Enhancement of antibiotic and secondary metabolite detection from filamentous fungi by growth on nutritional arrays.

AIMS: We asked to what extent does the application of the OSMAC (one strain, many compounds) approach lead to enhanced detection of antibiotics and secondary metabolites in fungi? Protocols for bacterial microfermentations were adapted to grow fungi in nutritional arrays. METHODS AND RESULTS: Protocols for microfermentations of non-sporulating fungi were validated using known antifungal-producing fungi. Detection of antifungal activity was often medium dependent. The effects of medium arrays and numbers of strains on detection of antifungal signals were modelled by interpolation of rarefaction curves derived from matrices of positive and negative extracts. Increasing the number of fermentation media for any given strain increased the probability of detection of growth inhibition of Candida albicans. Increasing biodiversity increased detection of antifungal phenotypes, however, nutritional arrays could partly compensate for lost antibiotic phenotypes when biodiversity was limiting. CONCLUSIONS: Growth and extraction in microtiter plates can enable a discovery strategy emphasizing low-cost medium arrays that can better exploit the metabolic potential of strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing fermentation parameters raise the probability of detecting bioactive metabolites from strains. The protocols can be used to pre-select strains and their growth conditions for scale up that will most likely yield antibiotics and secondary metabolites.

High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease.

African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.

Enantiospecific semisynthesis of (+)-almuheptolide-A, a novel natural heptolide inhibitor of the mammalian mitochondrial respiratory chain.

The development of novel styryl lactone derivatives as bioactive compounds and the semisynthesis of both 4,5-dialkoxylated eight-membered-ring lactones with a heptolide skeleton (almuheptolide-A (1) type) and 7-alkoxylated delta-lactones with a saturated furanopyrone skeleton (etharvensin (8) type) have been successfully achieved from the chiral unsaturated alpha-pyrone altholactone (7). This new method is a direct and one-step enantiospecific alkoxylation of altholactone (7) in concentrated acid medium, followed by formation of the eight-membered-ring zeta-lactone. The reaction mechanism operating in the synthesis of the heptolide skeleton is postulated to be a direct Michael-type addition. Concerted opening of both the alpha-pyrone and tetrahydrofuran rings and subsequent intramolecular rearrangement with the ring closure lead to almuheptolide-A (1). This compound (1) and its diacetated derivative (1a) showed potent and selective inhibitory activity toward mammalian mitochondrial respiratory chain complex I. This mechanism of action, reported here for the first time, provides a possible explanation for the cytotoxic and antitumor activities previously described for related natural compounds.

Semisynthesis of antitumoral acetogenins: SAR of functionalized alkyl-chain bis-tetrahydrofuranic acetogenins, specific inhibitors of mitochondrial complex I.

The acetogenins of Annonaceae are known by their potent cytotoxic activity. In fact, they are promising candidates as a new future generation of antitumoral drugs to fight against the current chemiotherapic resistant tumors. The main target enzyme of these compounds is complex I (NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain, a key enzymatic complex of energy metabolism. In an attempt to characterize the relevant structural factor of the acetogenins that determines the inhibitory potency against this enzyme, we have prepared a series of bis-tetrahydrofuranic acetogenins with different functional groups along the alkyl chain. They comprise several oxo, hydroxylimino, mesylated, triazido, and acetylated derivatives from the head series compounds rolliniastatin-1, guanacone, and squamocin. Our results suggest a double binding point of acetogenins to the enzyme involving the alpha,alpha'-dihydroxylated tetrahydrofuranic system as well as the alkyl chain that links the terminal alpha, beta-unsaturated-gamma-methyl-gamma-lactone. The former mimics and competes with the ubiquinone substrate. The latter modulates the inhibitory potency following a complex outline in which multiple structural factors probably contribute to an appropriate conformation of the compound to penetrate inside complex I.

Specific interactions of monotetrahydrofuranic annonaceous acetogenins as inhibitors of mitochondrial complex I.

Annonaceous acetogenins (ACG) are a wide group of cytotoxic compounds isolated from plants of the Annonaceae family. Some of them are promising candidates to be a future new generation of antitumor drugs due to the ability to inhibit the NADH:ubiquinone oxidoreductase of the respiratory chain (mitochondrial complex I), main gate of the energy production in the cell. ACG are currently being tested on standard antitumor trials although little is known about the structure activity relationship at the molecular level. On recent studies, the relevance of several parts of the molecule for the inhibitory potency has been evaluated. Due to the great diversity of skeletons included in this family of natural products, previous studies on the presence and distribution of oxygenated groups along the alkyl chain only covered the compounds with different bis-tetrahydrofuranic (bis-THF) relative configurations. Therefore, we have investigated the inhibitory action of all the mono-tetrahydrofuranic (mono-THF) acetogenins available, which differ in the oxygenated arrangements along the molecule. Our results show that the hydroxyl and carbonyl groups, placed in the aliphatic chain that links the initial gamma-lactone moiety with the dihydroxylated tetrahydrofuranic ring system, significantly contribute for modulating the inhibitory potency of the ACG through specific effects.

