Figure 2 - available via license: Creative Commons Attribution 4.0 International
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A) Schematic representation of the (reverse) prenylation of all available positions in the indole ring of tryptophan catalyzed by different prenyltransferases (PTs). (B) Selection of structurally complex and unusual DKP-containing natural products. Unusual functionalities are highlighted in red.
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Modified cyclic dipeptides represent a diverse family of microbial secondary metabolites. They display a broad variety of biological and pharmacological activities and have long been recognized as privileged structures with the ability to bind to a wide range of receptors. This is due to their conformationally constrained 2, 5-diketopiperazine (DKP...
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Context 1
... of the indole side chain using either isopentenyl pyrophosphate (IPP) or dimethylallyl pyrophosphate (DMAPP) as cofactors carried out by various types of prenyltransferases (PTs) may represent the most common modification found in tryptophan-containing CDPs. Over the last ten years, PTs that are able to modify all positions of the indole heterocycle in DKPs have been characterized and represent a valuable repertoire of diversification catalysts (Figure 2A) (Yu et al., 2012). Prenylations that expand the carbon skeleton of a natural product and may hugely influence its lipophilicity are very often crucial for their biological activity and ability to diffuse through cell membranes (Li, 2009a,b). ...
Context 2
... the last ten years, PTs that are able to modify all positions of the indole heterocycle in DKPs have been characterized and represent a valuable repertoire of diversification catalysts (Figure 2A) (Yu et al., 2012). Prenylations that expand the carbon skeleton of a natural product and may hugely influence its lipophilicity are very often crucial for their biological activity and ability to diffuse through cell membranes (Li, 2009a,b). The recent identification of a cyclo(L-Trp-L-Trp) producing CDPS in Actinosynnema mirum, a very small high-yielding biocatalyst, might further encourage the engineering of synthetic pathways for the production of various prenylated and further modified "unnatural" natural products which so far has relied on using very large NRPSs (vide infra; Giessen et al., 2013b). ...
Context 3
... the various tailoring enzymes discussed above and described in the literature, many highly complex modified CDPs have been isolated where the responsible enzymes haven't been identified or characterized so far. To highlight the hidden diversity of DKP-modifying enzymes a number of structurally intriguing natural products are shown in Figure 2B. Modifications found in those secondary metabolites include unusual oxidative cyclization reactions (norgeamides A; Grubbs et al., 2007), carbonyl (norgeamides A), oxoether (aspirochlorine; Klausmeyer et al., 2005), and peroxide (fumitremorgin A; Deveau et al., 2001) formations as well as hydroxylations (gypsetin etc.; Nuber et al., 1994), all likely introduced by oxidoreductase class enzymes. ...
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Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the nu...
Citations
... Cytochrome P450 genes are frequently located close to a cyclodipeptide synthase gene in microbes (Li 2010;Xu et al. 2014;Giessen and Marahiel 2015;Canu et al. 2020). Cyclodipeptides are an important family of iron-chelating precursors in bacteria. ...
... Cyclodipeptides are an important family of iron-chelating precursors in bacteria. The nearby P450 gene is primarily involved in oxidising the diketopiperazine nitrogen atoms in cyclodipeptide to be the corresponding N-oxides, resulting in two hydroxamate cores for chelating iron (Cryle et al. 2010;Giessen and Marahiel 2015;Canu et al. 2020). For instance, the cytochrome P450 gene CYP134A1 from Bacillus subtilis is responsible for transforming cyclodipeptide cyclo-leu-leu into pulcherriminic acid, a precursor of an extracellular iron chelate pulcherrimin (Cryle et al. 2010). ...
... In fungi, cyclodipeptides were commonly synthesised from tryptophan and a small group of amino acids (glycine, alanine, proline, and its derivatives). These cyclodipeptides were further prenylated to form prenylated indole alkaloids (PIAs) (Li 2010;Giessen and Marahiel 2015). PIAs are a well-known class of fungal metabolites with a wide range of biological and pharmacological activities and are widely distributed in filamentous fungi, especially in the genera Penicillium and Aspergillus of Ascomycota (Li 2010;Giessen and Marahiel 2015). ...
Thermomyces dupontii harbors two P450 paralogs (P450S and P450L) in the gene cluster for the biosynthesis of prenylated indole alkaloids (PIAs) and correponding iron chelators with P450L assigned as one protein containing a CYP like domain fused with a FAD-binding domain-containing oxidoreductase. Genetic manipulation and metabolic profile analysis indicated both P450S and P450L were involved in transforming simple PIAs to their corresponding iron chelators. Moreover, P450S is responsible for bolstering simple PIAs to complex PIAs, and P450L for reinforcing conjugating unsaturated systems in complex PIAs. Chemical investigation led to isolation and characterization of novel complex PIA metabolites with more oxidations. P450L also contributed to forming the third iron-chelating core in iron chelators. A series of iron bioassays and infrastructure analysis revealed that lack of these P450 genes caused strongly elevated Fe 3+ levels but attenuated Fe 2+ levels, together with abnormal mitochondria in mycelia and lipid droplets and vacuoles in conidia. Phenotype analysis revealed that P450S and P450L facilitated fungal colony pigments, conidial formation and germination via bolstering conidio-phores and cell walls in response to temperature reduction.
... Biosynthetically, in fungi, the core cyclic dipeptide nucleus of DKPs is mostly originated from the condensation of two tryptophan amino acids or one tryptophan molecule and another amino acid, assembled by non-ribosomal peptide synthetases (NRPSs) [8,10]. Different putative tailoring enzymes contribute to the structural diversity of DKP scaffolds, including oxidoreductases, hydrolases, methyl transferases, prenyltransferases, and ligases [11,12]. Prenylated indole diketopiperazines are constituted of an indole diketopiperazine backbone and isoprenoid moieties, in which prenylation is catalyzed by prenyltransferases acting as tailoring enzymes [7,8,13]. ...
A new prenylated indole diketopiperazine alkaloid, rubrumline P (1), was isolated along with six more analogues and characterized from the fermentation culture of a marine sediment-derived fungus, Aspergillus chevalieri, collected at a depth of 15 m near the lighthouse in Dahab, Red Sea, Egypt. In the current study, a bioassay-guided fractionation allowed for the identification of an active fraction displaying significant cytotoxic activity against the human pancreatic adenocarcinoma cell line PANC-1 from the EtOAc extract of the investigated fungus compared to the standard paclitaxel. The structures of the isolated compounds from the active fraction were established using 1D/2D NMR spectroscopy and mass spectrometry, together with comparisons with the literature. The absolute configuration of the obtained indole diketopiperazines was established based on single-crystal X-ray diffraction analyses of rubrumline I (2) and comparisons of optical rotations and NMR data, as well as on biogenetic considerations. Genome sequencing indicated the formation of prenyltransferases, which was subsequently confirmed by the isolation of mono-, di-, tri-, and tetraprenylated compounds. Compounds rubrumline P (1) and neoechinulin D (4) confirmed preferential cytotoxic activity against PANC-1 cancer cells with IC50 values of 25.8 and 23.4 µM, respectively. Although the underlying mechanism-of-action remains elusive in this study, cell cycle analysis indicated a slight increase in the sub-G1 peak after treatment with compounds 1 and 4.
... Nonribosomal peptide synthetases (NRPSs) are large, modular multidomain enzymes that give rise to a plethora of structurally and functionally diverse peptides that may be assembled from as many as 25 individual building blocks. [1][2][3] While nonribosomal peptides (NRPs) can act as virulence factors or neurotoxins, their pharmaceutical benefit is invaluable as exemplified by several marketed drugs including the immunosuppressant cyclosporin, the anti-tumor agent bleomycin and antibiotics such as vancomycin, cephalosporins and penicillins. [4] The vast structural complexity of NRPs, often challenging to achieve by synthetic chemistry methods, is introduced by only a few core domains and optional tailoring steps. ...
Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange.
... (+)-Verticillin A (8) was later isolated from wheat solid-substrate fermentation of Gliocladium roseum 1A, together with (+)-gliocladine A (25, Figure 4), (+)-gliocladine B (26, Figure 4), (+)-12'-deoxyverticillin A (9, Figure 2), (+)-Sch 52900 (18), and (+)-Sch 52901 (19, Figure 3), and found to display cytotoxic activity against a panel of human cancer cell lines, with IC 50 values of 20 to 370 nM. In addition, they also were effective nematicidal agents towards Caenorhabditis elegans and Panagrellus redivivus (vide infra) [80]. ...
... (+)-Sch 52900 (18) and (+)-Sch 52901 (19, Figure 3) were first isolated, together with (+)verticillin A (8, Figure 2) from the fermentation broth of the fungal culture Gliocladium sp. (SCF-1168) [91]. ...
... The identification relied on the structural similarities noted with previously described (+)-verticillin A (8) [20]. The nonsymmetrical nature of (+)-Sch 52900 (18) was concluded by the presence of apparently split signals in the 13 C NMR spectrum. The identification of signals for the secondary alcohol in place of the methyl group of (+)verticillin A (8) suggested the structure for (+)-Sch 52900 (18). ...
Within the 2,5-dioxopiperazine-containing natural products generated by “head-to-tail” cyclization of peptides, those derived from tryptophan allow further structural diversification due to the rich chemical reactivity of the indole heterocycle, which can generate tetracyclic fragments of hexahydropyrrolo[2,3-b]indole or pyrrolidinoindoline skeleton fused to the 2,5-dioxopiperazine. Even more complex are the dimeric bispyrrolidinoindoline epi(poly)thiodioxopiperazines (BPI-ETPs), since they feature transannular (poly)sulfide bridges connecting C3 and C6 of their 2,5-dioxopiperazine rings. Homo- and heterodimers composed of diastereomeric epi(poly)thiodioxopiperazines increase the complexity of the family. Furthermore, putative biogenetically generated downstream metabolites with C11 and C11’-hydroxylated cores, as well as deoxygenated and/or oxidized side chain counterparts, have also been described. The isolation of these complex polycyclic tryptophan-derived alkaloids from the classical sources, their structural characterization, the description of the relevant biological activities and putative biogenetic routes, and the synthetic efforts to generate and confirm their structures and also to prepare and further evaluate structurally simple analogs will be reported.
... CDPs contain a diketopiperazine ring, a scaffold that has been coined as privileged due to remarkable properties such as proteolytic resistance, blood-brain barrier permeability and the ability to mimic functional pharmacophores [2][3][4] . CDPSs act in combination with tailoring enzymes, adding significant complexity to the types of natural products that can be produced 5,6 . Because computational prediction of the specificity of CDPSs is challenging 7 , the determination of substrates and products of each enzyme requires experimental testing, in a time consuming and low throughput process [8][9][10] . ...
Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.
... Unfortunately, all of them were found to be inactive, displaying IC 50 values >40 μM. Complex structural and stereoisomeric modification might influence the biological effects of the target compounds through the alteration of the shape or rigidity of the scaffold (Giessen et al. 2013;Giessen and Marahiel 2015). As some members of dimeric diketopiperazine encompass a vast spectrum of biological properties, it is suggested that the four compounds' probability possesses an alternative and undefined biology that can be studied in further research. ...
