Volume 13 Supplement 7
Blocking Protein kinase C signaling pathway: mechanistic insights into the anti-leishmanial activity of prospective herbal drugs from Withania somnifera
- Abhinav Grover†1,
- Shashank Prakash Katiyar†1,
- Jeyaraman Jeyakanthan2,
- Vikash Kumar Dubey3 and
- Durai Sundar1Email author
© Grover et al.; licensee BioMed Central Ltd. 2012
Published: 13 December 2012
Leishmaniasis is caused by several species of leishmania protozoan and is one of the major vector-born diseases after malaria and sleeping sickness. Toxicity of available drugs and drug resistance development by protozoa in recent years has made Leishmaniasis cure difficult and challenging. This urges the need to discover new antileishmanial-drug targets and antileishmanial-drug development.
Tertiary structure of leishmanial protein kinase C was predicted and found stable with a RMSD of 5.8Å during MD simulations. Natural compound withaferin A inhibited the predicted protein at its active site with -28.47 kcal/mol binding free energy. Withanone was also found to inhibit LPKC with good binding affinity of -22.57 kcal/mol. Both withaferin A and withanone were found stable within the binding pocket of predicted protein when MD simulations of ligand-bound protein complexes were carried out to examine the consistency of interactions between the two.
Leishmanial protein kinase C (LPKC) has been identified as a potential target to develop drugs against Leishmaniasis. We modelled and refined the tertiary structure of LPKC using computational methods such as homology modelling and molecular dynamics simulations. This structure of LPKC was used to reveal mode of inhibition of two previous experimentally reported natural compounds from Withania somnifera - withaferin A and withanone.
Leishmaniasis is an endemic disease prevalent in many parts of the world; mostly in countries like India, Bangladesh, Pakistan, Afghanistan, Nepal, East and North Africa, and Deserts in western Asia . Leishmaniasis is responsible for the death of approximately 70,000 people each year worldwide . It is caused by various species of intramacrophage protozoan Leishmania like Leishmania donovani, Leishmania major, Leishmania mexicana and Leishmania panamensis to name a few, and spread by the bite of sandfly . Leishmaniasis is becoming the disease of attention and concern because in the last few decades L.donovani has developed drug-resistance and toxicity towards available drugs [3, 4]. Hence, it has become inevitable to identify new drug targets and to develop novel drugs against L.donovani to cure Leishmaniasis.
Previous experimental study has shown that methanolic compounds from Withania somnifera (ashwagandha) possess in vitro anti-leishmanial activity [5, 6]. Withaferin A has been identified as one of ashwagandha's prominent phytocompounds. It is a cell permeable steroidal lactone which has been shown to possess anti-leishmanial property  apart from many other pharmacological properties. Withaferin A belongs to a class of compounds from Withania somnifera collectively known as withanolides. These exhibit number of other therapeutic activities like anticancer [7–11], anti-herpetic  and neuronal regeneration property . Unlike higher eukaryotes, withaferin A has been reported to induce apoptosis in leishmanial cells by targeting its protein kinase .
Protein kinases in mammalian cells are associated with many important cellular processes like gene activation, cell differentiation and release of neurotransmitters [6, 14, 15]. On one hand, the types and role of protein kinases are well studied in mammalian cells, while on the other hand, only scarce information is available about protein kinases of protozoans. Previous studies have proven that protozoan protein kinases differ from mammalian protein kinases both structurally and functionally . These differences between mammalian and protozoan protein kinases render these kinases as potential drug targets . For the purpose of ease, protein kinase in Leishmania has been termed as leishmanial protein kinase C (LPKC) . Although previous studies have reported the inhibition of LPKC by methanolic compounds of ashwagandha plant, so far no study has been carried out which provides the mechanism of action and structural insights of the inhibition. Structure of LPKC has not yet been solved experimentally and unavailability of this structure of LPKC further limits the development of drugs against it. Structure-based drug designing is a popular approach to search inhibitors against a target protein but it requires information of three dimensional structure of the target [19, 20]. In the absence of experimental tertiary structures of a protein, computational methods such as homology modeling and threading are capable of predicting protein structures . In such scenario, computational methods can be used to predict the structure and active site of LPKC. Probing LPKC's mode of inhibition by pharmacologically active compounds of ashwagandha will broaden the prospects of drug development against leishmaniasis and this information can be used to screen large number of inhibitors against it more accurately and rapidly. Ashwagandha also contains another important compound known as withanone which is known to possess antitoxic activity against methoxyacetic acid in addition to its prominent anticancer properties [22, 23]. Though withanone has not yet been tested against leishmaniasis experimentally, this study provides a computational proof of its possible inhibitory activity against LPKC.
