Identification and study of actinomycete strains producers of a new family of spirotetronates

Motivation: New natural products are required to develop new antibiotics against the emergence of antibiotic-resistant pathogens. A family of three new spirotetronates with antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA) and Mycobacterium was identified in a Micromonospora strain from MEDINA culture collection. Interrogation of the Liquid chromatography–mass spectrometry (LC-MS) database with a LC-MS fingerprint of these compounds allowed the identification of 27 strains which potentially biosynthesize these family of compounds. The aims of this study are i) to confirm the production of the spirotetronates in the 27 strains; ii) identify best fermentation conditions to maximize their productions; iii) identify possible new derivatives of this spirotetronate family in any of the strains. Methods: The 27 strains were taxonomically identified by 16S rRNA and were subjected to fermentation in an array of 10 cultivation media. The resulting fermentation broths were extracted and the corresponding extracts analysed by LC-MS and subjected to High-throughput screening(HTS) against MRSA and Mycobacterium.Results: The analysis of the LC-MS data, together with the HTS data, allowed us to identify producers of the family of spirotetronates. Moreover, the best conditions (i.e fermentation medium) for their production were established. A potential new spirotetronate derivative was also identified based in the LC-MS data

Drug Discovery from Natural Products for Pancreatic Cancer

Since ancient times, natural products (NPs) have been used as anti-infectives, anti-inflammatories, antioxidants, analgesics and antitumorals and many compounds derived from NPs are in clinical use. The use of plants in traditional medicine for multiple purposes is well known, and throughout recent history, metabolites of microbial origin have had an extraordinary impact on the welfare of humanity. There is an outstanding diversity of chemical structures that nature, and especially microorganisms, are able to produce, due to millenniums of evolution. Since only a small amount of the world’s biodiversity has been evaluated for potential biological activity, many more useful natural lead compounds await discovery, the challenge being how to access this natural chemical diversity. However, the validation and selection of primary screening assays, both phenotypic and target-based, are vital to guaranteeing a selection of extracts or molecules with relevant pharmacological action. The screening of antitumor agents against pancreatic cancer (PC) involves the use of established cell lines, cancer stem cells and spheroids that mimic the patient’s tumor. Improvements in the discovery of natural products along with the emergence of new technologies in cancer screening assays, promise the discovery of new and valuable drugs to tackle pancreatic cancer in the coming years

Nuevos fármacos basados en productos naturales de origen microbiano

Nuevos fármacos basados en productos naturales de origen microbiano

Novel Biomarkers to Distinguish between Type 3c and Type 2 Diabetes Mellitus by Untargeted Metabolomics

Pancreatogenic diabetes mellitus (T3cDM) is a highly frequent complication of pancreatic disease, especially chronic pancreatitis, and it is often misdiagnosed as type 2 diabetes mellitus (T2DM). A correct diagnosis allows the appropriate treatment of these patients, improving their quality of life, and various technologies have been employed over recent years to search for specific biomarkers of each disease. The main aim of this metabolomic project was to find di erential metabolites between T3cDM and T2DM. Reverse-phase liquid chromatography coupled to high-resolution mass spectrometry was performed in serum samples from patients with T3cDM and T2DM. Multivariate Principal Component and Partial Least Squares-Discriminant analyses were employed to evaluate between-group variations. Univariate and multivariate analyses were used to identify potential candidates and the area under the receiver-operating characteristic (ROC) curve was calculated to evaluate their diagnostic value. A panel of five di erential metabolites obtained an area under the ROC curve of 0.946. In this study, we demonstrate the usefulness of untargeted metabolomics for the di erential diagnosis between T3cDM and T2DM and propose a panel of five metabolites that appear altered in the comparison between patients with these diseases.Junta de Andalucía PIN-0474-2016 PC-0549-2017 PC-0498-2017Instituto de Salud Carlos III DTS17/00081Fundación MEDINA, a public-private partnership of Merck Sharp and Dohme de España S.A./Universidad de Granada/Junta de Andalucía PIN-0474-201

Streptocyclinones A and B ameliorate Alzheimer's disease pathological processes in vitro

