Advances in decomposing complex metabolite mixtures using substructure- and network-based computational metabolomics approaches

Covering: up to the end of 2020 Recently introduced computational metabolome mining tools have started to positively impact the chemical and biological interpretation of untargeted metabolomics analyses. We believe that these current advances make it possible to start decomposing complex metabolite mixtures into substructure and chemical class information, thereby supporting pivotal tasks in metabolomics analysis including metabolite annotation, the comparison of metabolic profiles, and network analyses. In this review, we highlight and explain key tools and emerging strategies covering 2015 up to the end of 2020. The majority of these tools aim at processing and analyzing liquid chromatography coupled to mass spectrometry fragmentation data. We start with defining what substructures are, how they relate to molecular fingerprints, and how recognizing them helps to decompose complex mixtures. We continue with chemical classes that are based on the presence or absence of particular molecular scaffolds and/or functional groups and are thus intrinsically related to substructures. We discuss novel tools to mine substructures, annotate chemical compound classes, and create mass spectral networks from metabolomics data and demonstrate them using two case studies. We also review and speculate about the opportunities that NMR spectroscopy-based metabolome mining of complex metabolite mixtures offers to discover substructures and chemical classes. Finally, we will describe the main benefits and limitations of the current tools and strategies that rely on them, and our vision on how this exciting field can develop toward repository-scale-sized metabolomics analyses. Complementary sources of structural information from genomics analyses and well-curated taxonomic records are also discussed. Many research fields such as natural products discovery, pharmacokinetic and drug metabolism studies, and environmental metabolomics increasingly rely on untargeted metabolomics to gain biochemical and biological insights. The here described technical advances will benefit all those metabolomics disciplines by transforming spectral data into knowledge that can answer biological questions

Using of techniques of mass spectrometry in the analysis of natural compounds

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Bc. Alžběta Vopelková Supervisor: doc. Ing. Lucie Cahlíková, Ph.D. Title of diploma thesis: Using of techniques of mass spectrometry in the analysis of natural compounds This diploma thesis deals with the topic " Using of techniques of mass spectrometry in the analysis of natural compounds ". The aim of the work was to create a research work and describe the profile of alkaloids in individual genera of the family Amaryllidaceae. The introduction to the theoretical part summarizes the analyzes gas chromatography (GC). Alkaloids have been classified according to their heterocyclic parts and the family Amaryllidaceae has been described. The work was based on professional texts by Czech and foreign authors. Used sources focused on the analysis of alkaloids in bulbs, or in the aboveground parts of plants in this family, using gas chromatography with mass detection (GC-MS). Key words: mass spectometry, analysis, natural compoundsUniverzita Karlova v Praze Farmaceutická fakulta v Hradci Králové Katedra farmaceutické botaniky a ekologie Kandidát: Bc. Alžběta Vopelková Školitel: doc. Ing. Lucie Cahlíková, Ph.D. Název diplomové práce: Využití technik hmotnostní spektrometrie v analýze přírodních látek Tato diplomová práce se zabývá tématem "Využití technik hmotnostní spektrometrie v analýze přírodních látek". Cílem práce bylo vytvořit rešeršní práci a přiblížit profil alkaloidů u různých rodů čeledi Amaryllidaceae. V úvodu teoretické části je rozebrána plynová chromatografie (GC). Jsou zde klasifikovány alkaloidy podle svých heterocyklických částí a v neposlední řadě je popsána čeleď Amaryllidaceae. Práce vycházela z odborných textů českých i zahraničních autorů, které se zaměřovaly na analýzu alkaloidů v cibulích, nebo v nadzemních částech rostlin u této čeledi, pomocí plynové chromatografie s hmotnostní detekcí (GC-MS). Klíčová slova: hmotnostní spektrometrie, analýza, přírodní látkyDepartment of Pharmaceutical BotanyKatedra farmaceutické botanikyFarmaceutická fakulta v Hradci KrálovéFaculty of Pharmacy in Hradec Králov

Natural Product Genomics and Metabolomics of Marine Bacteria

Marine organisms are a treasure trove for the discovery of novel natural products, and, thus, marine natural products have been a focus of interest for researchers for decades. Some marine bacteria are prolific producers of natural products, occurring either free-living or, as recently shown, in symbiosis with marine animals. Recent advances in DNA sequencing have led to an enormous increase in published bacterial genomes and bioinformatics tools to analyze natural product biosynthetic potential by various “genome mining” approaches. Similarly, analytical NMR and MS methods for the characterization and comparison of metabolomes of natural product producers have advanced. Novel interdisciplinary approaches combine genomics and metabolomics data for accelerated and targeted natural product discovery. This Special Issue invites articles from both genomics- and metabolomics-driven studies on marine bacteria with a focus on natural product discovery and characterization. We particularly welcome articles that combine genomics and metabolomic approaches for the dereplication and characterization of marine bacterial natural products

