Machine Learning-Based Virtual Screening of Antibacterial Agents against Methicillin-Susceptible and Resistant Staphylococcus aureus.

The application of computer-aided drug discovery (CADD) approaches has enabled the discovery of new antimicrobial therapeutic agents in the past. The high prevalence of methicillin-resistantStaphylococcus aureus(MRSA) strains promoted this pathogen to a high-priority pathogen for drug development. In this sense, modern CADD techniques can be valuable tools for the search for new antimicrobial agents. We employed a combination of a series of machine learning (ML) techniques to select and evaluate potential compounds with antibacterial activity against methicillin-susceptible S. aureus (MSSA) and MRSA strains. In the present study, we describe the antibacterial activity of six compounds against MSSA and MRSA reference (American Type Culture Collection (ATCC)) strains as well as two clinical strains of MRSA. These compounds showed minimal inhibitory concentrations (MIC) in the range from 12.5 to 200 µM against the different bacterial strains evaluated. Our results constitute relevant proven ML-workflow models to distinctively screen for novel MRSA antibiotics.

Nematostatic activity of isoprenylated guanidine alkaloids from Pterogyne nitens and their interaction with acetylcholinesterase.

Although new nematicides have appeared, the demand for new products less toxic and more efficient for the control of plant-parasitic nematodes are still high. Consequently, studies on natural secondary metabolites from plants, to develop new nematicides, have increased. In this work, nineteen extracts from eleven Brazilian plant species were screened for activity against Meloidogyne incognita. Among them, the extracts of Piterogyne nitens showed a potent nematostatic activity. The alkaloid fraction obtained from the ethanol extract of leaves of P. nitens was more active than the coming extract. Due to the promising activity from the alkaloid fraction, three isoprenylated guanidine alkaloids isolated from this fraction, galegine (1), pterogynidine (2), and pterogynine (3) were tested, showing similar activity to the alkaloid fraction, which was comparable to that of the positive control Temik at 250 µg/mL. At lower concentrations (125-50 µg/mL), compound 2 showed to be the most active one. As several nematicides act through inhibition of acetylcholinesterase (AChE), the guanidine alkaloids were also employed in two in vitro AChE assays. In both cases, compound 2 was more active than compounds 1 and 3. Its activity was considered moderated compared to the control (physostigmine). Compound 2 was selected for an in silico study with the electric eel (Electrophorus electricus) AChE, showing to bind mostly to the same site of physostigmine in the AChEs, pointing out that this could be the mechanism of action for this compound. These results suggested that the guanidine alkaloids 1,2 and 3 from P. nitens are promising for the development of new products to control M. incognita, especially guanidine 2, and encourage new investigations to confirm the mechanism of action, as well as to determine the structure-activity relationship of the guanidine alkaloids.

Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

Crotalaria spectabilis as a source of pyrrolizidine alkaloids and phenolic compounds: HPLC-MS/MS dereplication and monocrotaline quantification of seed and leaf extracts.

INTRODUCTION: Crotalaria spectabilis is an important species used as a pre-plant cover for soybean crops to control the proliferation of endoparasitic nematodes. Species from the Crotalaria genus are known for presenting pyrrolizidine alkaloids (PAs) in their composition, however, C. spectabilis is still considered chemically under-explored. OBJECTIVE: The goal of this manuscript is the development and validation of a method for PAs and flavonoids identification and quantification of C. spectabilis seeds and leaves, a toxic plant used for nematode proliferation control in soil, especially in soybean crops. MATERIALS AND METHODS: Seeds and leaves extracts were analysed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the identification of the compounds. RESULTS: PAs and phenolic compounds could be identified in both samples based on the MS/MS fragmentation pattern. Molecular formulas of the annotated compounds were confirmed by ultra-high-performace liquid chromatography-quadrupole time-of-flight (UHPLC-QToF), and monocrotaline could also be confirmed by standard comparison. The quantification of monocrotaline was performed by HPLC-MS/MS, resulting in 123 times higher monocrotaline content in seeds than in the leaves, which could explain its efficiency in combating nematode proliferation in soil. CONCLUSION: This was the first report of phenolic compounds in C. spectabilis. The current study highlights the importance of C. spectabilis for nematode control due to the presence of toxic PAs, and the employment of analytical techniques for identification and quantification of compounds present in the extracts.

