Biology subjects

Dranchak, P.

Publications and source records attributed to Dranchak, P..

4 recordsLinked to original sources

bioRxiv2023

Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity

Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five-to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/563088v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@a21f94org.highwire.dtl.DTLVardef@1c76751org.highwire.dtl.DTLVardef@500930org.highwire.dtl.DTLVardef@8b6c05_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

pharmacology and toxicology↗

bioRxiv2022

In vivo quantitative high-throughput screening for drug discovery and comparative toxicology

Quantitative high-throughput screening (qHTS) evaluates the pharmacology of drug and investigational agent libraries for potential therapeutic uses, toxicological risk assessment, and increasingly for academic chemical tool discovery. Phenotypic HTS assays aim to interrogate molecular pathways and networks, often relying on cell culture systems, historically with less emphasis on multicellular organisms. C. elegans has served as a powerful eukaryotic model organism for human biology and disease by virtue of genetic conservation and experimental tractability. Here we describe a paradigm to enable C. elegans in qHTS using 384-well microtiter plate laser scanning cytometry. GFP-expressing organisms are used to reveal phenotype-modifying structure-activity relationships to guide subsequent life stages and proteomic analysis. E. coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over 7-days spanning two life cycles to mitigate complications from bacterial overgrowth. We demonstrate the method with a library composed of anti-infective agents, or molecules of general toxicological concern. Each was tested in 7-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations having a physiological impact. We anticipate a broader application of this qHTS-coupled proteomics approach will enable the analysis of C. elegans orthologous transgenic phenotypes of human pathologies to facilitate drug and probe profiling from high-impact chemical libraries for a range of therapeutic indications and study of potential toxicological signatures. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/505462v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@7ef320org.highwire.dtl.DTLVardef@8bc35corg.highwire.dtl.DTLVardef@1c6f446org.highwire.dtl.DTLVardef@cf56f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

Bioluminescence-based reporters for characterizing inhibitors and activators of human Sonic Hedgehog protein autoprocessing in live cells at high throughput.

The Sonic hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and cholesterylate the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursors enzymatic domain. Here we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing non-invasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a wild-type (WT) SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. A conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the substrate activity of cholesterol. Inducibility of the D46A reporter was established using a synthetic sterol, 2- carboxy cholestanol, designed to bypass the defect through intra-molecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel membrane-impermeable nanoluciferase substrate, CLZ-2P. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/497760v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@63c641org.highwire.dtl.DTLVardef@6cee38org.highwire.dtl.DTLVardef@18bd310org.highwire.dtl.DTLVardef@cdfdc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

High-content quantitative high-throughput screening identifies a cell cycle-associated signaling cascade that regulates a multienzyme metabolic assembly for glucose metabolism

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, the glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism in human cells. We developed a high-content quantitative high-throughput screening (qHTS) assay to evaluate the impact of small molecule libraries on the formation of PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three active compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling cascade that regulates the formation of PFK1-mediated glucosome assembly in human cells.

biochemistry↗