Search

In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete Lentzea aerocolonigenes to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask.

The interaction of Rebeccamycin with calf thymus (ctDNA) in the absence and presence of H1 was investigated by molecular dynamics, multi-spectroscopic, and cellular techniques. According to fluorescence and circular dichroism spectroscopies, Rebeccamycin interacted with ctDNA in the absence of H1 through intercalator or binding modes, while the presence of H1 resulted in revealing theintercalator, as the dominant role, and groove binding modes of ctDNA-Rebeccamycin complex. The binding constants, which were calculated to be 1.22 × 104 M−1 and 7.92 × 105 M−1 in the absence and presence of H1, respectively, denoted the strong binding of Rebeccamycin with ctDNA. The binding constants of Rebeccamycin with ct DNA in the absence and presence of H1 were calculated at 298, 303 and 308 K. Considering the thermodynamic parameters (ΔH0 and ΔS0), both vander waals forces and hydrogen bonds played predominant roles throughout the binding of Rebeccamycin to ctDNA in the absence and presence of H1. The outcomes of circular dichroism suggested the lack of any major conformational changes in ctDNA upon interacting with Rebeccamycin, except some perturbations in native B-DNA at local level. Additionally, the effect of NaCl and KI on ctDNA-Rebeccamycin complex provided further evidence for the reliance of their interaction modes on substituted groups. The observed increase in the relative viscosity of ctDNA caused by the enhancement of Rebeccamycin confirmed their intercalation and groove binding modes in the absence and presence of H1. Moreover, the assessments of molecular docking simulation corroborated these experimental results and also elucidated the effectiveness of Rebeccamycinin inhibiting and proliferating T24 and 5637 cells. Meanwhile, the ability of Rebeccamycin in inhibiting cell proliferation and tumor growth through the induction of apoptosis by down regulating the PI3K/AKT signaling pathway were provided. [ABSTRACT FROM AUTHOR]

Lentzeaaerocolonigenes, as an actinomycete, is a natural producer of the antibiotic and antitumoral drug rebeccamycin. Due to the filamentous cellular morphology handling in cultivations is challenging; therefore, morphology engineering techniques are mandatory to enhance productivity. One promising approach described in the literature is the addition of mineral particles in the micrometer range to precisely adjust cellular morphology and the corresponding product synthesis (microparticle-enhanced cultivation, MPEC). Glass microparticles are introduced in this study as a novel supplementation type for bioprocess intensification in filamentous organisms. Several investigations were conducted to screen for an optimal particle setup, including particle size and concentration regarding their impact and effects on enhanced productivity, microparticle incorporation behavior into the biopellets, the viability of pellets, and morphological changes. Glass microparticles (10 g·L−1) with a median diameter of 7.9 µm, for instance, induced an up to fourfold increase in product synthesis accompanied by overall enhanced viability of biomass. Furthermore, structural elucidations showed that biopellets isolated from MPEC tend to have lower hyphal density than unsupplemented control pellets. In this context, oxygen microprofiling was conducted to better understand how internal structural changes interwind with oxygen supply into the pellets. Here, the resulting oxygen profiles are of a contradictive trend of steeper oxygen consumption with increasing glass microparticle supplementation. Eventually, MPEC was combined with another promising cultivation strategy, the supplementation of soy lecithin (7.5 g·L−1), to further increase the cultivation performance. A combination of both techniques in an optimized setup resulted in a rebeccamycin concentration of 213 mg·L−1 after 10 days of cultivation, the highest value published so far for microparticle-supplemented shake flask cultivations of L. aerocolonigenes.

