Development of novel anti-malarial from structurally diverse library of molecules, targeting plant-like Calcium Dependent Protein Kinase 1, a multistage growth regulator of P. falciparum

Upon Plasmodium falciparum merozoites exposure to low [K+] environment in blood plasma, there is escalation of cytosolic [Ca2+] which activates Ca2+-Dependent Protein Kinase 1 (CDPK1), a signaling hub of intra-erythrocytic proliferative stages of parasite. Given its high abundance and multidimensional attributes in parasite life-cycle, this is a lucrative target for desiging antimalarials. Towards this, we have virtually screened MyriaScreenII diversity collection of 10,000 drug-like molecules, which resulted in 18 compounds complementing ATP-binding pocket of CDPK1. In vitro screening for toxicity in mammalian cells revealed that these compounds are non-toxic in nature. Further, SPR analysis demonstrated differential binding affinity of these compounds towards recombinantly purified CDPK1 protein. Selection of lead compound 1 was performed by evaluating their inhibitory effects on phosphorylation and ATP binding activities of CDPK1. Further, in vitro biophysical evaluations by ITC and FS revealed that binding of compound 1 is driven by formation of energetically favorable non-covalent interactions, with different binding constants in presence and absence of Ca2+, and TSA authenticated stability of compound 1 bound CDPK1 complex. Finally, compound 1 strongly inhibited intra-erythrocytic growth of P. falciparum in vitro. Concievably, we propose a novel CDPK1-selective inhibitor, step towards developing pan-CDPK kinase inhibitors, prerequisite for cross-stage anti-malarial protection.


INTRODUCTION
Plasmodium falciparum is the most prevalent malaria parasite which accounted for majority of the total showing reduction in the gliding motility phenotype. (3,4) Similarly, PbCDPK4 regulates sexual differentiation into male gametocytes and therefore, being crucial for transmission to the mosquito host and sexual reproduction. (5) Likewise, PfCDPK5 guides egress of merozoites from the host erythrocytes. (6) However, besides these members of cdpk multigene family (7,8), one of the most widely studied member is CDPK1, which is a signaling hub responsible for multistage cellular processes involved in schizogony, merozoite invasion, gametogenesis, and transmission to the mosquito host. (9)(10)(11)(12)(13) Mechanistic studies revealed that Protein Kinase G (PKG)-mediated phosphorylation of CDPK1 allows its localization to apical structures. (14) Another piece of study also depicted that CDPK1 regulates actinmyosin motor during gliding and invasion of the parasite by phosphorylating a 50-kDa Glideosome-Associated Protein (GAP-45) and Myosin Tail domain Interacting Protein (MTIP), two important components of Inner Membrane Complex (IMC) of P falciparum merozoites. Along with the proteins of IMC, it also phosphorylates regulatory subunit of cAMP-dependent Protein Kinase A (PKAr), thereby regulating cAMP-mediated signaling cascade module. (13) With this understanding of an indispensable role of CDPK1 in the parasite life cycle, we pursued this as a potential target for development of novel specific inhibitors that can block parasite growth targeting multiple stages in malarial infection. In this study, we have identified small molecule ligands that bind to and inhibit the functional (kinase) activity of CDPK1 in vitro and parasite growth ex vivo, by combining 5 high-throughput virtual screening strategy, followed by experimental evaluation by adopting several biochemical and biophysical assays in vitro. This study can in principle be extended towards developing novel drug candidates against any eukaryotic parasite by systematic high-throughput screening of small moleculebased drug-like libraries.

Purification of 6xHis-CDPK1, Homology modeling and Virtual screening
Cloning, over-expression and purification of 6xHis-CDPK1 was done as described previously. (11) To raise anti-sera, mice were immunized by injecting the emulsified protein. Comparative or homology modeling was employed to model 3D structure of CDPK1 from P. falciparum strain 3D7. To accomplish this, amino acid sequence of PfCDPK1 protein (PF3D7_0217500) was retrieved from PlasmoDB database. (16) To search for a suitable template for homology modeling, BLASTp search was performed using amino acid sequence of PfCDPK1 as query sequence, against Protein Data Bank (PDB) database. (17,18) Amino acid sequence identity between CDPK1 orthologs from P. falciparum 3D7 and P. berghei strain ANKA was found to be 93%, thus rendering X-Ray diffraction based structural model of PbCDPK1 (PDB ID: 3Q5I; resolution: 2.1 Å) as a suitable template to model 3D structure of PfCDPK1. (19) Homology modeling was done by using Modeller v9.17 software. (20) Generated model was further subjected to structural refinement by using ModRefiner. (21) Reliability of the refined structural model of CDPK1 was assessed by examining backbone dihedral angles: phi (Ø) and psi (Ψ) of the amino acid residues lying in the energetically favourable regions of Ramachandran space. (22) This was done by using PROCHECK v.3.5. (23) Percentage quality assessment of the protein structure was evaluated by utilizing four sorts of occupancies: core, additional allowed, generously allowed and disallowed regions. The refined 3D structural model of PfCDPK1, thus generated, was subsequently used for docking studies.

