Mixed fermentation for natural product drug discovery

Natural products continue to play a major role in drug discovery and development. However, chemical redundancy is an ongoing problem. Genomic studies indicate that certain groups of bacteria and fungi have dozens of secondary metabolite pathways that are not expressed under standard laboratory growth conditions. One approach to more fully access the metabolic potential of cultivatable microbes is mixed fermentation, where the presence of neighboring microbes may induce secondary metabolite synthesis. Research to date indicates that mixed fermentation can result in increased antibiotic activity in crude extracts, increased yields of previously described metabolites, increased yields of previously undetected metabolites, analogues of known metabolites resulting from combined pathways and, importantly, induction of previously unexpressed pathways for bioactive constituents.     

Diversifying microbial natural products for drug discovery

Historically, nature has provided the source for the majority of the drugs in use today. More than 20,000 microbial secondary metabolites have been described, but only a small percentage of these have been carried forward as natural product drugs. Natural products are in tough competition with large chemical libraries and with combinatorial chemistries. Hence, each step of a natural product program has to be more efficient than ever, starting from the collection of environmental samples and the selection of strains, to metabolic expression, genetic exploitation, sample preparation and chemical dereplication. This review will focus on approaches for diversifying microbial natural product strains and extract libraries, while decreasing genetic and chemical redundancy.V. Knight and J.-J. Sanglier contributed equally to this work     

An array of target-specific screening strains for antibacterial discovery

As the global threat of drug- and antibiotic-resistant bacteria continues to rise, new strategies are required to advance the drug discovery process. This work describes the construction of an array of Escherichia coli strains for use in whole-cell screens to identify new antimicrobial compounds. We used the recombination systems from bacteriophages lambda and P1 to engineer each strain in the array for low-level expression of a single, essential gene product, thus making each strain hypersusceptible to specific inhibitors of that gene target. Screening of nine strains from the array in parallel against a large chemical library permitted identification of new inhibitors of bacterial growth. As an example of the target specificity of the approach, compounds identified in the whole-cell screen for MurA inhibitors were also found to block the biochemical function of the target when tested in vitro.     

Bioactive natural products from marine cyanobacteria for drug discovery

The prokaryotic marine cyanobacteria continue to be an important source of structurally bioactive secondary metabolites. A majority of these molecules are nitrogen-containing compounds biosynthesized by large multimodular nonribosomal polypeptide (NRP) or mixed polyketide-NRP enzymatic systems. A total of 128 marine cyanobacterial alkaloids, published in the literature between January 2001 and December 2006, are presented in this review with emphasis on their biosynthesis and biological activities. In addition, a number of highly cytotoxic compounds such as hectochlorin, lyngbyabellins, apratoxins, and aurilides have been identified as potential lead compounds for the development of anticancer agents. A brief coverage on the distribution of natural product biosynthetic genes as well as the mechanisms of tailoring enzymes involved in the biosynthesis of cyanobacterial compounds will also be given.     

Exploring natural microbial resources for the discovery of anti-malarial compounds

Microorganisms in nature are highly diverse biological resources, which can be explored for drug discovery. Some countries including Brazil, Columbia, Indonesia, China, and Mexico, which are blessed with geographical uniqueness with diverse climates and display remarkable megabiodiversity, potentially provide microorganismal resources for such exploitation. In this review, as an example of drug discovery campaigns against tropical parasitic diseases utilizing microorganisms from such a megabiodiversity country, we summarize our past and on-going activities toward discovery of new antimalarials. The program was held in a bilateral collaboration between multiple Indonesian and Japanese research groups. In order to develop a new platform of drug discovery utilizing Indonesian bioresources under an international collaborative scheme, we aimed at: 1) establishment of an Indonesian microbial depository, 2) development of robust enzyme-based and cell-based screening systems, and 3) technology transfer necessary for screening, purification, and identification of antimalarial compounds from microbial culture broths. We collected, characterized, and deposited Indonesian microbes. We morphologically and genetically characterized fungi and actinomycetes strains isolated from 5 different locations representing 3 Indonesian geographical areas, and validated genetic diversity of microbes. Enzyme-based screening was developed against two validated mitochondrial enzymes from Plasmodium falciparum, dihydroorotate dehydrogenase and malate:quinone oxidoreductase, while cell-based proliferation assay was developed using the erythrocytic stage parasite of 3D7 strain. More than 17 thousands microbial culture extracts were subjected to the enzyme- and cell-based screening. Representative anti-malarial compounds discovered in this campaign are discussed, including a few isolated compounds that have been identified for the first time as anti-malarial compounds. Our antimalarial discovery campaign validated the Indonesian microbial library as a powerful resource for drug discovery. We also discuss critical needs for selection criteria for hits at each stage of screening and hit deconvolution such as preliminary extraction test for the initial profiling of the active compounds and dereplication techniques to minimize repetitive discovery of known compounds.     

