Expression and characterization of functional domains of FK506-binding protein 35 from Plasmodium knowlesi.

The FK506-binding protein of Plasmodium knowlesi (Pk-FKBP35) is considerably a viable antimalarial drug target, which belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) protein family member. Structurally, this protein consists of an N-terminal FK506-binding domain (FKBD) and a C-terminal tetratricopeptide repeat domain (TPRD). This study aims to decipher functional properties of these domains as a platform for development of novel antimalarial drugs. Accordingly, full-length Pk-FKBP35 as well as its isolated domains, Pk-FKBD and Pk-TPRD were overexpressed, purified, and characterized. The results showed that catalytic PPIase activity was confined to the full-length Pk-FKBP35 and Pk-FKBD, suggesting that the catalytic activity is structurally regulated by the FKBD. Meanwhile, oligomerization analysis revealed that Pk-TPRD is essential for dimerization. Asp55, Arg60, Trp77 and Phe117 in the Pk-FKBD were considerably important for catalysis as underlined by significant reduction of PPIase activity upon mutations at these residues. Further, inhibition activity of Pk-FKBP35 towards calcineurin phosphatase activity revealed that the presence of FKBD is essential for the inhibitory property, while TPRD may be important for efficient binding to calcineurin. We then discussed possible roles of FKBP35 in Plasmodium cells and proposed mechanisms by which the immunosuppressive drug, FK506, interacts with the protein.

Anti-malarial Activities of Two Soil Actinomycete Isolates from Sabah via Inhibition of Glycogen Synthase Kinase 3ß.

Exploiting natural resources for bioactive compounds is an attractive drug discovery strategy in search for new anti-malarial drugs with novel modes of action. Initial screening efforts in our laboratory revealed two preparations of soil-derived actinomycetes (H11809 and FH025) with potent anti-malarial activities. Both crude extracts showed glycogen synthase kinase 3ß (GSK3ß)-inhibitory activities in a yeast-based kinase assay. We have previously shown that the GSK3 inhibitor, lithium chloride (LiCl), was able to suppress parasitaemia development in a rodent model of malarial infection. The present study aims to evaluate whether anti-malarial activities of H11809 and FH025 involve the inhibition of GSK3ß. The acetone crude extracts of H11809 and FH025 each exerted strong inhibition on the growth of Plasmodium falciparum 3D7 in vitro with 50% inhibitory concentration (IC50) values of 0.57 ± 0.09 and 1.28 ± 0.11 µg/mL, respectively. The tested extracts exhibited Selectivity Index (SI) values exceeding 10 for the 3D7 strain. Both H11809 and FH025 showed dosage-dependent chemo-suppressive activities in vivo and improved animal survivability compared to non-treated infected mice. Western analysis revealed increased phosphorylation of serine (Ser 9) GSK3ß (by 6.79 to 6.83-fold) in liver samples from infected mice treated with H11809 or FH025 compared to samples from non-infected or non-treated infected mice. A compound already identified in H11809 (data not shown), dibutyl phthalate (DBP) showed active anti-plasmodial activity against 3D7 (IC50 4.87 ± 1.26 µg/mL which is equivalent to 17.50 µM) and good chemo-suppressive activity in vivo (60.80% chemo-suppression at 300 mg/kg body weight [bw] dosage). DBP administration also resulted in increased phosphorylation of Ser 9 GSK3ß compared to controls. Findings from the present study demonstrate that the potent anti-malarial activities of H11809 and FH025 were mediated via inhibition of host GSK3ß. In addition, our study suggests that DBP is in part the bioactive component contributing to the anti-malarial activity displayed by H11809 acting through the inhibition of GSK3ß.