Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis.

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Vincazalidine A, a unique bisindole alkaloid from Catharanthus roseus.

A new dimeric indole alkaloid, vincazalidine A consisting of an aspidosperma type and a modified iboga type with 1-azatricyclo ring system consisting of one azepane and two piperidine rings coupled with an oxazolidine ring was isolated from Catharanthus roseus, and the structure including absolute stereochemistry was elucidated on the basis of spectroscopic data as well as DP4 statistical analysis. Vincazalidine A induced G2 arrest and subsequent apoptosis in human lung carcinoma cell line, A549 cells.

A natural small molecule aspidosperma-type alkaloid, hecubine, as a new TREM2 activator for alleviating lipopolysaccharide-induced neuroinflammation in vitro and in vivo.

Neuroinflammation and oxidative stress play a crucial role in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease. The triggering receptor expressed on myeloid cells 2 (TREM2), highly expressed by microglia in the central nervous system (CNS), can modulate neuroinflammatory responses. Currently, there are no approved drugs specifically targeting TREM2 for CNS diseases. Aspidosperma alkaloids have shown potential as anti-inflammatory and neuroprotective agents. This study aimed to elucidate the potential therapeutic effect of Hecubine, a natural aspidosperma-type alkaloid, as a TREM2 activator in lipopolysaccharide (LPS)-stimulated neuroinflammation in in vitro and in vivo models. In this study, molecular docking and cellular thermal shift assay (CTSA) were employed to investigate the interaction between Hecubine and TREM2. Enzyme-linked immunosorbent assay (ELISA), quantitative PCR, immunofluorescence, Western blotting, and shRNA gene knockdown were used to assess the anti-neuroinflammatory and antioxidant effects of Hecubine in microglial cells and zebrafish. Our results revealed that Hecubine directly interacted with TREM2, leading to its activation. Knockdown of TREM2 mRNA expression significantly abolished the anti-inflammatory and antioxidant effects of Hecubine on LPS-stimulated proinflammatory mediators (NO, TNF-α, IL-6, and IL-1ß) and oxidative stress in microglia cells. Furthermore, Hecubine upregulated Nrf2 expression levels while downregulating TLR4 signaling expression levels both in vivo and in vitro. Silencing TREM2 upregulated TLR4 and downregulated Nrf2 signaling pathways, mimicking the effect of Hecubine, further supporting TREM2 as the drug target by which Hecubine inhibits neuroinflammation. In conclusion, this is the first study to identify a small molecule, namely Hecubine directly targeting TREM2 to mediate anti-neuroinflammation and anti-oxidative effects, which serves as a potential therapeutic agent for the treatment of neural inflammation-associated CNS diseases.

Antitumor Activity of New Olivacine Derivatives.

Olivacine is an alkaloid-containing pyridocarbazole structure. It is isolated from the bark of the evergreen timber tree, Aspidosperma olivaceum. Its well-documented anticancer activity led to the synthesis of new derivatives, which are semisynthetic and fully synthetic pyridocarbazoles. This study aimed to evaluate the potential antineoplastic activity of four newly synthesized olivacine derivatives. Multidrug resistance is a common phenomenon causing failure in the chemotherapy of many tumors. It is mainly related to increased function of P-glycoprotein, an efflux pump removing cytostatic out of the cells. The cell lines used in the study were colorectal carcinoma cell lines: LoVo (doxorubicin-sensitive) and LoVo/DX (doxorubicin-resistant). The NHDF cell line was used to assess cell viability. First, the cells were incubated with olivacine derivatives. In the next step, the following assays were performed: DCF-DA assay, MTT assay, rhodamine 123 assay, detection of apoptosis, proliferation inhibition-mitotic index. The tested compounds showed higher antineoplastic potential and lower toxicity than the reference compound ellipticine. The results indicate that the new olivacine derivatives are good candidates for future anticancer drugs.