The synthesis of biologically active indolocarbazole natural products has garnered significant attention due to their diverse pharmacological properties. This review provides a comprehensive overview of synthetic methodologies employed in the preparation of indolocarbazole derivatives, highlighting key strategies and recent advancements. Emphasis is placed on the isolation and synthesis of naturally occurring sugar based indolocarbazoles, such as rebeccamycin, staurosporine and K252a. Furthermore, the biological activities of these compounds and their potential applications in drug discovery are discussed. The present review aims to enhance understanding of sugar based indolocarbazole synthesis and inspire further research in this area. [Display omitted] [ABSTRACT FROM AUTHOR]

The actinomycete Lentzea aerocolonigenes produces the antitumor antibiotic rebeccamycin. In previous studies the rebeccamycin production was significantly increased by the addition of glass beads during cultivation in different diameters between 0.5 and 2 mm and the induced mechanical stress by the glass beads was proposed to be responsible for the increased production. Thus, this study was conducted to be a systematic investigation of different parameters for macroparticle addition, such as bead diameter, concentration, and density (glass and ceramic) as well as shaking frequency, for a better understanding of the particle‐induced stress on L. aerocolonigenes. The induced stress for optimal rebeccamycin production can be estimated by a combination of stress energy and stress frequency. In addition, the macroparticle‐enhanced cultivation of L. aerocolonigenes was combined with soy lecithin addition to further increase the rebeccamycin concentration. With 100 g L−1 glass beads in a diameter of 969 µm and 5 g L−1 soy lecithin a concentration of 388 mg L−1 rebeccamycin was reached after 10 days of cultivation, which corresponds to the highest rebeccamycin concentrations achieved in shake flask cultivations of L. aerocolonigenes stated in literature so far. [ABSTRACT FROM AUTHOR]

Lentzea aerocolonigenes produces the antitumor antibiotic rebeccamycin. For a medical application maximized titers and appropriate downstream processing are necessary. The addition of the adsorbents XAD 4, XAD 17 HP, and XAD 16 N was investigated for increased productivity and in situ product recovery. An increase in product titer was observed for XAD 7 HP and XAD 16 N. A combination of these particles with glass beads led to a further increase. Moreover, rebeccamycin can be adsorbed to these particles which facilitate the extraction since the liquid phase can be dispensed with. [ABSTRACT FROM AUTHOR]

The antibiotic AT2433-B1 belongs to a therapeutically important class of antitumor agents. This natural product contains an indolocarbazole aglycone connected to a unique disaccharide consisting of a methoxyglucose and an amino sugar subunit, 2,4-dideoxy-4-methylamino-l-xylose. The configuration of the amino sugar distinguishes AT2433-B1 from its diastereoisomer iso-AT2433-B1. Here we have investigated the interaction of these two disaccharide indolocarbazole derivatives with different DNA sequences by means of DNase I footprinting and surface plasmon resonance (SPR). Accurate binding measurements performed at 4 and 25°C using the BIAcore SPR method revealed that AT2433-B1 binds considerably more tightly to a hairpin oligomer containing a [CG]4 block than to an oligomer with a central [AT]4 tract. The kinetic analysis shows that the antibiotic dissociates much more slowly from the GC sequence compared to the AT one. Preferential binding of AT2433-B1 to GC-rich sequences in DNA was independently confirmed by DNase I footprinting experiments performed with a 117 bp DNA restriction fragment. The specific binding sequence 5′-AACGCCAG identified from the footprints was then converted into a biotin-labeled DNA hairpin duplex and compound interactions with this specific sequence were characterized by high resolution BIAcore SPR experiments. Such a combined approach provided a detailed understanding of the molecular basis of DNA recognition. The discovery that the glycosyl antibiotic AT2433-B1 preferentially recognizes defined sequences offers novel opportunities for the future design of sequence-specific DNA-reading small molecules.

