Novel chemical scaffold as potential drug against Leishmania donovani: Integrated computational and experimental approaches.

In this study, we have screened a large number of Food and Drug Administration-approved compounds for novel anti-leishmanial molecules targeting the citrate synthase enzyme of the parasite. Based on their docking and molecular dynamic simulation statistics, five compounds were selected. These compounds followed Lipinski's rule of five. Additionally, in vitro, antileishmanial and cytotoxicity studies were performed. The three compounds, Abemaciclib, Bazedoxifene, and Vorapaxar, had shown effective anti-leishmanial activities with IC50 values of 0.92 ± 0.02, 0.65 ± 0.09, and 6.1 ± 0.91 against Leishmania donovani promastigote and with EC50 values of 1.52 ± 0.37, 2.11 ± 0.38, 10.4 ± 1.27 against intramacrophagic amastigote without significantly harming macrophage cells. Among them, from in silico and antileishmanial activities studies, Abemaciclib had been selected based on their less binding energy, good antileishmanial activities, and also a significant difference in their binding energy with human citrate synthase for cell death mechanistic studies using flow cytometry and a DNA fragmentation assay. The action of this compound resulted in an increased reactive oxygen species production, depolarization of mitochondrial membrane potential, DNA damage, and an increase in the sub-G1 cell population. These properties are the hallmarks of apoptosis which were further confirmed by apoptotic assay. Based on the above result, this anticancer compound Abemaciclib could be employed as a potential treatment option for leishmaniasis after further confirmation.

Drug Encapsulated Lipid-Polymeric Nanohybrid as a Chemo-therapeutic Platform of Cancer.

The focus of this research is to design a bioengineered drug delivery vehicle that is efficient in anti-cancer drug delivery in a controlled manner. The experimental work focuses on constructing a methotrexate-loaded nano lipid polymer system (MTX-NLPHS) that can transport methotrexate (MTX) in MCF-7 cell lines in a controlled manner through endocytosis via phosphatidylcholine. In this experiment, MTX is embedded with polylactic-co-glycolic acid (PLGA) in phosphatidylcholine, which acts as a liposomal framework for regulated drug delivery. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and dynamic light scattering (DLS) were utilized to characterize the developed nanohybrid system. The particle size and encapsulation efficiency of the MTX-NLPHS were found to be 198 ± 8.44 nm and 86.48 ± 0.31 %, respectively, which is suitable for biological applications. The polydispersity index (PDI) and zeta potential of the final system were found to be 0.134 ± 0.048 and -28 ± 3.50 mV, respectively. The lower value of PDI showed the homogenous nature of the particle size, whereas higher negative zeta potential prevented the system from agglomeration. An in vitro release kinetics was conducted to see the release pattern of the system, which took 250 h for 100% drug release This kind of system may carry the drug for a long time in the circulatory system and prevent the drug discharge. Other cell culture assays such as 3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) and reactive oxygen species (ROS) monitoring were used to see the effect of inducers on the cellular system. MTT assay showed cell toxicity of MTX-NLPHS reduced at the lower concentration of the MTX, however, toxicity increased at the higher concentration of the MTX as compared to free MTX. ROS monitoring c revealed more scavenging of ROS using MTX-NLPHS as compared to free MTX. Confocal microscopy suggested the MTX-NLPHS induced more nuclear elongation with cell shrinkage comparatively.

Ligand conjugated lipid-based nanocarriers for cancer theranostics.