Aspidosperma species: A review of their chemistry and biological activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of Aspidosperma are known popularly as "peroba, guatambu, carapanaúba, pau-pereiro" and "quina". The genus can be found in the Americas, mainly between Mexico and Argentina. Many species of Aspidosperma are used by the population in treating cardiovascular diseases, malaria, fever, diabetes and rheumatism. The phytochemical aspects of the species of the genus Aspidosperma have been studied extensively. The monoterpene indole alkaloids are the main secondary metabolites in Aspidosperma species, and about 250 of them have been isolated showing a considerable structural diversity. Several of them have showed some important pharmacological activities. Aspidosperma subincanum Mart. and Aspidosperma tomentosum Mart. (Apocynaceae) are Brazilian species widely used by the population to treat diabetes mellitus, hypercholesterolemia. The pharmacological activities of both species have been investigated and the biological properties described can be related to their isolated indole alkaloids. However, more pharmacological studies are needed in order to justify the use of these species in folk medicine. In this review, we present reports mainly focused on chemical and biological studies and their relationship with the ethnopharmacological use of both Aspidosperma species. AIM OF THE STUDY: The aim of this review is to present their ethnopharmacological use as correlated to their biological activities as described for the extracts and isolated compounds from Aspidosperma subincanum Mart. and Aspidosperma tomentosum Mart. In addition, some aspects related to the biosynthetic pathways are discussed, also NMR assignments and some synthesis information about indole alkaloids from both Aspidosperma species are included. MATERIAL AND METHODS: The bibliographic search was made in theses and dissertations using some databases such as NDLTD (Networked Digital Library of Theses and Dissertations), OATD (Open Access Theses and Dissertations) and Google Scholar. More data were gathered from books, Brazilian journals and articles available on electronic databases such as, Google Scholar, PubChem, Scifinder, Web of Science, SciELO, PubMed and Science Direct. Additionally, the Google Patents and Espacenet Patent Search (EPO) were also consulted. The keywords Aspidosperma, A. subincanum, A. tomentosum, indole alkaloids were used in the research. The languages were restricted to Portuguese, English and Spanish and references were selected according to their relevance. RESULTS: A. subincanum Mart. and A. tomentosum Mart. (Apocynaceae) are Brazilian species widely used by the population to treat a few diseases. Extracts and isolated compounds of both species have shown antitumor and antimalarial activities. The antitumor activity of isolated compounds has been extensively studied. However, the antiplasmodial activity needs to be investigated further as well as the anti-inflammatory, anti-hyperlipidemic and anorexigenic activities. From A. subincanum twenty-one indole alkaloids were isolated and some of them have been extensively studied. From the leaves and bark of A. tomentosum four alkaloids and one flavonoid were isolated. Furthermore, CG-MS analysis of seeds, branches, leaves and arils identified nine indole alkaloids. Stemmadenine has been proposed as a precursor of indole alkaloids obtained from some species of Aspidosperma. Many of the biosynthetic steps have been characterized at the enzymatic level and appropriate genes have been identified, however, other steps have yet to be investigated and they are still controversial. Some isolated alkaloids from A. subincanum and A. tomentosum were identified only by mass spectrometry. In many cases, their NMR data was either not available or was incomplete. The described meta-analysis of the available NMR data revealed that the chemical shifts belonging to the indole ring might be used to characterize this class of alkaloids within complex matrices such as plant extracts. The biological activities and the structural complexity of these compounds have stimulated the interest of many groups into their synthesis. In this review, some information about the synthesis of indole alkaloids and their derivatives was presented. CONCLUSIONS: A. subincanum and A. tomentosum are used by the population of Brazil to treat many diseases. A few biological activities described for the extracts and isolated compounds of both species are in agreement with the ethnopharmacological use for others species of Aspidosperma, such as, antimalarial, the treatment of diabetes and other illnesses. These species are sources of leading compounds which can be used for developing new drugs. In addition, other biological activities reported and suggested by ethnopharmacological data have yet to be investigated and could be an interesting area in the search for new bioactive compounds.

Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidosperma monoterpenoid indole alkaloids from 19E-geissoschizine.

Monoterpenoid indole alkaloids (MIAs) possess a diversity of alkaloid skeletons whose biosynthesis is poorly understood. A bioinformatic search of candidate genes, combined with their virus-induced gene silencing, targeted MIA profiling and in vitro/in vivo pathway reconstitution identified and functionally characterized six genes as well as a seventh enzyme reaction required for the conversion of 19E-geissoschizine to tabersonine and catharanthine. The involvement of pathway intermediates in the formation of four MIA skeletons is described, and the role of stemmadenine-O-acetylation in providing necessary reactive substrates for the formation of iboga and aspidosperma MIAs is described. The results enable the assembly of complex dimeric MIAs used in cancer chemotherapy and open the way to production of many other biologically active MIAs that are not easily available from nature.