• Shake flask cultivations of pellets of a filamentous microorganism with glass beads. • Increased formation of the product due to glass bead induced mechanical stress. • CFD-DEM-simulations as a method for the calculation of mechanical stress energy. • Pellet Feret diameter is effected by the particle induced mechanical stress. The production of chemical and pharmaceutical substances by filamentous microorganisms is a wide and challenging field of study. The morphology of filamentous microorganisms, which can vary between loose mycelia and dense pellets, is influenced by process parameters like mechanical stress. The addition of glass particles of different sizes (0.2-2.1 mm) on the cultivation of Lentzea aerocolonigenes pellets was investigated. The glass bead-induced mechanical stress is made responsible for the increased product concentration of rebeccamycin. By coupling the discrete element method (DEM) with the computational fluid dynamics (CFD), a method for the calculation of the mechanical stress energy was developed and applied on first experimental results. As the mean stress energy was increased, the pellet diameter raised up to a maximum pellet size. This effect was explained by an increased pellet loosening and hence, a better oxygen supply. If the pellets were subject to even higher mean stress energies, the diameter decreased due to the assumed pellet fragmentation. In conclusion, to ensure high product concentrations, an optimal particle induced mechanical stress is necessary, which can be calculated with the developed method and validated with experimental cultivations. In future, this method enables the investigation of other process parameters and a process scale-up. [ABSTRACT FROM AUTHOR]

Rebeccamycin is an antibiotic and antitumor substance isolated from the filamentous bacterium Lentzea aerocolonigenes. After its discovery, investigations of rebeccamycin focused on elucidating its structure, biological activity, and biosynthetic pathway. For potential medical application, a sufficient drug supply has to be ensured, meaning that the production process of rebeccamycin plays a major role. In addition to the natural production of rebeccamycin in L. aerocolonigenes, where the complex cell morphology is an important factor for a sufficient production, rebeccamycin can also be heterologously produced or chemically synthesized. Each of these production processes has its own challenges, and first approaches to production often lead to low final product concentrations, which is why process optimizations are performed. This review provides an overview of the production of rebeccamycin and the different approaches used for rebeccamycin formation including process optimizations. [ABSTRACT FROM AUTHOR]

Abstract It is still a matter of worldwide continuous concern about how to obtain efficient, stable, pure blue light emitting materials and apply them in industrialized OLED devices. Here, a novel deep blue fluorescent material 2-(4'-(9 H -carbazol-9-yl)-[1,1′-biphenyl]-4-yl)-1-(4-methoxyphenyl)-1 H -phenanthro [9,10-d]imidazole(CzB-MOPPI) was firstly designed, synthesized and employed as emitters for deep-blue OLEDs. CzB-MOPPI, with carbazole as donor, twisted biphenyl bonded phenanthroimidazole which contains rigid plane structure as acceptor, exhibits high thermal stability with decomposition temperature (T d) of 480 °C. Weak donor carbazole unit and twist biphenyl both make the compound emit less red shift meanwhile maintain in deep blue region. The non-doped OLEDs based on the CzB-MOPPI as emitter show maximum emission at 435 nm, maximum luminance of 6450 cd/m2, maximum current efficiency (CE) of 3.34 cd/A. The chromaticity coordinate is (0.16, 0.08) which is extremely close to the NTSC standard blue. In Particular, the devices show an impressive external quantum efficiency (EQE) as high as 5.97%. Graphical abstract Image 1 Highlights • A donor-acceptor type deep blue fluorescent material was firstly designed and synthesized. • Compound exhibits high thermal stability and bipolar properties. • OLED devices were fabricated, showing deep blue emission and impressive high EQE of 5.97%. [ABSTRACT FROM AUTHOR]

In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete Lentzea aerocolonigenes to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schrader, Schrinner, Polomsky, Ivanov, Kampen, Schilde, Krull and Kwade.)

Extensive effort has been devoted to engineering flavin-dependent halogenases (FDHs) with improved stability, expanded substrate scope, and altered regioselectivity. Here, we show that variants of rebeccamycin halogenase (RebH) catalyze enantioselective desymmetrization of methylenedianilines via halogenation of these substrates distal to their pro-stereogenic center. Structure-guided engineering was used to increase the conversion and selectivity of these reactions, and the synthetic utility of the halogenated products was shown via conversion of to a chiral α-substituted indole. These results constitute the first reported examples of asymmetric catalysis by FDHs. [ABSTRACT FROM AUTHOR]