[Crohn's disease and ascites]. / Enfermedad de Crohn y ascitis.

Crohn's disease is not usually included among the causes of ascites. Although cases of Crohn's disease presenting as ascites have occasionally been described, the appearance of ascites during follow-up, with no relationship with other complications of the disease, such as thrombosis of the suprahepatic veins, has not been previously described. We present the case of a 36-year-old female patient with Crohn's disease who presented ascites during evolution. Evacuative paracenteses were required. No causes apart from Crohn's disease were found. Ascites followed a parallel course to acute episodes of this disease.

[Esophageal Crohn's disease]. / Enfermedad de Crohn esofágica.

Crohn's disease is characterized by chronic inflammation of the digestive tract which may be localized at any level. Nonetheless, esophageal disease is infrequent. Two cases of esophageal involvement in this disease in 2 young women with a history of previous involvement at an intestinal level are presented. Esophageal involvement by Crohn's disease should be suspected in any patient previously diagnosed with this disease who presents symptoms suggestive of esophagitis, using endoscopy with multiples biopsies of the affected areas and other of normal endoscopic appearance for diagnosis.

The discovery of moriniafungin, a novel sordarin derivative produced by Morinia pestalozzioides.

A novel sordarin derivative, moriniafungin (1), containing a 2-hydroxysebacic acid residue linked to C-3' of the sordarose residue of sordarin through a 1,3-dioxolan-4-one ring was isolated from the fungus Morinia pestalozzioides. Isolation of moriniafungin employed a highly specific bioassay consisting of a panel of Saccharomyces cerevisiae strains containing chimeric eEF2 for Candida glabrata, Candida krusei, Candida lusitaniae, Crytpococcus neoformans, and Aspergillus fumigatus as well as wild type and human eEF2. Moriniafungin exhibited an MIC of 6 microg/mL versus Candida albicans and IC(50)'s ranging from 0.9 to 70 microg/mL against a panel of clinically relevant Candida strains. Moriniafungin was shown to inhibit in vitro translation in the chimeric S. cerevisae strains at levels consistent with the observed IC(50). Moriniafungin has the broadest antifungal spectrum and most potent activity of any natural sordarin analog identified to date.

New evidence for the multiplicity of ubiquinone- and inhibitor-binding sites in the mitochondrial complex I.

Determination of the number of ubiquinone- and inhibitor-binding sites in the mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a controversial question with a direct implication for elaborating a suitable model to explain the bioenergetic mechanism of this complicated enzyme. We have used combinations of both selective inhibitors and common ubiquinone-like substrates to demonstrate the multiplicity of the reaction centers in the complex I in contrast with competition studies that have suggested the existence of a unique binding site for ubiquinone. Our results provide new evidence for the existence of at least two freely exchangeable ubiquinone-binding sites with different specificity for substrates, as well as for a different kinetic interaction of inhibitors with the enzyme.

Cherimolin-1, new selective inhibitor of the first energy-coupling site of the NADH: ubiquinone oxidoreductase (complex I).

The mechanism linking electron transport to proton translocation in the NADH:ubiquinone oxidoreductase (complex I of the mitochondrial respiratory chain) is still unclear. Inhibitors acting at different sites of the enzyme are powerful tools to clarify this mechanism. Up to now, a unique inhibitor, the Annonaceous acetogenin rolliniastatin-2, selectively blocks the most internal proton-translocation site. This study introduces cherimolin-1, a new acetogenin that inhibits the complex I with this special mode of action, which is more easily available from the plant material. Moreover, the mode of action of this scarce type of complex I inhibitor is further characterized.

A deficiency in respiratory complex I in heart mitochondria from vitamin A-deficient rats is counteracted by an increase in coenzyme Q.

Defects of NADH:coenzyme Q oxidoreductase (complex I) of mitochondria have been described in many congenital and acquired diseases. Administration of coenzyme Q (CoQ, ubiquinone) has been shown to benefit patients with some of these diseases. However, the mechanisms by which CoQ exerts the therapeutic effects are not clearly understood. A reason could be the lack of saturation of CoQ, in kinetic terms, for complex I activity. However, this hypothesis has not been proved in vivo because of the difficulty to incorporate CoQ into the mitochondrial membranes. We have found a deficiency in respiratory complex I in heart mitochondria from vitamin A-deficient rats which was accompanied by high CoQ content. The defect in complex I activity was compensated by the increase in CoQ to maintain the mitochondrial electron transfer rate. This finding supports, for the first time in an in vivo experimental approach, the kinetic hypothesis to explain the short-term therapeutic effects of CoQ.

