Design and synthesis of biologically active peptides: a 'tail' of amino acids can modulate activity of synthetic cyclic peptides

Abstract

In earlier work, we synthesized a cyclic 9-amino acid peptide (AFPep, cyclo[EKTOVNOGN]) and showed it to be useful for prevention and therapy of breast cancer. In an effort to explore the structure-function relationships of AFPep, we have designed analogs that bear a short 'tail' (one or two amino acids) attached to the cyclic peptide distal to its pharmacophore. Analogs that bore a tail of either one or two amino acids, either of which had a hydrophilic moiety in the side chain (e.g., cyclo[EKTOVNOGN]FS) exhibited greatly diminished biological activity (inhibition of estrogen-stimulated uterine growth) relative to AFPep. Analogs that bore a tail of either one or two amino acids which had hydrophobic (aliphatic or aromatic) side chains (e.g., cyclo[EKTOVNOGN]FI) retained (or had enhanced) growth inhibition activity. Combining in the same biological assay a hydrophilic-tailed analog with either AFPep or a hydrophobic-tailed analog resulted in decreased activity relative to that for AFPep or for the hydrophobic-tailed analog alone, suggesting that hydrophilic-tailed analogs are binding to a biologically active receptor. An analog with a disrupted pharmacophore (cyclo[EKTOVGOGN]) exhibited little or no growth inhibition activity. An analog with a hydrophilic tail and a disrupted pharmacophore (cyclo[EKTOVGOGN]FS) exhibited no growth inhibition activity of its own and did not affect the activity of a hydrophobic-tailed analog, but enhanced the growth inhibition activity of AFPep. These results are discussed in the context of a two-receptor model for binding of AFPep and ring-and-tail analogs. We suggest that tails on cyclic peptides may comprise a useful method to enhance diversity of peptide design and specificity of ligand-receptor interactions.

Figure 1. Inhibition of estrogen-stimulated uterine growth by analogs of AFPep

Panel A: AFPep (open squares) exhibits slight loss of inhibition at higher doses; cyclo[EKTOVNOGN]FI (open circles, indicated as N-FI) exhibits sigmoidal dose-response curve without loss of activity at higher doses. cyclo[EKTOVNOGN]FS (open triangles, indicated as N-FS) exhibits low activity at low dose, even less activity at higher doses. Panel B: Analogs with a disrupted pharmacophore exhibit very low activity. cyclo[EKTOVGOGN] (an analog of AFPep with a single point mutation) (closed squares, indicated as G) exhibited modest activity at low dose, no activity at higher doses, whereas cyclo[EKTOVGOGN]FS (closed triangles, indicated as G-FS) had no activity at any dose.

Figure 2. Combination of analogs affect biological responses

The activity of AFPep (Panel A) or of cyclo[EKTOVNOGN]FI (Panel B, indicated as N-FI) is decreased in the presence of an analog with an intact pharmacophore and a hydrophilic tail (cyclo[EKTOVNOGN]FS, indicated as N-FS) which may bind preferentially to, and activate, a putative low affinity receptor. The activity of AFPep is enhanced (Panel C), while the activity of cyclo[EKTOVNOGN]FI (indicated as N-FI) is unaffected (Panel D) in the presence of an analog with a disrupted pharmacophore and a hydrophilic tail, (namely cyclo[EKTOVGOGN]FS, indicated as G-FS) which may bind preferentially to, but not activate, the low affinity receptor.

Scheme 1

Synthetic process for generating ring-and-tail cyclic peptides.