Background/Aims: Although proinflammatory cytokine-induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α. Methods: To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER) and paracellular permeability were measured. Production levels and localization of tight junction (TJ) proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC) and MLC kinase (MLCK) mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively. Results: Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition. Conclusion: Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation. [ABSTRACT FROM AUTHOR]

Rebeccamycin is a promising antibiotic synthesized by the filamentous bacterium Lechevalieria aerocolonigenes . To date, the main investigations were focused on the biological effect or the structure elucidation of rebeccamycin. The aim of this study was to develop an efficient cultivation process based on shake flask experiments. The relationship between morphology and productivity and the positive effects of micro- and macroparticle enhanced cultivation on rebeccamycin production with this strain in the pellet-like morphology was investigated. The addition of talc microparticles increased the rebeccamycin concentration to 120 mg L −1 and 3-fold compared to the control without microparticles. An up to 9-fold increase in rebeccamycin concentration was achieved using surface modified talc particles. Through the addition of glass beads as macroparticles, the highest rebeccamycin concentration of approximately 120 mg L −1 was achieved by the induction of mechanical stress. Cultivation with glass beads enhanced the rebeccamycin concentration 22-fold compared to the control without macroparticles. By further increasing the diameter of the glass beads and increasing mechanical stress, the morphology was driven toward mycelial growth and the productivity was decreased. An adjusted mechanical stress, through the addition of macro-shaped glass bead particles, led to the desired increase in productivity. [ABSTRACT FROM AUTHOR]

Lentzea , as an actinomycete, is a natural producer of the antibiotic and antitumoral drug rebeccamycin. Due to the filamentous cellular morphology handling in cultivations is challenging; therefore, morphology engineering techniques are mandatory to enhance productivity. One promising approach described in the literature is the addition of mineral particles in the micrometer range to precisely adjust cellular morphology and the corresponding product synthesis (microparticle-enhanced cultivation, MPEC). Glass microparticles are introduced in this study as a novel supplementation type for bioprocess intensification in filamentous organisms. Several investigations were conducted to screen for an optimal particle setup, including particle size and concentration regarding their impact and effects on enhanced productivity, microparticle incorporation behavior into the biopellets, the viability of pellets, and morphological changes. Glass microparticles (10 g·L aerocolonigenes , as an actinomycete, is a natural producer of the antibiotic and antitumoral drug rebeccamycin. Due to the filamentous cellular morphology handling in cultivations is challenging; therefore, morphology engineering techniques are mandatory to enhance productivity. One promising approach described in the literature is the addition of mineral particles in the micrometer range to precisely adjust cellular morphology and the corresponding product synthesis (microparticle-enhanced cultivation, MPEC). Glass microparticles are introduced in this study as a novel supplementation type for bioprocess intensification in filamentous organisms. Several investigations were conducted to screen for an optimal particle setup, including particle size and concentration regarding their impact and effects on enhanced productivity, microparticle incorporation behavior into the biopellets, the viability of pellets, and morphological changes. Glass microparticles (10 g·L -1 ), to further increase the cultivation performance. A combination of both techniques in an optimized setup resulted in a rebeccamycin concentration of 213 mg·L -1 ), to further increase the cultivation performance. A combination of both techniques in an optimized setup resulted in a rebeccamycin concentration of 213 mg·L -1 after 10 days of cultivation, the highest value published so far for microparticle-supplemented shake flask cultivations of L. aerocolonigenes ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dinius, Schrinner, Schrader, Kozanecka, Brauns, Klose, Weiß, Kwade and Krull.)

Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine. [ABSTRACT FROM AUTHOR]

We provide the first experimental evidence for intermediates being involved in catalysis by RebD in generating the chromopyrrolic acid (CPA) scaffold of rebeccamycin. In the presence of its substrates (indole pyruvate imine – IPAI – and H 2 O 2 both produced by the flavoprotein oxidase RebO that oxidizes tryptophan), RebD reacts as a peroxidase forming two IPAI radicals that recombine as a C–C bond in the CPA. When catalase is included to remove H 2 O 2 , CPA can still be formed because the IPAI rapidly reduces RebD, which reacts with O 2 , utilizing oxidase–peroxidase chemistry to produce CPA. Reduced RebD can also react with H 2 O 2 forming Cpd II directly, which can oxidize IPAI. Stopped-flow spectrophotometric studies demonstrated that during the reaction of RebO and RebD with Trp and oxygen, a species with a red-shifted Soret band at 424.5 nm appeared. This species can react with either guaiacol or ABTS to form ferric RebD, suggesting that it is Cpd II of RebD involved in the formation of CPA. In summary, the studies reveal new and unusual aspects peroxidase and peroxygenase chemistry used by RebD in catalyzing carbon–carbon oxidative coupling reactions that are involved in biosynthesis of indolocarbazoles. [ABSTRACT FROM AUTHOR]

A chemoenzymatic platform for the synthesis of S-adenosyl- L-methionine (SAM) analogues compatible with downstream SAM-utilizing enzymes is reported. Forty-four non-native S/Se-alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non-native SAM analogues. As a proof of concept for the feasibility of natural product 'alkylrandomization', a small set of differentially-alkylated indolocarbazole analogues was generated by using a coupled hMAT2-RebM system (RebM is the sugar C4′- O-methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes. [ABSTRACT FROM AUTHOR]

Abstract: Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important class of antitumour drugs. In the course of our structure–activity relationship studies, new rebeccamycin analogues modified at the imide moiety were synthesised. The antiproliferative activity of the compounds was evaluated on three human cancer cell lines, A2780 (ovarian cancer), H460 (lung cancer), and GLC4 (small-cell lung cancer). The in vitro cytotoxicity of compounds 2 and 4, characterised respectively by a 1,3-dioxolan and (1,3-dioxolan-4-yl)methylene groups linked to the imide moiety, was higher than the reference compound, edotecarin. The effect of compound 2 in inducing tumour regression in the A2780 xenograft model was also investigated. [Copyright &y& Elsevier]

Abstract: Naphtho[2,1-α]pyrrolo[3,4-c]carbazole-5,7(6H,12H)-dione (NPCD) is known to be a very potent and selective cyclin D1-CDK4 inhibitors and could induce strong G1 phase arrest in breast tumor cell lines. In this work, the synthesis of five NPCD glycosides and their cytotoxic activities against eight tumor cell lines are presented, as well as the investigation of their cell cycle arrest profiles. The results showed that the introduction of a sugar moiety onto NPCD did not affect much of their cytotoxic activities, while the subtle structure of the sugar moiety affected the underlying mechanism strongly. In addition, NPCD showed distinct cell-cycle arrest profiles in BxPC3 prostate cells and MCF-7 breast cells, while NPCD glycosides shared similar cell cycle arrest profiles in MCF-7 and BxPC3 cells, which also indicated that not only the indolocarbazole framework as well known before but the sugar moiety can have a profound impact on the mechanism of action for these types of compounds. [Copyright &y& Elsevier]

The article presents a study on the biosynthesis of indolocarbazole and goadsporin, two different heterocyclic antibiotics that are produced by actinomycetes. The study aims to understand the basic mechanisms of these antibiotics on the genetic and biochemical level. Gene disruption experiments and integration of mutated godA genes and godA disruptant chromosomal DNA were conducted for the biosynthesis process. The study has produced a variety of indolocarbazole and goadsporin analogs.

Abstract: Rebeccamycin derivative 1 bearing a sugar moiety linked to both indole nitrogens and an amino substituent on the carbohydrate unit was synthesized in three steps from the bacterial metabolite. This compound was found to be a highly potent checkpoint kinase 1 inhibitor with an IC50 value of 2.8nM. [Copyright &y& Elsevier]