Chemical Space and Diversity of the NuBBE Database: A Chemoinformatic Characterization.

NuBBEDB is the first library of natural products of Brazilian biodiversity. It includes a large variety of classes of compounds and structural types of secondary metabolites of plants, fungi, insects, marine organisms, and bacteria. So far the chemical diversity and complexity of NuBBEDB have not been characterized in a systematic and detailed manner. Herein, we report a comprehensive chemoinformatic analysis of the most current version of NuBBEDB. As part of the characterization, NuBBEDB was compared with several databases of natural products in terms of structural diversity and complexity. Results of the analysis showed that NuBBEDB is diverse in terms of structural fingerprints, distribution of chemical scaffolds, and molecular properties. In addition, the results of the visualization of chemical space support quantitatively that NUBBEDB is a promising source of molecules for drug discovery and medicinal chemistry.

Improvement of bioactive metabolite production in microbial cultures-A systems approach by OSMAC and deconvolution-based 1 HNMR quantification.

Traditionally, the screening of metabolites in microbial matrices is performed by monocultures. Nonetheless, the absence of biotic and abiotic interactions generally observed in nature still limit the chemical diversity and leads to "poorer" chemical profiles. Nowadays, several methods have been developed to determine the conditions under which cryptic genes are activated, in an attempt to induce these silenced biosynthetic pathways. Among those, the one strain, many compounds (OSMAC) strategy has been applied to enhance metabolic production by a systematic variation of growth parameters. The complexity of the chemical profiles from OSMAC experiments has required increasingly robust and accurate techniques. In this sense, deconvolution-based 1 HNMR quantification have emerged as a promising methodology to decrease complexity and provide a comprehensive perspective for metabolomics studies. Our present work shows an integrated strategy for the increased production and rapid quantification of compounds from microbial sources. Specifically, an OSMAC design of experiments (DoE) was used to optimize the microbial production of bioactive fusaric acid, cytochalasin D and 3-nitropropionic acid, and Global Spectral Deconvolution (GSD)-based 1 HNMR quantification was carried out for their measurement. The results showed that OSMAC increased the production of the metabolites by up to 33% and that GSD was able to extract accurate NMR integrals even in heavily coalescence spectral regions. Moreover, GSD-1 HNMR quantification was reproducible for all species and exhibited validated results that were more selective and accurate than comparative methods. Overall, this strategy up-regulated important metabolites using a reduced number of experiments and provided fast analyte monitor directly in raw extracts.

Natural deep eutectic solvents as the major mobile phase components in high-performance liquid chromatography-searching for alternatives to organic solvents.

Over the past six decades, acetonitrile (ACN) has been the most employed organic modifier in reversed-phase high-performance liquid chromatography (RP-HPLC), followed by methanol (MeOH). However, from the growing environmental awareness that leads to the emergence of "green analytical chemistry," new research has emerged that includes finding replacements to problematic ACN because of its low sustainability. Deep eutectic solvents (DES) can be produced from an almost infinite possible combinations of compounds, while being a "greener" alternative to organic solvents in HPLC, especially those prepared from natural compounds called natural DES (NADES). In this work, the use of three NADES as the main organic component in RP-HPLC, rather than simply an additive, was explored and compared to the common organic solvents ACN and MeOH but additionally to the greener ethanol for separating two different mixtures of compounds, one demonstrating the elution of compounds with increasing hydrophobicity and the other comparing molecules of different functionality and molar mass. To utilize NADES as an organic modifier and overcome their high viscosity monolithic columns, temperatures at 50 °C and 5% ethanol in the mobile phase were used. NADES are shown to give chromatographic performances in between those observed for ACN and MeOH when eluotropic strength, resolution, and peak capacity were taken into consideration, while being less environmentally impactful as shown by the HPLC-Environmental Assessment Tool (HPLC-EAT) metric. With the development of proper technologies, DES could open a new class of mobile phases increasing the possibilities of new separation selectivities while reducing the environmental impact of HPLC analyses. Graphical abstract Natural deep eutectic solvents versus traditional solvents in HPLC.

