Lawrence, NicoleHandley, Thomas N.G.de Veer, Simon J.Harding, Maxim D.Andraszek, AlicjaHall, LachlanRaven, Karoline D.Duffy, SandraAvery, Vicky M.Craik, David J.Malins, Lara R.McMorran, Brendan J.2025-05-312025-05-31PubMed:39087267ORCID:/0000-0003-1845-8872/work/171935138http://www.scopus.com/inward/record.url?scp=85200422034&partnerID=8YFLogxKhttps://hdl.handle.net/1885/733755827The control of malaria, a disease caused by Plasmodium parasites that kills over half a million people every year, is threatened by the continual emergence and spread of drug resistance. Therefore, new molecules with different mechanisms of action are needed in the antimalarial drug development pipeline. Peptides developed from host defense molecules are gaining traction as anti-infectives due to theood of inducing drug resistance. Human platelet factor 4 (PF4) has intrinsic activity against P. falciparum, and a macrocyclic helix-loop-helix peptide derived from its active domain recapitulates this activity. In this study, we used a stepwise approach to optimize first-generation PF4-derived internalization peptides (PDIPs) by producing analogues with substitutions to charged and hydrophobic amino acid residues or with modifications to terminal residues including backbone cyclization. We evaluated the in vitro activity of PDIP analogues against P. falciparum compared to their overall helical structure, resistance to breakdown by serum proteases, selective binding to negatively charged membranes, and hemolytic activity. Next, we combined antiplasmodial potency-enhancing substitutions that retained favorable membrane and cell-selective properties onto the most stable scaffold to produce a backbone cyclic PDIP analogue with four-fold improved activity against P. falciparum compared to first-generation peptides. These studies demonstrate the ability to modify PDIP to select for and combine desirable properties and further validate the suitability of this unique peptide scaffold for developing a new molecule class that is distinct from existing antimalarial drugs.This work was supported by funding from the Australian National Health and Medical Research Council (1183927 to B. J. M., N. L., and L. R. M.), US Department of Defense grant (PR210354 to D. J. C. and N. L.), and the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012). D.J.C. was supported by NHMRC grant (2009564). S.D. was supported by a Griffith University Postdoctoral Fellowship (GUPF_23_24). The authors would like to thank Yen-Hua Huang, Bhavesh Khatri, and Lai Yue Chan for peptide synthesis; and Megan Drew, Huma Sohail, and Kiran Javed for malaria parasite culture and support in performing preliminary activity assays that guided analogue design.14enPublisher Copyright: © 2024 The Authors. Published by American Chemical Society.drug developmenthost defense peptidemalariaPlasmodiumrational designtargeted cell-penetration2024-08-0910.1021/acsinfecdis.4c0027685200422034