It is stated that the work described in this thesis entitled “Studies on redox system of Leishmania donovani: Understanding drug resistance process and discovery of novel drug candidate” by Ms.
Review of literature on Leishmania redox metabolism, drug resistance and drug discovery.*
Abstract
Introduction
- Drug Targeting Enzymes
It is also the second most neglected parasitic disease after malaria (Singh et al., 2012). Thus, the polyamine synthesis pathway ultimately aids in parasite growth and survival (Heby et al., 2003). Leishmania donovani ornithine decarboxylase (LdODC) is essential for parasite survival in the mammalian host (Boitz et al., 2009).
One report showed that Entamoeba histolytica ODC (EhODC) functions in a dimeric form (Preeti et al., 2012). Pentavalent Sb(V) is reduced to trivalent antimony Sb(III), which is the active form of the drug (Roberts et al., 1995). 5 Pentamidine Nephrotoxicity, heart problems, low blood pressure, low or high blood sugar (Silva et al., 2013).
Leishmania morphological change, rapid collapse of mitochondrial inner membrane potential (Loiseau et al., 2011).
Miltefosine unresponsive Leishmania donovani has better ability of resist reactive oxygen species.*
- Abstract
- Introduction
- Materials and methods
- Results and discussion
- Conclusion
Apoptotic death was mediated by inhibition of cytochrome c-oxidase and mitochondrial dysfunction (Verma et al., 2007; . Luque-Ortega et al., 2007). It is more effective (induces apoptosis) against the arsenite-resistant strain (Verma et al., 2007) and has the ability to degrade DNA. Unfortunately, the emergence of a Leishmania donovani strain resistant to miltefosine was first reported elsewhere (Seifert et al., 2003).
Overexpression of mitochondrial iron superoxide dismutase-A (LdFeSODA) from Leishmania donovani parasites has been reported to protect against miltefosine ( Getachew et al., 2012 ). It has been suggested that LdFeSODA protects the mitochondria of Leishmania from oxidative stress, thereby inhibiting programmed cell death (Getachew et al., 2012). The miltefosine transporter LdMT and its specific ß-subunit LdRos3 form the miltefosine translocation machinery at the Leishmania plasma membrane (Figure 2.2) (Pérez-Victoria et al., 2006).
Mutations in miltefosine transporters result in reduced uptake, increased efflux and faster metabolism of the drug (Pérez-Victoria et al., 2006; Seifert et al., 2007). The superoxide dismutase (SOD) assay was performed using the riboflavin-mediated Nitro Blue tetrazolium reduction method (García-Limones et al., 2002). Ascorbate peroxidase (APX) assay was performed as previously described (Gunes et al., 2009; Singh et al., 2012).
An efficient electron transport chain plays an important role in keeping the ROS concentration low (Pramanik et al., 2011). Several studies on miltefosine-nonresponsive Leishmania have been performed (Pérez-Victoria et al., 2006; Seifert et al., 2007; Rakotomanga et al., 2005). The widely used antimony-based drugs have also been reported to act via important enzymes involved in redox homeostasis (Baiocco et al., 2009).
Ornithine decarboxylase of Leishmania donovani: Biochemical properties and possible role of N-terminal extension.*
Abstract
Introduction
All other chemicals used in the experiments were of the highest quality, sourced from Sigma-Aldrich, USA and Merck, USA. The purified PCR fragments were digested with NheI and XhoI restriction endonucleases and cloned into the NheI and and pET28a-T- were generated. LdODC. Our current method is based on the reaction between SAO (soybean amine oxidase) and putrescine, the product of the ornithine decarboxylase catalyzed reaction (Figure 3.2).
The concentration of putrescine formed by LdODC catalysis was calculated by measuring the absorbance of the colored complex (505 nm) formed as a result of the reaction of H2O2 with 4-aminoantipyrine and phenol catalyzed by horseradish peroxidase. The authenticity of the gene was first confirmed by PCR of the ODC vector containing pSNBR showing an intense band size of 2121 bps (Figure 3.4A). Similarly, amplification of the 1371 bp T-LdODC was done using the ODC vector containing pSNBR (Figure 3.4D).
To optimize the expression of recombinant proteins, the transformed culture was grown at different temperatures ranging from 18°C to 25°C, but most of the recombinant protein was found in pellets. Kinetic parameters such as Km and Vmax of purified LdODC were estimated using Line-weaver Burk plot (Figure 3.6B). The secondary structure of the two forms of enzyme was studied by fluorescence spectra in native and 6 M GuHCl-containing buffer.
The data suggest a change in the tryptophan environment in T-LdODC, as evidenced by large decreases in fluorescence intensity and less change in wavelength maxima (Figure 3.8A). While removal of the N-terminal extension leads to significant increase in the stability of the enzyme, the activity of the enzyme is lost after truncation. The data are plotted as unfolded fraction for stability comparison of LdODC and T-LdODC.
Conclusion
The N-terminal extension of LdODC helps fold the enzyme into the active conformation at the cost of activity. In the experimental condition, when the N-terminal extension in LdODC is removed (T-LdODC), the enzyme is more stable but cannot fold into its original conformation.
Inhibition of ornithine decarboxylase of Leishmania donovani shows inhibitor specific effect: Mystery remains unsolved *
- Abstract
- Introduction
- Results and discussion
- Conclusion
Visceral leishmaniasis, caused by Leishmania donovani in the Indian subcontinent, is the most severe and fatal form of the disease (Shukla et al. 2010). Polyamines play an important role in the cellular homeostasis of most of the living organisms, including Leishmania parasite (Coleman et al. 2004; Krauth-Siegel and Comini 2008). Furthermore, DL-α-difluoromethylornithine (DFMO), a non-specific inhibitor of ornithine decarboxylase, is able to kill Leishmania promastigotes (Coons et al., 1990).
Some other LdODC inhibitors have shown anti-Leishmania effects (Hazra et al. 2013; Singh et al., 2014). We sorted the ligands according to the descending order of the difference in the binding free energy, i.e. the compound showing the greatest difference in the free. The mode of inhibition was found to be non-competitive as shown by the decrease in Vmax in the presence of 50 µM inhibitor concentration (Figure 4.1C).
It is worth noting that the initial 255 residues of LdODC (N-terminal extension) were excluded when generating the model in our previous report (Chakraborty et al., 2013), so residue 256 in the sequence corresponds to residue 1 in the modeled structure. As shown in Figure 4.5, treatment with compound M-5 increased the gene by twofold. Leishmania promastigotes treated with compound M-2 showed a nearly 27% reduction in thiol levels at 24 hours (Figure 4.5A), and mangiferin showed a 30% reduction in thiol levels at 3 hours.
However, in the case of mangiferin-treated cells, a 10% recovery of the thiol level was observed after the next 3 hours (Figure 4.5B). Ornithine decarboxylase (ODC) is involved in thiol (glutathione and trypanothion) biosynthesis (Krauth-Siegel and Comini 2008; Rai et al. 2013). Treatment with mangiferin has shown an increased ROS level of up to ~11% in 3 hours, which did not increase after further treatment (Figure 4.6C).
Novel leads against miltefosine unresponsive Leishmania donovani.*
Abstract
Introduction
Drug combinations
Results and discussion
The data reconfirms that the strain is miltefosine and is unresponsive even under laboratory conditions. Recently, a report from our laboratory has proven that the oxabicyclo derivative (for simplicity we called it PS-203). In vivo studies in hamster models further confirmed the anti-leishmanial effect of the compound (Saugar et al., 2014).
The effect of PS-203 was also evaluated in miltefosine-nonresponsive Leishmania donovani (BHU-1155) as well as miltefosine-responsive Leishmania donovani (BHU-1081) under similar laboratory conditions for a comparison (Figure 5.3). The data show that compound PS-203 is equally effective against the miltefosine-unresponsive strain with an IC50 value of 4.0 ± 0.5 μM. We have also explored the combination of PS-203 with miltefosine in both miltefosine-responsive and non-responsive strains of Leishmania (Figure 5.4).
Interestingly, as shown in Figure 5.4A, 25 µM miltefosine without PS-203 (0 µM on the x-axis) can kill only 60% Leishmania promastigote, but in the presence of 1 µM PS-203 the efficacy is significantly higher . Similarly, the combination of PS-203 with miltefosine reduced the promastigote viability of non-responsive strain, thereby improving the efficacy of PS-203 (Figure 5.4B). Another report also proved that mitomycin C is also a potential anti-leishmanian agent (Shukla et al., 2011).
Therefore, we tested the effect of the compound on the miltefosine non-responsive strain and our data demonstrated that mitomycin C is equally effective against the miltefosine non-responsive strain (Figure 5.5). The toxicity issue associated with mitomycin C limits its use, so using this compound in combination with other effective compounds will reduce the toxic effect of mitomycin. After using a mixture of mitomycin C, miltefosine and PS-203, a significant inhibition of the miltefosine non-responsive strain was observed (Figure 5.6).
Conclusion
The combination of miltefosine, PS-203 and mitomycin C has shown a significant decrease in promastigote viability, indicating a good combinatorial regimen for the treatment of the miltefosine-resistant strain of Leishmania.
SUMMARY AND FUTURE SCOPE
Thinking
Publications in Peer Reviewed Journals
Mousumi Das, Prakash Saudagar, Shyam Sundar and Vikash Kumar Dubey (2013) Miltefosine unresponsive Leishmania donovani has better ability to resist reactive oxygen species, FEBS Journal. Mousumi Das, Ritesh Kumar and Vikash Kumar Dubey (2015) Ornithine decarboxylase of Leishmania donovani: Biochemical properties and possible role of N-terminal extension, Protein & Peptide Letters. Mousumi Das, Shalini Singh and Vikash Kumar Dubey (2015) Novel inhibitors of Leishmania parasite ornithine decarboxylase (LdODC): The parasite resists LdODC inhibition by spermidine synthase overexpression (posted 14 April 2015, Molecular Parasite and Biochemical Parasite).
Mousumi Das, Gundappa Saha, Anil Saikia en Vikash Dubey (2015) Roman lei teen miltefosien nie-reageer Leishmania donovani. Ingedien op 21 April, Antimikrobiese Middels en Chemoterapie kort kommunikasie). Sudipta Hazra, Subhalakshmi Ghosh, Madhushree Das Sarma, Smriti Sharma, Mousumi Das, Prakash Saudagar, Vijay Kumar Prajapati, Vikash Kumar Dubey, Shyam Sundar, Banasri Hazra (2013). van Leishmania donovani, Eksperimentele Parasitologie. Neha Sharma, Anil Kumar Shukla, Mousumi Das en Vikash Kumar Dubey (2012) Evaluering van plumbagin en sy afgeleide as potensiële modulator van redokstiolmetabolisme van Leishmania-parasiet, Parasitology Research, 110:341–.
शालिनी सिंग, श्यामली सरमा, शशांक कटियार, मौसमी दास, रुचिका भारद्वाज, दुरई सुंदर आणि विकास कुमार दुबे (2015) हायपरिसिन-प्रेरित परजीवी मृत्यूच्या आण्विक यंत्रणेची तपासणी करणे: रेडॉक्स सीबोलिझम थेरपीच्या पलीकडे स्पर्मिडीनच्या भूमिकेत अंतर्दृष्टी , ५९:१५–२४. मौसमी दास, शालिनी सिंग, रुचिका भारद्वाज आणि विकास कुमार दुबे (2015) दुर्लक्षित उष्णकटिबंधीय रोग लीशमॅनियासिस: एक संक्षिप्त पुनरावलोकन (बुलेटिन).
Publications in conference Proceedings
Biological Chemists, held at the Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, November. Novel drug candidate discovery by targeting the enzyme ornithine decarboxylase: Biochemical analysis of the cellular effects of identified drug candidates. Participated in "BioSangam-2013, International Conference on Health, Environment & Environmental Biotechnology" organized by Motilal Nehru National Institute of Technology.
Actively participated in the Research Conclave from 23 - 26 March 2015 organized by the PhD Council of the Students' Academic Board (SAB), Indian Institute of Technology Guwahati.
Workshop / training attended