6
Novel small molecule inhibitors of CDPK1 were identified by virtual screening of MyriaScreen II diversity collection of 10,000 drug-like screening compounds. (24)  in the absence of any compound were maintained as positive control. After treatment, MTT labeling reagent was added to each well to a final concentration of 0.5 mg/ml and incubated at 37°C for 4 hrs. Purple colored formazan crystals, thus formed, were dissolved in 100 ul DMSO solvent and optical density was measured spectrophotometrically by taking absorbance at 570 nm using Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific™). The absorbance of untreated cells was taken as 100%.

Surface Plasmon Resonance (SPR) analysis of binding affinity between compounds and CDPK1
To determine binding strength of compounds (N=18) procured from virtual screening of MyriaScreen II library, real-time biomolecular interaction analysis with SPR was carried out at physiologically relevant concentrations by using AutoLab Esprit SPR. Kinetic rate constants: K a and K d (association and dissociation rate constants, respectively) as well as affinity constant, K D were measured at room temperature (RT, 293K). AutoLab SPR Kinetic Evaluation software provided with the instrument. K D value was calculated using the Integrated Rate Law (IRL) equation. Two independent experiments were performed.

In vitro kinase assay with CDPK1
Functional activity of 6xHis-CDPK1 was monitored by utilizing ADP-Glo™ Kinase Assay (Promega Corporation), which is ATP regeneration based luciferase reaction system resulting from nascent ADP phosphorylation. The luminescence signal, thus generated, is proportional to the amount of ADP released in a given kinase reaction. Phosphorylation experiments were performed with 100 ng of 6xHis-CDPK1, per reaction, in assay buffer (100 mM Tris-Cl, pH 7.4; 2.5 mM DTT; 50 mM MgCl 2 and 2.5 mM MnCl 2 ), by following previously described protocol. (29) Enzymatic reaction was carried out in the absence and presence of calcium ions. 10 μ g of dephosphorylated casein from bovine milk, per reaction, was used as exogenous substrate for the enzyme. 2.5 mM EGTA was added for conditions requiring absence of Ca 2+ ions. Kinase reactions were initiated by adding 1 µM ATP and allowed to take place at 30 o C for 1 hr. To test for any inhibitory effect of compounds (N=18) screened from MyriaScreen II Diversity Collection, CDPK1 was allowed to pre-incubate in the reaction buffer with 50 µM of each compound at room temperature for 1 hr, high resolution scanner. Estimates of band intensities from both Pro-Q Diamond and Coomassie stained gel images were made using ImageJ 1.52a. The experiment was done thrice.

Competitive ATP affinity chromatography assay
To confirm if the lead compounds 1 (ST092793) and 2 (S344699) inhibit functional activity of CDPK1 by directly binding to its active site (ATP-binding pocket), affinity chromatography of CDPK1 pre-incubated with the compounds was performed by utilizing ATP Affinity Test Kit (Jena Bioscience). Protocol was followed as recommended by the manufacturer. Briefly, 10 μ g of purified CDPK1 protein per reaction was incubated with 100 μ M of compound 1 (ST092793) and/or 2 (S344699) in binding solution for 1 hr at RT. This interaction was competed with 8AH-ATP-agarose {8-[(6-Amino) Hexyl]-amino-ATP-agarose} in which ATP is immobilized on the agarose matrix via the adenine base through C8 linker. Blank agarose served as negative control. Elution fractions were analyzed by SDS-PAGE and immunobloting by probing recombinant CDPK1 with monoclonal poly-Histidine-peroxidase antibody. CDPK1 present in flowthroughs of control and test samples served as loading controls for normalization. Signals were detected with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific™). Estimates of band intensities of CDPK1 from both control and treated reactions were made using ImageJ 1.52a. The experiment was done twice.