1262 amino acid-long protein sequence of LPKC (Accession no. CBZ31403) was retrieved from NCBI protein database in FASTA format. Position-Specific Iterated BLAST against PDB database was used to identify homologous protein structures of LODC [24–27]. There was complete absence of any homologous structure for the residual range ~0-660 and ~1030-1262 amino acids. LPKC sequence ranging from 650-1025 is a conserved protein sequence and contains the catalytic domain of serine/threonine protein kinase. Because of unavailability of homologous structure for initial and last region of LPKC and highly conserved nature of 650-842 amino acids, only conserved stretch was considered for modeling purpose using homology modeling approach. Crystal structures of human calcium/calmodulin-dependent protein kinase type-IV (2W4O) at 2.17 Å resolution and death-associated protein kinase-1 (2Y0A) at 2.6 Å resolution were selected as templates for homology modeling. 2W4O had an e-score of 3 × 10-11 and 27% identity with protein sequence of query with an 87% coverage and 2Y0A showed 2 × 10-5 e-core and 30% identity with 46% coverage of query. Homology model of LPKC using crystal structure of selected templates was built using multi-template protocol of MODELLER version 9.10 [28, 29]. Discrete Optimized Protein Energy (DOPE )  was applied to refine the loops of the generated models. Models were accessed on the basis of Modeler Objective Function, DOPE scores, verify3D score [31, 32] and ERRAT score . To select a model out of the several models generated by MODELLER, dope energy profile was generated for templates and models. Model possessing closest DOPE energy profile with template was selected for further studies. Selected model was further refined and stabilized using Molecular Dynamics (MD) simulations .
Molecular dynamics simulations
Desmond Molecular Dynamics system [35, 36] with Optimized Potentials for Liquid Simulations (OPLS) all-atom force field 2005 [37, 38] was used to perform MD simulations of all proteins and ligand-bound complexes. Modeled protein structure and structures of protein-ligand complexes were first prepared using protein preparation wizard of Maestro interface . Prepared structures were then uploaded in Desmond set up wizard for MD simulations. Preparation of protein structure includes the addition and optimization of hydrogens, generation of disulphide bonds, and removal of water molecules and capping of terminals. Prepared protein molecules were solvated with TIP4P water model in a cubic periodic boundary box to generate required systems for MD simulations. Systems were neutralized using appropriate number of counterions. The distance between box wall and protein complex was set to greater than 10Å to avoid direct interaction with its own periodic image. Energy of prepared systems for MD simulations was minimized up to maximum 5000 steps using steepest descent method until a gradient threshold ( 25 kcal/mol/Å) is reached, followed by L-BFGS (Low-memory Broyden-Fletcher-Goldfarb-Shanno quasi-Newtonian minimizer) until a convergence threshold of 1 kcal/mol/Å was met. The systems were equilibrated with the default parameters provided in Desmond. Further MD simulations were carried on the equilibrated systems for desired period of time at constant temperature of 300 K and constant pressure of 1 atm with a time step of 2fs. During the MD simulations smooth particle mesh Ewald method was used to calculate long range electrostatic interactions. Nine Å cut-off radius was used for coulombic short range interaction cutoff method. The modeled LPKC protein was prepared for MD simulations using the parameters described above. The system was then continuously simulated for a long time period of 15ns. Stability of docking of ligands into the modeled proteins were also investigated using MD simulations. All protein-ligand complexes were simulated for 10ns time period using similar parameters as described above.
