Larisa V. Avramova

A “Genome-to-Lead” Approach for Insecticide Discovery: Pharmacological Characterization and Screening of Aedes aegypti D1-like Dopamine Receptors

Background Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides. Methodology/Principal Findings We describe a “genome-to-lead” approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR) mined from a mosquito genome. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (AaDOP1 and AaDOP2) from the yellow fever mosquito, Aedes aegypti. Sequence analyses indicated that these receptors are orthologous to arthropod D1-like (Gαs-coupled) receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of AaDOP1 and AaDOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC50: AaDOP1 = 3.1±1.1 nM; AaDOP2 = 240±16 nM). Interestingly, only AaDOP1 exhibited sensitivity to epinephrine (EC50 = 5.8±1.5 nM) and norepinephrine (EC50 = 760±180 nM), while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at AaDOP2. Fifty-one compounds were identified as “hits,” and follow-up validation assays confirmed the antagonistic effect of selected compounds at AaDOP2. In vitro comparison studies between AaDOP2 and the human D1 dopamine receptor (hD1) revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as AaDOP2-selective compounds. In subsequent Ae. aegypti larval bioassays, significant mortality was observed for amitriptyline (93%) and doxepin (72%), confirming these chemistries as “leads” for insecticide discovery. Conclusions/Significance This research provides a “proof-of-concept” for a novel approach toward insecticide discovery, in which genome sequence data are utilized for functional characterization and chemical compound screening of GPCRs. We provide a pipeline useful for future prioritization, pharmacological characterization, and expanded chemical screening of additional GPCRs in disease-vector arthropods. The differential molecular and pharmacological properties of the mosquito dopamine receptors highlight the potential for the identification of target-specific chemistries for vector-borne disease management, and we report the first study to identify dopamine receptor antagonists with in vivo toxicity toward mosquitoes.

Differential Mitochondrial Toxicity Screening and Multi- Parametric Data Analysis

Early evaluation of new drug entities for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. Multi-parametric high-content screening (mp-HCS) of mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In this study, we have developed a mp-HCS and multi-parametric data analysis scheme for assessing cell responses to induced mitochondrial perturbation. The mp-HCS measurements are shown to be robust enough to allow for quantitative comparison of biological systems with different metabolic pathways simulated by alteration of growth media. Substitution of medium glucose for galactose sensitized cells to drug action and revealed novel response parameters. Each compound was quantitatively characterized according to induced phenotypic changes of cell morphology and functionality measured by fluorescent biomarkers for mitochondrial activity, plasma membrane permeability, and nuclear morphology. Descriptors of drug effects were established by generation of a SCRIT (Specialized-Cell-Response-to-Induced-Toxicity) vector, consisting of normalized statistical measures of each parameter at each dose and growth condition. The dimensionality of SCRIT vectors depends on the number of parameters chosen, which in turn depends on the hypothesis being tested. Specifically, incorporation of three parameters of response into SCRIT vectors enabled clustering of 84 training compounds with known pharmacological and toxicological activities according to the degree of toxicity and mitochondrial involvement. Inclusion of 6 parameters enabled the resolution of more subtle differences between compounds within a common therapeutic class; scoring enabled a ranking of statins in direct agreement with clinical outcomes. Comparison of drug-induced changes required variations in glucose for separation of mitochondrial dysfunction from other types of cytotoxicity. These results also demonstrate that the number of drugs in a training set, the choice of parameters used in analysis, and statistical measures are fundamental for specific hypothesis testing and assessment of quantitative phenotypic differences.

Revisiting absorbance at 230 nm as a protein unfolding probe

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230nm (A(230)) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A(230) as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A(230). Significant changes in A(230) were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A(230) in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A(230) under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A(230) allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A(230) is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.

A yeast two-hybrid smart-pool-array system for protein-interaction mapping

We present here a new two-hybrid smart pool array (SPA) system in which, instead of individual activation domain strains, well-designed activation domain pools are screened in an array format that allows built-in replication and prey-bait deconvolution. Using this method, a Saccharomyces cerevisiae genome SPA increases yeast two-hybrid screening efficiency by an order of magnitude.

Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells

BACKGROUND Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear. METHODS The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselens antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model. RESULTS Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2μg/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drugs antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselens in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load. CONCLUSION Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent. GENERAL SIGNIFICANCE The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development.

Phenotypic Profiling of Raf Inhibitors and Mitochondrial Toxicity in 3D Tissue Using Biodynamic Imaging

The existence of phenotypic differences in the drug responses of 3D tissue relative to 2D cell culture is a concern in high-content drug screening. Biodynamic imaging is an emerging technology that probes 3D tissue using short-coherence dynamic light scattering to measure the intracellular motions inside tissues in their natural microenvironments. The information content of biodynamic imaging is displayed through tissue dynamics spectroscopy (TDS) but has not previously been correlated against morphological image analysis of 2D cell culture. In this article, a set of mitochondria-affecting compounds (FCCP, valinomycin, nicardipine, ionomycin) and Raf kinase inhibitors (PLX4032, PLX4720, GDC, and sorafenib) are applied to multicellular tumor spheroids from two colon adenocarcinoma cell lines (HT-29 and DLD-1). These were screened by TDS and then compared against conventional image-based high-content analysis (HCA). The responses to the Raf inhibitors PLX4032 and PLX4720 are grouped separately by cell line, reflecting the Braf/Kras difference in these cell lines. There is a correlation between TDS and HCA phenotypic clustering for most cases, which demonstrates the ability of dynamic measurements to capture phenotypic responses to drugs. However, there are significant 2D versus 3D phenotypic differences exhibited by several of the drugs/cell lines.