Alzheimer's disease (AD) is a pathology characterized by the abnormal accumulation of amyloid-beta (Aβ) and hyperphosphorylated tau. Oxidative stress and neuroinflammation are also strongly related to this disease. The ability of two new glycosylated angucyclinones, streptocyclinones A and B (1 and 2), isolated from Streptomyces sp to improve AD hallmarks was evaluated. Compounds were able to protect SH-SY5Y neuroblastoma cells from H2O2-induced oxidative injury by activating the nuclear factor E2-related factor (Nrf2). Their capacity to modulate neuroinflammation was tested in lipopolysaccharide-activated BV2 microglial cells. Compounds reduced the release of pro-inflammatory factors, inhibited the activation of NFκB and mitogen activated kinases (MAPK), and induced the translocation of Nrf2 to the nucleus of microglial cells. A trans-well co-culture was established to determine the effect of microglia treated with streptocyclinones on the survival of SH-SY5Y cells. The cell viability of neuroblastoma cells increased when the compounds were added to BV2 cells. SH-SY5Y-TMHT441 cells were used to determine the effect of compounds on tau phosphorylation. Both compounds reduced tau hyperphophorylation by targeting MAPK kinases. Moreover, streptocyclinone B (2) was able to inhibit the activity of β-secretase 1 and decrease the release of reactive oxygen species in BV2 cells stimulated with Aβ. With the same co-culture trans-well system, the treatment of Aβ-stimulated microglia with compound 2 augmented the viability of SH-SY5Y-TMHT441 cells. The results presented in this work provide evidences of the multitarget activities displayed by these new Streptomyces compounds, making them good candidates for further studies in the treatment of ADThe research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072. From European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, H2020 778069-EMERTOX and FP7 PharmaSea (Grant Agreement 312184).S

Caniferolide A, a Macrolide from Streptomyces caniferus, Attenuates Neuroinflammation, Oxidative Stress, Amyloid-Beta, and Tau Pathology in Vitro

The macrolide caniferolide A was isolated from extracts of a culture of the marine-derived actinomyceteStreptomyces caniferus, and its ability to ameliorate Alzheimer’s disease (AD) hallmarks was determined. The compound reducedneuroinflammatory markers in BV2 microglial cells activated with lipopolysaccharide (LPS), being able to block NFκB-p65translocation to the nucleus and to activate the Nrf2 pathway. It also produced a decrease in pro-inflammatory cytokines (IL-1β,IL-6, and TNF-α), reactive oxygen species (ROS) and nitric oxide release and inhibited iNOS, JNK, and p38 activities.Moreover, the compound blocked BACE1 activity and attenuated Aβ-activation of microglia by drastically diminishing ROSlevels. The phosphorylated state of the tau protein was evaluated in SH-SY5Y tau441 cells. Caniferolide A reduced Thr212 andSer214 phosphorylation by targeting p38 and JNK MAPK kinases. On the other side, the antioxidant properties of themacrolide were determined in an oxidative stress model with SH-SY5Y cells treated with H2O2. The compound diminishedROS levels and increased cell viability and GSH content by activating the nuclear factor Nrf2. Finally, the neuroprotectiveability of the compound was confirmed in two trans-well coculture systems with activated BV2 cells (both with LPS and Aβ)and wild type and transfected SH-SY5Y cells. The addition of caniferolide A to microglial cells produced a significant increase inthe survival of neuroblastoma in both cases. These results indicate that the compound is able to target many pathologicalmarkers of AD, suggesting that caniferolide A could be an interesting drug lead for a polypharmacological approach to theillnessThe research leading to these results has received funding from the following FEDER cofunded-grants, Consellería de Cultura, Educación e Ordenación Universitaria Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01); CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830, RTC-2016-5507-2, and ITC-20161072; and European Union POCTEP 0161-Nanoeaters-1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, H2020 778069-EMERTOX, and FP7 PharmaSea (Grant Agreement 312184).This document is the Accepted Manuscript version of a Published Work that appeared in final form in Molecular pharmaceutics, copyright © 2019 American Chemical Society, after peer review and technical editing by the publisher.S

A High-Throughput Screening Plattform of Microbial Natural Products for the Discovery of Moleculaes with Antibiofilm Properties against Salmonella

In this report, we describe a High-Throughput Screening (HTS) to identify compounds that inhibit biofilm formation or cause the disintegration of an already formed biofilm using the Salmonella Enteritidis 3934 strain. Initially, we developed a new methodology for growing Salmonella biofilms suitable for HTS platforms. The biomass associated with biofilm at the solid-liquid interface was quantified by staining both with resazurin and crystal violet, to detect living cells and total biofilm mass, respectively. For a pilot project, a subset of 1120 extracts from the Fundación MEDINA's collection was examined to identify molecules with antibiofilm activity. This is the first validated HTS assay of microbial natural product extracts which allows for the detection of four types of activities which are not mutually exclusive: inhibition of biofilm formation, detachment of the preformed biofilm and antimicrobial activity against planktonic cells or biofilm embedded cells. Currently, several extracts have been selected for further fractionation and purification of the active compounds. In one of the natural extracts patulin has been identified as a potent molecule with antimicrobial activity against both, planktonic cells and cells within the biofilm. These findings provide a proof of concept that the developed HTS can lead to the discovery of new natural compounds with antibiofilm activity against Salmonella and its possible use as an alternative to antimicrobial therapies and traditional disinfectants