Cancer is one of the major health-related issues affecting the population worldwide and subsequently accounts for the second-largest death. Genetic and epigenetic modifications in oncogenes or tumor suppressor genes affect the regulatory systems that lead to the initiation and progression of cancer. Conventional methods, including chemotherapy/radiotherapy/appropriate combinational therapy and surgery, are being widely used for theranostics of cancer patients. Surgery is useful in treating localized tumors, but it is ineffective in treating metastatic tumors, which spread to other organs and result in a high recurrence rate and death. Also, the therapeutic application of free drugs is related to substantial issues such as poor absorption, solubility, bioavailability, high degradation rate, short shelf-life, and low therapeutic index. Therefore, these issues can be sorted out using nano lipid-based carriers (NLBCs) as promising drug delivery carriers. Still, at most, they fail to achieve site-targeted drug delivery and detection. This can be achieved by selecting a specific ligand/antibody for its cognate receptor molecule expressed on the surface of the cancer cells. In this review, we have mainly discussed the various types of ligands used to decorate NLBCs. A list of the ligands used to design nanocarriers to target malignant cells has been extensively undertaken. The approved ligand-decorated lipid-based nanomedicines with their clinical status have been explained in tabulated form to provide a wider scope to the readers regarding ligand-coupled NLBCs.

Beclin1-mediated interplay between autophagy and apoptosis: New understanding.

The definition for autophagy holds a 'single' meaning as a conserved cellular process that constitutes a recycling pathway for damaged organelles and long-lived proteins to maintain nutrient homeostasis and mediate quality control within the cell. But this process of autophagy may behave ambiguously depending on the physiological stress as the stress progresses in the cellular microenvironment; the 'single' meaning of the autophagy changes from the 'cytoplasmic turnover process' to 'tumor suppressive' and a farther extent, 'tumor promoter' process. In a tumorigenic state, the chemotherapy-mediated resistance and intolerance due to upregulated autophagy in cancer cells have become a significant concern. This concern has provided insight to the scientific community to enter into the arena of cross-talk between autophagy and apoptosis. Recent findings and ongoing research have provided insights on some of the key regulators of this cross-talk; one of them is Beclin1 and their involvement in the physiological and the pathophysiological processes; however, reconciliation of these two forms of death remains an arena to be explored extensively. This review sheds light on the interplay between autophagy and apoptosis, emphasizing one of the key players, Beclin1, and its importance in health and diseases.

Virtual screening and repurposing of FDA-approved drugs from ZINC database to identify potential autophagy inhibitors exploiting autophagy related 4A cysteine peptidase as a target: potential as novel anti-cancer molecule.

Cancer cells utilize extensive autophagy in effort to adapt to high metabolic stress. This indicates that impairing the high autophagic flux might be an attractive target for cancer therapy. Autophagy related gene 4A (ATG4A) is a key player for autophagy and its inhibition may help in tumor clearance. The present study aims to screen candidate drugs from FDA-approved drugs, a subset of Zinc database, to identify potential ATG4A inhibitors that may have anti-cancer activity. Computer aided drug design approach was applied for the study using the virtual screening tools Raccoon and MGLTools-1.5.6. We have identified the drug Lumacaftor as a potent inhibitor of ATG4A on the basis of computational approaches viz. molecular docking, molecular dynamics simulation and MM/PBSA method. The drug is likely to be a potent regimen candidate to be used as an anti-cancer molecule. However, this potent inhibitor against ATG4A as anti-cancer molecule needs further investigation and validation.Communicated by Ramaswamy H. Sarma.

Repurposing of FDA-approved drugs as autophagy inhibitors in tumor cells.

Autophagy and apoptosis are the two crucial processes of programmed cell death found in all eukaryotic cells; however, the elevated physiological stress in the tumor microenvironment leads to uncontrolled up-regulation in the process of autophagy. Available literatures suggest that inhibiting up-regulated autophagy in the cancerous cells may lead to the apoptosis and thereby culminate to tumor clearance. Several studies have been performed to design autophagy-inhibitors using either Beclin-1 or Bcl-2 as a target in isolation. However, to overcome the constraints of the availability of small and potent autophagy inhibitors, we have attempted extensive computational approach of repurposing the FDA-approved drugs from the ZINC database in order to inhibit the interaction between the Beclin1 and Bcl-2. Out of 1565 FDA-approved drugs used in our computational work, we sorted the drugs Ponatinib, Simeprevir, and Nilotinib through various methods viz. molecular docking, Lipinski's filter, MD simulation and MM/PBSA, and we found these aforementioned drugs to show good binding energy and favorable interaction with the BH3 domain of Beclin1. We anticipate from our computational results that these drugs may become potent candidates to inhibit autophagy and exhibit the apoptosis in the tumor microenvironment and combat the current limitation of potent autophagy inhibitors; however, to substantiate our in-silico results, further experimental validations of these drug molecules are currently in progress.Communicated by Ramaswamy H. Sarma.

Molecular docking, binding mode analysis, molecular dynamics, and prediction of ADMET/toxicity properties of selective potential antiviral agents against SARS-CoV-2 main protease: an effort toward drug repurposing to combat COVID-19.

The importance of the main protease (Mpro) enzyme of SARS-CoV-2 in the digestion of viral polyproteins introduces Mpro as an attractive drug target for antiviral drug design. This study aims to carry out the molecular docking, molecular dynamics studies, and prediction of ADMET properties of selected potential antiviral molecules. The study provides an insight into biomolecular interactions to understand the inhibitory mechanism and the spatial orientation of the tested ligands and further, identification of key amino acid residues within the substrate-binding pocket that can be applied for structure-based drug design. In this regard, we carried out molecular docking studies of chloroquine (CQ), hydroxychloroquine (HCQ), remdesivir (RDV), GS441524, arbidol (ARB), and natural product glycyrrhizin (GA) using AutoDock 4.2 tool. To study the drug-receptor complex's stability, selected docking possesses were further subjected to molecular dynamics studies with Schrodinger software. The prediction of ADMET/toxicity properties was carried out on ADMET Prediction™. The docking studies suggested a potential role played by CYS145, HIS163, and GLU166 in the interaction of molecules within the active site of COVID-19 Mpro. In the docking studies, RDV and GA exhibited superiority in binding with the crystal structure of Mpro over the other selected molecules in this study. Spatial orientations of the molecules at the active site of Mpro exposed the significance of S1-S4 subsites and surrounding amino acid residues. Among GA and RDV, RDV showed better and stable interactions with the protein, which is the reason for the lesser RMSD values for RDV. Overall, the present in silico study indicated the direction to combat COVID-19 using FDA-approved drugs as promising agents, which do not need much toxicity studies and could also serve as starting points for lead optimization in drug discovery.

IFN-γ+ CD4+T cell-driven prophylactic potential of recombinant LDBPK_252400 hypothetical protein of Leishmania donovani against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL.

Interaction of selected biomolecules and metabolites with amyloidogenic proteins.

The current manuscript reports docking and molecular interaction analyses of three FDA approved acetylcholinesterase inhibitors, nitrogenous bases and nucleotides with amyloidogenic proteins like hen egg white lysozyme (HEWL) and amyloid ß peptide. After prediction of aggregation-prone regions in hen egg-white lysozyme and amyloid ß peptide, grid boxes were defined for docking purposes covering these regions. We analyzed vital interactions and binding modes of molecules that dock near aggregation-prone regions of these proteins with acceptable statistics. The data hints toward the possibility that these molecules may bind to aggregation-prone regions and prevent amyloid/aggregation formation. We have also compared the binding energy and interactions of these molecules with certain other natural molecules viz. Curcumin, Coumarin and Resveratrol that have been previously reported to show anti-amyloidogenic activity as positive controls.Communicated by Ramaswamy H. Sarma.

Purines and Pyrimidines: Metabolism, Function and Potential as Therapeutic Options in Neurodegenerative Diseases.

