A new short template-designed peptide with good antitumor potential by an alternative modification of threonine and its phosphorylation.

Anticancer peptides (ACPs) are widely considered a promising antitumor alternative due to their unique action mechanism. A new type of ACPs with short sequence, excellent antitumor activity, low toxicity and good stability is urgently needed. In this work, a series of new short ACPs were designed and synthesized based on the novel short template (KLLK)3W by altering its hydrophobicity, net charge, and amino acid structure. By analyzing the structure-bioactivity relationship, the results showed that P1 with one Trp in the C-terminal showed stronger antitumor activity and relatively greater cell selectivity than the other P series peptides. After the further structural optimization, the T derivatives, particularly 2pT with pThr substitution at the amphipathic interface 2nd site showed desirable antitumor activity while maintaining significantly decreased hemolytic activity (an 8.5-fold reduction) and an improved serum half-life (a 2.4-fold increase) compared with P1. Mechanistic studies revealed the multiple action modes involving potent membrane disruption and intramolecular effects, implying a potential in combination with PTX. Moreover, the in vivo studies confirmed that 2 pT had excellent antitumor efficacy comparable to that of P1, exhibiting substantial superiority over the control groups (2T and PTX). Taken together, this study offers the valuable insights into developing promising short ACPs with good potential for tumor treatment.