Fig. 4. Design of antimicrobial cyclotide MCo-PG2 using molecular grafting. A topologically modified version of protegrin 1 (PG-1) was grafted into loop 6 of trypsin inhibitor cyclotide MCoTI-II. The resulting engineered cyclotide precursor was readily produced by solid-phase peptide synthesis. The crude linear thioester was then cyclized and oxidatively folded in a “one-pot reaction”.⁷⁷ The resulting cyclotide, MCo-PG2, displayed broad-spectrum antimicrobial activity in vitro against different ESKAPE pathogens (P. aeruginosa, S. aureus, K. pneumoniae, and E. coli), including 20 MDR clinical isolates for the human pathogens S. aureus and P. aeruginosa. Cyclotide MCo-PG2 also improved the survival rate in a peritonitis mice model using the clinical isolate of P. aeruginosa (ATCC 27853) from 0% for the untreated mice group to 90% for the mice group treated with 25 mg kg⁻¹ of cyclotide.⁷⁷.