Characterization of Actinomycetes Strains Isolated from the Intestinal Tract and Feces of the Larvae of the Longhorn Beetle Cerambyx welensii

[EN]Actinomycetes constitute a large group of Gram-positive bacteria present in different habitats. One of these habitats involves the association of these bacteria with insects. In this work, we have studied twenty-four actinomycetes strains isolated from the intestinal tract and feces from larvae of the xylophagous coleopteran Cerambyx welensii and have shown that seventeen strains present hydrolytic activity of some of the following substrates: cellulose, hemicellulose, starch and proteins. Fourteen of the isolates produce antimicrobial molecules against the Gram-positive bacteria Micrococcus luteus. Analysis of seven strains led us to identify the production of a wide number of compounds including streptanoate, alpiniamide A, alteramides A and B, coproporphyrin III, deferoxamine, demethylenenocardamine, dihydropicromycin, nocardamine, picromycin, surugamides A, B, C, D and E, tirandamycins A and B, and valinomycin. A significant number of other compounds, whose molecular formulae are not included in the Dictionary of Natural Products (DNP), were also present in the extracts analyzed, which opens up the possibility of identifying new active antibiotics. Molecular identification of ten of the isolated bacteria determined that six of them belong to the genus Streptomyces, two of them are included in the genus Amycolatopsis and two in the genus Nocardiopsis

Discovery of Pancreatic Adenocarcinoma Biomarkers by Untargeted Metabolomics

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers, with a 5-year survival rate of less than 5%. In fact, complete surgical resection remains the only curative treatment. However, fewer than 20% of patients are candidates for surgery at the time of presentation. Hence, there is a critical need to identify diagnostic biomarkers with potential clinical utility in this pathology. In this context, metabolomics could be a powerful tool to search for new robust biomarkers. Comparative metabolomic profiling was performed in serum samples from 59 unresectable PDAC patients and 60 healthy controls. Samples were analyzed by using an untargeted metabolomics workflow based on liquid chromatography, coupled to high-resolution mass spectrometry in positive and negative electrospray ionization modes. Univariate and multivariate analysis allowed the identification of potential candidates that were significantly altered in PDAC patients. A panel of nine candidates yielded excellent diagnostic capacities. Pathway analysis revealed four altered pathways in our patients. This study shows the potential of liquid chromatography coupled to high-resolution mass spectrometry as a diagnostic tool for PDAC. Furthermore, it identified novel robust biomarkers with excellent diagnostic capacities.This research was funded by JUNTA DE ANDALUCIA, grant number PIN-0474-2016 and PC-0549-2017 and INSTITUTO DE SALUD CARLOS III (FEDER), grant number DTS17/00081

Discovery of New Compounds Active against Plasmodium falciparum by High Throughput Screening of Microbial Natural Products

Due to the low structural diversity within the set of antimalarial drugs currently available in the clinic and the increasing number of cases of resistance, there is an urgent need to find new compounds with novel modes of action to treat the disease. Microbial natural products are characterized by their large diversity provided in terms of the chemical complexity of the compounds and the novelty of structures. Microbial natural products extracts have been underexplored in the search for new antiparasitic drugs and even more so in the discovery of new antimalarials. Our objective was to find new druggable natural products with antimalarial properties from the MEDINA natural products collection, one of the largest natural product libraries harboring more than 130,000 microbial extracts. In this work, we describe the optimization process and the results of a phenotypic high throughput screen (HTS) based on measurements of Plasmodium lactate dehydrogenase. A subset of more than 20,000 extracts from the MEDINA microbial products collection has been explored, leading to the discovery of 3 new compounds with antimalarial activity. In addition, we report on the novel antiplasmodial activity of 4 previously described natural productsThis work was supported by the Junta de Andalucía [BIO-199, P09-CVI- 5367], the VI Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011, Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET FIS Network: RD12/0018/0017),the Plan Nacional (SAF2013-48999-R), the FEDER funds from the EU and the PARAMET network (FP7-PEOPLE-2011-ITN. GA290080) to DG-P. Research of FV and OG was supported by the Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET FIS Network: RD12/0018/0005) and the FEDER funds from the EU and the PARAMET network (FP7-PEOPLE-2011-ITN. GA290080). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe