Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections

Background A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. Methodology/ Principal Findings Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. Conclusions The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites

Virtual screening identifies a novel piperazine-based insect juvenile hormone agonist

Juvenile hormone (JH) agonists constitute a subclass of insect growth regulators and play important roles in insect pest management. In this work, a multi-step virtual screening program was executed to find novel JH agonists. A database of 5 million purchasable compounds was sequentially processed with three computational filters: (i) shape and chemical similarity as compared to known JH-active compounds; (ii) molecular docking simulations against a Drosophila JH receptor, methoprene-tolerant; and (iii) free energy calculation of ligand–receptor binding using a modified MM/PBSA (molecular mechanics/Poisson–Boltzmann surface area) protocol. The 11 candidates that passed the three filters were evaluated in a luciferase reporter assay, leading to the identification of a hit compound that contains a piperazine ring system (EC₅₀=870 nM). This compound is structurally dissimilar to known JH agonists and synthetically easy to access; therefore, it is a promising starting point for further structure optimization

Identification of Potential Inhibitors from Pyriproxyfen with Insecticidal Activity by Virtual Screening

Aedes aegypti is the main vector of dengue fever transmission, yellow fever, Zika, and chikungunya in tropical and subtropical regions and it is considered to cause health risks to millions of people in the world. In this study, we search to obtain new molecules with insecticidal potential against Ae. aegypti via virtual screening. Pyriproxyfen was chosen as a template compound to search molecules in the database Zinc_Natural_Stock (ZNSt) with structural similarity using ROCS (rapid overlay of chemical structures) and EON (electrostatic similarity) software, and in the final search, the top 100 were selected. Subsequently, in silico pharmacokinetic and toxicological properties were determined resulting in a total of 14 molecules, and these were submitted to the PASS online server for the prediction of biological insecticide and acetylcholinesterase activities, and only two selected molecules followed for the molecular docking study to evaluate the binding free energy and interaction mode. After these procedures were performed, toxicity risk assessment such as LD50 values in mg/kg and toxicity class using the PROTOX online server, were undertaken. Molecule ZINC00001624 presented potential for inhibition for the acetylcholinesterase enzyme (insect and human) with a binding affinity value of -10.5 and -10.3 kcal/mol, respectively. The interaction with the juvenile hormone was -11.4 kcal/mol for the molecule ZINC00001021. Molecules ZINC00001021 and ZINC00001624 had excellent predictions in all the steps of the study and may be indicated as the most promising molecules resulting from the virtual screening of new insecticidal agents.Federal University of Amapá, Program in Biotechnology and Biodiversity-Network BIONORTE, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for funding in the publication of this article

Identification of Potential Insect Growth Inhibitor against Aedes aegypti: A Bioinformatics Approach

Aedes aegypti is the main vector that transmits viral diseases such as dengue, hemorrhagic dengue, urban yellow fever, zika, and chikungunya. Worldwide, many cases of dengue have been reported in recent years, showing significant growth. The best way to manage diseases transmitted by Aedes aegypti is to control the vector with insecticides, which have already been shown to be toxic to humans; moreover, insects have developed resistance. Thus, the development of new insecticides is considered an emergency. One way to achieve this goal is to apply computational methods based on ligands and target information. In this study, sixteen compounds with acceptable insecticidal activities, with 100% larvicidal activity at low concentrations (2.0 to 0.001 mg center dot L-1), were selected from the literature. These compounds were used to build up and validate pharmacophore models. Pharmacophore model 6 (AUC = 0.78; BEDROC = 0.6) was used to filter 4793 compounds from the subset of lead-like compounds from the ZINC database; 4142 compounds (dG < 0 kcal/mol) were then aligned to the active site of the juvenile hormone receptor Aedes aegypti (PDB: 5V13), 2240 compounds (LE < -0.40 kcal/mol) were prioritized for molecular docking from the construction of a chitin deacetylase model of Aedes aegypti by the homology modeling of the Bombyx mori species (PDB: 5ZNT), which aligned 1959 compounds (dG < 0 kcal/mol), and 20 compounds (LE < -0.4 kcal/mol) were predicted for pharmacokinetic and toxicological prediction in silico (Preadmet, SwissADMET, and eMolTox programs). Finally, the theoretical routes of compounds M01, M02, M03, M04, and M05 were proposed. Compounds M01-M05 were selected, showing significant differences in pharmacokinetic and toxicological parameters in relation to positive controls and interaction with catalytic residues among key protein sites reported in the literature. For this reason, the molecules investigated here are dual inhibitors of the enzymes chitin synthase and juvenile hormonal protein from insects and humans, characterizing them as potential insecticides against the Aedes aegypti mosquito.Laboratory of Cellular Immunology Applied to Health of the Oswaldo Cruz Foundation (FIOCRUZ)Department of Pharmaceutical and Organic Chemistry, Faculty of Pharmacy of the University of Granada (Spain)Researcher Assistance Program-PAPESQ/UNIFA