Cheminformatics Tools to Explore the Chemical Space of Peptides and Natural Products

Cheminformatics facilitates the analysis, storage, and collection of large quantities of chemical data, such as molecular structures and molecules' properties and biological activity, and it has revolutionized medicinal chemistry for small molecules. However, its application to larger molecules is still underrepresented. This thesis work attempts to fill this gap and extend the cheminformatics approach towards large molecules and peptides. This thesis is divided into two parts. The first part presents the implementation and application of two new molecular descriptors: macromolecule extended atom pair fingerprint (MXFP) and MinHashed atom pair fingerprint of radius 2 (MAP4). MXFP is an atom pair fingerprint suitable for large molecules, and here, it is used to explore the chemical space of non-Lipinski molecules within the widely used PubChem and ChEMBL databases. MAP4 is a MinHashed hybrid of substructure and atom pair fingerprints suitable for encoding small and large molecules. MAP4 is first benchmarked against commonly used atom pairs and substructure fingerprints, and then it is used to investigate the chemical space of microbial and plants natural products with the aid of machine learning and chemical space mapping. The second part of the thesis focuses on peptides, and it is introduced by a review chapter on approaches to discover novel peptide structures and describing the known peptide chemical space. Then, a genetic algorithm that uses MXFP in its fitness function is described and challenged to generate peptide analogs of peptidic or non-peptidic queries. Finally, supervised and unsupervised machine learning is used to generate novel antimicrobial and non-hemolytic peptide sequences

Study of bacterial interactions using comparative metabolomics for accelerared antibiotic discovery

Bacteria produce approximately 70% of microbial natural products with the bacterial class Actinobacteria being the major producer. They can contain over 30 Biosynthetic Gene Clusters (BGCs) encoding specialised metabolites. However, only a portion (up to 10%) of these BGCs are transcribed under normal laboratory conditions. Interspecies interactions play a role in the elicitation of specialised metabolites. Metabolites are often produced as a defence mechanism to kill or communicate with other strains. Therefore, co-culture techniques represent a potential method to elicit specialised metabolites that are not produced under mono-culture. In order to understand the chemical exchange between strains, the impact of bacterial interactions was assessed on the strains’ ability to produce specialised metabolites resulting in altered phenotypes. The study comprised a total of 51 strains (48 Actinobacteria, two Pseudomonas and one Bacillus) across 72 tri-cultures (three strains) and 63 one-to-one cultures (two strains). Four co-culture techniques were used; tri-cultures, one-to-one cultures, pre-conditioned media and mixed fermentations. A total of 21 tri-cultures and 40 one-to-one cultures resulted in altered phenotypes as a result of bacterial interactions. Pre-conditioned media revealed that specialised metabolites were responsible for these alterations. The antibacterial screening showed that seven bioactive strains displayed larger inhibition zones under co-culture. The LC/MS-based metabolomics of five strains’ mono-culture, six one-to-one cultures and two tri-cultures extracts revealed the production of interaction-specific metabolites. One interaction was subjected to Imaging Mass Spectrometry (IMS) in collaboration with CMAC and the National Physical Laboratory (NPL). The results demonstrated that bacterial interactions increase chemical diversity and that mass spectrometry-based comparative metabolomics represent an exciting strategy to prioritise novel chemistry.Bacteria produce approximately 70% of microbial natural products with the bacterial class Actinobacteria being the major producer. They can contain over 30 Biosynthetic Gene Clusters (BGCs) encoding specialised metabolites. However, only a portion (up to 10%) of these BGCs are transcribed under normal laboratory conditions. Interspecies interactions play a role in the elicitation of specialised metabolites. Metabolites are often produced as a defence mechanism to kill or communicate with other strains. Therefore, co-culture techniques represent a potential method to elicit specialised metabolites that are not produced under mono-culture. In order to understand the chemical exchange between strains, the impact of bacterial interactions was assessed on the strains’ ability to produce specialised metabolites resulting in altered phenotypes. The study comprised a total of 51 strains (48 Actinobacteria, two Pseudomonas and one Bacillus) across 72 tri-cultures (three strains) and 63 one-to-one cultures (two strains). Four co-culture techniques were used; tri-cultures, one-to-one cultures, pre-conditioned media and mixed fermentations. A total of 21 tri-cultures and 40 one-to-one cultures resulted in altered phenotypes as a result of bacterial interactions. Pre-conditioned media revealed that specialised metabolites were responsible for these alterations. The antibacterial screening showed that seven bioactive strains displayed larger inhibition zones under co-culture. The LC/MS-based metabolomics of five strains’ mono-culture, six one-to-one cultures and two tri-cultures extracts revealed the production of interaction-specific metabolites. One interaction was subjected to Imaging Mass Spectrometry (IMS) in collaboration with CMAC and the National Physical Laboratory (NPL). The results demonstrated that bacterial interactions increase chemical diversity and that mass spectrometry-based comparative metabolomics represent an exciting strategy to prioritise novel chemistry