Abstract: Lately, Quantitative Structure–Activity Relationship (QSAR) studies have been afar used to predict anticancer activity taking into account different molecular descriptors, statistical techniques, cell lines and data set of congeneric and non-congeneric compounds. Herein we report a QSAR study based on a TOPological Sub-structural Molecular Design (TOPS-MODE) approach, aiming at predicting the anticancer leukemia activity of a diverse data set of indolocarbazoles derivatives. Finally, several aspects of the structural activity relationships are discussed in terms of the contribution of different bonds to the anticancer activity, thereby making the relationship between structure and biological activity more transparent. [Copyright &y& Elsevier]

Staurosporine isolated from Streptomyces sp. TP-A0274 is a member of the family of indolocarbazole alkaloids that exhibit strong antitumor activity. A key step in staurosporine biosynthesis is the formation of the indolocarbazole core by intramolecular C-C bond formation and oxidative decarboxylation of chromopyrrolic acid (CPA) catalyzed by cytochrome P450 StaP (StaP, CYP245A1). In this study, we report x-ray crystal structures of CPA-bound and -free forms of StaP. Upon substrate binding, StaP adopts a more ordered conformation, and conformational rearrangements of residues in the active site are also observed. Hydrogen-bonding interactions of two carboxyl groups and T-shaped π-π interactions with indole rings hold the substrate in the substrate-binding cavity with a conformation perpendicular to the heme plane. Based on the crystal structure of StaP-CPA complex, we propose that C-C bond formation occurs through an indole cation radical intermediate that is equivalent to cytochrome c peroxidase compound I [Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245:738-740]. The subsequent oxidative decarboxylation reaction is also discussed based on the crystal structure. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of staurosporine biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of a new family of 7-aza-rebeccamycin analogues in which the sugar moiety is attached to the nitrogen of the pyridine ring is described. The capacity of the newly synthesized compounds to bind to DNA and to inhibit topoisomerase I has been evaluated. Their cytotoxicities toward four tumor cell lines, one murine leukemia L1210 and three human tumor cell lines, one prostate carcinoma DU145, one colon carcinoma HT29, and one non-small cell lung carcinoma A549, have been determined. Their abilities to inhibit the checkpoint kinase Chk1 have been evaluated. [Copyright &y& Elsevier]

The indolocarbazole family of natural products is a source of lead compounds with potential therapeutic applications in the treatment of cancer and neurodegenerative disorders. Rebeccamycin and staurosporine are two members of this family, which are produced by different actinomycete strains. Although both compounds display antitumor activity, their distinct structural features determine different modes of action: rebeccamycin targets DNA topoisomerase I, while staurosporine is a protein kinase inhibitor. Here we examine the biosyntheses of rebeccamycin and staurosporine while we summarize our recent work concerning (a) identification and characterization of genes involved in the biosynthesis of indolocarbazoles in actinomycetes, and (b) generation of novel indolocarbazole derivatives in microorganisms by combinatorial biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of a family of rebeccamycin analogues in which one indole unit has been replaced by a 7-azaindole moiety is described. Substitutions have been carried out on the imide nitrogen, on the carbazole framework and on the sugar part. Compounds with a lactam upper heterocycle have also been prepared. The cytotoxicities of the newly synthesized compounds toward four tumor cell lines, one murine leukemia (L1210) and three human tumor cell lines (prostate carcinoma DU145, colon carcinoma HT29, and non-small cell lung carcinoma A549) have been evaluated and compared to those of rebeccamycin and parent non-aza and aza compounds. [Copyright &y& Elsevier]

Abstract: The syntheses of rebeccamycin analogues possessing a 7-azaindole moiety instead of an indole unit, and with both indole and azaindole moieties linked to the carbohydrate are described. In these bridged aza compounds, the oxygen of the pyranose heterocycle is oriented towards either the indole, or the azaindole unit. In these series, compounds bearing a free imide nitrogen were synthesized by coupling the corresponding aglycones with a sugar pre-tosylated in 2-position via a Mitsunobu reaction. To obtain a precursor for bridged aza-rebeccamycin analogues substituted in 6-position on the sugar moiety, a 2,6-ditosylated sugar was used. [Copyright &y& Elsevier]

