Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates

Varache, Mathieu; Powell, Lydia; Aarstad, Olav A.; Williams, Thomas L.; Wenzel, Margot N.; Thomas, David W.; Ferguson, Elaine L.

doi:10.1021/acs.molpharmaceut.9b00393

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Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates

Mathieu Varache

, Lydia Powell

, Olav A. Aarstad

, Thomas L. Williams, Margot N. Wenzel, David W. Thomas, Elaine L. Ferguson

Molecular Pharmaceutics, Volume: 16, Issue: 7, Pages: 3199 - 3207

Swansea University Author: Lydia Powell

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DOI (Published version): 10.1021/acs.molpharmaceut.9b00393

Abstract

Polymer masked–unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide’s activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a co...

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Published in:	Molecular Pharmaceutics
ISSN:	1543-8384 1543-8392
Published:	American Chemical Society (ACS) 2019
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URI:	https://cronfa.swan.ac.uk/Record/cronfa61616
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Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin–colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography–mass spectrometry (UPLC–MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. 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spelling	2022-11-09T12:41:17.8153919 v2 61616 2022-10-20 Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates 0e7e702952672bcbfdfd4974199202fb 0000-0002-8641-0160 Lydia Powell Lydia Powell true false 2022-10-20 BMS Polymer masked–unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide’s activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin–colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography–mass spectrometry (UPLC–MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. UPLC–MS revealed that the fully “unmasked” dextrin–colistin conjugate composed of colistin bound to at least one linker, whereas larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully “unmasked” conjugate compared to the partially degraded species (MIC = 0.25 and 2–8 μg/mL, respectively), whereas dextrin conjugation reduced colistin’s in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure–antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC–MS to aid the design of biodegradable polymer–antibiotic conjugates. Journal Article Molecular Pharmaceutics 16 7 3199 3207 American Chemical Society (ACS) 1543-8384 1543-8392 colistin polymer therapeutics mass spectrometry infection Gram-negative bacteria 1 7 2019 2019-07-01 10.1021/acs.molpharmaceut.9b00393 COLLEGE NANME Biomedical Sciences COLLEGE CODE BMS Swansea University This work was supported by a research grant from the UK Medical Research Council (MR/N023633/1). 2022-11-09T12:41:17.8153919 2022-10-20T14:32:52.4061661 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Mathieu Varache 0000-0001-7166-2253 1 Lydia Powell 0000-0002-8641-0160 2 Olav A. Aarstad 0000-0003-3671-9060 3 Thomas L. Williams 4 Margot N. Wenzel 5 David W. Thomas 6 Elaine L. Ferguson 7 61616__25710__0566ffac83884a3299e64c05c21a3996.pdf 61616.pdf 2022-11-09T12:40:14.3475434 Output 2095597 application/pdf Version of Record true This is an open access article published under a Creative Commons Attribution (CC-BY) License true eng http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html
title	Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates
spellingShingle	Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates Lydia Powell
title_short	Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates
title_full	Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates
title_fullStr	Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates
title_full_unstemmed	Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates
title_sort	Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates
author_id_str_mv	0e7e702952672bcbfdfd4974199202fb
author_id_fullname_str_mv	0e7e702952672bcbfdfd4974199202fb_***_Lydia Powell
author	Lydia Powell
author2	Mathieu Varache Lydia Powell Olav A. Aarstad Thomas L. Williams Margot N. Wenzel David W. Thomas Elaine L. Ferguson
format	Journal article
container_title	Molecular Pharmaceutics
container_volume	16
container_issue	7
container_start_page	3199
publishDate	2019
institution	Swansea University
issn	1543-8384 1543-8392
doi_str_mv	10.1021/acs.molpharmaceut.9b00393
publisher	American Chemical Society (ACS)
college_str	Faculty of Medicine, Health and Life Sciences
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hierarchy_top_title	Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id	facultyofmedicinehealthandlifesciences
hierarchy_parent_title	Faculty of Medicine, Health and Life Sciences
department_str	Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
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description	Polymer masked–unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide’s activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin–colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography–mass spectrometry (UPLC–MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. UPLC–MS revealed that the fully “unmasked” dextrin–colistin conjugate composed of colistin bound to at least one linker, whereas larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully “unmasked” conjugate compared to the partially degraded species (MIC = 0.25 and 2–8 μg/mL, respectively), whereas dextrin conjugation reduced colistin’s in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure–antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC–MS to aid the design of biodegradable polymer–antibiotic conjugates.
published_date	2019-07-01T04:20:33Z
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score	11.013148

Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates

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