Isothermal Titration Calorimetry (ITC) and Steady-state fluorescence spectroscopy
To calculate kinetic parameters such as binding affinity constant, K a for the interaction of CDPK1 with relatively more potent inhibitor (i.e., compound 1 (ST092793) ), ITC experiments were performed by using MicroCal iTC200 (Malvern Instruments Ltd, UK). For this purpose, recombinant CDPK1 protein was dialyzed extensively against HEPES-NaCl buffer (10 mM HEPES-NaOH, pH 7.4 and 150 mM NaCl) using Amicon TM Ultra-15 Centrifugal Filter Unit (10 kDa cutoff) before its subsequent use in ITC. Dilutions of CDPK1 and compound 1 (ST092793) were prepared in the same buffer to ignore contribution from buffer-buffer interaction, and degassed by vacuum for 10 minutes prior to use. ITC analysis was done in the absence and presence of 2.5 mM μ M in the presence of calcium). The emission spectra for CDPK1 were recorded in the range of 300-450 nm after excitation at 295 nm to selectively excite tryptophan residues of the protein. Excitation and emission slits were set at 10 nm. All spectra were corrected by subtracting the corresponding buffer baseline, obtained under similar conditions. The experiment was done twice. Data obtained from CDPK1 quenching experiments were fitted using Kaleidagraph 4.0.

Thermal Stability Assay (TSA)
Thermostability of CDPK1 was monitored for the detection of binding events between CDPK1 and Compound 1 (ST092793) by following the protocol as described earlier. Thermal Cycler (Eppendorf™) at different temperatures ranging from 40°C to 90°C for 6 min and then cooled down to room temperature for 4 min. Following centrifugation at 16,800g for 40 min at 4°C, SDS-PAGE of supernatants was performed. Estimates of band intensities of CDPK1 protein from Coomassie stained gel images were made using ImageJ 1.52a. For analysis of melting shift, CDPK1 signal intensity was normalized to the respective intensity at 40°C and quantified protein levels were analyzed.

Evaluation of growth inhibition of malaria parasite
Lead compound 1 (ST092793) ) with potent CDPK1-inhibitory activity was subjected to dose-dependent cytotoxic evaluation on parasite growth at concentrations ranging from 1.56 to 100μM. Briefly, trophozoites at 1% initial parasitaemia were treated with the compounds for one complete intra-erythrocytic life cycle of the parasite. Untreated parasites served as control. Post incubation, parasites were stained with Ethidium Bromide

Statistical analysis
In the bar graphs, data are expressed as the mean ± standard deviation (s.d.) of three independent experiments done in duplicates. Unless stated, data analysis of the assay plots and calculation of biochemical parameters were done by using both Microsoft Excel and Origin® 2018b Graphing and Analysis Software (OriginLab Corporation) were used. Unless indicated, the differences were considered to be statistically significant at P < 0.05.

Purification of 6xHis-CDPK1, three-dimensional structural model of PfCDPK1 and its quality assessment
Coomassie-stained gel shows the level of purity of recombinant PfCDPK1 used for all assays (Fig. 1a).
Anti-sera raised in mice was used to probe recombinantly purified CDPK1 protein and native protein from schizonts lysate (Fig. 1a). PfCDPK1 contains an N-terminal KD tethered to a C-terminal CamLD via JD (Fig.   1b). Three-dimensional structure of a protein can provide us with precise information about its single, most stable conformation, as dictated by its sequence. X-Ray diffraction based structural model of PbCDPK1 was used as a template to generate 3D coordinates of PfCDPK1 by homology modeling, one of the most common structure prediction methods in structural genomics and proteomics (Fig. S1a). After optimal rigid-body superimposition of PbCDPK1 with the generated structural model of PfCDPK1, overall Root-Mean-Square Deviation (RMSD) value of the C-alpha atomic coordinates was found to be 0.31 Å, suggesting a reliable 3D structure of PfCDPK1 (Fig. S1b). Assessment of stereochemical quality and accuracy of the generated homology model displayed 89.2% of amino acid residues lying in the most favored ("core") regions, with 8.1%, 1.9%, and 0.8% residues in "additional allowed", "generously allowed" and "disallowed regions" of Ramachandran plot, respectively (Fig. S1c). Also, protein structure with ≥ 90% of its amino acid residues lying in the most favored regions of Ramachandran plot is considered to be as accurate as a crystal structure at 2Åresolution. This indicated that the backbone dihedral angles: phi and psi of the generated PfCDPK1 model were reasonably accurate. The comparable Ramachandran plot characteristics and RMSD value confirmed the reliability of the 3D-model of PfCDPK1 to be taken further for virtual screening.