Purpose: The rebeccamycins, indolocarbazole topoisomerase I poisons originally discovered in actinomycetes, have shown activity in vitro against a range of adult and pediatric tumors. The derivative NSC 655649 (diethylaminoethyl analog of rebeccamycin, or DEAE rebeccamycin) is currently undergoing early-phase human studies and has shown some signs of antitumor activity. We studied the plasma and cerebrospinal fluid (CSF) pharmacokinetics of NSC 655649 after systemic administration in a nonhuman primate model that is predictive of anticancer drug behavior in humans. Design: A dose of 400 mg/m² was infused over I h to three rhesus monkeys. Serial blood and CSF samples were collected. Rebeccamycin concentrations were measured by high-pressure liquid chromatography. Pharmacokinetic analysis was performed using compartmental and noncompartmental methods. Results: A two-compartment or three-compartment model described rebeccamycin pharmacokinetics in plasma adequately. In two animals, the three-compartment model provided a better fit, and in one animal, the two-compartment model was better. The terminal half-life was 730±410 mm, the AUC was 3130± 425 μM min, and the clearance was 190±25 ml/min/m². Rebeccamycin was below the limit of quantitation in all CSF samples The animals had some nausea and agitation during and shortly after the infusion that responded to treatment with prochlorperazine or diazepam. Otherwise, rebeccamycin was well tolerated with minimal toxicity. Conclusion: Rebeccamycin penetrates poorly into the CSF following an intravenous infusion. Therefore, systemically administered rebeccamycin is unlikely to be an important agent for the treatment of leptomeningeal tumors. Because the drug is associated with local irritation at injection sites, it is not an ideal candidate for development as an intrathecal agent. However, the role of rebeccamycin in the treatment of parenchymal brain tumors should be determined in clinical trials. [ABSTRACT FROM AUTHOR]

The synthesis of a new staurosporine analogue possessing a 7-azaindole unit instead of an indole moiety is described. This synthesis could be achieved by coupling a sugar moiety previously tosylated in 2′ position to the azaindolocarbazole aglycone. Nucleophilic substitution on the carbon bearing the tosyl group yielded to the key cyclization leading to a compound in which the carbohydrate part is linked to both indole and azaindole nitrogens. [Copyright &y& Elsevier]

Analogues of antifungal tjipanazoles were obtained by semi-synthesis from rebeccamycin, an antitumor antibiotic isolated from cultures of Saccharothrix aerocolonigenes. The antiproliferative activities of the new compounds were evaluated in vitro against nine tumor cell lines. The effect on the cell cycle of murine leukemia L1210 cells was examined and the antimicrobial activities against two Gram positive bacteria, a Gram negative bacterium and a yeast were determined. The inhibitory properties toward four kinases and toward topoisomerase I were evaluated. The most cytotoxic compound in the series was a dinitro derivative characterized as a potent topoisomerase I inhibitor. [Copyright &y& Elsevier]

Using blot hybridization, it has been shown that microorganisms producing halogen-containing antibiotics—Pseudomonas pyrrocinia, P. aureofaciens ACN, P. aureofaciens Pa1, P. fluorescens CHA0, Actinoplanes sp., Kitasatasporia sp., Sacharothrix aerocolonigenes, Actinomadura melliaura, and Streptomyces albogriseolus—contain the genes of the halogenating enzymes related to tryptophan-7-halogenase and monodechloroaminopyrrolnitrin halogenase from P. fluorescens BL 915. [ABSTRACT FROM AUTHOR]

The synthesis of a new family of isogranulatimides analogues is described in which a 7-azaindole replaces the indole moiety. The key step is the photocyclization of the aza didemnimide intermediates which leads to two isomeric analogues of isogranulatimides A and B. A derivative bearing a carbohydrate part linked to the azaindole via a β-N-glycosidic bond was also prepared. [Copyright &y& Elsevier]

