Functional Characterization of the GATA-Type Transcription Factor PaNsdD in the Filamentous Fungus Podospora anserina and Its Interplay with the Sterigmatocystin Pathway.

The model ascomycete Podospora anserina, distinguished by its strict sexual development, is a prolific but yet unexploited reservoir of natural products. The GATA-type transcription factor NsdD has been characterized by the role in balancing asexual and sexual reproduction and governing secondary metabolism in filamentous fungi. In the present study, we functionally investigated the NsdD ortholog PaNsdD in P. anserina. Compared to the wild-type strain, vegetative growth, ageing processes, sexual reproduction, stress tolerance, and interspecific confrontations in the mutant were drastically impaired, owing to the loss of function of PaNsdD. In addition, the production of 3-acetyl-4-methylpyrrole, a new metabolite identified in P. anserina in this study, was significantly inhibited in the ΔPaNsdD mutant. We also demonstrated the interplay of PaNsdD with the sterigmatocystin biosynthetic gene pathway, especially as the deletion of PaNsdD triggered the enhanced red-pink pigment biosynthesis that occurs only in the presence of the core polyketide synthase-encoding gene PaStcA of the sterigmatocystin pathway. Taken together, these results contribute to a better understanding of the global regulation mediated by PaNsdD in P. anserina, especially with regard to its unexpected involvement in the fungal ageing process and its interplay with the sterigmatocystin pathway. IMPORTANCE Fungal transcription factors play an essential role in coordinating multiple physiological processes. However, little is known about the functional characterization of transcription factors in the filamentous fungus Podospora anserina. In this study, a GATA-type regulator PaNsdD was investigated in P. anserina. The results showed that PaNsdD was a key factor that can control the fungal ageing process, vegetative growth, pigmentation, stress response, and interspecific confrontations and positively regulate the production of 3-acetyl-4-methylpyrrole. Meanwhile, a molecular interaction was implied between PaNsdD and the sterigmatocystin pathway. Overall, loss of function of PaNsdD seems to be highly disadvantageous for P. anserina, which relies on pure sexual reproduction in a limited life span. Therefore, PaNsdD is clearly indispensable for the survival and propagation of P. anserina in its complex ecological niches.

Cytotoxic compounds from the leaves and stems of the endemic Thai plant Mitrephora sirikitiae.

Context: Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders (Annonaceae) is a plant endemic to Thailand. Its constituents and their biological activities are unknown.Objective: Isolation and identification of the compounds in the leaves and stems of M. sirikitiae and determination of their cytotoxicity.Materials and methods: Methanol extracts of the leaves and stems of M. sirikitiae were separated by chromatography, and spectroscopic methods were used to determine the structures of the components. The cytotoxicity of the extracts and pure compounds was evaluated using the sulforhodamine B assay with several cell lines. The cells were treated with the compounds at concentrations of 0.16-20 µg/mL for 48 or 72 h.Results: The investigation of the extracts of M. sirikitiae leaves and stems resulted in the isolation of a new lignan, mitrephoran, and 15 known compounds. Among these compounds, 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, ciliaric acid, 6-methoxymarcanine A, and stepharanine were isolated from this genus for the first time. The alkaloids liriodenine and oxoputerine exhibited strong cytotoxicity against all tested cells (IC50 values of 6.59-11.02 µM). In contrast, magnone A, 3',4-O-dimethylcedrusin, and 6-methoxymarcanine A inhibited the growth of some of the tested cells (IC50 values of 2.03-19.73 µM). Magnone A and 6-methoxymarcanine A showed low toxicity for Hek 293 cells (IC50 >20 µM).Discussion and conclusions: M. sirikitiae is a source of cytotoxic lignans and alkaloids. Among the cytotoxic compounds, magnone A and 6-methoxymarcanine A are potentially useful lead compounds for the further development of anticancer agents because of their selective inhibitory effects on cancer cell lines.

Functional characterization of the sterigmatocystin secondary metabolite gene cluster in the filamentous fungus Podospora anserina: involvement in oxidative stress response, sexual development, pigmentation and interspecific competitions.