Marine Microbial Diversity as Source of Bioactive Compounds

Over 70% of the Earth’s surface is covered by oceans and seas, which are massively complex and consist of diverse assemblages of life forms. Marine bacteria, fungi, and other microorganisms develop unique metabolic and physiological capabilities that enable them to survive in extreme habitats and to produce compounds that might not be produced by their terrestrial counterparts. In the last few decades, the systematic investigations of marine/marine-derived microorganisms as sources of novel biologically active agents has exponentially increased. This Special Issue will focus on aspects relating to new bioactive metabolites from marine microorganisms including the isolation, taxonomy, and/or dereplication of microorganisms and the corresponding isolation, structure elucidation, biosynthesis, and/or biological activities of the new compounds. Comprehensive topical review articles relating to marine metabolites will also be considered

A phylogeny-based comparative study of the phytochemical and pharmacological characteristics of Croton species occurring in KwaZulu-Natal.

Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.Ethnobotanical enquiries often lead to the discovery of phytocompounds with pharmacological activities. Against this background a comparative and quantitative evaluation of the phytochemical and antioxidant activity of extracts from six Croton species; C. gratissimus Burch., C. sylvaticus Hochst., C. menyhartii Pax, C. pseudopulchellus Pax, C. steenkampianus Gerstner and C. rivularis Müll.Arg., all collected from KwaZulu-Natal (KZN), South Africa, was conducted. The analysis included a comparison of the different plant organs to explore the possibility of using leaves rather than bark for medicinal purposes. The latter would result in less destructive harvesting and would contribute to sustainable use of these medicinal plant resources. Extraction of the different plants and their organs were done in water and, in different organics solvents, including methanol (MeOH), dichloromethane (DCM), and petroleum ether (PE). The extracts were screened for antibacterial and antifungal activities using the microdilution technique. All of the tested plant samples showed some notable antibacterial activity in one or two of their organs, except for C. rivularis, which was the only species in the list that had no record of medicinal use. The most potent antibacterial activity was exhibited by the dichloromethane (DCM) extracts of C. steenkampianus leaves and the petroleum ether (PE) extracts of C. pseudopulchellus stem bark, both sampled from the Durban Botanic Gardens, which yielded a minimum inhibition concentration (MIC) value of 0.04 mg/ml against Enterococcus faecalis (E. faecalis). The DCM stem bark and leaf extracts, as well as the PE twig extracts of C. pseudopulchellus, (Durban Botanic Gardens) also exhibited noteworthy activities against S. aureus (MIC value of 0.08 mg/ml). A broad spectrum of activity was observed in the DCM and PE twig extracts of C. sylvaticus collected from Umdoni Park, with a MIC ranging from 0.31-0.94 mg/ml. This activity was against E. faecalis, Staphylococcus aureus (S. aureus) and Klebsiella pneumoniae (K. pneunomiae). Noteworthy antifungal activity against Candida albicans was only observed in one extract, the MeOH leaf extract of C steenkampianus collected at Kosi Bay. The MIC of this extract was 0.6 mg/ml. Water extracts did not show any antimicrobial activity. The results of the pharmacological study suggested that the aerial plant organs, such as the leaves and the twigs, could replace bark as they exhibited significant antimicrobial activity when compared to the preferred bark. The study also revealed that the same species collected from different regions may not necessarily exhibit similar biological activities, as pharmacological activities are as a result of the phytochemicals present in the plant, which are triggered by environmental stimuli. The phenolic profiles of aqueous (50%) methanol extracts obtained from the plants were assessed using the Folin and Ciocalteu (Folin C), butanol-hydrochloric acid and aluminium chloride assays. Aqueous (50%) methanol extracts were also run on thin layer chromatography plates and the plates were later stained with the Dragendorff reagent to determine the possible presence of alkaloids. Antioxidant activity was determined with two different assays, β-carotene /Linoleic model system and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay. The overall EC50 values of the different Croton species displayed by the DPPH assay ranged from 1.76 to 5.35 μg/ml. The β-carotene /Linoleic model system displayed antioxidant activity that ranged between 48.66 to 81.97%. In the phytochemical study; the leaf extracts of C. pseudopulchellus from Mkuze exhibiting the highest phenolic content at 23.8±1.1 mg GAE/g Dry Weight. The highest condensed tannin content was from the leaf extracts of C. gratissimus from Southport at a concentration of 31.3± 0.1 mg CCE/g Dry Weight. The highest flavonoid content observed in the leaf extracts of C. gratissimus from Southport at a concentration of 31.2±0.7 mg CE/g Dry Weight. Higher phytochemical contents were also observed in the leaves and twigs. These phytochemicals are believed to be the reason for most of the notable antimicrobial activities exhibited by these plant organs. The mutagenic potential of the most biologically active Croton extracts was tested. An Ames with two Salmonella tester strains (TA98 and TA102) revealed that the species are not toxic as they did not produce His+ revertant colonies in Salmonella tester strains that were more than twice the number of His+ revertant produced by the positive control, Nitroquinoline-N-oxide (4-NQO). Standard DNA barcodes of the Croton species occurring in KZN were generated, since these are useful in plant identification and authentication. These were used to run a phylogenetic analysis in order to assess whether the phytochemical profile is clade-specific. The results showed that the quantity and quality of phytochemicals closely related species may vary. DNA barcoding may be a useful tool in medicinal plant identification, especially where fragments of the plants are traded and morphological identification is not possible. In this study, the biological activity of different parts of six Croton species occurring in KZN, was investigated with the aim of replacing bark as the source of local medicine with other plant parts (leave and twigs) that can be more sustainably harvested, as this will contribute to the conservation of these species.No submissions form available