Rebeccamycin, a microbial metabolite possessing a maleimide indolo[2,3-a]carbazole framework with a carbohydrate moiety attached to one of the indole nitrogens, is a well-known topoisomerase I inhibitor. This review reports the various total syntheses of rebeccamycin and structure–activity relationship studies on rebeccamycin analogues. Rebeccamycin analogues were prepared either by semi-synthesis from the natural metabolite or by total synthesis. Different families of rebeccamycin analogues were obtained by modifications at the imide heterocycle, dechlorination and substitutions on the indole moieties, modifications of the sugar residue, construction of dimers, coupling the sugar unit to the second indole nitrogen, changing indolo[2,3-a]carbazole skeleton to indolo[2,3-c]carbazole, replacing one or both indole moieties by 7-azaindole units. The biological activities of the rebeccamycin analogues are described. According to their chemical structure, the analogues can inhibit topoisomerase I and/or kinases. From the structure–activity relationships, some important rules were established. Several compounds exhibit stronger antiproliferative activities than the natural metabolite with IC50 values in the nanomolar range. Some analogues, especially those possessing azaindole moieties, are much more selective than rebeccamycin toward the tumour cell lines tested. [Copyright &y& Elsevier]

In recent years, several glycosyl indolocarbazole derivatives have been developed as antitumor agents targeting the topoisomerase I-DNA complex and a few of them were evaluated in clinical trials. The lead drug in the series is compound A which bears a formylamino substituent on the N-imide F-ring. This compound has shown promising antitumor activities in vivo and was tested clinically but it has been recently replaced with a more active analogue, J-107088, bearing a (hydroxymethyl-2-hydroxy) ethylamino substituent on the N-imide F-ring. We have compared the plasma stability of two molecules in this series, compounds A and D, which only differ by the nature of the group on the imide ring. The conversion of the compounds into the anhydride species B was studied by HPLC and the resulting metabolite, formed both in human plasma ultrafiltrate and in water, was characterized by NMR and mass spectrometry. Absorption measurements provided a facile method to follow the conversion of compounds A and D into their metabolite product B. Altogether, the experimental data demonstrate that the replacement of the NHCHO substituent of compound A with a hydrophilic NHCH(CH2OH)2 chain preserves the intact imide function that is known to be essential for topoisomerase I inhibition and cytotoxicity. The transformation of compound A into the anhydride metabolite B (or its diacid open form) occurs much more slowly compared to compound D. Half-life parameter t1/2 of 67 and 245 min−1 were calculated for compounds A and D, respectively. A molecular modeling analysis, performed to compare the conformation and electronic properties of compounds A and D, offers a rational explanation for the gain of chemical stability of the indolocarbazole derivative D. The data provide important information for the rational design of antitumor indolocarbazole derivatives. [Copyright &y& Elsevier]

In the course of structure–activity relationships on rebeccamycin analogues, two dimers of dechlorinated rebeccamycin were synthesised with the aim to improve the interaction with DNA and in vitro antiproliferative activities. The synthesis of two dimeric compounds obtained by joining two molecules of dechlorinated rebeccamycin via the imide nitrogen is described. Melting temperature and DNase I footprinting studies were performed to investigate their interaction with DNA. Four tumour cell lines, murine L1210 leukaemia, human HT29 colon carcinoma, A549 non-small cell lung carcinoma and K-562 leukaemia, were used to evaluate the cytotoxicity of the drugs. Their effects on the cell cycle of L1210 cells were also investigated. [ABSTRACT FROM AUTHOR]

Bacterial symbionts of fungus-growing ants occupy a highly specialized ecological niche and face the constant existential threat of displacement by another strain of ant-adapted bacteria. As part of a systematic study of the small molecules underlying this fraternal competition, we discovered an analog of the antitumor agent rebeccamycin, a member of the increasingly important indolocarbazole family. While several gene clusters consistent with this molecule's newly reported modification had previously been identified in metagenomic studies, the metabolite itself has been cryptic. The biosynthetic gene cluster for 9-methoxyrebeccamycin is encoded on a plasmid in a manner reminiscent of plasmidderived peptide antimicrobials that commonly mediate antagonism among closely related Gram-negative bacteria. [ABSTRACT FROM AUTHOR]