Filamentous fungi are known as prolific untapped reservoirs of diverse secondary metabolites, where genes required for their synthesis are organized in clusters. The bioactive properties of these compounds are closely related to their functions in fungal biology, which are not well understood. In this study, we focused on the Podospora anserina gene cluster responsible for the biosynthesis of sterigmatocystin (ST). Deletion of the PaStcA gene encoding the polyketide synthase and overexpression (OE) of the PaAflR gene encoding the ST-specific transcription factor in P. anserina were performed. We showed that growth of PaStcAΔ was inhibited in the presence of methylglyoxal, while OE-PaAflR showed a little inhibition, indicating that ST production may enhance oxidative stress tolerance in P. anserina. We also showed that the OE-PaAflR strain displayed an overpigmented thallus mediated by the melanin pathway. Overexpression of PaAflR also led to sterility. Interspecific confrontation assays showed that ST-overexpressed strains produced a high level of peroxides and possessed a higher competitiveness against other fungi. Comparative metabolite profiling demonstrated that PaStcAΔ strain was unable to produce ST, while OE-PaAflR displayed a ST overproduction. This study contributes to a better understanding of ST in P. anserina, especially with regard to its involvement in fungal physiology.

Complementary Strategies to Unlock Biosynthesis Gene Clusters Encoding Secondary Metabolites in the Filamentous Fungus Podospora anserina.

The coprophilous ascomycete Podospora anserina is known to have a high potential to synthesize a wide array of secondary metabolites (SMs). However, to date, the characterization of SMs in this species, as in other filamentous fungal species, is far less than expected by the functional prediction through genome mining, likely due to the inactivity of most SMs biosynthesis gene clusters (BGCs) under standard conditions. In this work, our main objective was to compare the global strategies usually used to deregulate SM gene clusters in P. anserina, including the variation of culture conditions and the modification of the chromatin state either by genetic manipulation or by chemical treatment, and to show the complementarity of the approaches between them. In this way, we showed that the metabolomics-driven comparative analysis unveils the unexpected diversity of metabolic changes in P. anserina and that the integrated strategies have a mutual complementary effect on the expression of the fungal metabolome. Then, our results demonstrate that metabolite production is significantly influenced by varied cultivation states and epigenetic modifications. We believe that the strategy described in this study will facilitate the discovery of fungal metabolites of interest and will improve the ability to prioritize the production of specific fungal SMs with an optimized treatment.

Influence of the stereoisomeric position of the reactive acetate groups of the benzo[b]acronycine derivative S23906-1 on its DNA alkylation, helix-opening, cytotoxic, and antitumor activities.

S23906-1 is a benzo[b]acronycine derivative acting as a DNA-alkylating agent through covalent bonding to the exocyclic amino group of guanines and subsequent local opening of the DNA helix. This compound was selected for phase I clinical trials based on its efficient antitumor activity in experimental models and its unique mode of action. S23906-1 is the racemate of cis-1,2-diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one. Here, we evaluated the cytotoxic and antitumor activities of the two pure cis-enantiomers and investigated the mechanism of action of both cis- and trans-racemates and their enantiomers in terms of DNA alkylation potency and locally drug-induced DNA helix opening process. Reaction with glutathione, as a detoxification process, was also studied. The trans-compounds, both as racemate or separated enantiomers, were found less potent than the corresponding cis-derivatives. Among the cis-enantiomers, the most efficient one regarding DNA alkylation bears the acetate on the reactive C1 position in the R configuration, both on purified DNA and genomic DNA extracted from cell cultures. By contrast, the most cytotoxic and tumor-active enantiomer bears the C1-acetate in the S configuration. Distinct cellular DNA-alkylation levels or covalent bonding to glutathione could not explain the differences. However, we showed that the S and R orientations of the acetate on C1 asymmetric carbon lead to different local opening of the DNA, as visualized using nuclease S1 mapping. These different interactions could lead to modulated DNA-repair, protein/DNA interaction, and apoptosis processes.

Synthesis, cytotoxic activity, and DNA binding properties of antitumor cis-1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine cinnamoyl esters.

Monocinnamoyl esters at position 2 of (+/-)-cis-1,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one and their acetyl derivatives at position 1 were prepared as stabilized analogues of the anticancer alkylating agent S23906-1. Monocinnamoyl esters at position 2 were slower DNA alkylators than the reference 2-monoacetate. Mixed esters bearing an acetyl ester group at position 1 and a cinnamoyl ester group at position 2 alkylated DNA slower than S23906-1. A strong correlation was observed between cytotoxicity and DNA alkylation kinetics, with slower alkylators displaying more potent antiproliferative activities. The most cytotoxic compounds proved to be significantly active in vivo against murine C-38 adenocarcinoma implanted in mice, but less potent than S23906-1.

Health risk associated with the oral consumption of "Chiniy-tref", a traditional medicinal preparation used in Martinique (French West Indies): Qualitative and quantitative analyses of aristolochic acids contained therein.