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Plant natural fragments,an innovative approach for drug discovery

Plant natural products (PNP) (e.g., secondary vegetal metabolites and their derivatives) have been a productive source of active ingredients for the pharmaceutical industry. The High Throughput Screening of Plant Natural Products (PNP-HTS) with extracts or isolated compounds has shown to be time consuming, expensive, and not as successful as expected. Recently building upon the innovative fragment-based drug discovery (FBDD) a disruptive approach was developed based on PNP. The fragment approach involves elaboration and/or isolation of weakly binding small molecules with molecular weights between 150 and 250 Da. This method is fundamentally different from HTS in almost every aspect (i.e., size of the compound library, screening methods, and optimization steps from hit to lead). Due to their nature, vegetal natural fragments have unique three-dimensional (3D) properties, high Fsp³, low aromaticity, and large chemo-diversities which represent potential opportunities for developing novel drugs. Preliminary results using vegetal natural fragments appear to be a promising and emerging field which offers valuable prospects for developing new drugs.

     

New natural products as new leads for antibacterial drug discovery

Natural products have been a rich source of antibacterial drugs for many decades, but investments in this area have declined over the past two decades. The purpose of this review article is to provide a recent survey of new natural product classes and the mechanisms by which they work.     

Venomics: a new paradigm for natural products-based drug discovery

The remarkable potency and pharmacological diversity of animal venoms has made them an increasingly valuable source of lead molecules for drug and insecticide discovery. Nevertheless, most of the chemical diversity encoded within these venoms remains uncharacterized, despite decades of research, in part because of the small quantities of venom available. However, recent advances in the miniaturization of bioassays and improvements in the sensitivity of mass spectrometry and NMR spectroscopy have allowed unprecedented access to the molecular diversity of animal venoms. Here, we discuss these technological developments in the context of establishing a high-throughput pipeline for venoms-based drug discovery.     

New aspects of natural products in drug discovery

During the past 15 years, most large pharmaceutical companies have decreased the screening of natural products for drug discovery in favor of synthetic compound libraries. Main reasons for this include the incompatibility of natural product libraries with high-throughput screening and the marginal improvement in core technologies for natural product screening in the late 1980s and early 1990 s. Recently, the development of new technologies has revolutionized the screening of natural products. Applying these technologies compensates for the inherent limitations of natural products and offers a unique opportunity to re-establish natural products as a major source for drug discovery. Examples of these new advances and technologies are described in this review.     

Sulfur-containing marine natural products as leads for drug discovery and development

Among the large series of marine natural products (MNPs), sulfur-containing MNPs have emerged as potential therapeutic agents for the treatment of a range of diseases. Herein, we reviewed 95 new sulfur-containing MNPs isolated during the period between 2021 and March 2023. In addition, we discuss that the widely used strategies and the emerging technologies including natural product-based antibody drug conjugates (ADCs), small-molecule-based proteolysis targeting chimeras (PROTACs), nanotechnology-based drug carriers, artificial intelligence (AI)-driven drug discovery have been used for improving the efficiency and success rate of NP-based drug development. We also provide perspectives regarding the challenges and opportunities in sulfur-containing MNPs based drug discovery and development and future research directions.     

