High-Throughput Identification of Crystalline Natural Products from Crude Extracts Enabled by Microarray Technology and microED.

The structural determination of natural products (NPs) can be arduous because of sample heterogeneity. This often demands iterative purification processes and characterization of complex molecules that may be available only in miniscule quantities. Microcrystal electron diffraction (microED) has recently shown promise as a method to solve crystal structures of NPs from nanogram quantities of analyte. However, its implementation in NP discovery remains hampered by sample throughput and purity requirements, akin to traditional NP-discovery workflows. In the methods described herein, we leverage the resolving power of transmission electron microscopy (TEM) and the miniaturization capabilities of deoxyribonucleic acid (DNA) microarray technology to address these challenges through the establishment of an NP screening platform, array electron diffraction (ArrayED). In this workflow, an array of high-performance liquid chromatography (HPLC) fractions taken from crude extracts was deposited onto TEM grids in picoliter-sized droplets. This multiplexing of analytes on TEM grids enables 1200 or more unique samples to be simultaneously inserted into a TEM instrument equipped with an autoloader. Selected area electron diffraction analysis of these microarrayed grids allows for the rapid identification of crystalline metabolites. In this study, ArrayED enabled structural characterization of 14 natural products, including four novel crystal structures and two novel polymorphs, from 20 crude extracts. Moreover, we identify several chemical species that would not be detected by standard mass spectrometry (MS) or ultraviolet-visible (UV/vis) spectroscopy and crystal forms that would not be characterized using traditional methods.

Potent Antifungal Activity of Penta-O-galloyl-ß-d-Glucose against Drug-Resistant Candida albicans, Candida auris, and Other Non-albicans Candida Species.

Among fungal pathogens, infections by drug-resistant Candida species continue to pose a major challenge to healthcare. This study aimed to evaluate the activity of the bioactive natural product, penta-O-galloyl-ß-d-glucose (PGG) against multidrug-resistant (MDR) Candida albicans, MDR Candida auris, and other MDR non-albicans Candida species. Here, we show that PGG has a minimum inhibitory concentration (MIC) of 0.25-8 µg mL-1 (0.265-8.5 µM) against three clinical strains of C. auris and a MIC of 0.25-4 µg mL-1 (0.265-4.25 µM) against a panel of other MDR Candida species. Our cytotoxicity studies found that PGG was well tolerated by human kidney, liver, and epithelial cells with an IC50 > 256 µg mL-1 (>272 µM). We also show that PGG is a high-capacity iron chelator and that deletion of key iron homeostasis genes in C. albicans rendered strains hypersensitive to PGG. In conclusion, PGG displayed potent anti-Candida activity with minimal cytotoxicity for human cells. We also found that the antifungal activity of PGG is mediated through an iron-chelating mechanism, suggesting that the compound could prove useful as a topical treatment for superficial Candida infections.

Recent advances in the discovery of plant-derived antimicrobial natural products to combat antimicrobial resistant pathogens: insights from 2018-2022.

Covering: 2018 to 2022Antimicrobial resistance (AMR) poses a significant global health threat. There is a rising demand for innovative drug scaffolds and new targets to combat multidrug-resistant bacteria. Before the advent of antibiotics, infections were treated with plants chosen from traditional medicine practices. Of Earth's 374 000 plant species, approximately 9% have been used medicinally, but most species remain to be investigated. This review illuminates discoveries of antimicrobial natural products from plants covering 2018 to 2022. It highlights plant-derived natural products with antibacterial, antivirulence, and antibiofilm activity documented in lab studies. Additionally, this review examines the development of novel derivatives from well-studied parent natural products, as natural product derivatives have often served as scaffolds for anti-infective agents.

Cryptic Chemical Variation in a Marine Red Alga as Revealed by Nontargeted Metabolomics.

