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From Bioinspired Scaffolds to Genetically Encoded Libraries: The Quest for Next-Generation Chelating Agents for Lead


Sauser, Luca. From Bioinspired Scaffolds to Genetically Encoded Libraries: The Quest for Next-Generation Chelating Agents for Lead. 2024, University of Zurich, Faculty of Science.

Abstract

Lead (Pb) is a toxic metal that has been utilized by humanity for millennia. Particularly from the 20th century onwards, vast amounts of Pb have been released into the environment, causing persistent environmental pollution. This abundant and cumulative toxicant is a global health hazard, with an estimated 1 million deaths being attributed to Pb poisoning annually. Chelation therapy, which has been the mainstay for treating Pb poisoning since the middle of the last century, seeks to form eliminable complexes with this toxic metal, to reduce the Pb body burden. This is achieved by using small molecules, called chelating agents, as ligands. The current benchmark drugs, albeit being the default treatment, possess several drawbacks, among them being a lack of metal selectivity that leads to the depletion of essential metal ions. Therefore, they are prescribed only at high Pb levels, which is among the most severe drawbacks. In this work we explored the capability of short peptides to act as chelating agents for Pb2+ ions in three different projects. In the first project, glutathione (GSH), a naturally abundant tripeptide with low affinity and detoxification ability for Pb2+, was incorporated into a rationally-designed cyclic octapeptide scaffold with the goal of enhancing these traits. Members of the scaffold were computationally screened to identify promising ligands. Five peptides were chosen for synthesis and subsequent experimental investigations, which started with the evaluation of their capability to recover Pb-poisoned human cells. The two most successful peptides produced recovery rates that were approximately two times higher than the one of GSH. These lead candidates were subjected to a thorough analytical investigation to characterize their Pb-complexes and the reasons by which they succeeded in detoxifying this ion. In the second project, the influence of thiolation and carboxylation of GSH on its affinity for Pb2+ ions and its Pb-detoxification abilities were investigated. The additional functional groups were incorporated into the scaffold by using novel non-canonical amino acids (ncAAs). Three analogues of GSH were therefore investigated. This encompassed the assessment of their ability to detoxify Pb-poisoned human cells and the characterization of their respective Pb-complexes, metal selectivity, and binding modes. The results reveal the beneficial influence of the two ncAAs on Pb- detoxification and affinity in the given scaffold. In the third project, we used a genetically-encoded peptide library that was expressed in E.coli. A survival selection strategy (SSS) was employed to test individual, fully-proteinogenic phenotypes for their ability to enhance bacterial survival above the minimal inhibitory concentration (MIC) of Pb. Two identified peptides were subsequently applied as surface-displayed octa-repeats in bacterial bioremediation, which pointed towards a favorable impact of one of the sequences on removing Pb from medium. Secondly, one of the identified peptides was synthesized and tested in its linear and head-to-tail cyclic forms for its capabilities of recovering Pb-poisoned human cells, which demonstrated a desired efficacy and therefore the versatility for Pb-detoxification of the peptide identified by SSS.

Abstract

Lead (Pb) is a toxic metal that has been utilized by humanity for millennia. Particularly from the 20th century onwards, vast amounts of Pb have been released into the environment, causing persistent environmental pollution. This abundant and cumulative toxicant is a global health hazard, with an estimated 1 million deaths being attributed to Pb poisoning annually. Chelation therapy, which has been the mainstay for treating Pb poisoning since the middle of the last century, seeks to form eliminable complexes with this toxic metal, to reduce the Pb body burden. This is achieved by using small molecules, called chelating agents, as ligands. The current benchmark drugs, albeit being the default treatment, possess several drawbacks, among them being a lack of metal selectivity that leads to the depletion of essential metal ions. Therefore, they are prescribed only at high Pb levels, which is among the most severe drawbacks. In this work we explored the capability of short peptides to act as chelating agents for Pb2+ ions in three different projects. In the first project, glutathione (GSH), a naturally abundant tripeptide with low affinity and detoxification ability for Pb2+, was incorporated into a rationally-designed cyclic octapeptide scaffold with the goal of enhancing these traits. Members of the scaffold were computationally screened to identify promising ligands. Five peptides were chosen for synthesis and subsequent experimental investigations, which started with the evaluation of their capability to recover Pb-poisoned human cells. The two most successful peptides produced recovery rates that were approximately two times higher than the one of GSH. These lead candidates were subjected to a thorough analytical investigation to characterize their Pb-complexes and the reasons by which they succeeded in detoxifying this ion. In the second project, the influence of thiolation and carboxylation of GSH on its affinity for Pb2+ ions and its Pb-detoxification abilities were investigated. The additional functional groups were incorporated into the scaffold by using novel non-canonical amino acids (ncAAs). Three analogues of GSH were therefore investigated. This encompassed the assessment of their ability to detoxify Pb-poisoned human cells and the characterization of their respective Pb-complexes, metal selectivity, and binding modes. The results reveal the beneficial influence of the two ncAAs on Pb- detoxification and affinity in the given scaffold. In the third project, we used a genetically-encoded peptide library that was expressed in E.coli. A survival selection strategy (SSS) was employed to test individual, fully-proteinogenic phenotypes for their ability to enhance bacterial survival above the minimal inhibitory concentration (MIC) of Pb. Two identified peptides were subsequently applied as surface-displayed octa-repeats in bacterial bioremediation, which pointed towards a favorable impact of one of the sequences on removing Pb from medium. Secondly, one of the identified peptides was synthesized and tested in its linear and head-to-tail cyclic forms for its capabilities of recovering Pb-poisoned human cells, which demonstrated a desired efficacy and therefore the versatility for Pb-detoxification of the peptide identified by SSS.

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Additional indexing

Item Type:Dissertation (cumulative)
Referees:Sigel Roland K O, Shoshan Michal Mazal, Holland Jason Philip, Rulíšek Lubomír
Communities & Collections:07 Faculty of Science > Department of Chemistry
UZH Dissertations
Dewey Decimal Classification:540 Chemistry
Language:English
Place of Publication:Zürich
Date:24 April 2024
Deposited On:24 Apr 2024 13:12
Last Modified:03 May 2024 03:30
Number of Pages:253
OA Status:Closed