Microbial Secondary Metabolites and Biotechnology

Many research teams are working to demonstrate that microorganisms can be our daily partners, due to the great diversity of biochemical transformations and molecules they are able to produce. This Special Issue highlights several facets of the production of microbial metabolites of interest. From the discovery of new strains or new bioactive molecules issued from novel environments, to the increase in their synthesis by traditional or innovative methods, different levels of biotechnological processes are addressed. Combining the new dimensions of "Omics" sciences, such as genomics, transcriptomics or metabolomics, microbial biotechnologies are opening up incredible opportunities for discovering and improving microorganisms and their production

Development and implementation of in silico molecule fragmentation algorithms for the cheminformatics analysis of natural product spaces

Computational methodologies extracting specific substructures like functional groups or molecular scaffolds from input molecules can be grouped under the term “in silico molecule fragmentation”. They can be used to investigate what specifically characterises a heterogeneous compound class, like pharmaceuticals or Natural Products (NP) and in which aspects they are similar or dissimilar. The aim is to determine what specifically characterises NP structures to transfer patterns favourable for bioactivity to drug development. As part of this thesis, the first algorithmic approach to in silico deglycosylation, the removal of glycosidic moieties for the study of aglycones, was developed with the Sugar Removal Utility (SRU) (Publication A). The SRU has also proven useful for investigating NP glycoside space. It was applied to one of the largest open NP databases, COCONUT (COlleCtion of Open Natural prodUcTs), for this purpose (Publication B). A contribution was made to the Chemistry Development Kit (CDK) by developing the open Scaffold Generator Java library (Publication C). Scaffold Generator can extract different scaffold types and dissect them into smaller parent scaffolds following the scaffold tree or scaffold network approach. Publication D describes the OngLai algorithm, the first automated method to identify homologous series in input datasets, group the member structures of each group, and extract their common core. To support the development of new fragmentation algorithms, the open Java rich client graphical user interface application MORTAR (MOlecule fRagmenTAtion fRamework) was developed as part of this thesis (Publication E). MORTAR allows users to quickly execute the steps of importing a structural dataset, applying a fragmentation algorithm, and visually inspecting the results in different ways. All software developed as part of this thesis is freely and openly available (see https://github.com/JonasSchaub)