Anti-apoptotic effects of decyl gallate on the induction of apoptosis in A549 pneumocytes by Paracoccidioides brasiliensis gp43.

Apoptosis is considered an escape mechanism from the host immune system for the fungus Paracoccidioides spp, and it serves as a vehicle for entry into macrophages without stimulating microbicidal activities. Recently, gp43 of P. brasiliensis was demonstrated to be involved in this process. Therefore, as a new therapeutic alternative, it is very important to study compounds that could reduce the modulation of the induction of apoptosis caused by this fungus. Decyl gallate (G14) is a known antifungal compound, and we decided to investigate its anti-apoptotic properties. Our results demonstrate that G14 was effective against apoptosis induced by gp43, as observed in epithelial cells, and led to a reduction in DNA damage, Bak down-regulation and Bcl-2 up-regulation. Together, these data show that G14 presents promising anti-apoptotic activity.

In vitro evaluation of the schistosomicidal effect of the extracts, fractions and major 3-hydroxy-2,6-dialkyl-substituted piperidine alkaloids from the flowers of Senna spectabilis (Fabaceae).

In this work, we present the in vitro schistosomicidal activity evaluation of the most active dichloromethane fraction (FDm) (ED50=83.5µg/mL) and of a mixture of the major alkaloids ((-)-cassine/(-)-spectaline, C/E) (ED50=37.4µg/mL) from the flowers of Senna spectabilis against adult worms and cercariae. We also demonstrate other toxic effects including paralysis of the adult worms, inhibition of the secretory activity, tegument lesions and cercaricidal activity. In the association test of Praziquantel (PZQ)-C/E, we observed up to 80% mortality of Schistosoma mansoni in comparison to PZQ monotherapy. Due to the diversity of the toxic effects, the schistosomicidal activity of C/E is likely a result of a multitarget mechanism involving the tegument, secretory system and neuromotor action.

Partial least squares model and design of experiments toward the analysis of the metabolome of Jatropha gossypifolia leaves: Extraction and chromatographic fingerprint optimization.

A major challenge in metabolomic studies is how to extract and analyze an entire metabolome. So far, no single method was able to clearly complete this task in an efficient and reproducible way. In this work we proposed a sequential strategy for the extraction and chromatographic separation of metabolites from leaves Jatropha gossypifolia using a design of experiments and partial least square model. The effect of 14 different solvents on extraction process was evaluated and an optimized separation condition on liquid chromatography was estimated considering mobile phase composition and analysis time. The initial conditions of extraction using methanol and separation in 30 min between 5 and 100% water/methanol (1:1 v/v) with 0.1% of acetic acid, 20 µL sample volume, 3.0 mL min(-1) flow rate and 25°C column temperature led to 107 chromatographic peaks. After the optimization strategy using i-propanol/chloroform (1:1 v/v) for extraction, linear gradient elution of 60 min between 5 and 100% water/(acetonitrile/methanol 68:32 v/v with 0.1% of acetic acid), 30 µL sample volume, 2.0 mL min(-1) flow rate, and 30°C column temperature, we detected 140 chromatographic peaks, 30.84% more peaks compared to initial method. This is a reliable strategy using a limited number of experiments for metabolomics protocols.

In Vitro Antibacterial Activity of Prenylated Guanidine Alkaloids from Pterogyne nitens and Synthetic Analogues.

The present investigation deals with the antibiotic activity of eight natural guanidine alkaloids and two synthetic analogues against a variety of clinically relevant methicillin-resistant Staphylococcus aureus strains. Galegine (1) and pterogynidine (2) were the most potent compounds, with a minimum inhibitory concentration of 4 mg/L, to all tested strains. The preliminary chemical features correlating to anti-MRSA activity showed that the size of the side chain and the substitution pattern in the guanidine core played a key role in the antibacterial activity of the imino group. Guanidine alkaloids 1 and 2 are promising molecular models for further synthetic derivatives and, thus, for medicinal chemistry studies.

Chemical composition of the bark of Tetrapterys mucronata and identification of acetylcholinesterase inhibitory constituents.

The secondary metabolite content of Tetrapterys mucronata, a poorly studied plant that is used occasionally in Brazil for the preparation of a psychotropic plant decoction called "Ayahuasca", was determined to establish its chemical composition and to search for acetylcholinesterase (AChE) inhibitors. The ethanolic extract of the bark of T. mucronata exhibited in vitro AChE inhibition in a TLC bioautography assay. To localize the active compounds, biological profiling for AChE inhibition was performed using at-line HPLC-microfractionation in 96-well plates and subsequent AChE inhibition bioautography. The analytical HPLC-PDA conditions were transferred geometrically to a preparative medium-pressure liquid chromatography column using chromatographic calculations for the efficient isolation of the active compounds at the milligram scale. Twenty-two compounds were isolated, of which six are new natural products. The structures of the new compounds (9, 10, 16-18, and 20) were elucidated by spectroscopic data interpretation. Compounds 1, 5, 6, 9, and 10 inhibited AChE with IC50 values below 15 µM.

Free radical scavenging activity of Kielmeyera variabilis (Clusiaceae).

As part of our ongoing research on antioxidant agents from Brazilian flora, we screened the free radical scavenging activity of two extracts and eight fractions of Kielmeyera variabilis (Clusiaceae) using DPPH· (2,2-diphenyl-1-picrylhydrazyl-hydrate) and ABTS·+ [2,2'-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid)] colorimetric assays. The ethyl acetate and n-butanol fractions of the leaves of K. variabilis displayed the strongest activity (IC50 of 3.5 ± 0.3 and 4.4 ± 0.2 µg mL⁻¹ for DPPH· and 6.6 ± 0.4 and 3.1 ± 0.1 µg mL⁻¹ for ABTS·+, respectively). Chromatographic fractionation of the most potent fractions led to identification of three flavonols with previously described antioxidant activity, quercitrin (1), quercetin-3-O-ß-glucoside (3), and quercetin-3-O-ß-galactoside (4), and of one biflavone, podocarpusflavone A (2). This is the first time that the presence of these flavonoids in Kielmeyera variabilis has been reported.

In vitro antiviral activity of piperidine alkaloids from Senna spectabilis flowers on Chikungunya virus infection.

BACKGROUND: Chikungunya fever is an endemic disease caused by the Chikungunya virus (CHIKV). To date there is no antiviral treatment against this infection or licensed vaccine to prevent it. Our study aims to evaluate whether (-)-cassine (1) and (-)-spectaline (2), the main alkaloids of Senna spectabilis, display anti-CHIKV activity. Both compounds have been described to be biologically active against neglected tropical diseases, including malaria, leishmaniasis, and schistosomiasis, which emphasizes that these molecules could be repurposed for chikungunya fever treatment. METHODS: The structures of the isolated compounds 1 and 2 were identified by NMR and HRESIMS analyses, and their antiviral activity against CHIKV was assessed by a dose-response assay employing BHK-21 cells and CHIKV-nanoluc, a recombinant virus carrying the nanoluciferase gene reporter. RESULTS: Compound 1 presented CC50 of 126.5 µM and EC50 of 14.9 µM, while compound 2 presented CC50 of 91.9 µM and EC50 of 8.3 µM. The calculated selectivity index (SI) was 8.5 for 1 and 11.3 for 2. CONCLUSION: The data presented herein show that compounds 1 and 2 have potential for being repurposed as anti-CHIKV drug. Our promising in vitro results encourage further in vitro and in vivo assays. This is the first description of the antiviral activity of compounds 1 and 2 against CHIKV infection, which can impact the development of antiviral drug candidates against chikungunya fever, which sometimes can be debilitating.

Chemical composition and chromatographic fingerprint of three strains of Agaricus subrufescens cultivated with handmade and commercial supplements.

