Contents
Molecular Events Leading To Death of Leishmania Donovani Under Spermidine Starvation
Proteome Analysis of Leishmania donovani Under Spermidine Starvation
135 6.5 Proteome profiling of Leishmania donovani during spermidine starvation 135 6.6 Structure-based virtual screening studies of CAAX prenyl protease I and II of.
Leishmaniasis: Introduction, Challenges and Scope of the Current Work*
Abstract
Introduction
- Vectors of Leishmania: Leishmania is transmitted via the bite of infected sandflies belonging to the genus Phlebotomus and Lutzomyia. Phlebotomus is known to spread the
- Eukaryotic initiation factor 5A (eIF5A): eIF5A is an acidic protein found to be highly conserved and essential in organisms ranging from archaebacteria to mammals
In Leishmania, it has been found to be crucial for parasite virulence (Gilroy et al., 2011). Certain growth factors are also known to regulate autophagy, such as interleukin-3 (IL-3), which inhibits autophagy (Lum et al. 2005b).
Prenylated proteins
Ras super-
Lamins
Yeast a- factor
The CAAX motif: The presence of a CAAX motif at the carboxyl terminus is an essential requirement for the prenyl dependent post-translational processing of a protein
Prenylated cysteine Aliphatic amino acids Any amino acid
Processing of proteins containing CAAX motif: The consensus sequence CAAX is known to direct a series of post-translational processing initiated by addition of lipid
It was found that variants of the prenyl protease CAAX type I (Afc1) in yeast showed reduced production of factor a below the detectable limit even in the presence of the type II prenyl protease CAAX, Rce1 (a-factor can also be processed by Rce1) . Overall, we have identified new potential drug candidates against CAAX prenyl protease I and II of Leishmania donovani.
Probing the Molecular Mechanism of Hypericin Induced Parasite Death*
- Abstract
- Introduction
- Materials and methods
- Results
- Trypanothione supplementation was a futile attempt to reduce cell death; however spermidine supplementation displays survival of the parasite: Promastigotes were
- Hypericin stimulates necrosis like death in parasite: In order to check the mode of cell death after hypericin treatment, flow cytometric analysis of Leishmania donovani was
- Discussion
Thus, to monitor the change of the thiol pool of the parasite after treatment with hypericin, a Leishmania promastigotes culture (10 ml) with a cell density of 2.5 x 10 cells/ml was treated with an IC50 dose of hypericin for 12 hours and 24 hours. Supplementing the parasite with trypanothione prior to hypericin treatment should result in reversal of the parasite's altered thiol levels. To monitor the effect of trypanothione on the in vivo thiol pool of the parasite, a 10 ml culture of Leishmania donovani (2.5 x 106 cells/ml) was supplemented with 0.5 µM trypanothione, followed by treatment with an IC50 dose of hypericin for 12 hours. and 24 hours.
Interestingly, target specificity of hypericin was evident from insignificant toxicity of hypericin towards macrophages and decrease in spermidine pool of the parasite after hypericin treatment. This change in the thiol pool of the parasite after hypericin treatment was reversed with trypanothione supplementation. Furthermore, the specificity of hypericin was also assessed by measuring the intracellular thiol and spermidine pool of the parasite.
Spermidine supplementation of the media recovered parasite survival after hypericin treatment confirming that parasite death is solely dependent on spermidine starvation due to hypericin.
Molecular Events Leading to Death of Leishmania donovani Under Spermidine Starvation*
- Abstract
- Introduction
- Materials and Methods
- Results
- Change in expression of gene after hypericin treatment: Spermidine starvation of Leishmania donovani by hypericin treatment generated reactive oxygen species (ROS)
- Polysome profiling of Leishmania promastigotes showed translational arrest after hypericin treatment: eIF5A has well established role in protein synthesis. Alteration in
For autophagy assay, Leishmania promastigotes (1X106 cells/ml) were also treated with 10 mM 3-methyl adenine, an autophagy inhibitor (Seglen and Gordon, 1982; McFarland et al., 2012) for 2 h followed by treatment with hypericin (18. μM) for 24 hours. To control for defects in translation initiation, promastigotes were treated with 100 μg/ml cycloheximide for 10 min to block translation elongation and termination (Cloutier et. al., 2012). Next, promastigotes were washed with PBS (pH 7.4) and analyzed using BD FACSCalibur flow cytometer (Wang et. al., 2008).
To analyze defect in translation initiation, promastigotes were treated with cycloheximide to block translation elongation. This decrease in polysome to monosome ratio indicates the change in translation initiation due to spermidine starvation after hypericin treatment. Here, we observed that increase in polysome to monosome ratio is not significant after treatment with hypericin compared to untreated promastigotes.