Many marine algae occupy habitats that are dark, deep, or encrusted on other organisms and hence are frequently overlooked by natural product chemists. However, exploration of less-studied organisms can lead to new opportunities for drug discovery. Genetic variation at the individual, species, genus, and population levels as well as environmental influences on gene expression enable expansion of the chemical repertoire associated with a taxonomic group, enabling natural product exploration using innovative analytical methods. A nontargeted LC-MS and 1H NMR spectroscopy-based metabolomic study of 32 collections of representatives of the calcareous red algal genus Peyssonnelia from coral reef habitats in Fiji and the Solomon Islands revealed significant correlations between natural products' chemistry, phylogeny, and biomedically relevant biological activity. Hierarchical cluster analysis (HCA) of LC-MS data in conjunction with NMR profiling and MS/MS-based molecular networking revealed the presence of at least four distinct algal chemotypes within the genus Peyssonnelia. Two Fijian collections were prioritized for further analysis, leading to the isolation of three novel sulfated triterpene glycosides with a rearranged isomalabaricane carbon skeleton, guided by the metabolomic data. The discovery of peyssobaricanosides A-C (15-17) from two Fijian Peyssonnelia collections, but not from closely related specimens collected in the Solomon Islands that were otherwise chemically and phylogenetically very similar, alludes to population-level variation in secondary metabolite production. Our study reinforces the significance of exploring unusual ecological niches and showcases marine red algae as a chemically rich treasure trove.

Synthesis, Stereochemical Confirmation, and Derivatization of 12(S),16ϵ-Dihydroxycleroda-3,13-dien-15,16-olide, a Clerodane Diterpene That Sensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

Over the past decades, antibiotic resistance has grown to a point where orthogonal approaches to combating infections caused by resistant bacteria are needed. One such approach is the development of non-microbicidal small molecules that potentiate the activity of conventional antibiotics, termed adjuvants. The diterpene natural product 12(S),16ϵ-dihydroxycleroda-3,13-dien-15,16-olide, which we refer to as (-)-LZ-2112, is known to synergize with oxacillin against methicillin-resistant Staphylococcus aureus (MRSA). To explore this activity, (-)-LZ-2112 was synthesized and the structure confirmed through X-ray analysis. Preliminary structure-activity relationship studies following the synthesis of several analogs identified key structural elements responsible for activity and indicate that scaffold simplification is possible. A preliminary mode of action study suggests mecA plays a role in the adjuvant activity of (-)-LZ-2112.

Castaneroxy A From the Leaves of Castanea sativa Inhibits Virulence in Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) represents one of the most serious infectious disease concerns worldwide, with the CDC labeling it a "serious threat" in 2019. The current arsenal of antibiotics works by targeting bacterial growth and survival, which exerts great selective pressure for the development of resistance. The development of novel anti-infectives that inhibit quorum sensing and thus virulence in MRSA has been recurrently proposed as a promising therapeutic approach. In a follow-up of a study examining the MRSA quorum sensing inhibitory activity of extracts of Italian plants used in local traditional medicine, 224C-F2 was reported as a bioactive fraction of a Castanea sativa (European chestnut) leaf extract. The fraction demonstrated high activity in vitro and effective attenuation of MRSA pathogenicity in a mouse model of skin infection. Through further bioassay-guided fractionation using reverse-phase high performance liquid chromatography, a novel hydroperoxy cycloartane triterpenoid, castaneroxy A (1), was isolated. Its structure was established by nuclear magnetic resonance, mass spectrometry and X-ray diffraction analyses. Isomers of 1 were also detected in an adjacent fraction. In a series of assays assessing inhibition of markers of MRSA virulence, 1 exerted activities in the low micromolar range. It inhibited agr::P3 activation (IC50 = 31.72 µM), δ-toxin production (IC50 = 31.72 µM in NRS385), supernatant cytotoxicity to HaCaT human keratinocytes (IC50 = 7.93 µM in NRS385), and rabbit erythrocyte hemolytic activity (IC50 = 7.93 µM in LAC). Compound 1 did not inhibit biofilm production, and at high concentrations it exerted cytotoxicity against human keratinocytes greater than that of 224C-F2. Finally, 1 reduced dermonecrosis in a murine model of MRSA infection. The results establish 1 as a promising antivirulence candidate for development against MRSA.

Ethnobotany and the Role of Plant Natural Products in Antibiotic Drug Discovery.

The crisis of antibiotic resistance necessitates creative and innovative approaches, from chemical identification and analysis to the assessment of bioactivity. Plant natural products (NPs) represent a promising source of antibacterial lead compounds that could help fill the drug discovery pipeline in response to the growing antibiotic resistance crisis. The major strength of plant NPs lies in their rich and unique chemodiversity, their worldwide distribution and ease of access, their various antibacterial modes of action, and the proven clinical effectiveness of plant extracts from which they are isolated. While many studies have tried to summarize NPs with antibacterial activities, a comprehensive review with rigorous selection criteria has never been performed. In this work, the literature from 2012 to 2019 was systematically reviewed to highlight plant-derived compounds with antibacterial activity by focusing on their growth inhibitory activity. A total of 459 compounds are included in this Review, of which 50.8% are phenolic derivatives, 26.6% are terpenoids, 5.7% are alkaloids, and 17% are classified as other metabolites. A selection of 183 compounds is further discussed regarding their antibacterial activity, biosynthesis, structure-activity relationship, mechanism of action, and potential as antibiotics. Emerging trends in the field of antibacterial drug discovery from plants are also discussed. This Review brings to the forefront key findings on the antibacterial potential of plant NPs for consideration in future antibiotic discovery and development efforts.

Pentagalloyl glucose from Schinus terebinthifolia inhibits growth of carbapenem-resistant Acinetobacter baumannii.

The rise of antibiotic resistance has necessitated a search for new antimicrobials with potent activity against multidrug-resistant gram-negative pathogens, such as carbapenem-resistant Acinetobacter baumannii (CRAB). In this study, a library of botanical extracts generated from plants used to treat infections in traditional medicine was screened for growth inhibition of CRAB. A crude extract of Schinus terebinthifolia leaves exhibited 80% inhibition at 256 µg/mL and underwent bioassay-guided fractionation, leading to the isolation of pentagalloyl glucose (PGG), a bioactive gallotannin. PGG inhibited growth of both CRAB and susceptible A. baumannii (MIC 64-256 µg/mL), and also exhibited activity against Pseudomonas aeruginosa (MIC 16 µg/mL) and Staphylococcus aureus (MIC 64 µg/mL). A mammalian cytotoxicity assay with human keratinocytes (HaCaTs) yielded an IC50 for PGG of 256 µg/mL. Mechanistic experiments revealed iron chelation as a possible mode of action for PGG's activity against CRAB. Passaging assays for resistance did not produce any resistant mutants over a period of 21 days. In conclusion, PGG exhibits antimicrobial activity against CRAB, but due to known pharmacological restrictions in delivery, translation as a therapeutic may be limited to topical applications such as wound rinses and dressings.

Quantifying synergy in the bioassay-guided fractionation of natural product extracts.

Mixtures of drugs often have greater therapeutic value than any of their constituent drugs alone, and such combination therapies are widely used to treat diseases such as cancer, malaria, and viral infections. However, developing useful drug mixtures is challenging due to complex interactions between drugs. Natural substances can be fruitful sources of useful drug mixtures because secondary metabolites produced by living organisms do not often act in isolation in vivo. In order to facilitate the study of interactions within natural substances, a new analytical method to quantify interactions using data generated in the process of bioassay-guided fractionation is presented here: the extract fractional inhibitory concentration index (EFICI). The EFICI method uses the framework of Loewe additivity to calculate fractional inhibitory concentration values by which interactions can be determined for any combination of fractions that make up a parent extract. The EFICI method was applied to data on the bioassay-guided fractionation of Lechea mucronata and Schinus terebinthifolia for growth inhibition of the pathogenic bacterium Acinetobacter baumannii. The L. mucronata extract contained synergistic interactions (EFICI = 0.4181) and the S. terebinthifolia extract was non-interactive overall (EFICI = 0.9129). Quantifying interactions in the bioassay-guided fractionation of natural substances does not require additional experiments and can be useful to guide the experimental process and to support the development of standardized extracts as botanical drugs.

A Clerodane Diterpene from Callicarpa americana Resensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

The rise of antibiotic resistance presents a significant healthcare challenge and precludes the use of many otherwise valuable antibiotics. One potential solution to this problem is the use of antibiotics in combination with resistance-modifying agents, compounds that act synergistically with existing antibiotics to resensitize previously resistant bacteria. In this study, 12(S),16ξ-dihydroxycleroda-3,13-dien-15,16-olide, a clerodane diterpene isolated from the medicinal plant Callicarpa americana, was found to synergize with oxacillin against methicillin-resistant Staphylococcus aureus. This synergy was confirmed by checkerboard (fractional inhibitory concentration index (FICI) = 0.125) and time-kill assays, with a subinhibitory dose of 12(S),16ξ-dihydroxycleroda-3,13-dien-15,16-olide causing the effective concentration of oxacillin to fall below the susceptibility breakpoint for S. aureus, a >32-fold decrease in both cases.

Prevalence and Therapeutic Challenges of Fungal Drug Resistance: Role for Plants in Drug Discovery.

Antimicrobial resistance is a global issue that threatens the effective practice of modern medicine and global health. The emergence of multidrug-resistant (MDR) fungal strains of Candida auris and azole-resistant Aspergillus fumigatus were highlighted in the Centers for Disease Control and Prevention's (CDC) 2019 report, Antibiotic Resistance Threats in the United States. Conventional antifungals used to treat fungal infections are no longer as effective, leading to increased mortality. Compounding this issue, there are very few new antifungals currently in development. Plants from traditional medicine represent one possible research path to addressing the issue of MDR fungal pathogens. In this commentary piece, we discuss how medical ethnobotany-the study of how people use plants in medicine-can be used as a guide to identify plant species for the discovery and development of novel antifungal therapies.

A Systematic Review of Plants With Antibacterial Activities: A Taxonomic and Phylogenetic Perspective.

Background: Antimicrobial resistance represents a serious threat to human health across the globe. The cost of bringing a new antibiotic from discovery to market is high and return on investment is low. Furthermore, the development of new antibiotics has slowed dramatically since the 1950s' golden age of discovery. Plants produce a variety of bioactive secondary metabolites that could be used to fuel the future discovery pipeline. While many studies have focused on specific aspects of plants and plant natural products with antibacterial properties, a comprehensive review of the antibacterial potential of plants has never before been attempted. Objectives: This systematic review aims to evaluate reports on plants with significant antibacterial activities. Methods: Following the PRISMA model, we searched three electronic databases: Web of Science, PubMed and SciFinder by using specific keywords: "plant," "antibacterial," "inhibitory concentration." Results: We identified a total of 6,083 articles published between 1946 and 2019 and then reviewed 66% of these (4,024) focusing on articles published between 2012 and 2019. A rigorous selection process was implemented using clear inclusion and exclusion criteria, yielding data on 958 plant species derived from 483 scientific articles. Antibacterial activity is found in 51 of 79 vascular plant orders throughout the phylogenetic tree. Most are reported within eudicots, with the bulk of species being asterids. Antibacterial activity is not prominent in monocotyledons. Phylogenetic distribution strongly supports the concept of chemical evolution across plant clades, especially in more derived eudicot families. The Lamiaceae, Fabaceae and Asteraceae were the most represented plant families, while Cinnamomum verum, Rosmarinus vulgaris and Thymus vulgaris were the most studied species. South Africa was the most represented site of plant collection. Crude extraction in methanol was the most represented type of extraction and leaves were the main plant tissue investigated. Finally, Staphylococcus aureus was the most targeted pathogenic bacteria in these studies. We closely examine 70 prominent medicinal plant species from the 15 families most studied in the literature. Conclusion: This review depicts the current state of knowledge regarding antibacterials from plants and provides powerful recommendations for future research directions.