The impact of natural products upon modern drug discovery

In the period 1970-2006, a total of 24 unique natural products were discovered that led to an approved drug. We analyze these successful leads in terms of drug-like properties, and show that they can be divided into two equal subsets. The first falls in the 'Lipinski universe' and complies with the Rule of Five. The second is a 'parallel universe' that violates the rules. Nevertheless, the latter compounds remain largely compliant in terms of logP and H-bond donors, highlighting the importance of these two metrics in predicting bioavailability. Natural products are often cited as an exception to Lipinski's rules. We believe this is because nature has learned to maintain low hydrophobicity and intermolecular H-bond donating potential when it needs to make biologically active compounds with high molecular weight and large numbers of rotatable bonds. In addition, natural products are more likely than purely synthetic compounds to resemble biosynthetic intermediates or endogenous metabolites, and hence take advantage of active transport mechanisms. Interestingly, the natural product leads in the Lipinski and parallel universe had an identical success rate (50%) in delivering an oral drug.     

Unlocking the potential of natural products in drug discovery

The natural product specialized metabolites produced by microbes and plants are the backbone of our current drugs. Despite their historical importance, few pharmaceutical companies currently emphasize their exploitation in new drug discovery and instead favour synthetic compounds as more tractable alternatives. Ironically, we are in a Golden Age of understanding of natural product biosynthesis, biochemistry and engineering. These advances have the potential to usher in a new era of natural product exploration and development taking full advantage of the unique and favourable properties of natural products compounds in drug discovery.     

Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery

Abstract

Endophytes, microorganisms that colonize internal tissues of all plant species, create a huge biodiversity with yet unknown novel natural products, presumed to push forward the frontiers of drug discovery. Next to the clinically acknowledged antineoplastic agent, paclitaxel, endophyte research has yielded potential drug lead compounds with antibacterial, antiviral, antioxidant, insulin mimetic, anti-neurodegenerative and immunosuppressant properties. Furthermore, while being implicated in livestock neurotoxicosis, some endophyte-produced alkaloids have been shown to display insecticidal activity. The endophyte-host relationship is postulated to be a ‘balanced antagonism’. Moreover, the plausibility of horizontal gene transfer (HGT) hypothesis is taken into account. Knowledge of the genetic background of endophytic natural product biosynthesis is discussed on the basis of loline alkaloids, ergopeptines, lolitrems and maytansinoids. The current dynamic progress in genomics will contribute to a better understanding of endophytic microbes and to further exploiting them as a source of pharmaceutically relevant compounds.     

A new approach to drug discovery: high-throughput screening of microbial natural extracts against Aspergillus fumigatus using resazurin

Natural products are an inexhaustible source for drug discovery. However, the validation and selection of primary screening assays are vital to guarantee a selection of extracts or molecules with relevant pharmacological action and worthy of following up. The assay must be rapid, simple, easy to implement, and produce quick results and preferably at a low cost. In this work, we developed and validated a colorimetric microtiter assay using the resazurin viability dye. The parameters of the resazurin method for high-throughput screening (HTS) using natural extracts against Aspergillus fumigatus were optimized and set up. The extracts plus RPMI-1640 modified medium containing the spores and 0.002% resazurin were added per well. The fluorescence was read after 24 to 30 h of incubation. The resazurin proved to be as suitable as Alamar Blue for determining the minimal inhibitory concentration of different antifungals against A. fumigatus and effective to analyze fungicidal and fungistatic compounds. An HTS of 12 000 microbial extracts was carried out against two A. fumigatus strains, and 2.7% of the extracts displayed antifungal activity. Our group has been the first to use this methodology for screening a collection of natural extracts to identify compounds with antifungal activity against the medically important human pathogen A. fumigatus.     

Hydrosoluble formazan XTT: its application to natural products drug discovery for Leishmania

A colorimetric method for measuring the viability of Leishmania promastigotes is described that is based on the reduction of the tetrazolium salt, XTT, to a water-soluble formazan. Values obtained by the XTT method correlated well with parasite number (r=0.965) and with methods that rely upon the reduction of MTT or MTS (r=0.96 and 0.97, respectively). The IC(50) values obtained by XTT method with amphotericin-B, miltefosine and ketoconazole were similar to those previously reported by other methods. The XTT method proved to be a reliable and convenient method for the screening of methanolic extracts from 1059 plants and was used for the bioassay-guided fractionation of the alkaloid aegeline from Sarcorhachis naranjoana.