Enhancing the Intrinsic Antiplasmodial Activity and Improving the Stability and Selectivity of a Tunable Peptide Scaffold Derived from Human Platelet Factor 4

Abstract

The 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.

Keywords: Plasmodium; drug development; host defense peptide; malaria; rational design; targeted cell-penetration.

Figure 1

First-generation PF4-derived internalization peptide (PDIP) analogue (cPF4PD, 1) targets and selectively enters RBCs infected with P. falciparum. (A) Normal uninfected RBCs (uRBC) have an asymmetric distribution of lipids, maintaining negatively charged phosphatidylserine (PS) headgroups on the inner leaflet of the cell plasma membrane, whereas PS is also present on the outer leaflet of infected RBCs (iRBC). Percentage RBC phospholipid data from ref (32). PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine. (B) PDIP analogue (1) labeled with AlexaFluor-488 (A488) has rapid (<1 h) selective entry into iRBC and accumulates inside the parasite. Scale bars are 5 μm.

Figure 2

Cartoon Representation of the Structures of PDIP Parent Peptides and Analogues.

Figure 3

Sequence Diversity of the C-terminal 14 Amino Acids of PF4 Informed the Design of PDIP Analogues. (A) C-terminal 14 amino acids of PF4 subunits (PDB: 1F9Q) arrange into paired α-helices. Representative sequence of PF4 C-terminal amino acids showing naturally occurring substitutions (full alignment from BLASTP search of nonredundant protein sequences is shown in Figure S3). (B) Helical wheel diagram showing the predicted position of residues in the two helices of PDIP analogue 3; heptad position is shown (a–g), and the diagram was generated using DrawCoil 1.0 (https://grigoryanlab.org/drawcoil). (C) A cartoon of PDIP (3) shows the location of amino acid substitutions.

Figure 4

Characteristics of PDIP Base Peptides and Set 1 Analogues. (A) CD spectra were collected for 50 μM peptides in aqueous solution (100 mM NaF, 10 mM KH₂PO₄ pH 7.5). Peptides with spectral minima at 208 and 222 nm have an overall helical structure. The MRE at 222 nm was used to calculate percentage helicity (Table 1). (B) Resistance to breakdown by serum proteases was determined from the amount of peptide remaining after 24 h of incubation with 25% (v/v) human serum. Peptides were quantified relative to time zero samples from area under the curve of intact mass m/z peaks using TOF-MS. (C) Peptide–lipid binding was compared using surface plasmon resonance (SPR) sensorgrams collected for 16 μM peptides binding to POPC/POPS (4:1) and POPC lipid bilayers. Response units (RU) were converted to P/L using (RU_peptide/mw_peptide)/(RU_lipid/mw_lipid). P/L at the end of the association phase (170 s) was used for comparing the peptide–lipid binding affinity for POPC/POPS (4:1) compared to that of POPC membranes in (D) (Table 1). Dashed lines or shaded regions provide a comparison to peptide 3 in each of the plots. Structure cartoons show the location of substitutions (in blue) relative to the parent scaffold.

Figure 5

Characteristics of Set 2 Analogues. (A) CD spectra were collected for 50 μM peptides in aqueous solution (100 mM NaF, 10 mM KH₂PO₄ pH 7.5) as above. (B) Resistance to breakdown by serum proteases was determined from the amount of peptide remaining after 24 h incubation with 25% (v/v) human serum as above. (C) Peptide–lipid binding was compared using SPR sensorgrams collected for 16 μM peptides binding to POPC/POPS (4:1) and POPC lipid bilayers as above. P/L at the end of the association phase (170 s) was used for comparing peptide–lipid binding affinity for POPC/POPS (4:1) compared to POPC membranes in (D). Peptide 3 is included, with dashed lines or shaded regions providing a comparison in each of the plots.

Figure 6

Characteristics of PDIP Analogues with Combined Modifications. (A) CD spectra were collected for 50 μM peptides in aqueous solution (100 mM NaF, 10 mM KH₂PO₄ pH 7.5) as above. (B) Resistance to breakdown by serum proteases was determined from the amount of peptide remaining after 24 h incubation with 25% (v/v) human serum as above. (C) Peptide–lipid binding was compared using SPR sensorgrams collected for 16 μM peptides binding to POPC/POPS (4:1) and POPC lipid bilayers as above. P/L at the end of the association phase (170 s) was used for comparing peptide–lipid binding affinity for POPC/POPS (4:1) compared to POPC membranes in (D). Peptide 3 is included, with dashed lines or shaded regions providing comparison in each of the plots.

Figure 7

Flow diagram showing the rational improvement of selective antiplasmodial activity and stability for PDIPs.