Scaffold repositioning of spiro-acridine derivatives as fungi chitinase inhibitor by target fishing and in vitro studies.

The concept of "one target, one drug, one disease" is not always true, as compounds with previously described therapeutic applications can be useful to treat other maladies. For example, acridine derivatives have several potential therapeutic applications. In this way, identifying new potential targets for available drugs is crucial for the rational management of diseases. Computational methodologies are interesting tools in this field, as they use rational and direct methods. Thus, this study focused on identifying other rational targets for acridine derivatives by employing inverse virtual screening (IVS). This analysis revealed that chitinase enzymes can be potential targets for these compounds. Subsequently, we coupled molecular docking consensus analysis to screen the best chitinase inhibitor among acridine derivatives. We observed that 3 compounds displayed potential enhanced activity as fungal chitinase inhibitors, showing that compound 5 is the most active molecule, with an IC50 of 0.6 ng/µL. In addition, this compound demonstrated a good interaction with the active site of chitinases from Aspergillus fumigatus and Trichoderma harzianum. Additionally, molecular dynamics and free energy demonstrated complex stability for compound 5. Therefore, this study recommends IVS as a powerful tool for drug development. The potential applications are highlighted as this is the first report of spiro-acridine derivatives acting as chitinase inhibitors that can be potentially used as antifungal and antibacterial candidates.

Intra-hemocoel injection of pseurotin A from Metarhizium anisopliae, induces dose-dependent reversible paralysis in the Greater Wax Moth (Galleria mellonella).

Species from the Metarhizium genus are the causal agents of the green muscardine disease of insects. These fungi have been successfully employed for the biological control of pests over decades. Besides the biocontrol applications, recent efforts for genome sequencing of species in this genus have revealed a great diversity of biosynthetic gene clusters potentially associated with secondary metabolite synthesis. Amongst such molecules are the pseurotins, compounds with several activities, as chitin synthase inhibitors, and immunoglobulin E suppressors. Here, we report, for the first time, the isolation of pseurotin A from the culture broth of M. anisopliae, as well as the characterization of the effects of this compound over the model-arthropod Galleria mellonella. Pseurotin A displayed dose-dependent reversible paralysis effects when injected into the larvae hemocoel. However, the posterior challenge of the treated insects with M. anisopliae conidia did not lead to increased mortality, suggesting that pseurotin A treatment did not increase larvae susceptibility to the green muscardine disease. Although apparent insecticidal effects were not observed for pseurotin A, the paralysis effect observed can be important in M. anisopliae infection development.

A Plumieridine-Rich Fraction From Allamanda polyantha Inhibits Chitinolytic Activity and Exhibits Antifungal Properties Against Cryptococcus neoformans.

Cryptococcosis is a fungal infection caused mainly by the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii. The infection initiates with the inhalation of propagules that are then deposited in the lungs. If not properly treated, cryptococci cells can disseminate and reach the central nervous system. The current recommended treatment for cryptococcosis employs a three-stage regimen, with the administration of amphotericin B, flucytosine and fluconazole. Although effective, these drugs are often unavailable worldwide, can lead to resistance development, and may display toxic effects on the patients. Thus, new drugs for cryptococcosis treatment are needed. Recently, an iridoid named plumieridine was found in Allamanda polyantha seed extract; it exhibited antifungal activity against C. neoformans with a MIC of 250 µg/mL. To address the mode of action of plumieridine, several in silico and in vitro experiments were performed. Through a ligand-based a virtual screening approach, chitinases were identified as potential targets. Confirmatory in vitro assays showed that C. neoformans cell-free supernatant incubated with plumieridine displayed reduced chitinase activity, while chitinolytic activity was not inhibited in the insoluble cell fraction. Additionally, confocal microscopy revealed changes in the distribution of chitooligomers in the cryptococcal cell wall, from a polarized to a diffuse cell pattern state. Remarkably, further assays have shown that plumieridine can also inhibit the chitinolytic activity from the supernatant and cell-free extracts of bacteria, insect and mouse-derived macrophage cells (J774.A1). Together, our results suggest that plumieridine can be a broad-spectrum chitinase inhibitor.

Genetic diversity of 16S rRNA and mcrA genes from methanogenic archaeas

Methanogenic archaeas are found in aquatic and terrestrial environments and are fundamental in the conversion of organic matter into methane, a gas that has a potential use as renewable source of energy, which is also considered as one of the main agents of the greenhouse effect. The vast majority of microbial genomes can be identified by a conservative molecular marker, the 16S ribosomal gene. However, the mcrA gene have been using in studies of methanogenic archaea diversity as an alternative marker, highly conserved and present only in methanogens. This gene allows the expression of the enzyme Methyl-coenzyme M reductase, the main agent in converting by-products of anaerobic digestion into methane. In this context, we aimed to study the genetic diversity of mcrA and 16S rRNA genes sequences available in databases. The nucleotide sequences were selected from the NCBI. The heterozygosity and molecular diversity indexes were calculated using the Arlequin 3.5 software, with plots generated by package R v3.0. The diversity and heterozygosity indices for both genes may have been influenced by the number and size of the sequences. Descriptive analysis of genetic diversity generated by sequences deposited in databases allowed a detailed study of these molecules. It is known that the organisms in a population are genetically distinct, and that, despite having similarities in their gene composition, the differences are essential for their adaptation to different environments.