Biology subjects

Zboray, K.

Publications and source records attributed to Zboray, K..

4 recordsLinked to original sources

bioRxiv2023

High-throughput ligand profile characterization in novel biosensor cell lines expressing seven heterologous olfactory receptors for the detection of volatile plant biomarkers

Agriculturally important crop plants emit a multitude of volatile organic compounds (VOCs), which are excellent indicators of their health status and/or their interaction with pathogens and pests. Here, we present the generation of a novel cellular biosensor panel for the recognition of fungal pathogen-related VOCs we had identified in the field as well as during controlled inoculations of several crop plants. The panel consists of seven stable HEK293 cell lines each expressing a functional Drosophila olfactory receptor as a biosensing element and a genetically encoded fluorescent calcium indicator protein. For high-throughput measurement of odorant binding by the cells, fluorescence response was detected in an automated 384-well microplate reader upon the injection of tester VOCs. Biosensor cell lines were characterized for their reference ligand binding in more detail, then a set of 66 VOCs was profiled on all cell lines over a concentration range of three orders of magnitude (1 M to 100 M). Forty-six VOCs (70%) evoked a response in at least one biosensor cell line and certain VOCs could activate the cell lines already from the nanomolar (ppb) concentration. Interaction profiles mapped in this study will support biosensor development for agricultural applications, but the olfactory receptor proteins may also be purified from these cell lines at sufficient yields for further processing including structure determination or coupling with artificial sensor devices.

molecular biology↗

bioRxiv2021

An advanced automated patch clamp protocol design to investigate drug - ion channel binding dynamics.

Standard high throughput screening projects using automated patch-clamp instruments often fail to grasp essential details of the mechanism of action, such as binding/unbinding dynamics and modulation of gating. In this study, we aim to demonstrate that depth of analysis can be combined with acceptable throughput on such instruments. Using the microfluidics-based automated patch clamp, IonFlux Mercury, we developed a method for a rapid assessment of the mechanism of action of sodium channel inhibitors, including their state-dependent association and dissociation kinetics. The method is based on a complex voltage protocol, which is repeated at 1 Hz. Using this time resolution we could monitor the onset and offset of both channel block and modulation of gating upon drug perfusion and washout. Our results show that the onset and the offset of drug effects are complex processes, involving several steps, which may occur on different time scales. We could identify distinct sub-processes on the millisecond time scale, as well as on the second time scale. Automated analysis of the results allows collection of detailed information regarding the mechanism of action of individual compounds, which may help the assessment of therapeutic potential for hyperexcitability-related disorders, such as epilepsies, pain syndromes, neuromuscular disorders, or neurodegenerative diseases.

pharmacology and toxicology↗

bioRxiv2021

Compound-specific, concentration-independent biophysical properties of sodium channel inhibitor mechanism of action

We have developed an automated patch-clamp protocol that allows high information content screening of sodium channel inhibitor compounds. We have observed that individual compounds had their specific signature patterns of inhibition, which were manifested irrespective of the concentration. Our aim in this study was to quantify these properties. Primary biophysical data, such as onset rate, the shift of the half inactivation voltage, or the delay of recovery from inactivation, are concentration-dependent. We wanted to derive compound-specific properties, therefore, we had to neutralize the effect of concentration. This study describes how this is done, and shows how compound-specific properties reflect the mechanism of action, including binding dynamics, cooperativity, and interaction with the membrane phase. We illustrate the method using four well-known sodium channel inhibitor compounds, riluzole, lidocaine, benzocaine, and bupivacaine. Compound-specific biophysical properties may also serve as a basis for deriving parameters for kinetic modeling of drug action. We discuss how knowledge about the mechanism of action may help to predict the frequency-dependence of individual compounds, as well as their potential persistent current component selectivity. The analysis method described in this study, together with the experimental protocol described in the accompanying paper, allows screening for inhibitor compounds with specific kinetic properties, or with specific mechanisms of inhibition.

pharmacology and toxicology↗

bioRxiv2020

Mechanism of non-blocking inhibition of sodium channels revealed by conformation-selective photolabeling

Sodium channel inhibitor drugs can exert their effect by either blocking, or modulating the channel. The extent of modulation versus channel block is crucial regarding the therapeutic potential of drug candidates. Modulation can be selective for pathological hyperactivity, while channel block affects vital physiological function as much as pathological activity. Previous results indicated that riluzole, a drug with neuroprotective and antiepileptic effects, may have a unique mechanism of action, where modulation is predominant, and channel block is negligible. We studied the effects of riluzole on rNaV1.4 channels expressed in HEK cells. We observed that inhibition by riluzole disappeared and reappeared at a rate that could not be explained by association/dissociation dynamics. In order to verify the mechanism of non-blocking modulation, we synchronized photolabeling with the voltage clamp protocol of patch-clamp experiments. Using this method, we could bind a photoreactive riluzole analog covalently to specific conformations of the channel. Photolabeling was ineffective at resting conformation, but effective at inactivated conformation, as judged from persisting modulated gating after removal of unbound photoactive drug from the solution. Mutation of the key residue of the local anesthetic binding site (F1579A) did not fully prevent ligand binding and inhibition, however, it eliminated most of the modulation caused by ligand binding. Our results indicate that riluzole binds with highest affinity to the local anesthetic binding site, which transmits inhibition by the unique non-blocking modulation mechanism. Our results also suggest the existence of one or more additional binding sites, with lower affinity, and different inhibition mechanism.

pharmacology and toxicology↗