"Chiniy-tref" (CT) is a traditional preparation used in folk medicine in Martinique Island (French West Indies) that is nowadays mainly taken orally to prevent or act against any "manifestation of evil". CT is easily prepared at home by macerating larvae of the endemic swallowtail Battus polydamas (ssp.) cebriones (Dalman, 1823), sometimes accompanied by a leaf of its host-plant Aristolochia trilobata L., in commercial rum. We have previously reported the detection of nephrotoxic and carcinogenic aristolochic acids (AAs) I and II in CT, leading the Regional Health Agency (ARS) of Martinique to issue an alert regarding the potential risks associated with its consumption in 2015. In order to complete the toxicity risk assessment for oral consumption of CT, a full qualitative analysis of AAs and their analogues (AAAs) was performed, as well as a quantitative determination of the major AAs, namely AAs I and II. The phytochemical profiling of AAAs present in CT, that also corresponds to that of B. polydamas cebriones larvae feeding on A. trilobata, has been established for the first time by ultra-high performance liquid chromatography/electrospray ionization quadrupole time of-flight tandem mass spectrometry. AAs I and II were quantified in a small panel of tinctures by using a validated UHPLC/UV method, allowing us to estimate the probable daily intakes of these toxins by CT consumers. The results proved the existence of a real risk of renal toxicity and carcinogenicity associated with the chronic oral consumption of CT in Martinique, and more generally of similar "snake bottles" throughout the Caribbean.

Collected mass spectrometry data on monoterpene indole alkaloids from natural product chemistry research.

This Data Descriptor announces the submission to public repositories of the monoterpene indole alkaloid database (MIADB), a cumulative collection of 172 tandem mass spectrometry (MS/MS) spectra from multiple research projects conducted in eight natural product chemistry laboratories since the 1960s. All data have been annotated and organized to promote reuse by the community. Being a unique collection of these complex natural products, these data can be used to guide the dereplication and targeting of new related monoterpene indole alkaloids within complex mixtures when applying computer-based approaches, such as molecular networking. Each spectrum has its own accession number from CCMSLIB00004679916 to CCMSLIB00004680087 on the GNPS. The MIADB is available for download from MetaboLights under the identifier: MTBLS142 ( https://www.ebi.ac.uk/metabolights/MTBLS142 ).

Chiroptical study and absolute configuration of securinine oxidation products.

Time-dependant density functional theory-electronic circular dichroism spectra prediction was carried out to study the absolute configuration of phyllanthidine-type derivatives 5 and 6, derived from securinine (1) and its enantiomer virosecurinine (2), respectively. This method demonstrated to be very reliable in this alkaloid series. Thus, 5 and 6 shared the same stereochemistry as their parent precursors, confirming the retentive nature of the oxidation sequence. In addition, this study highlighted the key role of the methylene bridge (BC ring) in the chiroptical activity of these compounds. These results fully clarified the stereochemical relationships between the phyllanthidine and the securinine subgroups.

Design of novel antitumor DNA alkylating agents: the benzacronycine series.

Acronycine, a natural alkaloid originally extracted from the bark of the Australian ash scrub Acronychia baueri, has shown a significant antitumor activity in animal models. Acronycine has been tested against human cancers in the early 1980s, but the clinical trials showed modest therapeutic effects and its development was rapidly discontinued. In order to optimize the antineoplastic effect, different benzoacronycine derivatives were synthesized. Among those, the di-acetate compound S23906-1 was recently identified as a promising anticancer drug candidate and a novel alkylating agent specifically reacting with the exocylic 2-NH2 group of guanines in DNA. The study of DNA bonding capacity of acronycine derivatives leads to the identification of the structural requirements for DNA alkylation. In nearly all cases, the potent alkylating agents, such as S23906-1, were found to be much more cytotoxic than the unreactive analogs such as acronycine itself or diol derivatives. Alkylation of DNA by the monoacetate derivative S28687-1, which is a highly reactive hydrolysis metabolite of S23906-1, occurs with a marked preference for the N2 position of guanine. Other bionucleophiles can react with S23906-1. The benzacronycine derivatives, which efficiently alkylate DNA, also covalently bind to the tripeptide glutathione (GSH) but not to the oxidized product glutathione disulfide. Here we review the reactivity of S23906-1 and some derivatives toward DNA and GSH. The structure-activity relationships in the benzacronycine series validate the reaction mechanism implicating DNA as the main molecular target. S23906-1 stands as the most promising lead of a medicinal chemistry program aimed at discovering novel antitumor drugs based on the acronycine skeleton.

