Use of lantibiotic synthetases for the preparation of bioactive constrained peptides

Abstract

Stabilization of biologically active peptides is a major goal in peptide-based drug design. Cyclization is an often-used strategy to enhance resistance of peptides towards protease degradation and simultaneously improve their affinity for targets by restricting their conformational flexibility. Amongst the various cyclization strategies, the use of thioether crosslinks has been successful for various peptides including enkephalin. The synthesis of these thioethers can be arduous, especially for longer peptides. Described herein is an enzymatic strategy taking advantage of the lantibiotic synthetase LctM that dehydrates Ser and Thr residues to the corresponding dehydroalanine and dehydrobutyrine residues and catalyzes the Michael-type addition of Cys residues to form thioether crosslinks. The use of LctM to prepare thioether containing analogs of enkephalin, contryphan, and inhibitors of human tripeptidyl peptidase II and spider venom epimerase is demonstrated.Interest in peptide-based materials for use in human therapeutics has greatly increased in recent years, and fully synthetic peptide drugs have increasingly reached the clinic. 1 The proteolytic instability of peptides still presents a limitation, however, for widespread utilization of peptide therapeutics. An often employed strategy for the design of peptide-based drugs with improved selectivity and decreased proteolytic susceptibility involves cyclization to constrain their conformational flexibility. 2 A notable example features the stabilization of enkephalin by the introduction of a thioether crosslink between two alanines (Figure 1), which increased the bioactivity of the compound by several orders of magnitude due to increased biostability. 3 Other studies have also shown the increased stability of peptides and proteins by thioether crosslinks. 4,5 Thioether crosslinks between two alanine residues are called lanthionines and their synthesis has received much attention. 6 Despite important recent advances, 7-12 at present it is still difficult to introduce these structures efficiently into synthetic peptides, especially for large peptides. One promising route to these structures is through the biosynthetic machinery for lantibiotics. These compounds are ribosomally synthesized and posttranslationally modified antimicrobial peptides. 13,14 The first step in the modification process of class II lantibiotics involves phosphorylation of Ser and Thr residues and subsequent elimination of the phosphate group to generate dehydroalanines and Z-dehydrobutyrines, respectively ( Figure 2). 15 In a succeeding step, intramolecular Michael-type addition by Cys residues to the dehydroamino acids forms the lanthionine (from Ser) and methyllanthionine (from Thr) crosslinks. Lacticin 481 synthetase (LctM) carries out both steps in a regio-and Correspondence to: Wilfred A. van der Donk. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of ...

Ribosomally Synthesized and Post‐Translationally Modified Peptide Natural Products: New Insights into the Role of Leader and Core Peptides during Biosynthesis

“…[53] Similarly, the class II lanthionine synthetase LctM was shown to dehydrate and cyclise non-lantibiotic peptides attached to the leader peptide of its substrate LctA. [54] Non-natural cyclic peptides have also been generated by the cyclization machinery of cyanobactins and lasso peptides by insertion of non-natural peptide sequence in pace of native core peptides, [31],[55] and several RiPP biosynthetic enzymes have been shown to accept chimeric peptides with the leader peptide of one compound and core peptide of another ( vide infra ). These investigations show the very high substrate tolerance with respect to the core peptide and seem to require a specific activation mechanism to prevent the biosynthetic enzymes to act on just any peptide in the RiPP-producing cell.…”

Mechanistic Dissection of the Enzyme Complexes Involved in Biosynthesis of Lacticin 3147 and Nisin

“…Lantibiotic mutants with modulated activity have previously been described (35). Furthermore, the application of lantibiotic enzymes can generate biostable thioether-bridged therapeutic peptides, which are resistant against proteolytic degradation (15,21) Several studies indicated that lantibiotic-modifying and -transporting enzymes are organized in multimeric complexes (14,30,41). It was demonstrated before that cells containing all lacticin 3147 biosynthesis enzymes, except LtnM1, still produced Ltn ␤ (27).…”

Microbial engineering of dehydro-amino acids and lanthionines in non-lantibiotic peptides

“…Glycines flanking dehydratable residues at both sides appear to preclude dehydration . Dehydration of nonlantibiotic peptides has been demonstrated for LctM Levengood and van der Donk 2008) and for LtnM2 . Importantly LctM has broad substrate specificity with large peptide-engineering possibilities.…”