Virtual screening of MyriaScreen II library against ATP binding pocket of CDPK1
MyriaScreen II diversity collection of drug-like screening compounds consists of 10,000 high-purity and diverse molecular candidates from Sigma-Aldrich and TimTec, Inc., created by combining medicinal chemistry and computational expertise, and selected on the basis of diversity and structural relevance for general screening. The collection is largely drug-like according to Lipinski's rule of five. Fast docking of MyriaScreen II library against the generated homology model of CDPK1 was done to computationally screen for ligands with propensity to bind catalytically active ATP binding pocket of the protein (Fig. 1c). Compounds were arranged on the basis of their most negative free binding energies or binding affinity to the ATP binding pocket of the protein, and subsequently ranked by decreasing value of their negative binding energies along the abscissa (Fig.   1d). Compounds were further filtered out on the basis of their propensity to follow Lipinski's rule of five pertaining to physicochemical properties including Molecular Weight (MW), Octanol/water partition coefficient (LogP), Hydrogen bond acceptors (H-acceptor) and Hydrogen bond donors (H-donors). 18 compounds were finally selected as probable hits and set to submit for experimental validation (Fig. 1d). Besides, all filtered out compounds (N=18) are stabilized by the formation of favourable molecular interactions with amino acid residues constituting ATP binding pocket of CDPK1. Their binding energies, Lipinski's properties and chemical structures are shown in Table S1.

Compounds 1 (ST092793) and 2 (S344699) inhibit functional activity of CDPK1 in vitro
Since MyriaScreen II library of drug-like small molecular candidates was virtually screened on the basis of probable propensity of the compounds to interact with ATP binding pocket of CDPK1, we utilized in vitro kinase assay to demonstrate enzymatic activity of the protein in presence of the filtered out candidates by measuring amount of ADP produced in the kinase reactions. All 18 compounds taken into consideration showed differential inhibition of CDPK1 activity which supports well with our in silico virtual screening approach and SPR interaction analysis (Fig. 3a). Notably, two compounds: ST092793 (assigned as compound 1) and S344699 (assigned as compound 2) significantly inhibited phosphorylation activity of CDPK1, accounting for 87.34% + 0.15% and 68.05% + 0.48% inhibition, respectively. DMSO, used as solvent for compounds, had no inhibitory effect on the enzymatic reactions and was taken as negative control. Dose-response curves for inhibitory activity of compounds 1 (ST092793) and 2 (S344699) against CDPK1 protein were found to be sigmoidal, depicting their IC 50 values to be 33.8 μ M and 42.6 μ M, respectively (Fig. 3b). Taken together, these results suggest compounds 1 (ST092793) & 2 (S344699) as potent inhibitors of CDPK1 enzymatic activity.
Auto-phosphorylation & trans-phosphorylation status of CDPK1 and dephosphorylated casein, respectively, were checked in the absence and presence of varying concentrations of compounds 1 (ST092793) and 2 (S344699) . Fig. 3c shows staining for enzymatic activity, after gel electrophoresis of reaction mixtures, with Pro-Q Diamond phosphoamino acid and Coomassie Brilliant Blue G-250 stains. Decrease in phosphorylation status of both CDPK1 and casein was readily detectable with corresponding increase in concentrations of the compounds, further reaffirming the observation that compounds 1 (ST092793) and 2 (S344699) are potent inhibitors of CDPK1 activity. We attribute minor differences in apparent size to the gel shift resulting from multiple phosphorylation sites on CDPK1 protein.