Exploratory factor analysis was applied to determine the chemical differences between fruitbodies of three Agaricus subrufescens mushroom strains [from Japan (JP), Brazil (ABZ), and Belgium (T2)] grown with handmade and commercial supplements. The composition of the ABZ strain cultivated with agro-industrial waste supplement presented a high nutritional composition regarding the amounts of fibre and protein, similar to mushrooms cultivated with the commercial supplement. The chromatographic fingerprints obtained for T2 and JP strains grown with commercial supplements presented similar profiles compared to those cultivated with the supplement based on peanut and the mix of supplements. The chromatographic analysis also showed that the similarities are correlated with the relative abundance of antioxidant compounds annotated by HPLC-MS, such as vanillic acid deoxyhexoside, caffeic acid hexoside, catechin hexosemalonate, digallic acid, cinnamic acid derivative, and p-coumaroylmalic acid. This study showed that handmade supplements based on agro-industrial waste could be viable alternatives for replacing high-cost supplements.

Computational Applications in Secondary Metabolite Discovery (CAiSMD): an online workshop.

We report the major conclusions of the online open-access workshop "Computational Applications in Secondary Metabolite Discovery (CAiSMD)" that took place from 08 to 10 March 2021. Invited speakers from academia and industry and about 200 registered participants from five continents (Africa, Asia, Europe, South America, and North America) took part in the workshop. The workshop highlighted the potential applications of computational methodologies in the search for secondary metabolites (SMs) or natural products (NPs) as potential drugs and drug leads. During 3 days, the participants of this online workshop received an overview of modern computer-based approaches for exploring NP discovery in the "omics" age. The invited experts gave keynote lectures, trained participants in hands-on sessions, and held round table discussions. This was followed by oral presentations with much interaction between the speakers and the audience. Selected applicants (early-career scientists) were offered the opportunity to give oral presentations (15 min) and present posters in the form of flash presentations (5 min) upon submission of an abstract. The final program available on the workshop website ( https://caismd.indiayouth.info/ ) comprised of 4 keynote lectures (KLs), 12 oral presentations (OPs), 2 round table discussions (RTDs), and 5 hands-on sessions (HSs). This meeting report also references internet resources for computational biology in the area of secondary metabolites that are of use outside of the workshop areas and will constitute a long-term valuable source for the community. The workshop concluded with an online survey form to be completed by speakers and participants for the goal of improving any subsequent editions.

In vitro trypanocidal activity of phenolic derivatives from Peperomia obtusifolia.

The trypanocidal activity of crude extracts and fractions from the leaves and stems of Peperomia obtusifolia (Piperaceae) was evaluated in vitro against the epimastigote forms of Trypanosoma cruzi. Bioactivity-guided fractionation of the most active extracts afforded seven known compounds, including three chromanes, two furofuran lignans and two flavone C-diglycosides. The most active compounds were the chromanes peperobtusin A and 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(2''-methyl-2''-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2 H-1-benzopyran-6-carboxylic acid, with IC (50) values of 3.1 microM (almost three times more active than the positive control benznidazole, IC (50) 10.4 microM) and 27.0 microM, respectively. Cytotoxicity assays using peritoneal murine macrophages indicated that the chromanes were not toxic at the level of the IC (50) for trypanocidal activity. This is the first report on the trypanocidal activity besides unspecific cytotoxicity of chromanes from Peperomia species. Additionally it represents the first time isolation of 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(2''-methyl-2''-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2 H-1-benzopyran-6-carboxylic acid from P. obtusifolia.

Validated HPLC method for the standardization of Phyllanthus niruri (herb and commercial extracts) using corilagin as a phytochemical marker.

Phyllanthus niruri L., commonly known in Brazil as 'quebra-pedra', has long been used in the treatment of diverse diseases and especially urolithiasis. The therapeutic effects of P. niruri are attributed to various compounds present in the plant, including the hydrolysable tannin corilagin. In the present study, high-performance liquid chromatography (HPLC-/PAD) profiles of leaves and commercial extracts of P. niruri were examined and three compounds, found to be present in all of the samples studied, were isolated by open column chromatography over C18)silica gel followed by preparative HPLC. These compounds were identified by nuclear magnetic resonance as corilagin, rutin and ethyl 3,4,5-trihydroxybenzoate. Corilagin, which has been proposed as a phytochemical marker for P. niruri, was employed as an external standard in the development and validation of a rapid and efficient qualitative and quantitative HPLC assay for the analyte. The method may be applied in the standardization of herbs and phytomedicines commercialized in Brazil as quebra-pedra.