Mode of action, concentrations and effects of tributyltin in common shrimp <i>Crangon crangon</i>

With an annual catch of 35,000 – 50,000 tons and a commercial value of roughly €100 million, common shrimp is one of the most valuable European fisheries and aquaculture commodities. Since the late 1960s, European landings strongly decreased until 1990, due to a strongly reduced shrimp abundance and reproduction. In the Wadden Sea, the shrimp stock and hence landed volumes recovered slowly after 1990 to record landings in 2010. In the Southern Bight (i.e. Northern France to Dutch Delta), one of the busiest international shipping lanes, the stock only recovered after 2003. Until now, no explanation was found for these abnormal long-term trends in the shrimp stock. The use of TBT as antifouling agent began in the 1960s, leading to exuberant TBT concentrations in the 1970s and 1980s, especially nearby recreational marinas, shipping lanes, and industrial harbours. At the end of the 1980s, the use of TBT on smaller ship ( 25 m) was only accepted in 2003 and finally entered-into-force in 2008. In 2004, Nishikawa et al. (2004) proved that TBT mimics vertebrate hormone signalling by activating the retinoid-X-receptor (RXR). As this receptor is strongly conserved in the animal kingdom, an identical mode of action (MOA) was generally accepted to have caused the observed reproductive and developmental defects in marine shellfish and gastropods. RXR is a nuclear receptor (NR) believed to directly modulate (i.e. by heterodimerization) the activity of many other NRs. NRs are the major targets of lipophilic hormones (e.g. steroids), and directly induce tissue specific expression of genes involved in development, reproduction, immune response, etc. Within this PhD-thesis, three main research questions were addressed. (1) Does TBT affect the functionality of the shrimp RXR receptor?, (2) what is the temporal and spatial distribution of TBT in North Sea shrimp, and finally (3) do the TBT concentrations observed in the North Sea disrupt shrimp endocrine functioning

Non-Canonical Odor Coding Ensures Robust Mosquito Attraction to Humans

Aedes aegypti mosquitoes spread deadly diseases, including dengue, Zika, yellow fever, and chikungunya. Only female mosquitoes bite, and they do so because they require a blood-meal for reproduction. Aedes aegypti prefer to bite human hosts, which contributes to their effectiveness as a deadly disease vector. Mosquitoes rely heavily on chemosensory cues, including carbon dioxide (CO2) emitted from breath and human body odor, which is a mixture of more than 200 different individual odorants. Although the exact odor profile of people varies considerably, Aedes aegypti are incredibly reliable in finding humans to bite, despite widespread efforts to by humans to mask our odor. Even mosquitoes with genetic mutations that eliminate entire families of chemosensory receptors are still able to find and bite humans. It remains unknown how the mosquito olfactory system is seemingly infallible in its ability to detect humans for taking a blood meal. In the well-studied olfactory systems of Drosophila melanogaster and Mus musculus, individual olfactory sensory neurons express a single type of olfactory receptor and project their axons to discrete regions, called glomeruli, in the antennal lobe or olfactory bulb, respectively. This organization is believed to be a widespread motif in olfactory systems and has been established dogma since the mid-2000s and is hypothesized to permit the brain to parse which subpopulation of olfactory neurons is activated by a given odor. To understand how human odor is encoded in the mosquito olfactory system, we developed a CRISPR-Cas9-based genetic knock-in strategy in Aedes aegypti and generated a suite of transgenic mosquito strains that label populations of olfactory sensory neurons. Surprisingly, we find that the olfactory system of Aedes aegypti does not have the expected “one-receptor-to-one-neuron-to-oneglomerulus” organization seen in other insects. Rather, there are many more receptors than glomeruli. We frequently observe co-expression of multiple chemosensory receptors within individual olfactory sensory neurons and individual glomeruli are commonly innervated by olfactory sensory neurons expressing different receptors. What is the functional consequence of this unconventional organization? To understand how co-expression of multiple chemosensory families affects human odor detection by mosquitoes, we examined a minimal mixture that drives host seeking behavior. Mosquitoes are attracted to the combination of the two human-derived, cues CO2 and lactic acid. We found that the same neurons that sense CO2 also sense volatile amines, including triethyl amine. These amines are detected by separate chemosensory receptor genes and we discovered that these cues can be interchanged to drive attraction in the presence of lactic acid. This sensory organization, in which multiple receptors that respond to very different types of chemicals are co-expressed, suggests a redundancy in the odor code at the level of the olfactory sensory neurons for cues that signal the presence of a human to bite. We speculate that this design supports the robust human host-seeking seen in this olfactory specialist

Insulin signaling regulates neurite growth during ecdysone-dependent neuronal remodeling