Structure-activity relationships and mechanism of action of antitumor benzo[b]pyrano[3,2-h]acridin-7-one acronycine analogues.

The cytotoxic and antitumor activities of cis-1,2-diacyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one derivatives 3, 6-9 were strongly correlated with their ability to give covalent adducts with purified, as well as genomic, DNA. Such adducts involve reaction between the exocyclic N-2 amino group of guanines exposed in the minor groove of double helical DNA and the leaving ester group at the benzylic position 1 of the drug. A transesterification process of the ester group from position 2 to position 1 in aqueous medium accounted for the intense activity of the cis-1-hydroxy-2-acyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one derivatives 10-13. Compounds without acyloxy or hydroxy group at position 1, such as 15, 17, 18, and 22, were inert with respect to DNA and almost devoid of significant cytotoxic activity. Condensation of 5-amino-2,2-dimethyl-2H-chromene (26) with 3-bromo-2-naphthoic acid (27), followed by cyclization, gave access to 6-demethoxy analogues. Diacetate 32 and cyclic carbonate 33, both belonging to the latter series, were less reactive toward DNA and less cytotoxic than their 6-methoxy counterparts 3 and 34. DNA alkylation appears thus to play an important role in the antitumor properties of benzo[b]pyrano[3,2-h]acridin-7-one derivatives.

Benzo[b]acronycine derivatives: a novel class of antitumor agents.

A hypothesis of bioactivation of the antitumor alkaloid acronycine by transformation of the 1,2-double bond into the corresponding epoxide in vivo and the suggestion that acronycine could interact with DNA, led to develop 1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine diesters (1,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one diesters) as new anticancer drug candidates. Compared to acronycine these compounds were markedly more potent, both in terms of cytotoxicity and antitumor activity. The biological activity of these compounds was strongly related with their ability to give covalent adducts with purified as well as genomic DNA. Formation of those adducts involves alkylation of the exocyclic N-2 amino groups of guanines exposed in the minor groove of double helical DNA by the carbocation produced by the elimination of the acyloxy leaving group at position 1 of the drug. A transesterification process of the ester group from position 2 to position 1 accounted for the intense activity of cis-1-hydroxy-2-acyloxy-1,2-dihydrobenzo[b]acronycine derivatives. Cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine, which displays a particularly impressive broad antitumor spectrum, is currently developed by Servier Laboratories under the code S23906-1.

Synthesis and cytotoxic activity of benzo[a]acronycine and benzo[b]acronycine substituted on the A ring.

The impact of substitutions at position 10 in the A ring of the cytotoxic benzo[a]acronycine and benzo[b]acronycine series has been explored. 10-Bromobenzo[a] and 10-bromobenzo[b]acronycine were prepared in 12% and 15% yield respectively from commercially available chemicals. Their 1,2-dihydro-1,2-dihydroxy diesters were synthesized. The different derivatives were tested against two cell lines KB-3-1 and L1210. Their cytotoxic activities were found in the same range of magnitude as their non-substituted counterparts. These structure-activity relationships permitted to conclude that the introduction of a substituent at position 10 maintains the activity in both the benzo[a] and [b]acronycine series and open the way to further pharmacomodulations.

Synthesis, biological activity, and evaluation of the mode of action of novel antitubercular benzofurobenzopyrans substituted on A ring.

The 8-, 9-, 10-, and 11-halo, hydroxy, and methoxy derivatives of the antimycobacterial 3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran were synthesized by condensation of the diazonium salts of 2-chloroanilines (13-17) with 1,4-benzoquinone (18), reduction of the intermediate phenylbenzoquinones 19-22 to dihydroxybiphenyls, cyclisation to halo-2-hydroxydibenzofurans 24-27, and construction of the pyran ring by thermal rearrangement of the corresponding dimethylpropargyl ethers 35-38. Palladium catalyzed nucleophilic aromatic substitution permitted conversion of the halo to the corresponding hydroxy derivatives which were methylated to methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran. All compounds substituted on the A ring were found more potent than the reference compound 1 against Mycobacterium bovis BCG and the virulent strain Mycobacterium tuberculosis H37Rv. The effect of the most active derivatives on mycolate synthesis was explored in order to confirm the preliminary hypothesis of an effect on mycobacterial cell wall biosynthesis. The linear 9-methoxy-2,2-dimethyl-2H-benzofuro[2,3-g][1]benzopyran (46) exhibiting a good antimycobacterial activity and devoid of cytotoxicity appeared to be the most promising compound.

