS axis. In contrast, in CKD kidneys higher levels of Ang II were recorded, which originated from mast cell chymase activity. CONCLUSIONS: Ang (1-7) is the dominant RAS peptide in healthy human kidneys with NEP rather than ACE2 being essential for its generation. Severe RAS dysregulation is present in CKD dictated by high chymase-mediated Ang II formation. Kidney RAS enzyme analysis might lead to novel therapeutic approaches for CKD.

Biochemical Characterization of the Fusarium graminearum Candidate ACC-Deaminases and Virulence Testing of Knockout Mutant Strains.

Fusarium graminearum is a plant pathogenic fungus which is able to infect wheat and other economically important cereal crop species. The role of ethylene in the interaction with host plants is unclear and controversial. We have analyzed the inventory of genes with a putative function in ethylene production or degradation of the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC). F. graminearum, in contrast to other species, does not contain a candidate gene encoding ethylene-forming enzyme. Three genes with similarity to ACC synthases exist; heterologous expression of these did not reveal enzymatic activity. The F. graminearum genome contains in addition two ACC deaminase candidate genes. We have expressed both genes in E. coli and characterized the enzymatic properties of the affinity-purified products. One of the proteins had indeed ACC deaminase activity, with kinetic properties similar to ethylene-stress reducing enzymes of plant growth promoting bacteria. The other candidate was inactive with ACC but turned out to be a d-cysteine desulfhydrase. Since it had been reported that ethylene insensitivity in transgenic wheat increased Fusarium resistance and reduced the content of the mycotoxin deoxynivalenol (DON) in infected wheat, we generated single and double knockout mutants of both genes in the F. graminearum strain PH-1. No statistically significant effect of the gene disruptions on fungal spread or mycotoxin content was detected, indicating that the ability of the fungus to manipulate the production of the gaseous plant hormones ethylene and H2S is dispensable for full virulence.

Peptide-based protease inhibitors from plants.

Proteases have an important role in homeostasis, and dysregulation of protease function can lead to pathogenesis. Therefore, proteases are promising drug targets in cancer, inflammation, and neurodegenerative disease research. Although there are well-established pharmaceuticals on the market, drug development for proteases is challenging. This is often caused by the limited selectivity of currently available lead compounds. Proteinaceous plant protease inhibitors are a diverse family of (poly)peptides that are important to maintain physiological homeostasis and to serve the innate defense machinery of the plant. In this review, we provide an overview of the diversity of plant peptide- and protein-based protease inhibitors (PIs), provide examples of such compounds that target human proteases, and discuss opportunities for these molecules in protease drug discovery and development.

Untargeted LC-MS based 13C labelling provides a full mass balance of deoxynivalenol and its degradation products formed during baking of crackers, biscuits and bread.

Deoxynivalenol (DON) is considered to be one of the most important contaminants in cereals and food commodities produced thereof. So far it is not clear i) to which extent DON is degraded during baking and ii) if a degradation results in reduced toxicity. We have elucidated the fate of DON during baking of crackers, biscuits and bread, which were produced from fortified dough and processed under pilot plant conditions. Untargeted stable isotope assisted liquid chromatography (LC) high resolution mass spectrometry was used to determine all extractable degradation products. Targeted LC - tandem mass spectrometry based quantification revealed that DON was partially degraded to isoDON (1.3-3.9%), norDON B (0.2-0.9%) and norDON C (0.3-1.2%). A DON degradation of 6% (crackers), 5% (biscuits) and 2% (bread), respectively, was observed. In vitro translation experiments indicate that isoDON is less toxic than DON.

Discovery of peptide probes to modulate oxytocin-type receptors of insects.

The oxytocin/vasopressin signalling system is conserved across the animal kingdom. In insects, the role of oxytocin-type (inotocin) neuropeptides has only been studied in locusts, beetles and ants, but their physiology continues to be poorly understood. One reason for this knowledge deficit is the lack of available research tools to complement functional genomics efforts. Consequently, ligands to probe insect inotocin receptors are essential. In this study, we sought to identify novel agonists and antagonists of the inotocin receptor from the representative model species Tribolium castaneum and Lasius niger. Drawing upon known ligands of the human receptors, we examined the pharmacology of the plant-derived cyclotide kalata B7 and the synthetic oxytocin analogue atosiban. Kalata B7 is a weak partial agonist of both inotocin receptors. This is the first reported direct interaction of cyclotides with an insect receptor, an observation that may explain their presumed role in herbivore defence. Furthermore, we discovered atosiban is an antagonist of the Tribolium receptor, which may provide a useful probe to investigate the functionality of inotocin signalling in beetles and related insect species. Our findings will enable further examination of insect inotocin receptor pharmacology and physiology, and may trigger studies to comprehend the interaction of plant cyclotides and insects.

Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins.

Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomally synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study, a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target for the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible functional modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.

Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum.

Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives.