What can we learn from commercial insecticides? Efficacy, toxicity, environmental impacts, and future developments.

Worldwide pesticide usage was estimated in up to 3.5 million tons in 2020. The number of approved products varies among different countries, however, in Brazil, there are nearly 5000 of such products available. Among them, insecticides correspond to a group of mounting importance for controlling crop pests and disease-associated vectors in public health. Unfortunately, resistance to commercially approved insecticides is commonly observed, limiting the use of these products. Thus, the search for more effective and environmentally friendly products is both a challenge and a necessity since several insecticides are no longer allowed in many countries. In this review, we discuss the historical strategies used in the development of modern insecticides, including chemical structure alterations, mechanism of action and their impact on insecticidal activity. The environmental impact of each pesticide class is also discussed, with persistence data and activity on non-target organisms, along with the human toxicological effect. By tracing the historical route of discovery and development of blockbuster pesticides like DDT, pyrethroids and organophosphates, we also aim to categorize and relate the successful chemical alterations and novel pesticide development strategies that resulted in safer alternatives. A brief discussion on the Brazilian registration procedure and a perspective of insecticides currently approved in the country was also included.

A Molecular Networking Strategy: High-Throughput Screening and Chemical Analysis of Brazilian Cerrado Plant Extracts against Cancer Cells.

Plants have historically been a rich source of successful anticancer drugs and chemotherapeutic agents, with research indicating that this trend will continue. In this contribution, we performed high-throughput cytotoxicity screening of 702 extracts from 95 plant species, representing 40 families of the Brazilian Cerrado biome. Activity was investigated against the following cancer cell lines: colon (Colo205 and Km12), renal (A498 and U031), liver (HEP3B and SKHEP), and osteosarcoma (MG63 and MG63.3). Dose-response tests were conducted with 44 of the most active extracts, with 22 demonstrating IC50 values ranging from <1.3 to 20 µg/mL. A molecular networking strategy was formulated using the Global Natural Product Social Molecular Networking (GNPS) platform to visualize, analyze, and annotate the compounds present in 17 extracts active against NCI-60 cell lines. Significant cytotoxic activity was found for Salacia crassifolia, Salacia elliptica, Simarouba versicolor, Diospyros hispida, Schinus terebinthifolia, Casearia sylvestris var. lingua, Magonia pubescens, and Rapanea guianensis. Molecular networking resulted in the annotation of 27 compounds. This strategy provided an initial overview of a complex and diverse natural product data set, yielded a large amount of chemical information, identified patterns and known compounds, and assisted in defining priorities for further studies.

Antileishmanial compounds from Connarus suberosus: Metabolomics, isolation and mechanism of action.

Leishmaniasis is a disease impacting public health worldwide due to its high incidence, morbidity and mortality. Available treatments are costly, lengthy and toxic, not to mention the problem of parasite resistance. The development of alternative treatments is warranted and natural products demonstrate promising activity. This study investigated the activity of Connarus suberosus extracts and compounds against Leishmania species. Several C. suberosus extracts were tested against L. amazonensis promastigotes. Active and inactive extracts were analyzed by UHPLC-MS and data evaluated using a metabolomics platform, revealing an unknown neoflavonoid (connarin, 3), isolated together with the pterocarpans: hemileiocarpin (1) and leiocarpin (2). The aforementioned compounds (1-3), together with the benzoquinones: rapanone (4), embelin (5) and suberonone (6) previously isolated by our group from the same species, were tested against: (i) L. amazonensis and L. infantum promastigotes, and (ii) L. amazonensis intracellular amastigotes, with the most active compound (3) also tested against L. infantum amastigotes. Cytotoxicity against murine peritoneal macrophages was also investigated. Compounds 2 and 3 presented an IC50 33.8 µM and 11.4 µM for L. amazonensis promastigotes; and 44.3 µM and 13.3 µM for L. infantum promastigotes, respectively. For L. amazonensis amastigotes, the IC50 of 2 was 20.4 µM with a selectivity index (SI) of 5.7, while the IC50 of 3 was 2.9 µM with an SI of 6.3. For L. infantum amastigotes, the IC50 of 3 was 7.7 µM. Compounds 2 and 3 presented activity comparable with the miltefosine positive control, with compound 3 found to be 2-4 times more active than the positive control, depending on the Leishmania species and form. The extracts and isolated compounds showed moderate toxicity against macrophages. Compounds 2 and 3 altered the mitochondrial membrane potential (ΔΨm) and neutral lipid body accumulation, while 2 also impacted plasma membrane permeabilization, culminating in cellular disorder and parasite death. Transmission electron microscopy of L. amazonensis promastigotes treated with compound 3 confirmed the presence of lipid bodies. Leiocarpin (2) and connarin (3) demonstrated antileishmanial activity. This study provides knowledge of natural products with antileishmanial activity, paving the way for prototype development to fight this neglected tropical disease.