Four dimeric diketopiperazine stereoisomers (1-4) including two new ones (1-2) had been isolated from the culture broth of one marine-derived fungus Aspergillus sp. Z3, which was found in the gut of a marine isopod Ligia exotica. The planner structures and absolute configurations of the new compounds were determined by combination of NMR, HRESIMS, electronic circular dichroism calculation, Marfey's method as well as single-crystal X-ray diffraction. Their cytotoxicity against the prostate cancer PC3 cell line was assayed by the MTT method. ARTICLE HISTORY
... [2][3][4] CDPSs act in combination with tailoring enzymes, adding significant complexity to the types of natural products that can be produced. 5,6 Because computational prediction of the specificity of CDPSs is challenging 7 , the determination of substrates and products of each enzyme requires experimental testing, in a time consuming and low throughput process. [8][9][10] Prior to this work there were no reports of successfully engineering CDPS enzymes to direct substrate selection. ...
Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we generated an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work pioneers the successful engineering a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.
... Although TRXs' reduction activity in converting dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS) has been proven, their oxidase activity in DMSO production has not been confirmed yet. DKPs are cyclic peptides that are often biosynthesized by non-ribosomal peptide synthase from two amino acids [42,43]. Thus, potential defense synthetic pathways for the AMMs that have been detected by GC/MS can be suggested and proposed based on the proteins detected in the MS/MS data (Figure 3). ...
We hypothesized that environmental microbiomes contain a wide range of bacteria that produce yet uncharacterized antimicrobial compounds (AMCs) that can potentially be used to control pathogens. Over 600 bacterial strains were isolated from soil and food compost samples, and 68 biocontrol bacteria with antimicrobial activity were chosen for further studies based on inhibition assays against a wide range of food and plant pathogens. For further characterization of the bioactive compounds, a new method was established that used living pathogens in a liquid culture to stimulate bacteria to produce high amounts of AMCs in bacterial supernatants. A peptide gel electrophoresis microbial inhibition assay was used to concurrently achieve size separation of the antimicrobial peptides. Fifteen potential bioactive peptides were then further characterized by tandem MS, revealing cold-shock proteins and 50S ribosomal proteins. To identify non-peptidic AMCs, bacterial supernatants were analyzed by HPLC followed by GC/MS. Among the 14 identified bioactive compounds, 3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2-acetyl-3-methyl-octahydropyrrolo[1,2-a]piperazine-1,4-dione were identified as new AMCs. Our work suggests that antimicrobial compound production in microbes is enhanced when faced with a threat from other microorganisms, and that this approach can rapidly lead to the development of new antimicrobials with the potential for upscaling.
... Although producing DKPs from microorganisms via an expression system is feasible; the optimization is long and not a straightforward process [58]. The biosynthesis of DKPs relies mainly on two enzymes, non-ribosomal peptide synthetases (NRPs) and tRNA-dependent cyclodipeptide synthases (CDPs) [59]. Both enzymes are part of a biosynthetic gene cluster that targets DKPs scaffold modifications necessary for the stability of the produced DKPs [60]. ...
... DKPs are heterocyclic compounds consisting of two amino acid residues linked to a central six-membered lactam ring core with (or without) various substituents, providing the control of the substituent's stereochemistry at up to four positions, chiral nature, three-dimensionality, and consequently leading to the promotion of the intermolecular Hbonding interactions with bio-target sites via the corresponding sites of donors and acceptors [17]. Thus, the rigid DKP core allows either a constrained or flexible behavior of amino acids, mimicking preferential peptide conformation [59]. It makes DKP molecules perfect to predicting properties of larger peptides with multiple H-bond acceptors, and donor functionality, and multiple sites for the structural elaboration of diverse functional groups. ...
... More specifically, 2,5-DKPs are common, naturally occurring peptide derivatives (and are frequently generated as unwanted by-products in the synthesis of oligopeptides). The 2,5-DKP core is present in the structure of known drugs, such as Tadalafil, phosphodiesterase-5 inhibitor for the treatment of pulmonary arterial hypertension and erectile dysfunction [78][79][80], Retosiban, an oxytocin antagonist for preterm labor [81], Epelsiban, an oxytocin antagonist in premature ejaculation in men [32], Aplaviroc against HIV [82], in the vascular disruption, and tubulin-depolymerizing Plinabulin, on the basis of marine fungal Halimide, a potential therapeutical drug in lung cancer [83,84], and other anticancer natural agents as Ambewelamide, Phenylahistin, Dehydrophenylahistin [85], Verticillin A [86], antiviral and immunosuppressive Sirodesmin, a type of phytotoxin, anti-inflammatory agents, e.g., FR106969 [59], antibacterial Bicyclomycin, Brevianamide S, Avrainvillamide [87] or Albonoursin, antifungal Maremycin, mycotoxins such as Roquefortine C [22] or Gliotoxin, which is also a potent inducer of apoptotic, and necrotic cell death [88,89]. Thaxtomin A [90], phytotoxin and insecticidal okaramine, and so on. ...
Cyclic dipeptides, also know as diketopiperazines (DKP), the simplest cyclic forms of pep-tides widespread in nature, are unsurpassed in their structural and bio-functional diversity. DKPs, especially those containing proline, due to their unique features such as, inter alia, extra-rigid con-formation, high resistance to enzyme degradation, increased cell permeability, and expandable ability to bind a diverse of targets with better affinity, have emerged in the last years as biologically pre-validated platforms for the drug discovery. Recent advances have revealed their enormous potential in the development of next-generation theranostics, smart delivery systems, and biomateri-als. Here, we present an updated review on the biological and structural profile of these appealing biomolecules, with a particular emphasis on those with anticancer properties, since cancers are the main cause of death all over the world. Additionally, we provide a consideration on supramolecular structuring and synthons, based on the proline-based DKP privileged scaffold, for inspiration in the design of compound libraries in search of ideal ligands, innovative self-assembled nanomaterials, and bio-functional architectures.
... In addition, Pro was reported as an activator of mTOR 100 signal pathway in concert with leucine. Cyclic dipeptides can bind to diverse targets due to conformationally constrained scaffolds and a vast 101 number of tailoring enzymes. Yan et al. reported that hydrophobic amino acids and proline are critical 102 for cyclic dipeptides' biological activity. ...
Background: Oxidative stress plays a key role in breast carcinogenesis. Cyclo (L-Leu-L-Pro) (CLP) is a homodetic cyclic dipeptide with 2,5-diketopiperazine scaffold isolated from marine actinobacteria. This study aimed to evaluate the protective activity of CLP and linear - (L-Leu-L-Pro) (LP) from tert-butyl hydroperoxide (tBHP)-induced damage using normal breast epithelial cell line model (MCF-12A). Methods: The cytoprotective activity was evaluated by detecting the changes in intracellular ROS, mitochondrial superoxide, hydroxyl radical, hydrogen peroxide, and lipid peroxidation detection assays as well as cytotoxic assays of MTT, LDH assays and phase contrast microscopy. Genoprotective activity was evaluated by (Apurinic/Apyrimidinic) AP site, alkaline Comet, and 8-hydroxy-2-deoxyguanosine assays. Results: The marine cyclic peptide, CLP, significantly protected MCF-12A cells by scavenging tBHP induced intracellular ROS such as super oxide, hydroxyl radicals and hydrogen peroxide, and by reducing the cytotoxicity and genotoxicity effect compared to LP. Moreover, the results showed that CD151 gene silencing by shRNA significantly reduced the overexpression of CD151, tBHP-induced ROS generation, cytotoxicity and genotoxicity in MCF-12A cells. The overexpression of CD151 caused increased levels of cytochrome P450, but was reduced following the application of CD151shRNA and CLP which led to elevated levels of intracellular ROS. Conclusion: In the present study we noticed that CD151 gene silencing by shRNA and treatment with CLP have similar effects on reducing the intracellular ROS. This study uncovers the protective activity of CLP against a CD151-mediated oxidative stress-induced cellular damage. Our observations suggest that the anti-stress and anti-inflammation properties of CLP might have implications in cancer and are worth testing in cancer cell lines and tumor cells.