High-Throughput Secondary Screening at the Single-Cell Level

We have developed an automated system for drug screening using a single-cell–multiple functional response technology. The approach uses a semiautomated preparatory system, high-speed sample collection, and a unique analytical tool that provides instantaneous results for compound dilutions using 384-well plates. The combination of automation and rapid robotic sampling increases quality control and robustness. High-speed flow cytometry is used to collect single-cell results together with a newly defined analytical tool for extraction of IC50 curves for multiple assays per cell. The principal advantage is the extreme speed of sample collection, with results from a 384-well plate being completed for both collection and data processing in less than 10 min. Using this approach, it is possible to extract detailed drug response information in a highly controlled fashion. The data are based on single-cell results, not populations. With simultaneous assays for different functions, it is possible to gain a more detailed understanding of each drug/compound interaction. Combined with integrated advanced data processing directly from raw data files, the process from sampling to analytical results is highly intuitive. Direct PubMed links allow review of drug structure and comparisons with similar compounds.

Repurposing Approach Identifies Auranofin with Broad Spectrum Antifungal Activity That Targets Mia40-Erv1 Pathway.

Current antifungal therapies have limited effectiveness in treating invasive fungal infections. Furthermore, the development of new antifungal is currently unable to keep pace with the urgent demand for safe and effective new drugs. Auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, inhibits growth of a diverse array of clinical isolates of fungi and represents a new antifungal agent with a previously unexploited mechanism of action. In addition to auranofins potent antifungal activity against planktonic fungi, this drug significantly reduces the metabolic activity of Candida cells encased in a biofilm. Unbiased chemogenomic profiling, using heterozygous S. cerevisiae deletion strains, combined with growth assays revealed three probable targets for auranofins antifungal activity—mia40, acn9, and coa4. Mia40 is of particular interest given its essential role in oxidation of cysteine rich proteins imported into the mitochondria. Biochemical analysis confirmed auranofin targets the Mia40-Erv1 pathway as the drug inhibited Mia40 from interacting with its substrate, Cmc1, in a dose-dependent manner similar to the control, MB-7. Furthermore, yeast mitochondria overexpressing Erv1 were shown to exhibit resistance to auranofin as an increase in Cmc1 import was observed compared to wild-type yeast. Further in vivo antifungal activity of auranofin was examined in a Caenorhabditis elegans animal model of Cryptococcus neoformans infection. Auranofin significantly reduced the fungal load in infected C. elegans. Collectively, the present study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antifungal agent and may offer a safe, effective, and quick supplement to current approaches for treating fungal infections.

A Chemogenomic Screening Platform Used to Identify Chemotypes Perturbing HSP90 Pathways

Compounds that modulate the heat shock protein (HSP) network have potential in a broad range of research applications and diseases. A yeast-based liquid culture assay that measured time-dependent turbidity enabled the high-throughput screening of different Saccharomyces cerevisae strains to identify HSP modulators with unique molecular mechanisms. A focused set of four strains, with differing sensitivities to Hsp90 inhibitors, was used to screen a compound library of 3680 compounds. Computed turbidity curve functions were used to classify strain responses and sensitivity to chemical effects across the compound library. Filtering based on single-strain selectivity identified nine compounds as potential heat shock modulators, including the known Hsp90 inhibitor macbecin. Haploid yeast deletion strains (360), mined from previous Hsp90 inhibitor yeast screens and heat shock protein interaction data, were screened for differential sensitivities to known N-terminal ATP site-directed Hsp90 inhibitors to reveal functional distinctions. Strains demonstrating differential sensitivity (13) to Hsp90 inhibitors were used to prioritize primary screen hit compounds, with NSC145366 emerging as the lead hit. Our follow-up biochemical and functional studies show that NSC145366 directly interacts and inhibits the C-terminus of Hsp90, validating the platform as a powerful approach for early-stage identification of bioactive modulators of heat shock–dependent pathways.

High Throughput Experimentation and Continuous Flow Validation of Suzuki-Miyaura Cross-Coupling Reactions

Traditional methods to discover optimal reaction conditions for small molecule synthesis is a time-consuming effort that requires large quantities of material and a significant expenditure of labor. High-throughput techniques are a potentially transformative approach for reaction condition screening, however, rapid validation of the reaction hotspots under continuous flow conditions remains necessary to build confidence in high throughput screening hits. Continuous flow technology offers the opportunity to upscale the screening hotspots and optimize their output of the target compounds due to the exceptional heat and mass transfer ability of flow reactions that are conducted in a smaller and safer reaction volume. We report a robotic high throughput technique to execute reactions in multi-well plates that were coupled with fast mass spectrometric analysis using an autosampler to accelerate the reaction screening process. Palladium-catalyzed Suzuki-Miyaura cross-coupling reactions were screened in this system and a heat map was generated to identify the best reaction conditions for downstream scale-up under continuous flow. Here, high throughput experimentation reactions in 96-well plates were performed for 1 h at 50 °C, 100 °C, 150 °C, and 200 °C before diluting them into 384-well plates for mass analysis. With the aid of high throughput tools, 648 unique experiments were conducted in duplicate. The cross-coupling reactions were evaluated as a function of stoichiometry, temperature, concentration, order of addition, and substrate type. The hotspots from high throughput experimentation were examined using a microfluidic Chemtrix system that confirmed the positive reaction leads as true positives. Negative outcomes identified by these experiments were found to be true negatives by microfluidic reaction evaluation. Quantitation of product yields was performed using high-performance liquid chromatography-mass spectrometry (HPLC/MS-MS).