Discovery of a P450-catalyzed step in vindoline biosynthesis: a link between the aspidosperma and eburnamine alkaloids.

Here we report the discovery of a cytochrome P450 that is required for the biosynthesis of vindoline, a plant-derived natural product used for semi-synthesis of several anti-cancer drugs. This enzyme catalyzes the formation of an epoxide that can undergo rearrangement to yield the vincamine-eburnamine backbone, thereby providing evidence for the long-standing hypothesis that the aspidosperma- and eburnamine-type alkaloids are biosynthetically related.

Germinação de sementes de Aspidosperma subincanumMart. ex A. DC em diferentes temperaturas / Germination of Aspidosperma subincanum Mart. ex A. DC seeds at different temperatures

RESUMO : Aspidosperma subincanum é uma espécie arbórea utilizada em projetos de recuperação de áreas degradadas e como planta medicinal. Levando-se em consideração a importância da espécie, o presente trabalho teve como objetivo definir a temperatura ideal para a germinação de suas sementes. O material vegetal foi coletado na região do Pantanal de Miranda, Mato Grosso do Sul. As sementes foram colocadas em caixas plásticas transparentes em câmara de germinação e submetidas às temperaturas constantes de 20, 25, 30 e 35°C e alternadas de 20-30 e 25-35°C. O delineamento experimental foi inteiramente casualizado, com quatro repetições por tratamento, sendo as médias comparadas pelo teste de Tukey a 5%. A temperatura ideal para a germinação de Aspidosperma subincanum foi a 25°C, com o maior índice de velocidade de germinação, 18,8. Temperaturas mais elevadas (30 e 35°C) causaram a morte dos embriões

ABSTRACT Germination of Aspidosperma subincanum Mart. ex A. DC seeds at different temperatures. The Aspidosperma subincanumem is a tree species used in the recovery of degraded areas projects and as a medicinal plant. Taking into account the importance of the species, the current study aimed to determine which would be the ideal temperature for the germination of their seeds. The plant material was collected in the Pantanal of Miranda, Mato Grosso do Sul. The seeds were placed in transparent boxes in germination chambers and submitted to constant temperatures of, 20, 25, 30 and 35°C and alternated at 20-30 and 25- 35°C, with four replicates per treatment. The statistical design was a completely randomized one. The averages were compared by Tukey test at 5%. The ideal temperature for germination of Aspidosperma subincanum was 25°C. The high temperatures (30 and 35°C) have caused the death of embryos

Is the oxidative stress caused by Aspidosperma spp. galls capable of altering leaf photosynthesis?

The generation of ROS (reactive oxygen species) in plant galls may induce the degradation of the membrane systems of a plant cell and increase the number of plastoglobules. This numerical increase has been related to the prevention of damage to the thylakoid systems, and to the maintenance of photosynthesis rates. To investigate this hypothesis in gall systems, a comparative study of the ultrastructure of chloroplasts in non-galled leaves and in leaf galls of A. australe and A. spruceanum was conducted. Also, the pigment composition and the photosynthetic performance as estimated by chlorophyll fluorescence measurements were evaluated. The ultrastructural analyses revealed an increase in the number and size of plastoglobules in galls of both species studied. The levels of total chlorophylls and carotenoids were lower in galls than in non-galled tissues. The chlorophyll a/b ratio did not differ between the non-galled tissues and both kinds of galls. The values of maximum electron transport rate (ETR(MAX)) were similar for all the samples. The occurrence of numerous large plastoglobules in the galled tissues seemed to be related to oxidative stress and to the recovery of the thylakoid membrane systems. The maintenance of the ETR(MAX) values indicated the existence of an efficient strategy to maintain similar photosynthetic rates in galled and non-galled tissues.

Quantification of metabolic flux in plant secondary metabolism by a biogenetic organizational approach.

Metabolic engineering represents a promising approach to enhance the yield of valuable natural products from plants. A method to quantify flux through metabolite measurements is necessary for the analysis of native and modified pathways. Rather than focusing only on the accumulation of the final products, analyzing a wide range of secondary metabolites has significant advantages. We propose a model that organizes the flux analysis by grouping metabolites of similar biosynthetic origin. To this end, we have quantified temporal profiles of metabolites from several branches of the indole alkaloid pathway in Catharanthus roseus hairy root cultures. By analyzing these data, we are able to examine the distribution of flux around key branchpoints. Furthermore, this analysis provides crucial information such as an estimate of total flux to secondary metabolism.