Compounds 1 (ST092793) and 2 (S344699) inhibit CDPK1 activity by interacting with its ATP binding pocket
Since compounds 1 (ST092793) and 2 (S344699) inhibit kinase activity of CDPK1 in vitro, we wanted to confirm if they accomplish this by directly binding to catalytically active ATP binding pocket of the protein.
For this purpose, recombinantly purified CDPK1 was allowed to interact with compound 1 (ST092793) and/or 2 (S344699) , followed by competing this interaction with ATP immobilized on the surface of agarose matrix.
Reduction in binding with the ATP-conjugated agarose matrix was observed in the presence of compound 1 (ST092793) (91.76%), compound 2 (S344699) (86.63%) and in combination of both compounds (89.65%) (Fig. 4a). It was therefore estimated that CDPK1 in complexation with the compound(s) possessed diminished ability to bind ATP, indicating that compounds 1 (ST092793) and 2 (S344699) inhibit in vitro kinase activity of CDPK1 by directly binding to its ATP-binding pocket. Schematic representation of complex formation between catalytically active ATP binding pocket of CDPK1 and compounds 1 (ST092793) & 2 (S344699) is shown in Fig. 4b.

Compound 1 (ST092793) interacts with CDPK1 favourably in the absence of calcium
After affirming CDPK1-inhibitory activity of compounds 1 (ST092793) and 2 (S344699) in vitro, we further sought to establish the knowledge of stoichiometry details and binding modes of complexation between CDPK1 13 and the relatively more potent inhibitor, i.e., compound 1 (ST092793) , by employing ITC. The representative binding isotherms resulting from titration of compound 1 (ST092793) with CDPK1 are represented in Fig. 5a. The compound showed differential binding with CDPK1 in the absence and presence of Ca 2+ . Binding isotherm in the absence of Ca 2+ was monophasic in nature, reaching a plateau phase at 1:1 stoichiometry (~1.0 molar ratio) which indicates that compound 1 (ST092793) interacts with only a single independent binding site (i.e., catalytically active ATP binding pocket) of CDPK1 protein. As further injections continued, gradual decline in exothermic heat resulted in sigmoidal curve ending near zero baseline, indicating saturation of binding sites. On the basis of the nature of the curve, the data were fitted using single-site binding model. In contrast, linear thermogram was observed in the presence of Ca 2+ , indicative of non-specific binding of compound 1 (ST092793) with CDPK1. In Overall, the resultant 1:1 stoichiometric complex of compound 1 (ST092793) and CDPK1 in the absence of Ca 2+ was found to be highly stable, supported by stronger K a and favourable binding enthalpy and entropy.

Fluorescence spectroscopy supports ITC interaction analysis of compound 1 (ST092793) and CDPK1
To confirm ITC interaction analysis of compound 1 (ST092793) and CDPK1, fluorescence spectroscopic study was performed.

Compound 1 (ST092793) confers thermal stability to CDPK1
Thermal Stability Assay (TSA) is a method for detecting target engagement by monitoring thermostability of a given protein in the presence of its ligand. (32) This is based on the principle that targetligand interactions result in alteration in the thermodynamic parameters of the protein, affecting its stability with corresponding increase in temperature. TSA was optimized for binding analysis between purified recombinant CDPK1 protein and Compound 1 (ST092793) . Melting curves at temperatures ranging from 40 o C to 90 o C demonstrated significant thermo-stabilization of CDPK1 in the presence of Ca 2+ (Fig. 6a). Importantly, in the opted temperature range, CDPK1 treated with Compound 1 (ST092793) (100 μ M) in the absence of Ca 2+ showed abundances of the protein higher than the abundances of those treated with DMSO as control, suggesting ligand-dependent thermo-stabilization of CDPK1. No alteration in thermal stability was observed upon treatment of the protein with Compound 1 (ST092793) in the presence of Ca 2+ (Fig. 6b). Our TSA analysis is in  (Fig. 6c), further supporting our in vitro findings. For quantification of thermostable CDPK1, the signal intensity was normalized to the respective intensity at 40°C.

Cytotoxic effect of compounds with high selectivity for parasite over host cells
Having observed no cytotoxic effect on HepG2 cell line, lead compound 1 (ST092793) with propensity to bind and inhibit the enzymatic activity of CDPK1 was subjected to dose response over 72 hours of intraerythrocytic life cycle of the parasite. Compound 1 (ST092793) showed IC 50 value of 9.5 μ M (Fig. 6d). Control parasites were healthy and completed its life cycle producing trophozoites at 72 hrs post-incubation.