Residual Larvicidal Activity of Quinones against Aedes aegypti.

The number of documented dengue cases has increased dramatically in recent years due to transmission through the Aedes aegypti mosquito bite. Vector control remains the most effective measure to protect against this and other arboviral diseases including Zika, chikungunya and (urban) yellow fever, with an established vaccine only available for yellow fever. Although the quinone class shows potential as leading compounds for larvicide development, limited information restricts the development of optimized structures and/or formulations. Thus, in this contribution we investigated the larvicidal and pupicidal activity of three quinone compounds isolated from a Connarus suberosus root wood ethyl acetate extract together with 28 quinones from other sources. Eight quinones demonstrated larvicidal activity, of which tectoquinone (4) proved to be the most active (LC50 1.1 µg/mL). The essential residual effect parameter of four of these quinones was evaluated in laboratory trials, with tectoquinone (4) and 2-ethylanthraquinone (7) presenting the most prolonged activity. In small-scale field residual tests, tectoquinone (4) caused 100% larvae mortality over 5 days, supporting its selection for formulation trials to develop a prototype larvicide to control Ae. aegypti.

Mass spectrometry-based metabolomics approach in the isolation of bioactive natural products.

Metabolomics is a powerful tool in the analysis and identification of metabolites responsible for biological properties. Regarding natural product chemistry, it constitutes a potential strategy to streamline the classic and laborious process of isolating natural products, which often involves the re-isolation and identification of known compounds. In this contribution, we establish a mass spectrometry-based metabolomics strategy to discover compounds with larvicidal activity against Aedes aegypti. We analyse the Brazilian plant Annona crassiflora using different platforms to annotate the active compounds in different extracts/fractions of various plant parts. The MetaboAnalyst and GNPS platforms, which consider LC-MS and LC-MS/MS data, respectively, were chosen to identify compounds that differentiate active and inactive samples. Bio-guided isolation was subsequently performed to confirm compound activity. Results proved the capacity of metabolomics to predict metabolite differences between active and inactive samples using LC-MS and LC-MS/MS data. Moreover, we discuss the limitations, possibilities, and strategies to have a broad view of vast data.

Cytotoxic Triterpenes from Salacia crassifolia and Metabolite Profiling of Celastraceae Species.

The new pentacyclic triterpene 11ß-hydroxypristimerin (1), along with the known metabolites pristimerin (2), 6-oxopristimerol (3) and vitideasin (4), were isolated from a Salacia crassifolia root wood extract, following a bioassay-guided fractionation approach. Both the extract and the purified triterpenes displayed pronounced cytotoxic activity against human cancer cell lines. The NCI-60 cell line screen revealed that compound 2 was the most active, with a mean GI50 of 0.17 µM, while compound 1 had a mean GI50 of 8.7 µM. A COMPARE analysis of the screening results showed that pristimerin is likely to be the main compound responsible for the cytotoxic activity of the extract (mean GI50 of 0.3 µg·mL−1). A targeted search for pristimerin and related derivatives using LC-MS/MS revealed the presence of pristimerin (2) and 6-oxopristimerol (3) in all Celastraceae species examined and in all plant parts tested, while vitideasin (4) was only detected in the genus Salacia.

The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry.

Euglena gracilis is a highly complex alga belonging to the green plant line that shows characteristics of both plants and animals, while in evolutionary terms it is most closely related to the protozoan parasites Trypanosoma and Leishmania. This well-studied organism has long been known as a rich source of vitamins A, C and E, as well as amino acids that are essential for the human diet. Here we present de novo transcriptome sequencing and preliminary analysis, providing a basis for the molecular and functional genomics studies that will be required to direct metabolic engineering efforts aimed at enhancing the quality and quantity of high value products from E. gracilis. The transcriptome contains over 30,000 protein-encoding genes, supporting metabolic pathways for lipids, amino acids, carbohydrates and vitamins, along with capabilities for polyketide and non-ribosomal peptide biosynthesis. The metabolic and environmental robustness of Euglena is supported by a substantial capacity for responding to biotic and abiotic stress: it has the capacity to deploy three separate pathways for vitamin C (ascorbate) production, as well as producing vitamin E (α-tocopherol) and, in addition to glutathione, the redox-active thiols nor-trypanothione and ovothiol.

Importance of bacillithiol in the oxidative stress response of Staphylococcus aureus.

In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human whole-blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.