Towards an effective approach to target plant Like Calcium Dependent Protein Kinase of Plasmodium falciparum PfCDPK1 for anti malarial drug discovery

Abstract

Clinical manifestations of malaria are attributed to the rapid intra-erythrocytic proliferation cycles of Plasmodium falciparum. A plant-like Calcium-Dependent Protein Kinase 1 (PfCDPK1) mediates signaling cascades that play key role in deriving the cellular processes regulating the parasite growth. We identify novel PfCDPK1-inhibitory compounds by adopting combinatorial approach including virtual screening of MyriaScreenII library of 10,000 drug-like molecules and their in vitro validation. Biochemical enzymatic assays were utilized, coupled with biophysical characterization of inhibition. Two lead compounds: ST092793 and S344699 with potent PfCDPK1-inhibitory activity were shown to directly bind to ATP-binding pocket of the protein, preferably in the absence of Calcium-ions; thus, conferring thermo stabilization to the protein. Cytotoxicity of the molecular candidates showed high selectivity for the parasite over host cells. PfCDPK1-inhibitory ligands, thus identified, may behave as pan-PfCDPK inhibitors owing to well conserved catalytic pocket across PfCDPK isoforms. Furthermore, analytical biochemistry and biophysical assays showed that PfCDPK1 interacts with a member of highly conserved regulatory adapter, Pf14-3-3 protein in a phosphorylation dependent manner. This finding was supported by Molecular Dynamics (MD) simulation studies. Blocking this interaction using consensus 14-3-3 binding peptides as CDPK1 mimetics, inhibits the parasite growth ex vivo. We not only characterize Pf14-3-3I as scaffold protein in Plasmodium falciparum and unveil PfCDPK1 as its target, but also define a new way of potentially targeting parasite kinases towards the development of anti-malarial chemotherapeutics.