A series of compounds containing either non-proteinogenic β-/γ-amino acids or N-substituted β-alanine residues (β-peptoid units) in P1 specificity position were synthesized based on the structure of sunflower trypsin inhibitor 1 (SFTI-1). The compounds were synthesized on a solid support; the N-substituted β-alanines (βNhlys and βNhphe) were introduced into a peptidomimetic chain via a two-step approach using acryloyl chloride and an appropriate primary amine. The inhibitory activities were characterized in vitro against bovine α-chymotrypsin or bovine β-trypsin. Three analogues displayed activity comparable to fully proteinogenic counterparts-monocyclic SFTI-1 and [Phe(5)]SFTI-1. Moreover, all active peptidomimetics were resistant toward proteolytic degradation, even after 24-h incubation at room temperature.

θ-defensins belong to the family of host defence peptides. They are the only known example of cyclic polypeptides in animal proteomes. This study presents the synthesis of simplified θ-defensin analogues with pairs of cysteine replaced either by alanine, leucine or serine residues. Cytotoxicity tests were performed on human mammary epithelial (HB2) and breast cancer (SKBR3, MDA-MB-231) cell lines to determine whether peptides are selectively targeting cancer cells. The effect of these peptides was also evaluated in 3D Matrigel cultures, which are based on extracellular matrix components and therefore closely represent in vivo conditions. Finally, to determine whether analogues are able to sensitize MDA-MB-231 triple-negative breast cancer cells to chemotherapeutics, we co-administrated peptides with cisplatin or doxorubicin hydrochloride also in 3D Matrigel cultures. Additionally, cytotoxicity towards peripheral blood mononuclear cells and haemolytic effect were examined for a chosen representative of synthesized compounds. The results showed that positively charged serine-containing θ-defensin derivatives were more cytotoxic towards breast cancer cells (SKBR3, MDA-MB-231) than towards mammary epithelial cells (HB2). Analogues enhanced the effect of cisplatin and doxorubicin hydrochloride on triple-negative breast cancer cell line (MDA-MB-231).