Epsilon subunit of T-complex protein-1 from Leishmania donovani: A tetrameric chaperonin.

The cytosolic T-complex protein-1 ring complex (TRiC), also referred as chaperonin containing TCP-1(CCT), comprising eight different subunits stacked in double toroidal rings, binds to around 10 % of newly synthesized polypeptides and facilitates their folding in ATP dependent manner. In Leishmania, among five subunits of TCP1 complex, identified either by transcriptome or by proteome analysis, only LdTCP1γ has been well characterized. It forms biologically active homo-oligomeric complex and plays role in protein folding and parasite survival. Lack of information regarding rest of the TCP1 subunits and its structural configuration laid down the necessity to study individual subunits and their role in parasite pathogenicity. The present study involves the cloning, expression and biochemical characterization of TCP1ε subunit (LdTCP1ε) of Leishmania donovani, the causative agent of visceral leishmaniasis. LdTCP1ε exhibited significant difference in primary structure as compared to LdTCP1γ and was evolutionary close to LdTCP1 zeta subunit. Recombinant protein (rLdTCP1ε) exhibited two major bands of 132 kDa and 240 kDa on native-PAGE that corresponds to the dimeric and tetrameric assembly of the epsilon subunit, which showed the chaperonin activity (ATPase and luciferase refolding activity). LdTCP1ε also displayed an increased expression upto 2.7- and 1.8-fold in the late log phase and stationary phase promastigotes and exhibited majorly vesicular localization. The study, thus for the first time, provides an insight for the presence of highly diverge but functionally active dimeric/tetrameric TCP1 epsilon subunit in Leishmania parasite.

Design, synthesis, and biological evaluation of quinoline-piperazine/pyrrolidine derivatives as possible antileishmanial agents.

In pursuance of our efforts to expand the scope of novel antileishmanial entities, a series of thirty-five quinoline-piperazine/pyrrolidine, and other heterocyclic amine derivatives were synthesized via a molecular hybridization approach and examined against intracellular amastigotes of luciferase-expressing Leishmania donovani. The preliminary in vitro screening suggests that twelve compounds in the series exhibited better inhibition against amastigote form with good IC50 values ranging from 2.09 to 8.89 µM and lesser cytotoxicity in contrast to the standard drug miltefosine (IC50 9.25 ± 0.17 µM). Based on the satisfactory selectivity index (SI), two compounds were tested for in vivo leishmanicidal efficacy against Leishmania donovani/golden hamster model. Compounds 33 and 46 have shown significant inhibition of 56.32%, and 49.29%, respectively, in vivo screening at a daily dose of 50 mg/kg for 5 days. The pharmacokinetic results confirmed that 33 and 46 have satisfactory IP exposure with adequate parameters. Collectively, Compound 33 was identified as the most significant potential lead that could be employed as a prototype for future optimizations.

Antileishmanial evaluation of triazole-butenolide conjugates: design, synthesis, in vitro screening, SAR and in silico ADME predictions.

In the quest to discover novel scaffolds with leishmanicidal effects, a series of 23 compounds containing the most promising 1,2,3-triazole and highly potent butenolide in one framework were synthesized. The synthesized conjugates were screened against Leishmania donovani parasite; five of them showed moderate antileishmanial activity against promastigotes (IC50 30.6 to 35.5 µM) and eight of them exhibited significant activity against amastigotes (IC50 ≤12 µM). Compound 10u was found to be the most active (IC50 8.4 ± 0.12 µM) with the highest safety index (20.47). The series was further evaluated against Plasmodium falciparum (3D7 strain) and seven compounds were found to be moderately active. Among them, again 10u emerged as the most active compound (IC50 3.65 µM). In antifilarial assays against adult female Brugia malayi, five compounds showed grade II inhibition (50-74%). Structure-activity relationship (SAR) analysis suggested a substituted phenyl ring, triazole and butenolide as essential structural features for bioactivity. Moreover, the results of in silico ADME parameter and pharmacokinetic studies indicated that the synthesized triazole-butenolide conjugates abide by the required criteria for the development of orally active drugs, and thus this scaffold can be used as a pharmacologically active framework that should be considered for the development of potential antileishmanial hits.

Design and synthesis of N-acyl and dimeric N-Arylpiperazine derivatives as potential antileishmanial agents.

