Proposed role of <i>P. falciparum</i> Hsp110 and other chaperones as capacitors for evolutionary change.

<p>Malaria parasite proteins with asparagine repeat–containing sequences have a greater risk of aggregation. <i>P. falciparum</i> Hsp110c, possibly with the help of other chaperones, negates much of this risk, thereby allowing these loop-like regions to mutate. Over time these mutations can give rise to novel protein domains, allowing the parasite to develop new functionalities such as drug resistance and new pathogenic factors.</p

The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism

<div><p>Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between <i>Hs</i>PKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against <i>Plasmodium falciparum</i> blood stage parasites <i>in vitro</i>. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC₅₀ ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.</p></div

Stapled Peptides.

<p>*Length in amino acids</p><p>Stapled Peptides.</p

STAD-2 uptake is largely independent of the PSAC.

<p>(<b>A</b>) 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>B</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 (<b>A</b>) while treatment in the presence of glycerol or AgNO₃ did not differ from STAD-2 alone (n = 2, mean ± S.E.).</p

STAD-2 is uniquely permeable to iRBC.

<p>iRBC were treated for 6 hours with 1 μM stapled peptides of varying charges (<b>A</b>) or variants of STAD-2 (<b>B</b>), analyzed by flow cytometry, and reported as median fluorescence intensity (<b>C</b>, n = 2–6, mean ± S.E.). Of the various stapled peptides analyzed, only STAD-2 was clearly permeable to iRBC.</p

STAD-2 peptides are selectively permeable to <i>Plasmodium</i>-infected red blood cells.

<p>(<b>A</b>) <i>Plasmodium</i>-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>B, C</b>) 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.).</p

STAD-2 rapidly localizes within the parasitophorous vacuole.

<p>(<b>A</b>) 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>B</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.</p

STAD-2 does not associate with PKA.

<p>(<b>A</b>) Late-stage iRBC were treated with 1 μM FITC-conjugated STAD-2 for 2 hours and probed for <i>P</i>. <i>falciparum</i> PKA-R (top panel) or <i>H</i>. <i>sapiens</i> PKA-RII (bottom panel). STAD-2 did not show clear colocalization with either of the regulatory subunits. (<b>B</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.</p

STAD-2 synthesis and function.

<p>(<b>A</b>) 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>B</b>) Fmoc chemistry was used to synthesize STAD-2 peptides containing the non-natural S₅ residues at <i>i</i>, <i>i</i>+4 positions. Ring-closing metathesis was performed using Grubbs I catalyst to generate the hydrocarbon staple. (<b>C</b>) The interaction between the <i>Hs</i>PKA 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129239#pone.0129239.ref041" target="_blank">41</a>]).</p