Abstract
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 HsPKA 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 Plasmodium falciparum blood stage parasites in vitro. 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.
Highlights
Malaria, caused by haemoprotozoan parasites of the genus Plasmodium, is endemic to nearly 100 countries, placing the lives of an estimated 3.2 billion people at risk each year
Stapled AKAP Disruptor 2 (STAD-2) was designed to target the regulatory subunit of HsPKA and occlude A Kinase Anchoring Proteins (AKAPs) binding interactions as previously described [29] (Fig 1)
Treatment of late-stage infected red blood cells (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. (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)
Summary
Malaria, caused by haemoprotozoan parasites of the genus Plasmodium, is endemic to nearly 100 countries, placing the lives of an estimated 3.2 billion people at risk each year. Despite widespread endemicity, intensified control efforts have reduced annual malaria-attributable deaths by approximately 47% from nearly 1 million in 2000 to 584 000 in 2013 [1]. Such advances in malaria control are critically dependent on effective disease diagnosis and efficacious drugs. Since 2002, artemisinin combination therapy (ACT) has been the recommended first line treatment for uncomplicated P. falciparum malaria [2], and chloroquine is recommended for P. vivax in regions where it remains efficacious [1]. Extensive resistance to all existing antimalarials, including growing resistance to artemisinin in the Greater Mekong Subregion, threatens to place control efforts, and millions of lives, in jeopardy [3,4,5]
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