Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	George V. Sharonov, Oksana V. Nekrasova, Ksenia S. Kudryashova, Mikhail P. Kirpichnikov, Alexey V. Feofanov
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	potassium channel channel blockers spheroplasts high throughput screening agitoxin charybdotoxin Technology T Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/d16d354f7ef944c1a6ecd7667f7027b7
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:d16d354f7ef944c1a6ecd7667f7027b7
record_format	dspace
spelling	oai:doaj.org-article:d16d354f7ef944c1a6ecd7667f7027b72021-11-25T16:46:42ZBioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers10.3390/bioengineering81101872306-5354https://doaj.org/article/d16d354f7ef944c1a6ecd7667f7027b72021-11-01T00:00:00Zhttps://www.mdpi.com/2306-5354/8/11/187https://doaj.org/toc/2306-5354Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the <i>E. coli</i> membrane. In order to make this channel accessible for the soluble compounds, <i>E. coli</i> were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay.George V. SharonovOksana V. NekrasovaKsenia S. KudryashovaMikhail P. KirpichnikovAlexey V. FeofanovMDPI AGarticlepotassium channelchannel blockersspheroplastshigh throughput screeningagitoxincharybdotoxinTechnologyTBiology (General)QH301-705.5ENBioengineering, Vol 8, Iss 187, p 187 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	potassium channel channel blockers spheroplasts high throughput screening agitoxin charybdotoxin Technology T Biology (General) QH301-705.5
spellingShingle	potassium channel channel blockers spheroplasts high throughput screening agitoxin charybdotoxin Technology T Biology (General) QH301-705.5 George V. Sharonov Oksana V. Nekrasova Ksenia S. Kudryashova Mikhail P. Kirpichnikov Alexey V. Feofanov Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers
description	Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the <i>E. coli</i> membrane. In order to make this channel accessible for the soluble compounds, <i>E. coli</i> were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay.
format	article
author	George V. Sharonov Oksana V. Nekrasova Ksenia S. Kudryashova Mikhail P. Kirpichnikov Alexey V. Feofanov
author_facet	George V. Sharonov Oksana V. Nekrasova Ksenia S. Kudryashova Mikhail P. Kirpichnikov Alexey V. Feofanov
author_sort	George V. Sharonov
title	Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers
title_short	Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers
title_full	Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers
title_fullStr	Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers
title_full_unstemmed	Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers
title_sort	bioengineered system for high throughput screening of kv1 ion channel blockers
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/d16d354f7ef944c1a6ecd7667f7027b7
work_keys_str_mv	AT georgevsharonov bioengineeredsystemforhighthroughputscreeningofkv1ionchannelblockers AT oksanavnekrasova bioengineeredsystemforhighthroughputscreeningofkv1ionchannelblockers AT kseniaskudryashova bioengineeredsystemforhighthroughputscreeningofkv1ionchannelblockers AT mikhailpkirpichnikov bioengineeredsystemforhighthroughputscreeningofkv1ionchannelblockers AT alexeyvfeofanov bioengineeredsystemforhighthroughputscreeningofkv1ionchannelblockers
_version_	1718412983856529408

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

Ejemplares similares