Densitometric analysis of the bands also reveals a significant decrease in hypozin modification after hypericin treatment.
Hypusine α tubulin
ATP determination after hypericin treatment has shown increase in intracellular ATP pool of Leishmania parasite: Intracellular ATP estimation was done in order to
Equal numbers of promastigotes were treated with hypericin supplemented with spermidine or trypanothione after treatment with the IC50 dose of hypericin for 24 h. Promastigotes were also pretreated with 3-methyl adenine before treatment with the IC50 dose of hypericin for 24 h. ATP estimation showed an increase in the intracellular ATP pool of the parasite after hypericin treatment.
However, this increase in the ATP pool reverts after supplementation with trypanothione or spermidine or in the case of pretreatment with 3-methyladenine before hypericin treatment. Intracellular ATP pool of parasites supplemented with trypanothione, spermidine, or 3-methyladenine followed by hypericin treatment was 33±2 pM, 36±4 pM, and 27±6 pM, respectively. ATG8 puncta formation was analyzed in (A) untreated (B) hypericin treated for 12 h (C) trypanothion supplemented and hypericin treated (D) spermidine supplemented and hypericin treated and (E) 3-methyladenine pretreated and hypericin treated Leishmania promastigotes.
Treatment with hypericin shows formation of ATG8 puncta, which is reversed by supplementation with trypanothione or spermidine or pretreatment with 3-methyhladenine.
Discussion
Interestingly, in our studies, we observed increased HAT expression and decreased SIR2RP (a type III histone deacetylase) expression of the parasite after spermidine starvation. We observed an increase in NAD+ levels after hypericin treatment, which was reversed by trypanothione or spermidine supplementation. Here we have seen increased expression of 5'-AMP-activated protein kinase (AMPK) after hypericin treatment.
In our previous study, we have also reported generation of ROS after hypericin treatment, which was reversed by both trypanothione and spermidine supplementation (Singh et al., 2015). Our previous work showed that trypanothione supplementation does not rescue parasite death after hypericin treatment. We have observed that hypericin-induced autophagy was a result of DNA damage response against ROS generation.
In our study, we observed an increase in the intracellular ATP pool of the parasite after hypericin treatment.
Proteome Analysis of Leishmania donovani Under Spermidine Starvation *
- Abstract
- Introduction
- Materials and Methods
- Protein protein interaction analysis: Differentially up regulated proteins above 1.5 fold and down regulated protein below 0.9 fold were taken to analyze the interaction
- Results
- Distribution of proteins being altered after hypericin treatment into major classes
- Differential regulation of proteins related to translation: There was 2.96 fold and 1.68 fold increase in elongation factor 1 alpha and eukaryotic translation initiation factor
- Differential expression of enzymes involved in protein turnover, processing and modification was observed after hypericin treatment: The enzymes involved in protein
- Protein-protein interaction of differentially modulated proteins: Differentially up regulated protein above 1.5 fold and down regulated proteins below 0.9 fold were
- Discussion
There is increased expression of poly(A) binding protein and threonyl-tRNA synthetase after treatment with hypericin (Table 4.1). It was shown that several enzymes involved in glucose metabolism show increased expression after treatment with hypericin. However, proteins involved in cellular motility were found to show reduced expression after hypericin treatment.
The expression of farnesyl pyrophosphate was also found to be higher (2.07 fold) after hypericin treatment (Table 4.1). Here represent the down regulated proteins after hypericin treatment with ANOVA value of less than 0.05. Here, we observed an increased expression of HSP 90 after hypericin treatment, suggesting a response to DNA damage caused by ROS generation.
Expression of stress-inducible protein 1 (STI1), which is thought to be a cochaperone of HSP 90, increased after hypericin treatment.
Molecular Docking and Structure Based Virtual Screening Studies of CAAX Prenyl Protease I and II
Abstract
Introduction
Yeast type I CAAX prenyl protease (AFC1p) and yeast type II CAAX prenyl protease have been reported to have distinct but overlapping substrate specificities ( Boyartchuk, Ashby, & Rine, 1997 ). In addition to its CAAX proteolysis activity, Ste24p, a type I CAAX prenyl protease from yeast, is found to have NH2-terminal proteolysis activity for processing yeast α-factor precursor (Tam et al., 2001). However, this cannot account for the importance of CAAX prenyl proteases and methyltransferase, as prenylation is a prerequisite for their activity.
RCE1 deficiency was found to be lethal in late embryonic development in mice, which also confirms the physiological importance of CAAX prenylated proteases (Kim et al., 1999). The CAAX prenyl protease STE24 of yeast type I was recently identified as having a role in chitin synthesis (Meissner et al., 2010). Leishmania CAAX prenyl proteases have a unique active site environment and low sequence similarity to human host CAAX prenyl proteases.