DISCUSSION
In Plasmodium falciparum, Ca 2+ -mediated signaling pathways are translated into cellular responses by CDPKs, among which CDPK1 is the most widely studied member of the cdpk multigene family (7,8,11,34,35). Insights into the physiological role of CDPK1 in cellular biology of P. falciparum depicts importance of this protein in late schizogony, subsequent invasion of merozoites into host RBCs, formation of male and female gametes, and transmission to the mosquito host (9)(10)(11)(12). During gliding and invasion of the parasite, CDPK1 regulates actin-myosin motor by phosphorylating components of IMC, i.e., GAP45 and MTIP. It also regulates cAMP signaling module by phosphorylating and activating PKAr (13). With high abundance, CDPK1 stands alone as a multistage signaling regulator of P falciparum and depicted as the most sought after molecular target for drug discovery against malaria.
Most of the small molecule kinase inhibitors have generally been identified either by high-throughput virtual or fragment-based screening of chemical libraries. Among the drug designing platforms, virtual or molecular docking screening utilizes in silico robotic platforms for identification of new lead compounds in the process of drug discovery. In this approach, large libraries of unique organic molecules are computationally screened for compounds, collectively referred to as biologically relevant chemical space, that complements 'structural code' (i.e., ligand binding pocket) of target protein(s), followed by predicting the binding affinity by experimental validation. Filters may be applied to ensure that the hit compounds meet standard of biological relevance or drug-likeness, exploting the druggability of the target proteins. Once promising candidates are identified, one can utilize medicinal chemistry reactions and synthetic strategies to dynamically generate more diverse compounds around the initial hits, with enhanced efficacy. This whole process of structure-based screening and identifying lead candidates frees medicinal chemistry from the tyranny of empirical screening which includes substrate-based design and incremental modification (36)(37)(38)(39). Previous studies by, Hou et al. Due to the emergence of parasite resistance to existing frontline antimalarial drugs, there is a pressing need to identify and characterize new anti-malarial molecules that would target the parasite at multiple proliferative stages during its life cycle. In this context, since PfCDPK1 has been previously identified as one such target, we have utilized a structurally diverse collection of 10,000 drug-like molecules from MyriaScreen II library to screen for ligands complementing ATP binding pocket of CDPK1, as represented schematically in Fig. 7. In the absence of an experimentally determined atomic structure of PfCDPK1, earlier attempts have been made to resolve homology modeling based three-dimensional structure of PfCDPK1 (9,11,44). In all these studies, distantly related orthologous sequences of PfCDPK1 either from humans (death-associated protein kinase, PDB ID: 1JKK) (9) or from another apicomplexan parasite, Cryptosporidium parvum (calcium/calmodulin-dependent protein kinase, PDB ID: 2qg5; and, CpCDPK3, PDB ID: 3LIJ) (11,44) was used as template for structural modeling of PfCDPK1. Owing to the high sequence identity (~93%) between CDPK1 orthologs from P. falciparum strain 3D7 and evolutionarily closely related species, P. berghei strain ANKA, we used X-Ray diffraction based structural model of PbCDPK1 (the most recently resolved crystal structure of CDPK1 protein, PDB ID: 3Q5I) as template to generate 3D structural coordinates of PfCDPK1.
Stereochemical quality and accuracy of the structurally refined model of PfCDPK1 (Fig. S1a and S1b) indicated that the backbone dihedral angles: phi and psi were reasonably accurate, thus confirming reliability of the generated model for performing docking-based virtual screening (Fig. S1c). The candidates were filtered and ranked from MyriaScreen II library on the basis of their most negative free binding energies or binding affinity towards ATP binding pocket of the protein (Fig. 1c). After applying additional filter of Lipinski's rule of five, 18 compounds were finally selected as probable hits and set to submit for experimental validation (Fig. 1d and Table S1). Also, at 100 To verify probable tendency of these compounds to inhibit functional activity of the kinase, compounds (N=18) were further subjected to CDPK1 functional activity assays in vitro. All 18 compounds showed differential inhibition of CDPK1 activity at 50 μ M concentrations, with compounds 1 (ST092793) and 2 (S344699) accounting for 87.34% + 0.15% and 68.05% + 0.48% inhibition, respectively (Fig. 3a). Dose-response curves for CDPK1-inhibitory activity of compound 1 (ST092793) or 2 (S344699) were found to