Various neurodegenerative disorders have various molecular origins but some common molecular mechanisms. In the current scenario, there are very few treatment regimens present for advanced neurodegenerative diseases. In this context, there is an urgent need for alternate options in the form of natural compounds with an ameliorating effect on patients. There have been individual scattered experiments trying to identify potential values of various intracellular metabolites. Purines and Pyrimidines, which are vital molecules governing various aspects of cellular biochemical reactions, have been long sought as crucial candidates for the same, but there are still many questions that go unanswered. Some critical functions of these molecules associated with neuromodulation activities have been identified. They are also known to play a role in foetal neurodevelopment, but there is a lacuna in understanding their mechanisms. In this review, we have tried to assemble and identify the importance of purines and pyrimidines, connecting them with the prevalence of neurodegenerative diseases. The leading cause of this class of diseases is protein misfolding and the formation of amyloids. A direct correlation between loss of balance in cellular homeostasis and amyloidosis is yet an unexplored area. This review aims at bringing the current literature available under one umbrella serving as a foundation for further extensive research in this field of drug development in neurodegenerative diseases.

LRSAM1 E3 ubiquitin ligase promotes proteasomal clearance of E6-AP protein.

Numerous proteins participate and actively contribute to the various cellular mechanisms, where several of them are crucial for regular metabolism, including survival. Thus, to maintain optimal cellular physiology, cells govern protein quality control functions with the assistance of comprehensive actions of molecular chaperones, the ubiquitin-proteasome system, and autophagy. In the ubiquitin-proteasome pathway, few quality control E3 ubiquitin ligases actively participate against misfolded protein aggregation generated via stress conditions. But how these quality control E3s active expression levels returned to basal levels when cells achieved re-establishment of proteostasis is still poorly understood. Our current study demonstrated that LRSAM1 E3 ubiquitin ligase promotes the proteasomal degradation of quality control E3 ubiquitin ligase E6-AP. We have observed the co-localization and recruitment of LRSAM1 with E6-AP protein and noticed that LRSAM1 induces the endogenous turnover of E6-AP. Partial depletion of LRSAM1 elevates the levels of E6-AP and affects overall cell cycle regulatory proteins (p53 and p27) expression, including the rate of cellular proliferation. The current finding also provides an excellent opportunity to better understand the basis of the E6-AP associated pathomechanism of Angelman Syndrome disorder. Additionally, this study touches upon the novel potential molecular strategy to regulate the levels of one quality control E3 ubiquitin ligase with another E3 ubiquitin ligase and restore proteostasis and provide a possible therapeutic approach against abnormal protein aggregation diseases.

Bacterial protein azurin and derived peptides as potential anti-SARS-CoV-2 agents: insights from molecular docking and molecular dynamics simulations.

The current pandemic SARS-CoV-2 has wreaked havoc in the world, and neither drugs nor vaccine is available for the treatment of this disease. Thus, there is an immediate need for novel therapeutics that can combat this deadly infection. In this study, we report the therapeutic assessment of azurin and its peptides: p18 and p28 against the viral structural S-protein and non-structural 3CLpro and PLpro proteins. Among the analyzed complexes, azurin docked relatively well with the S2 domain of S-protein compared to the other viral proteins. The derived peptide p18 bound to the active site domain of the PLpro protein; however, in other complexes, lesser interactions were recorded. The second azurin derived peptide p28, fared the best among the docked proteins. p28 interacted with all the three viral proteins and the host ACE-2 receptor by forming several electrostatic and hydrogen bonds with the S-protein, 3CLpro, and PLpro. MD simulations indicated that p28 exhibited a strong affinity to S-protein and ACE-2 receptor, indicating a possibility of p28 as a protein-protein interaction inhibitor. Our data suggest that the p28 has potential as an anti-SARS-CoV-2 agent and can be further exploited to establish its validity in the treatment of current and future SARS-CoV crisis.Communicated by Ramaswamy H. Sarma.

BLIMP-1 Mediated Downregulation of TAK1 and p53 Molecules Is Crucial in the Pathogenesis of Kala-Azar.

