Design and Optimisation of Bioactive Cyclic Peptides: Generation of a Down-Regulator of TNF Secretion

Abstract

Although strong binding interactions between protein receptor and ligand do not require the participation of a large number of amino acids in either site, short peptide chains are generally poor at recreating the types of protein-protein interactions which take place during cell recognition and signalling process, probably because their flexible backbones prevent the side chains from forming sufficiently rigid and stable epitopes, which can take part in binding with the desired strength and specificity. In a recently-reported study, it was shown that a proto-epitope containing F, R and S amino acids has the ability to down-regulate TNF secretion by macrophages. This paper extends these findings, putting those amino acids into a short cyclic peptide scaffold, and determining the optimal configuration required to overcome the problems of conformational instability, and give rise to molecules which have potential as therapeutic agents in human disease, such as rheumatoid arthritis.

Efficacy of Bioactive Cyclic Peptides in Rheumatoid Arthritis: Translation from In Vitro to In Vivo Models

“…The technology then seeks to develop a small oligo-peptide containing the same amino acids, but which can present them as a rigid ligand-binding epitope to interact with cell receptors without need for presentation on a micelle surface. The end result (an internally constrained cyclic peptide) is described elsewhere [ 2 ], and examples are shown in Figure 6 below. However, in certain circumstances, a “halfway house” is introduced into the process, in which peptides are constructed, but these peptides are still presented on the surface of micelles.…”

“…For this reason, peptides were created in which the same amino acid side-chains were presented on a planar surface similar to that presumed to pertain on the exterior of the micelle. The development process is described elsewhere [ 1 , 2 ], and the resultant peptides (described as ‘Lexicon’ peptides) are shown in Figure 6 , where two lipid chains (represented by ‘Σ’ on the opposite side from the S, F and R side-chain residues) are enclosed inside the cavity of a hydroxypropyl β-cyclodextran ring. Upper and lower case letters indicate L and D chiral forms, respectively.…”

“…The development programme described in the previous papers cited [ 1 , 2 ] indicated that a combination of F, S and R on a planar ring, arranged in a clockwise triangle was the motif which elicited the responses observed. We felt it was important to establish whether this could be presented on different types of ring, or was critically dependent on one sole conformation.…”

“…At the same time, the cyclodextrin contributes further to the rigidity, and helps the peptide chain to form a planar structure, where the amino acid side-chains are all located above the plane, capable of interacting with receptors, while the lipid chains and cyclodextrin molecule are located beneath the plane of the peptide chain. In this way, the peptide mimics the orientation of the sidechains on the planar surface of the micelle, the presentation system that was originally employed to identify which amino acid combinations were active in bringing about biological changes (in this case inhibition of TNF secretion) in in vitro models for disease (see References [ 1 ] and [ 2 ] for more information).…”

“…Confounding factors include the unusual behaviour of cultured animal cell lines, different time courses of activity between a cell monolayer and a whole organism, drug interactions with blood components absent from culture media, restricted access to relevant cells in vivo, and unwanted side effects in normal cells in vivo. In the case of the peptides studied here, proof-of-principle was demonstrated in both a murine cell line, and human primary isolates [ 1 , 2 ]. However, culture conditions reflected the in vivo situation only approximately, peptide concentrations being maintained at a high concentration for eighteen hours, the stimulation of cells taking place two hours after introduction of the peptide, and the culture medium containing a relatively low concentration of serum components (~10% foetal bovine serum).…”