be sigmoidal, depicting their IC 50 values to be 33.8 μ M and 42.6 μ M, respectively (Fig. 3b). Also, decrease in phosphorylation status of CDPK1 and casein was observed with increase in concentrations of both of these compounds (Fig. 3c), which suggests that compounds 1 (ST092793) and 2 (S344699) are potent inhibitors of CDPK1 protein. To further authenticate, if compounds 1 (ST092793) and 2 (S344699) inhibit functional activity of CDPK1 by directly competing with ATP for binding to catalytically active pocket of the protein, affinity chromatography of CDPK1 pre-incubated with compound 1 (ST092793) and/or 2 (S344699) was performed. By doing so, reduction in binding to ATPconjugated agarose matrix was observed in the presence of compounds 1 (ST092793) (91.76%) and 2 (S344699) (86.63%), indicating that both of these compounds directly bind to catalytically active ATP-binding pocket of CDPK1 and thus inhibits its enzymatic activity (Fig. 4).
Binding affinity and stoichiometry details of the complex formation between relatively more potent compound 1 (ST092793) and CDPK1 was further examined by utilizing ITC, in the absence and presence of Ca 2+ .
K a values demonstrated that compound 1 (ST092793) showed higher binding affinity towards CDPK1 in the absence of Ca 2+ (K a = 3.6 X 10 5 M -1 ) than in its presence (K a = 2.1 X 10 4 M -1 ), with the interactions mainly driven by enthalpy (ΔH = -8.1 kcal.mol -1 ). Binding isotherm in the absence of Ca 2+ reached a plateau phase at 1:1 molar ratio, indicating that the compound interacts with only a single independent binding site (ATP binding pocket) of the protein. In contrast, in the presence of Ca 2+ , non-specific interaction was observed as depicted by the linear isotherm (Fig. 5a). ITC interaction analysis was further confirmed by measuring intrinsic tryptophan fluorescence through spectroscopic studies in the presence of increasing concentrations of compound 1 (ST092793) . Fluorescence displacement titrations in the absence of Ca 2+ resulted in progressive decline in the tryptophan fluorescence intensity. However, lack of any trend in the presence of Ca 2+ indicated that compound 1 (ST092793) preferentially binds with CDPK1 in its open or 'inactive' conformation, as confirmed by ITC (Fig. 5b).
To further confirm the target engagement, TSA was applied to monitor thermostability of CDPK1 upon treatment with compound 1 (ST092793) , in the absence and presence of Ca 2+ . Melting curves at temperatures ranging from 40 o C to 90 o C demonstrated thermo-stabilization of CDPK1 in the presence of Ca 2+ (Fig. 6a).
More importantly, treatment of CDPK1 with Compound 1 (ST092793) resulted in enhanced thermo-stabilization of the protein in a Ca 2+ dependent manner (Fig. 6b). Further, TSA analysis with all 18 compounds procured from virtual screening of MyriaScreen II library demonstrated that thermal stability of CDPK1 gets relatively highly enhanced in the presence of Compound 1 (ST092793) , further supporting our in vitro findings (Fig. 6c). Finally, lead compound 1 (ST092793) obtained from virtual screening of MyriaScreen II library was subjected to dose response over 72 hours of intra-erythrocytic life cycle of the parasite. Compounds 1 (ST092793) with CDPK1 inhibitory activity in vitro showed IC 50 value of 9.5μM (Fig. 6d).
In conclusion, we adopted a structure-based virtual chemical library screening approach in combination with extensive biochemical and biophysical characterization based tools to identify novel lead candidates, Compound 1 (ST092793) and Compound 2 (S344699) capable of inhibiting CDPK1 activity in vitro. The inhibitory potency of these compounds was authenticated by favorable interactions with the catalytically active ATP binding pocket of the protein. These compounds exhibited potent cytotoxicity against malaria parasite ex vivo, indicating their promising candidacy for malaria treatment. Further studies can be planned to improve CDPK1 inhibitory activity and kinase selectivity of these compounds by designing new analogues of the same.
Altogether, our study proposes a novel CDPK1-selective inhibitor with strong anti-malarial activity and represents a combinatorial high-throughput platform involving in silico and in vitro applications as a blueprint for the rational identification of novel selective inhibitors.     Representative ITC binding isotherms resulting from titrations of CDPK1 with compound 1 (ST092793) . The compound showed differential binding with CDPK1 in the absence (K a = 3.6 X 10 5 + 2.4 X 10 5 M -1 ) and presence (K a = 2.6 X 10 3 + 25% M -1 ) of Ca 2+ . Binding isotherm in the absence of Ca 2+ reached a plateau phase at 1:1 stoichiometry indicating that compound 1 (ST092793) interacts with only a single independent binding site of CDPK1. Also, interaction of compound 1 (ST092793) with CDPK1 was enthalpically driven with