Precise regulation of inflammasome is critical during any pathogenic encounter. The whole innate immune system comprising of pattern recognition receptors (PRRs) relies on its ability to sense microbes. The fate of cellular death in infected cells depends mostly on the activation of these inflammasome, the dysregulation of which, due to functional manipulation by various pathogens, leads to be the cause of many human diseases. Here, an interesting finding has been observed which is related to how Leishmania donovani parasites exploit various host mediator molecules to cause immunosuppression. Here we report for the first time that the parasites check pyroptosis in the infected cells in-vitro by BLIMP-1 mediated suppression of TAK1 and p53 proteins. This might be one of the reasons how parasites evade the pro-inflammatory response of the host cells. Further understandings and validations are required to come up with better therapeutic approaches against kala-azar.

Advances in protein misfolding, amyloidosis and its correlation with human diseases.

Protein aggregation, their mechanisms and trends in the field of neurodegenerative diseases is still far from completely being decoded. It is mainly attributed to the complexity surrounding the interaction between proteins which includes various regulatory mechanisms involved with the presentation of abnormal conditions. Although most proteins are functional in their soluble form, they have also been reported to convert themselves into insoluble aggregates under certain conditions naturally. Misfolded protein forms aggregates which are mostly unwanted by the cellular system and are mostly involved in various pathophysiologies including Alzheimer's, Type II Diabetes mellitus, Kurus's etc. Challenges lie in understanding the complex mechanism of protein misfolding and its correlation with clinical evidence. It is often understood that due to the slowness of the process and its association with ageing, timely intervention with drugs or preventive measures will play an essential role in lowering the rate of dementia causing diseases and associated ailments in the future. Today approximately more than 35 proteins have been identified capable of forming amyloids under defined conditions, and nearly all of them have been associated with disease outcomes. This review incorporates a major understanding from the history of diseases associated with protein misfolding, to the current state of neurodegenerative diseases globally, highlighting challenges in drug development and current state of research in a comprehensive manner in the field of protein misfolding diseases. There is increasing clinical association of protein misfolding with regards to amyloids compelling us to thread questions solved and further helping us design possible solutions by generating a pathway-based research on which future work in this field could be driven.

Structure-guided approach to identify a novel class of anti-leishmaniasis diaryl sulfide compounds targeting the trypanothione metabolism.

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777-TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.

BLIMP-1 Plays Important Role in the Regulation of Macrophage Pyroptosis for the Growth and Multiplication of Leishmania donovani.

Visceral leishmaniasis, one of the fatal forms of the disease, is caused by Leishmania donovani and presents morbid clinical manifestations. The parasite evades pro-inflammatory immune responses by several reported mechanisms and modulates the host immune system to cause fatal symptoms. A plethora of reports related to the role of BLIMP-1 and its involvement in suppressing the immune response in various infectious diseases have been documented. Higher parasitic burden due to increased BLIMP-1 production has been reported earlier for malaria and leishmaniasis with no detailed information. We report for the first time the role of BLIMP-1 in suppressing macrophage pyroptosis during L. donovani infection and thereby tweaking the tight regulation of the NFκß-NLRP3 signaling pathway. Expression analyses of BLIMP-1 and NFκß have been measured using real-time PCR and Western blotting. The importance of BLIMP-1 has been validated using a siRNA-mediated experiment along with caspase 1 activity, LDH release assay, and infectivity index analyses. An inverse relationship between BLIMP-1 and NFκß expression has been highlighted during L. donovani infection, which is reversed in blimp-1 deficient cells infected with promastigotes. The above fact has been further validated with caspase 1 activity assay, and LDH release along with IFNγ and TNF-α release assay. Finally, resumption of pyroptosis has been concluded in infected blimp-1 deficient cells in contrast to wild type infected cells. We conjecture that parasites modulate the NFκß-NLRP3 signaling pathway by taking advantage of BLIMP-1 dependent IL-10 production and finally disrupting an inflammation-mediated pyroptosis cell death pathway in infected cells.

Geobacillus yumthangensis sp. nov., a thermophilic bacterium isolated from a north-east Indian hot spring.

