Fig 1.
STAD-2 synthesis and function.
(A) STAD-2 and STAD-2 scramble were synthesized by substituting S-pentenyl alanine (S5, shown as *) into positions that are opposite to the binding surface targeting PKA-R. A helical wheel represents the STAD-2 secondary structure wherein hydrophobic residues are shown in red, S5 in black, and other residues in grey. (B) Fmoc chemistry was used to synthesize STAD-2 peptides containing the non-natural S5 residues at i, i+4 positions. Ring-closing metathesis was performed using Grubbs I catalyst to generate the hydrocarbon staple. (C) The interaction between the HsPKA D/D domain (pale cyan) and the docking sequence of an AKAP (orange) can be inhibited by the stapled disruptor peptide STAD-2 (red). STAD-2 mimics the docking sequence of an AKAP and disrupts binding to the regulatory subunit of PKA. Images were created using the crystal structure of PKA-RII (PDB access code: 2HWN [41]).
Fig 2.
STAD-2 peptides are selectively permeable to Plasmodium-infected red blood cells.
(A) Plasmodium-iRBC were treated for 6 hours with 1 μM FITC-conjugated STAD-2 and analyzed by flow cytometry. iRBC showed selective permeability to STAD-2 relative to uRBC. (B, C) Treatment of synchronous ring-stage (black bars) or late-stage (grey bars) cultures with 1 μM FITC-conjugated STAD-2, unstapled STAD-2 parent, or STAD-2 scramble demonstrated significantly increased uptake of STAD-2 by late-stage relative to ring-stage iRBC. However, both ring-stage and late-stage iRBC were minimally permeable to STAD-2 parent and STAD-2 scramble controls (2way ANOVA with Bonferroni posttest, p<0.001, n = 3–6, mean ± S.E.).
Fig 3.
STAD-2 reduces viability of P. falciparum in vitro.
(A) Synchronous ring- or late-stage iRBC were treated with 0, 0.5, 1, 2, or 5 μM STAD-2 or its scramble control, and parasitemia was determined by flow cytometry at 24-hours post-treatment. STAD-2 IC50 ≈ 1 μM for late-stage and 1.5 μM for ring-stage parasites (n = 3, mean ± S.E., red = STAD-2/late, blue = STAD-2/ring, orange = STAD-2 scramble/late, green = STAD-2 scramble/ring). (B) Synchronous iRBC were treated with 1 μM STAD-2 or STAD-2 scramble, and parasitemia was determined by flow cytometry at 24, 48 and 72 hours post-treatment. A significant decrease in parasitemia was seen with STAD-2 treatment of both ring- and late-stage iRBC (2way ANOVA, p<0.001, n = 3, mean ± S.E., red = STAD-2/late, blue = STAD-2/ring, orange = STAD-2 scramble/late, green = STAD-2 scramble/ring). (C) Analysis of cells from (B) by light microscopy showed STAD-2 treated iRBC to be morphologically indistinguishable from untreated controls. (D) Late-stage iRBC of increasing parasitemia were treated with 1 μM STAD-2, 1 μM STAD-2 scramble, or DMSO control for 6 hours. Since the presence of oxyhemoglobin is indicative of red blood cell lysis, culture medium was removed and analyzed for evidence of oxyhemoglobin (A415) by UV-Vis spectroscopy. Linear regression demonstrates positive correlation of cell lysis with increasing parasitemia and significantly increased lysis in STAD-2 treated cells relative to STAD-2 scramble and DMSO controls (p<0.0001, n = 4).
Fig 4.
STAD-2 rapidly localizes within the parasitophorous vacuole.
(A) 3D7 iRBC were treated with 1 μM STAD-2 or STAD-2 scramble, stained with 2 μg/mL Hoechst 33342, and analyzed by fluorescence microscopy. STAD-2 peptides consistently localized within the intracellular parasite and at much higher levels than its scrambled control. (B) iRBC treated for 20 minutes, 3 hours, or 6 hours with 1 μM STAD-2 showed that STAD-2 traffics to the parasitophorous vacuole by 20 minutes post-treatment.
Fig 5.
STAD-2 does not associate with PKA.
(A) Late-stage iRBC were treated with 1 μM FITC-conjugated STAD-2 for 2 hours and probed for P. falciparum PKA-R (top panel) or H. sapiens PKA-RII (bottom panel). STAD-2 did not show clear colocalization with either of the regulatory subunits. (B) Late-stage iRBC were treated with 1 μM STAD-2, 30 μM H89 (small molecule inhibitor of PKA), or 0.001% DMSO and analyzed by light microscopy at 48 hours post-treatment. H89-treated iRBC demonstrated clear absence of parasite digestive vacuoles while STAD-2 treated iRBC were indistinguishable from DMSO controls.
Fig 6.
STAD-2 uptake is largely independent of the PSAC.
(A) Late-stage iRBC were treated with 1 μM STAD-2 in the presence of 200 μM furosemide following pre-treatment with complete culture medium (Fur/STAD-2) or 200 μM furosemide (Fur/Fur/STAD-2). Treatment of late-stage iRBC with STAD-2 in the presence of furosemide demonstrated a visible, yet insignificant, decrease in STAD-2 uptake only when iRBC were pre-treated with furosemide (two-tailed t test, p = 0.0557, n = 3, mean ± S.E.). (B) Late-stage iRBC were treated with 1 μM STAD-2 in the presence of 5% D-Sorbitol (PSAC solute), 130 mM glycerol (AQP3 solute), or 6 μM AgNO3 (AQP1 inhibitor). Treatment with STAD-2 in the presence of 5% D-Sorbitol yielded a decrease in STAD-2 uptake similar to that seen in (A) while treatment in the presence of glycerol or AgNO3 did not differ from STAD-2 alone (n = 2, mean ± S.E.).
Fig 7.
STAD-2 is uniquely permeable to iRBC.
iRBC were treated for 6 hours with 1 μM stapled peptides of varying charges (A) or variants of STAD-2 (B), analyzed by flow cytometry, and reported as median fluorescence intensity (C, n = 2–6, mean ± S.E.). Of the various stapled peptides analyzed, only STAD-2 was clearly permeable to iRBC.
Table 1.
Stapled Peptides.