The morphological rearrangement of neurons to accommodate new functions or activities is called "neuronal remodeling". Although neuronal remodeling is an important feature of nervous systems, the mechanisms governing the transition of neurons, from relatively stable states to more dynamic and differentiative remodeling states, are largely unknown. In holometabolous insects, there is a major transition from maintenance growth to organizational growth near the onset of metamorphosis, and these changes provide an unparalleled opportunity to explore the underlying mechanisms of neuronal remodeling. Many differentiated larval neurons are maintained throughout metamorphosis and undergo extensive remodeling, which involves the elimination of larval dendrites and axons (neurites) and the outgrowth and elaboration of adult-specific projections (Levine and Truman 1982; Brown, Cherbas et al. 2006). Here, I show that a metamorphosis-specific increase in insulin and insulin-like-growth factor signaling (IIS) promotes neuronal growth and axon branching after a long period of morphological stability during the larval stages. In a previous gain-of-function genetic screen, we found that overexpression of a negative effector in the IIS pathway, Forkhead box, sub-group O (FOXO), blocked the metamorphic growth of peptidergic neurons that secrete crustacean cardioactive peptide (CCAP) and bursicon. RNA interference (RNAi) and CCAP/bursicon cell-targeted expression of dominant negative constructs for other components of the IIS pathway [Insulin-like receptor (InR), Pi3K92E, Akt1, and S6K] also partially suppressed the growth of the CCAP/bursicon neuron somata and neurite arborization. In contrast, expression of wild-type or constitutively active forms of InR, Pi3K92E, Akt1, Rheb, and Target of rapamycin (TOR), as well as RNAi for negative regulators of the IIS pathway (PTEN and FOXO), stimulated overgrowth. Interestingly, InR displayed little effect on larval growth of the CCAP/bursicon neurons, but strong effects on the metamorphic outgrowth of these neurons. In addition, manipulations of IIS in a pan-peptidergic neuronal pattern revealed a general role in promoting organizational outgrowth of many neurons during metamorphosis. These results reveal that specific activation of IIS during metamorphosis facilitates renewed organizational growth in mature neurons. In order to further elucidate the molecular and cellular mechanisms governing IIS regulation of the metamorphic remodeling, I performed a genetic modifier screen to detect IIS-interacting genes. I screened 492 deficiency lines for modifiers of a foxo overexpression phenotype (wing expansion defects). A total of 14 deficiencies were confirmed as suppressors of foxo, and 19 were confirmed as enhancers. Two selected suppressors, Df(1)Exel6221 and Df(1)Exel6002, strongly reversed the effects of foxo on neuronal outgrowth. Df(1)Exel6221 also significantly rescued the phenotypes produced by expression of InRDN, suggesting that the gene(s) within Df(1)ExEL6221 might be involved in IIS-mediated growth during the neuronal remodeling process. The source of suppression in Df(1)Exel6002 was mapped to an individual locus, Su(z)2. Reduced expression of Su(z)2 by RNAi suppressed the effects of FOXO on neuronal outgrowth. Su(z)2 is a Zinc finger protein that belongs to the Drosophila Polycomb Group (PcG) protein family, the members of which function as negative regulators of transcription and chromatin modification (Brunk, Martin et al. 1991). This indicates that transcriptional regulation through chromatin modification by Su(z)2 may play an important role in reprogramming neuronal entry into the organizational growth phase, or in the execution of that growth program

Ecological and Chemical Studies on the Gq-protein Inhibitor FR900359

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, displays a strong and selective inhibition of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as corroborated by in vivo experiments on mice, affinity to insect Gq proteins and insect toxicity studies. The uncultured endosymbiont of A. crenata, 'Candidatus Burkholderia crenata' was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. In this study we provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time, paving the way for a biotechnological production of FR independent from time- and work-intensive plant cultivation, harvesting and extraction. Direct targeting of intracellular Gα subunits is a challenging task in pursuit of chemical tools for pharmacological studies and for developing novel therapeutic approaches. We isolated four new FR analogs (1-4) from A. crenata and elucidated their structures by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS2 analysis. Next, we analyzed all currently known inhibitors of Gq protein including YM-254890, FR900359, above mentioned novel FR-derivatives from A. crenata, and synthetic cyclic peptides to devise a strategy for the elucidation of characteristics that determine interaction with Gq. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocycles that govern specific binding to and inhibition of Gq. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed biological activity of FR or YM. This raises the question of whether nature has optimized these depsipeptides for specific inhibition of Gq. Thus, rather than attempting to enhance Gq activity of newly synthesized inhibitors, future synthetic efforts on FR/YM-analogs should target Gα subunits other than Gq. Additionally, FR was detected from leaves of five other Ardisia species, among them the non-nodulated A. lucida as well as from a soil bacterium, implicating a much broader distribution of FR as originally anticipated. Furthermore the first reported fluorescent FR analogs were synthesized, biologically evaluated and applied to study the mechanism of cellular uptake of FR