The present study will help discover new drug-like molecules that could be potential inhibitors of CAAX prenylproteases from Leishmania donovani.
Materials and Methods
- Sequence alignment and protein modelling: The protein sequence of CAAX prenyl protease I (Accession No.: E9BTI9) and CAAX prenyl protease II (Accession No
Recently, inhibitors against gBP21 protein that are essential for RNA editing in Leishmania donovani were identified using a computational approach (Sahoo et al., 2013). Also, imidazole analogs have been identified as potential inhibitors of trypanothione reductase from Leishmania donovani using molecular docking and virtual screening ( Pandey et al., 2015 ). It uses a template-based homology domain prediction method, Rosetta, to yield a high-quality 3D structure of the target protein (Kim et al., 2004).
At the initial stage of structure prediction, Ginzu, a domain prediction method was used to screen the query sequence for regions homologous to the experimentally characterized structure using BLAST, PSI-BLAST and Pcons2 (Kim et al., 2004; Mount, 2007 ) ). Other methods such as ERRAT and Verify 3D were also used from the SAVES server (Yadav et al., 2014). Finally, the ligands were rigidly docked in Glide XP mode into the lowest energy induced fit receptor structure with no scale (vdW scale of 1) and their final scores were obtained (Sherman et al., 2006; Suryanarayanan and Singh, 2014).
It also evaluates the acceptability of the entire known and screened compounds on the basis of Lipinski's rule of 5, which is significant for rational drug design (Suryanarayanan and Singh, 2014; Reddy et al., 2014).
Results
- Electrostatic potential Surface of protein: The electrostatic potential surface of the protein was generated using molecular mechanics method which reveals the
- Molecular docking studies: To carry out the docking studies we have taken two sets of inhibitors namely, bi substrate analogue inhibitors of protein farnesyl transferase of 16
ERRAT, Verify 3D, and Z-score for both CAAX prenylprotease I and CAAX prenylprotease II are tabulated. I was shown in Table 5.6 and the docking results of CAAX prenyl protease II were given in Table 5.7. The top screened compounds for CAAX prenyl protease I and II are shown in Tables 5.9 and 5.10, respectively.
The top fifteen compounds from the screening of CAAX prenyl proteases I and II were carried forward to further induced docking studies. The IFD results for CAAX prenyl protease I and II were shown in Table 5.11 and 5.12, respectively. Both CAAX prenyl proteases I and II were given novel and potent inhibitors by virtual screening as the compounds have a lower docking score.
QikProp results for CAAX prenyl protease I and II are shown in Table 5.15 and Table 5.16, respectively.
No. Compound Code SASA QP logHERG QP PMDCK QP logPC16
- Discussion
In this current study, we performed homology modeling for both CAAX prenylprotease I and II and validated using the SAVES and PROSA server. Active sites for both proteins were predicted using Sitemap, which reveals the key interacting residues in the active site. Further molecular docking studies with known compounds provided assistance in predicting the binding mode with both proteins.
Subsequently, structure-based virtual screening was performed to obtain new and potent inhibitors for both proteins. Then, the top 15 compounds were taken forward for IFD studies, which predict the exact bindings of the compounds to both proteins. Molecular dynamics simulation studies of the best complexes of the two proteins reveal binding stability and conformational stability.
Finally, the best compounds obtained from the screening can be used for further rational drug design of CAAX prenyl protease I and II inhibitors.
Summary of Work
- Abstract
- Introduction of Leishmaniasis
- Molecular mechanism of hypericin induced parasite death
- Molecular events leading to death of Leishmania donovani under spermidine starvation
- Proteome profiling of Leishmania donovani under spermidine starvation
- Structure based virtual screening studies of CAAX prenyl protease I and II of Leishmania donovani
Further, as an approach for multi-target drug discovery, we have also modeled CAAX prenyl protease I & II from Leishmania donovani and have identified potential inhibitors against these proteases using in silico analysis. In a quest to develop a new drug candidate, we have identified hypericin as a new drug candidate that specifically targets spermidine synthase from Leishmania donovani. So targeting CAAX prenyl proteases of Leishmania donovani may be a good approach to develop another drug candidate.
We have modeled the structure of CAAX prenyl protease I and II of Leishmania donovani using the homology modeling approach. Overall, we have identified inhibitors against Leishmania donovani CAAX prenyl protease I and II, whose potential as drug candidates can be further confirmed using biochemical and cellular studies. We have also identified potential inhibitors of CAAX prenyl protease I and II of Leishmania donovani.
The potential of the best identified inhibitors to inhibit Leishmania donovani recombinant CAAX prenyl protease I and II can be demonstrated.
Bibliography
Shalini Singh, Ekta Kumari, Ruchika Bharadwaj and Vikash Kumar Dubey