The root mean square deviation (RMSD) for both the modeled protein and the docked ligands within the binding pocket of protein were calculated for the entire simulations trajectory with reference to their respective first frames. ROG and H-bond analyses were carried out for all the frames of 15ns MD simulation of LPKCL. The hydrophobic interactions and H-bonds were calculated using Ligplot program  where H-bonds were defined as acceptor-donor atom distances of less than 3.3 Å, hydrogen-acceptor atom distance of maximum 2.7 Å and acceptor-H-donor angle greater than 90°. During the MD simulations, H-bond fluctuations of ligand with protein were calculated using VMD software .
Binding site identification
Binding site and catalytic site of LPKC were present in serine/threonine kinase domain, which is a conserved sequence. Same domain was also present in human calcium/calmodulin-dependent protein (2W4O) which has an experimentally solved tertiary structure along with an inhibitor. Template structure of 2W4O was superimposed over the modeled structure to know the location of conserved binding site of LPKC. To confirm the binding site of LPKCL, detected by superimposition, structure of LPKCL was submitted to CASTp server  and SiteMap module . Both CASTp and SiteMap confirmed the accuracy of predicted binding site as the binding site revealed by superimposition located within the largest and highest ranked cavity.
Virtual molecular docking of ligands with LPKC
Structure files of withaferin A [PubChem:265237] and withanone [PubChem: 21679027] were retrieved from the PubChem Compound database. Structure files of both the ligands were prepared using LigPrep's ligand preparation protocol . LigPrep improved the dataset of small molecules by generation of tautomeric, stereochemical and ionization variations, as well as by performing energy minimization and flexible filtering. Similarly, modeled protein structures were also prepared before the docking steps using Schrödinger's protein preparation wizard . Protein preparation implicated the addition and optimization of hydrogen atoms, removal of bad contacts, optimization of bond lengths, creation of disulphide bonds, capping of protein terminals, and conversion of selenomethionine to methionine. A grid was generated at the predicted binding site of modeled structure as an essential step for docking using the Glide docking module of Schrödinger [45, 46].
Prepared natural compounds were virtually docked against modeled LPKC protein at desired grid coordinates using Glide model's XP docking protocols . Stability of the top scoring docked conformations obtained from glide XP docking, was inspected using MD simulations. All the Glide docking studies were performed on Intel Core 2 Duo CPU @ 3 GHz of HP origin with 1 GB DDR RAM. Schrodinger 9 Maestro interface was compiled and run under Ubuntu 32 bits operating system. All the MD simulations studies were performed in GPU server Intel (R) Core (TM) i7 CPU 930, with 4 GB DDR RAM.
Prime/MM-GBSA binding-free energy calculation
where ER:L is the energy of the complex, ER + EL is sum of energies of the receptor and ligand in unbound state, ΔGsolv is the difference in the GBSA solvation energy of the complex and sum total of solvation energies of unbound receptor and ligand. ΔGSA is the difference in surface area energies of the complex and sum total of surface area energies of unbound receptor and inhibitor. OPLS-AA force field  and GB/SA continuum solvent model were used to calculate necessary energies of the complexes.
Results and discussion
LPKC protein structure modeling and active site prediction
Two homologous X-ray crystal structures of human calcium/calmodulin-dependant protein kinase and death associated protein kinase-1 were used as templates to predict the tertiary structure of LPKC protein. Predicted protein contained all the residues in allowed regions on Ramachandran plot and showed ~70% ERRAT score. Low resolutions of the template structures were the probable cause of slightly low ERRAT score of LPKC structure. Hence modeled protein was stabilized by MD simulations technique. LPKC is a kinase protein which possesses a conserved active site similar to other protein kinases. However, primary structure of LPKC is distinctly related to mammalian eukaryotic kinases, LPKC protein tertiary structure aligned well with human calcium/calmodulin-dependant protein kinase protein structure. Structural alignment of LPKC with 2W4O protein structure identified the plausible active site of LPKC protein which was further confirmed using cavity analysis server CASTp and active site identification software SiteMap. CASTp and Sitemap reported the presence of Asp666, Arg667, Gln669, Arg670, Glu687, Glu689, Gln691, Asn710, Val711, Thr712, Ala713, Leu714, Met728, Glu729, Ala731, Asp778, and Ser781 residues around the highest scoring cavity [Additional File 1].
