CHAPTER VI
and hypericin treated Leishmania promastigotes. Proteomics analysis has revealed alteration in several proteins involved in protein synthesis, stress related proteins, signalling and transporter protein, proteins of metabolic pathways and ubiquitin proteasome system and several hypothetical proteins. Further, as an approach towards multi target drug discovery, we have also modelled CAAX prenyl protease I & II of Leishmania donovani and have identified potential inhibitors against these proteases by using in silico analysis. A formulation of inhibitors identified against spermidine synthase and CAAX prenyl protease I and II could be a good strategy to cope up drug resistance problem and to identify a formulation effective against Leishmania donovani.
6.2 Introduction of Leishmaniasis:
Leishmaniasis, popularly known as kala azar, is caused by different species of digenetic parasite Leishmania. There are several drugs available in the market against Leishmaniasis.
However, these available drugs experience many disadvantages. These disadvantages include high cost, development of resistance of different strains of Leishmania against drugs, toxicity, difficult route of administration etc. Increasing burden of the disease and the limitations of available drugs, necessitate the requirement for the development of novel and potent drug candidates having specific drug targets.
6.3 Molecular mechanism of hypericin induced parasite death:
In a search for developing new drug candidate, we have identified hypericin as a novel drug candidate specifically targeting spermidine synthase of Leishmania donovani. Target specificity of hypericin was confirmed by both computational and biochemical method.
Further exploring the mechanism of action of hypericin we have identified that hypericin has caused generation of reactive oxygen species which is reverted back by either trypanothione or spermidine supplementation. Hypericin was found to cause necrosis like death of the parasite. The death of the parasite was reverted back by spermidine but not by trypanothione.
This suggests the role of spermidine in processes other than redox metabolism of the parasite.
6.4 Molecular events leading to death of Leishmania donovani under spermidine starvation:
To further explore the putative role of spermidine in pathways other than redox metabolism, we have checked the quantitative gene expression of several genes. We have found altered expression of genes involved in autophagy, hypusine modification, DNA repair and redox metabolism of the parasite. Hypericin was found to induce autophagy as a cytoprotective against DNA damage due ROS generation. Autophagy was reverted by either trypanothione or spermidine supplementation. Also, there was increase in ATP production after hypericin treatment which is reverted back either by trypanothione, spermidine or 3-methy adenine.
This confirms the cytoprotection response of cell against DNA damage. Defective hypusine modification of eIF5A was found to be the main cause of parasite death after hypericin treatment. This was evident from the altered translation and lowered hypusination which get reverted back by spermidine supplementation but not by trypanothione supplementation. This suggests the role of spermidine in hypusine modification of Leishmania donovani.
6.5 Proteome profiling of Leishmania donovani under spermidine starvation:
Proteome analysis is considered as an important approach to solve various biological challenges. To know the overall mechanism of hypericin induced parasite death, it is important to overview the global change in proteins of Leishmania after hypericin treatment.
We have utilized label free quantitation approach for proteome profiling of untreated and hypericin treatment Leishmania donovani. In line with our previous studies we have identified change in proteins related to protein synthesis and stress related proteins. This further confirms the occurrence of two major events i.e. DNA damage response and translational defects. This has ultimately led to the altered metabolic and signalling pathways of the parasite. We have also analyzed the relative distribution of total altered protein, up regulated proteins and down regulated proteins. Proteome profiling of Leishmania donovani has shown altered levels of proteins involved in signalling, transporters proteins and several membrane proteins. This also suggests the crucial role of these proteins in Leishmania donovani.
6.6 Structure based virtual screening studies of CAAX prenyl protease I and II of Leishmania donovani:
Multitarget drug discovery is a new approach for targeting several diseases. CAAX prenyl proteases are involved in modification of several proteins involved in signalling pathways.
So, targeting CAAX prenyl proteases of Leishmania donovani can be a good approach towards developing another drug candidate. We have modelled the structure of CAAX prenyl protease I and II of Leishmania donovani using homology modelling approach. The modelled structure was validated and further simulated in DPPC lipid membrane and TIP3P water molecule. Active site of both CAAX prenyl protease I and II was predicted using Schrodinger suite. Using structure based virtual screening, several known and new inhibitors were docked against CAAX prenyl protease I and II to get the best docked compounds. Best docked 15 compounds were subjected to induced fit docking to predict accurate binding poses with CAAX prenyl protease I and II. ADME properties of the best compounds were predicted which confirms that these compounds can be used for Human consumption.
Binding and conformational stability of the best protein inhibitor complex was confirmed by using molecular dynamics simulation studies. Overall, we have identified inhibitors against CAAX prenyl protease I and II of Leishmania donovani whose potential as drug candidate can be further confirmed by using biochemical and cellular studies.
Overall, we have identified a novel inhibitor of hypericin and we have also elucidated the mechanism of hypercin induced parasite death. Further, the potential of hypericin can be elucidated to check its in vivo effect on the infected animal models. Further, hypericin can be taken up for clinical trials on human. Several strains of Leishmania parasite are gaining resistance against the available drugs like sodium stibogluconate, amphotericin B and miltefosine. Hypericin can be a new drug molecule which can be used to treat patients unresponsive towards these drugs. We have also identified potential inhibitors of CAAX prenyl protease I and II of Leishmania donovani. The potential of best identified inhibitors to inhibit the recombinant CAAX prenyl protease I and II of Leishmania donovani can be validated. The inhibitor showing in vitro inhibition can be further taken up to check its potential as antileishmanial agent by monitoring its effects on the promastigotes of Leishmania donovani.