A thermophilic, spore-forming, rod-shaped bacterium isolated from the Yumthang hot spring in North Sikkim, India was subjected to taxonomic studies. The thermophilic bacterial isolate was designated as strain AYN2T. Cells were Gram-stain-positive, aerobic, motile, rod-shaped, catalase-positive and methyl red-negative. Strain AYN2T was able to grow in the pH range from 6 to 10 (optimum, pH 7.5-8.0), at 40-70 °C (60 °C) and in NaCl concentrations of 0-4 % (1 %). The major cellular fatty acids were iso-C15 : 0 (12.8 %), iso-C16 : 0 (13.9 %) and iso-C17 : 0 (13.8 %). No matches were found in the rtsba6 Sherlock libraries. The G+C content of the genomic DNA was 42.11 mol%. Based on phylogenetic analysis of the 16S rRNA gene sequences, strain AYNT showed highest sequence similarity to the type strain of Geobacillus toebii (96 %). However, the phenotypic properties of strain AYN2T were clearly distinct from those of G. toebii and related species. On the basis of polyphasic analysis, strain AYN2T represents a novel species in the genus Geobacillus, for which the name Geobacillus yumthangensis sp. nov. is proposed. The type strain is AYN2T(MTCC=12749=KCTC=33950= JCM 32596).

Synthesis and Evaluation of Methyl 4-(7-Hydroxy-4,4,8-Trimethyl-3-Oxabicyclo[3.3.1]Nonan-2-yl)Benzoate as an Antileishmanial Agent and Its Synergistic Effect with Miltefosine.

In our interest in oxabicyclic compounds as potent antileishmanial agents, the present work deals with the chemical synthesis of a new oxabicyclic derivative, methyl 4-(7-hydroxy-4,4,8-trimethyl-3-oxabicyclo[3.3.1]nonan-2-yl)benzoate (PS-207). This oxabicyclic derivative showed a good antileishmanial effect on the parasite, on both the promastigote and the amastigote. The mode of parasitic death from PS-207 seemed to be apoptosis-like. Interestingly, the combination of PS-207 with a low dose of miltefosine showed a synergistic effect against the parasite.

Molecular events leading to death of Leishmania donovani under spermidine starvation after hypericin treatment.

We have previously reported that the hypericin treatment caused spermidine starvation and death of Leishmania parasite. Here, we report different molecular events under spermidine starvation and potential role of spermidine in processes other than redox homeostasis of the parasite. We have analyzed changes in expression of several genes by using quantitative gene expression analysis. Further, these changes at molecular level were also confirmed by using biochemical and cellular studies. Altered expression of several genes involved in redox metabolism, hypusine modification of eIF5A, DNA repair pathway and autophagy was observed. There was decrease in Sir2RP expression after hypericin treatment and this decrease has been found to be associated with induced ROS due to hypericin treatment as it has been rescued by either trypanothione or spermidine supplementation. Translation initiation in the parasite was decreased upon spermidine starvation. We also observed increased AMPK expression upon hypericin treatment. The increase in intracellular ATP and NAD+ levels as well as decrease in Sir2RP expression of the parasite are cytoprotective mechanism towards generated ROS due to hypericin treatment possibly by inducing autophagy as indicated by increase in autophagy related gene expression and acridine orange staining. However, the autophagy needs to be established using more rigorous methodologies.

Evaluation of CAAX prenyl protease II of Leishmania donovani as potential drug target: Infectivity and growth of the parasite is significantly lowered after the gene knockout.

Prenylation pathway is responsible for post translational modification of various signal proteins, including proteins of Ras superfamily. CAAX prenyl proteases are known to be key players in prenylation pathway. In the current study, we have evaluated CAAX prenyl protease II as a possible drug target against Leishmania donovani parasite, the causative agent of visceral leishmaniasis. Gene knockout strategy was employed to target CAAX prenyl protease II and subsequent effects were studied. CAAX prenyl protease II knockout resulted in significant decrease in growth and infectivity.