Molecular dynamics simulations of modeled LPKC protein
Virtual docking of protein with withaferin A and withanone
XP Docking scores and Binding energies of LPKC with natural compounds
XP Glide Score
Prime/MM-GBSA binding-free energy (dG)(kcal/mol)
Withaferin A-LPKC complex
Molecular dynamics simulations of complexes
Stability of both the natural compounds in the binding pockets of LPKC was further analyzed by MD simulation. RMSD analysis of withaferin A during 10 ns simulations showed that withaferin A altered its configuration by 1Å at very beginning of the simulation and maximum RMSD of 1.36 Å was noticed at 7.7 ns and that too for just one frame. After the initial deviation, withaferin A did not deviate further and showed consistent RMSD of around 1Å throughout the simulation process indicating that withaferin A had acquired a very stable conformational state.
Interaction analysis of withaferin A and withanone with LPKC protein
Interaction profile of LPKC with natural compounds
Type of Interaction
Withaferin A (before MD)
Withaferin A (after MD)
Withanone (before MD)
Withanone (after MD)
Thr62, Ala63, Glu79, Asn128
Arg17, Leu53, Leu64, Met78, Ala81, Thr131, Cys139
Leu54, Thr62, Met78, Glu79, Ala81, Gln85, Thr131, Ala133, Cys139, Asp140
Arg17, Gln19, Glu39, Leu53, Thr62, Leu64, Met78, Ala81,
Gln19, Thr62, Met78, Ala81
Though both withaferin A and withanone were found stable within the binding pocket of LPKC during the MD simulations, higher RMSD was observed for withanone as compared to withaferin A during the MD simulation but both had acquired a conformation which was not deviating any longer at the end of MD simulation. There was not a noticeable difference between the docking score of Withaferin A and Withanone after the docking and after the MD simulations. Both withaferin A and withanone were also found to interact with same residues of the active site after the docking process either via H-bonds or hydrophobic interactions, thus validating the accuracy of predicted binding pocket of LPKC and also confirming the inhibitory nature of both natural compounds against the kinase. Comparison of final binding free energies of LPKC complexes with withaferin A and withanone suggested that both show almost similar free energies of binding [Table 2].
Withaferin A and withnone are two pharmacologically active natural products from the medicinal plant Withania somnifera. Withaferin A has been reported to exhibit antileishmanial properties in previous studies. We analyzed the inhibitory property of withaferin A as well as that of withanone at the molecular level. We modeled an important enzyme of leishmania - LPKC using comparative homology modeling and virtually docked withaferin A and withanone with it. Both withaferin A and withanone were found to inhibit LPKC protein with almost equal affinity. Withanone has not yet been experimently proven to inhibit LPKC protein before. The present study suggests that these two natural products can be potential candidates for checking Leishmaniasis by inhibiting LPKC. By this study we provide structural insights of the inhibitory action of withaferin A and withanone against LPKC.
The authors acknowledge: the Bioinformatics facility at the Distributed Information Sub Centre, Department of Biochemical Engineering and Biotechnology, IIT Delhi and at the Department of Bioinformatics at Alagappa University, Karaikudi. This study was made possible in part through the support of a grant from the Department of Biotechnology (DBT), Government of India, New Delhi to VKD and DS.
This article has been published as part of BMC Genomics Volume 13 Supplement 7, 2012: Eleventh International Conference on Bioinformatics (InCoB2012): Computational Biology. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcgenomics/supplements/13/S7.
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