Evaluation of novel N-terminal capping groups in peptide-based drug design: A SAR study utilizing neurotensin (8-13)

The use of peptides as therapeutic agents is limited by several factors including instability in biological fluids and inability to cross biological barriers. Modifications of peptide N-termini improve biostability by blocking the action of aminopeptidases. Judicious choice of N-terminal capping moieties also can lead to enhancement of barrier crossing and other parameters relevant to peptide-based therapeutics. Direct comparisons between the influence of N-terminal modifications on a variety of PK parameters have not been reported. In order to benchmark N-terminal capping groups, modifications were made to NT(8-13), the active fragment of the endogenous peptide neurotensin, as well as the related analogues KH28 and the Eisai peptide. These well-characterized peptide scaffolds are structurally distinct, but exhibit similar biological activities and feature progressively greater biostability.; Derivatives based on the three template peptides were synthesized with the following functional groups substituted for the N-terminal alpha-amine: amino-, azido-, Nalpha-acetyl-, Nalpha-methyl-, Calpha-methyl-, or hydro-. The peptides were tested in vitro for serum stability, receptor binding affinity, and ability to activate intracellular Ca 2+ signaling. The ability of the derivatives to partition into the CNS and induce hypothermia was also evaluated in vivo. Each of the N-terminal modifications drastically increased resistance of the parent peptides to aminopeptidase activity, resulting in distinct enzymatic degradation profiles at cleavage sites other than the N-terminus. Further, the capping moieties promoted different rates of degradation within each series, suggesting that the substitutions produced broad conformational effects. The modifications did not adversely affect binding affinity or functional agonism. However, these related parameters did not correlate with the effectiveness of induced hypothermia in vivo. In contrast, serum stability correlated significantly with the extent of hypothermia induced by each peptide. Hence, this work demonstrated that diminished binding affinity and agonist efficacy can be offset by enhanced molecular stability, a relationship likely due to increased bioavailability. Each of the N-terminal capping groups evaluated improves molecular stability without altering the backbone and amino acid side-chain functional groups. This work provides a direct comparison between N-terminal modifications, and validates N-terminal capping with each of these groups as an effective strategy that should find widespread use in peptide-based drug design.