Thiolation and Carboxylation of Glutathione Synergistically Enhance Its Lead-Detoxification Capabilities

Abstract

The natural tripeptide glutathione (GSH) is a ubiquitous compound harboring various biological tasks, among them interacting with essential and toxic metal ions. Yet, although weakly binding the poisonous metal lead (Pb), GSH poorly detoxifies it. β-Mercaptoaspartic acid is a new-to-nature novel amino acid that was found to enhance the Pb-detoxification capability of a synthetic cyclic tetrapeptide. Aiming to explore the advantages of noncanonical amino acids (ncAAs) of this nature, we studied the detoxification capabilities of GSH and three analogue peptides, each of which contains at least one ncAA that harbors both free carboxylate and thiolate groups. A thorough investigation that includes in vitro detoxification and mechanistic evaluations, metal-binding affinity, metal selectivity, and computational studies shows that these ncAAs are highly beneficial in additively enhancing Pb binding and reveals the importance of both high affinity and metal selectivity in synergistically reducing Pb toxicity in cells. Hence, such ncAAs join the chemical toolbox against Pb poisoning and pollution, enabling peptides to strongly and selectively bind the toxic metal ion.

Cyclic Octapeptides Composed of Two Glutathione Units Outperform the Monomer in Lead Detoxification

“…Significantly, all cyclic octapeptides, in particular, 8 and 9, outperform the two drugs against Pb poisoning; DMSA and EDTA, and GSH, even when the tripeptide is administered in twofold excess compared with the octamers (Figures 3A-B, S2). These results, once again, corroborate the potential of peptides to act as chelating agents [17,18] and of this scaffold specifically as a clever design that enables controlled complexation and detoxification.…”

“…As expected, in the most stable isomer of the [Pb‐ 2 ⋅(H 2 O) m ]⋅ n {H 2 O} complex (predicted computationally), both Cys participate in binding the ion (Figure 2). Likewise, the thiolato group of GSH also coordinates with Pb(II) ion [18,23,24] . We, therefore, inverted the stereochemistry of one of the Cys residues of 2 to form peptide 3 , aiming to further understand the effect of Cys configuration on Pb‐binding and stability.…”

“…To achieve the desired efficiency, not only a high binding affinity, but also an enhanced metal selectivity is required. [18] Due to its smaller macrocyclic cavity, we hypothesized that 5 might prioritize smaller metal ions such as Zn(II), Cu(II), etc. [32] This would thus reduce the peptide's Pb selectivity.…”

“…With an excellent alignment to the reported ΔG comp values (Table 1), both peptides bind the toxic ion stronger than GSH. [18] Depleting Zn(II) and Ca(II) ions as essential metals are frequently reported during chelation treatment for Pb-poisoning and represent a significant drawback of the current CAs. [9,42] Evaluating complex stability in the presence of such ions and metal selectivity is crucial in designing new CAs.…”

“…[9,14] Considering all these disadvantages, the standard CAs for Pb poisoning are only indicated for extremely high blood-lead levels (BLL) of 9-14 times the BLL determined to require intervention, [14,16] even though there is no known safe BLL. [1,3] Inspired by natural detoxification systems, and based on our recent accomplishments with cyclic tetrapeptides [17] and GSHanalogs, [18] we envisioned that two units of GSH cyclized together would provide the necessary macrocyclic cavity and enough binding moieties to strongly capture Pb(II) ions. The cyclic design was chosen to improve metabolic stability [19] and for imposing conformational constraints on the peptide to achieve a more defined three-dimensional structure.…”