PROTEOLYTIC EXCISION AND IN-SITU CYCLIZATION OF A BIOACTIVE LOOP FROM AN REI-V-L 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 V-L domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-V-L/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha(IIb)beta(3) The RGD-containing peptide excised by endo-LysC from the REI-V-L presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, an IC50 of about 10 nM. Significantly, both the N-alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-V-L, 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.