Structure-activity relationships in the acronycine and benzo[b]acronycine series: role of the pyran ring.

In order to explore the structure-activity relationships in the acronycine series, simplified analogues of cis-1,2-diacetoxy-1,2-dihydroacronycine and cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine (S23906-1, under clinical trials) lacking the fused pyran ring, but possessing an acetoxymethyl leaving group at position 4 were prepared. These new analogues only displayed marginal antiproliferative activity compared to the parent compounds. The presence of the angularly fused dimethylpyran ring appears as an indispensable structural requirement to observe significant cytotoxic activity in this series.

Synthesis and cytotoxic activity of dimeric analogs of acronycine in the benzo[b]pyrano[3,2-h]acridin-7-one series.

Coupling of 6-hydroxy-3,3-14-trimethyl-3,14-dihydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (6) with alpha,omega-diiodoalkanes of varying length under alkaline conditions gave dimers 7-10. Halogenated ethers 11-14, cyclization products 15-17, and compounds 18-22 were also isolated in small yield from the reaction mixtures. Compounds 7-10 were more potent than acronycine and benzo[b]acronycine in inhibiting L1210 cell proliferation. The length of the alkyl ether linkage between the two benzopyranoacridone units had a dramatic influence on the cytotoxic activity. Compound 9 (n=5) was the most active, with an IC(50) value against L1210 cells within the same range of magnitude as diacetate 5, currently under clinical development.

Design, synthesis, and cytotoxic activity of Michael acceptors and enol esters in the benzo[b]acronycine series.

A series of 2-acyl-6-methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]pyrano[3,2-h]acridin-7-ones (4-6) was prepared by treatment of 6-methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (3) with an excess of an appropriate acyl chloride in the presence of aluminum chloride. Treatment of (+/-)-cis-1-hydroxy-2-acyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-ones (9, 10) or (+/-)-cis-1,2-diacyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-ones (2, 11) with hydrochloric acid gave the corresponding 2-acyloxy-6-methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]pyrano[3,2-h]acridin-7-ones, exemplified by acetate 7 and butyrate 8. None of the Michael acceptors 4-6 showed significant antiproliferative activity. Enol esters 7 and 8 were markedly cytotoxic toward L1210 leukemia cells, with IC50 values within the same range of magnitude as (+/-)-cis-1,2-diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (S23906-1), currently under phase I clinical trials. In contrast with S23906-1, enol esters 7 and 8 were not reactive toward purified DNA.

Synthesis and cytotoxic and antitumor activity of 1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine diacid hemiesters and carbamates.

A series of cis-1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine diacid hemiesters and dicarbamates were prepared by acylation of cis-1,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one. The cytotoxicity of the dicarbamates depended on the steric hindrance of the esterifying groups at positions 1 and 2. Diacid hemiesters displayed significant in vitro cytotoxic activities and induced cell cycle perturbations similar to those obtained with cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine (S23906-1) currently under preclinical development. cis-1-Acetoxy-2-hemiglutaryloxy-1,2-dihydrobenzo[b]acronycine was the most promizing compound of the series, inducing complete inhibition of tumor growth when tested against C38 colon adenocarcinoma implanted in mice.

A transesterification reaction is implicated in the covalent binding of benzo[b]acronycine anticancer agents with DNA and glutathion.

The benzo[b]acronycine derivative S23906-1 has been recently identified as a promising antitumor agent, showing remarkable in vivo activities against a panel of solid tumors. The anticancer activity is attributed to the capacity of the drug to alkylate DNA, selectively at the exocyclic 2-amino group of guanine residues. Hydrolysis of the C-1 and C-2 acetate groups of S23906-1 provides the diol compound S28907-1 which is inactive whereas the intermediate C-2 monoacetate derivative S28687-1 is both highly reactive toward DNA and cytotoxic. The reactivity of this later compound S28687-1 toward two bionucleophiles, DNA and the tripeptide glutathion, has been investigated by mass spectrometry to identify the nature of the (type II) covalent adducts characterized by the loss of the acetate group at position 2. On the basis of NMR and molecular modeling analyses, the reaction mechanism is explained by a transesterification process where the acetate leaving group is transferred from position C-2 to C-1. Altogether, the study validates the reaction scheme of benzo[b]acronycine derivative with its target.