Proteolytic excision and in situ cyclization of a bioactive loop from an REI‐VL presentation scaffold

Covalent cyclization of peptides is an important tool in structure–function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor‐bound conformation. Previously we described a 2‐step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N‐terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533–539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo‐LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI‐VL) with an RGD‐containing sequence engineered into its CDR3 and flanked by Lys residues. This REI‐VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor αIIbβ3. The RGD‐containing peptide excised by endo‐LysC from the REI‐VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, an IC50 of about 10 nM. Significantly, both the Nα‐acylation and the cyclization reactions occur efficiently even in the context of the other endo‐LysC fragments of REI‐VL, which suggests that the reaction may prove useful in converting mixtures of endo‐LysC products of many proteins into the corresponding cyclic peptides in situ.