Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.

<h4>Background</h4>Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballi...

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Autores principales:	Sarah J Moore, Cheuk Lun Leung, Heidi K Norton, Jennifer R Cochran
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ca2be32f20cc460f80181c6f688ba77b
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spelling	oai:doaj.org-article:ca2be32f20cc460f80181c6f688ba77b2021-11-18T07:50:44ZEngineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.1932-620310.1371/journal.pone.0060498https://doaj.org/article/ca2be32f20cc460f80181c6f688ba77b2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23573262/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor.<h4>Methodology/principal findings</h4>We used a rational loop-grafting approach to engineer AgTx variants that bound to αvβ3 integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants. Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI. Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins.<h4>Conclusions/significance</h4>In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.Sarah J MooreCheuk Lun LeungHeidi K NortonJennifer R CochranPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 4, p e60498 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sarah J Moore Cheuk Lun Leung Heidi K Norton Jennifer R Cochran Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.
description	<h4>Background</h4>Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor.<h4>Methodology/principal findings</h4>We used a rational loop-grafting approach to engineer AgTx variants that bound to αvβ3 integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants. Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI. Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins.<h4>Conclusions/significance</h4>In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.
format	article
author	Sarah J Moore Cheuk Lun Leung Heidi K Norton Jennifer R Cochran
author_facet	Sarah J Moore Cheuk Lun Leung Heidi K Norton Jennifer R Cochran
author_sort	Sarah J Moore
title	Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.
title_short	Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.
title_full	Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.
title_fullStr	Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.
title_full_unstemmed	Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.
title_sort	engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/ca2be32f20cc460f80181c6f688ba77b
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Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.

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