Synthesis of N-diisopropyl phosphoryl benzyltetrahydroisoquinoline, a new class of mitochondrial complexes I and III inhibitors.

The synthesis of N-(O,O-diisopropylphosphoryl)-benzyltetrahydroisoquinoline (3) has been achieved in a 'one pot' procedure from imine (2) and diisopropyl-phosphorochloridate (1) generated in situ (POCl3 + iPrOH). Compound 3 is the first benzyltetrahydroisoquinoline derivative found to be a potent inhibitor of mitochondrial complexes I and III, and therefore it opens a new perspective with this series of compounds as they can be considered as new class of antitumor agents.

Effects of vitamin A deficiency on mitochondrial function in rat liver and heart.

The aim of this study was to investigate comparative effects of vitamin A deficiency on respiratory activity and structural integrity in liver and heart mitochondria. Male rats were fed a liquid control diet (control rats) or a liquid vitamin A-deficient diet (vitamin A-deficient rats) for 50 days. One group of vitamin-A deficient rats was refed a control diet for 15 days (vitamin A-recovered rats). To assess the respiratory function of mitochondria the contents of coenzyme Q (ubiquinone, CoQ), cytochrome c and the activities of the whole electron transport chain and of each of its respiratory complexes were evaluated. Chronic vitamin A deficiency promoted a significant increase in the endogenous coenzyme Q content in liver and heart mitochondria when compared with control values. Vitamin A deficiency induced a decrease in the activity of complex I (NADH-CoQ reductase) and complex II (succinate-CoQ reductase) and in the levels of complex I and cytochrome c in heart mitochondria. However, NADH and succinate oxidation rates were maintained at the control levels due to an increase in the CoQ content in accordance with the kinetic behaviour of CoQ as an homogeneous pool. On the contrary, the high CoQ content did not affect the electron-transfer rate in liver mitochondria, whose integrity was preserved from the deleterious effects of the vitamin A deficiency. Ultrastructural assessment of liver and heart showed that vitamin A deficiency did not induce appreciable alterations in the morphology of their mitochondria. After refeeding the control diet, serum retinol, liver and heart CoQ content and the activity of complex I and complex II in heart mitochondria returned to normality. However, the activities of both whole electron transfer chain and complex I in liver were increased over the control values. The interrelationships between physiological antioxidants in biological membranes and the beneficial effects of their administration in mitochondrial diseases are discussed.

Polyalthidin: new prenylated benzopyran inhibitor of the mammalian mitochondrial respiratory chain.

Polyalthidin (3), a new benzopyran derivative, was isolated from the stem bark of Polyalthia cerasoides. Its structure was established on the basis of chemical and spectral evidence. Polyalthidin has showed potent biological activity as an inhibitor of the mammalian mitochondrial respiratory chain.

Epoxy-acetogenins and other polyketide epoxy derivatives as inhibitors of the mitochondrial respiratory chain complex I.

Annonaceous acetogenins (ACG), an extensive group of cytotoxic natural products, are antitumor agents whose main mode of action is inhibition of the mammalian mitochondrial complex I. Herein we describe the importance of the different chemical groups along the alkyl chain for optimal inhibitory potency, discussing the structurally relevant factors present in these compounds. For this purpose, a series of epoxide derivatives from alpha-linolenic acid were prepared and their activity compared with that of epoxy-acetogenins and tetrahydrofuranic (THF) acetogenins isolated from Rollinia membranacea.

Kinetic characterization of mitochondrial complex I inhibitors using annonaceous acetogenins.

The NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain is by far the largest and most complicated of the proton-translocating enzymes involved in the oxidative phosphorylation. Many clues regarding the electron pathways from matrix NADH to membrane ubiquinone and the links of this process with the translocation of protons are highly controversial. Different types of inhibitors become valuable tools to dissect the electron and proton pathways of this complex enzyme. Therefore, further knowledge of the mode of action of complex I inhibitors is needed to understand the underlying mechanism of energy conservation. This study presents for the first time a detailed exploration of the inhibitory action of the Annonaceous acetogenins, the most powerful inhibitors of the mammalian enzyme, taking as the head-series rolliniastatin-1, rolliniastatin-2, and corossolin. Despite their close chemical resemblance, each of them inhibits the complex I with different kinetic features reflecting differential binding to the enzyme.

10-Oximeguanacone, the first nitrogenated acetogenin derivative found to be a potent inhibitor of mitochondrial complex I.

A new 10-keto bis-tetrahydrofuran acetogenin, guanacone (1), has been isolated from a cytotoxic extract of Annona aff. spraguei seeds. The 10-oximeguanacone derivative 1f is the first bioactive nitrogenated acetogenin found to be a very potent inhibitor of complex I. In addition, a SAR study of guanacone analogues is reported based on the titration of the NADH oxidase and NADH:ubiquinone oxidoreductase activities.