The current regime for leishmaniasis is associated with several adverse effects, expensive, parenteral treatment for longer periods and the emergence of drug resistance. To develop affordable and potent antileishmanial agents, a series of N-acyl and homodimeric aryl piperazines were synthesized with high purity, predicted druggable properties by in silico methods and investigated their antileishmanial activity. The in vitro biological activity of synthesized compounds against clinically validated intracellular amastigote and extracellular promastigote form of Leishmania donovani parasite showed eight compounds inhibited 50% amastigotes growth below 25 µM. The half maximal inhibitory concentration (IC50) and cytotoxicity assessment of eight active compounds, 4a, 4d and 4e demonstrated activity with an IC50 2.0 - 9.1 µM and selectivity index 10 - 42. Compound 4d (IC50 2.0 µM, SI = 42) found to be the best among them with four-folds more potent and eight-folds less toxic than the control drug miltefosine. Overall, results demonstrated that compound 4d is a promising lead candidate for further development as antileishmanial drug.

Design and synthesis of novel halogen rich salicylanilides as potential antileishmanial agents.

The available therapeutic treatment for leishmaniasis is inadequate and toxic due to side effects, expensive and emergence of drug resistance. Affordable and safe antileishmanial agents are urgently needed and toward this objective, we synthesized a series of 32 novel halogen rich salicylanilides including niclosamide and oxyclozanide and investigated their antileishmanial activity against amastigotes of Leishmania donovani. In vitro data showed fifteen compounds inhibited intracellular amastigotes with an IC50 of below 5 µM and selectivity index above 10. Among 15 active compounds, 14 and 24 demonstrated better activity with an IC50 of 2.89 µM and 2.09 µM respectively and selectivity index is 18. Compound 24 exhibited significant in vivo antileishmanial efficacy and reduced 65% of the splenic parasite load on day 28th post-treatment in the experimental visceral leishmaniasis golden hamster model. The data suggest that 24 can be a promising lead candidate possessing potential to be developed into a leishmanial drug candidate.

Leishmania donovani Dipeptidylcarboxypeptidase Inhibitor as a Potential Oral Treatment for Visceral Leishmaniasis.

Chemotherapy is the key intervention to control visceral leishmaniasis (VL), a neglected tropical disease. Current regimens include not only a few drugs but also present several drawbacks, including moderate to severe toxicity, cost, long-term administration, patient compliance, and growing drug resistance. Thus, the need for better treatment options against VL is a priority. In an endeavor to find an orally active and affordable antileishmanial agent, we evaluated the therapeutic potential of compounds belonging to the (2Z,2'Z)-3,3'-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-ones) series, identified as inhibitor(s) of Leishmania donovani dipeptidylcarboxypeptidase, a novel drug target. Among them, compound 3c exhibited best in vivo antileishmanial efficacy via both intraperitoneal and oral routes. Therefore, the present study led to the identification of compound 3c as the lead candidate for treating VL.

Downregulation of gamma subunit of TCP1 chaperonin of Leishmania donovani modulates extracellular vesicles-mediated macrophage microbicidal function.

T-complex protein-1 (TCP1) is a group II chaperonin, known to fold various proteins like actin and tubulin. In Leishmania donovani only one subunit that is gamma subunit (LdTCP1γ) has been functionally characterized as a homo-oligomeric complex that exhibits ATP-dependent protein folding. The gene is essential for the survival and infectivity of the parasite. Leishmania parasite releases extracellular vesicles (EVs) containing numerous virulence factors, which play an essential role in parasite pathogenesis and modulate host immune cell signaling. The present study demonstrates that LdTCP1γ is secreted in the EVs and modulates host macrophage functions. EVs isolated from LdTCP1γ single-allele-replacement mutants significantly upregulate the microbicidal function of LPS-induced macrophage as evident by increased levels of proinflammatory cytokines (TNF-α, IL-6), iNOS and NO production. Further, the comparative proteomics of wild-type and single-allele-replacement mutant EVs showed that out of 876 identified proteins, 207 were significantly modulated. Among them, the top 50 modulated and abundantly secreted proteins constitute ∼40% of the total identified protein intensity and include virulence factors such as GP63, peroxiredoxin, enolase, HSP70, elongation factor 2, amastin, eukaryotic translation initiation factor and α-tubulin. The comparative proteomic analysis revealed that the proteome enrichment of the EVs from LdTCP1γ single-allele replacement mutants significantly differs from wild-type EVs, which may be responsible for the altered host microbicidal responses. Thus, our data provide new insight into the role of LdTCP1γ in EVs-mediated host-parasite interactions.

Dipeptidylcarboxypeptidase of Leishmania donovani: A potential vaccine molecule against experimental visceral leishmaniasis.

Visceral leishmaniasis is one of the deadliest parasitic diseases in the world. In the absence of an efficient and cost-effective drugs, development of an effective vaccine is the need of the day. In spite of several efforts, a successful vaccine against the disease has been elusive. We have evaluated immunoprophylactic efficacy of recombinant dipeptidycarboxypeptidase (rLdDCP), predominantly expressed in amastigotes, in chronic hamster model. rLdDCP induced in vitro lymphoproliferation and NO production in cured hamsters. Immunization with rLdDCP alone, or with BCG, caused significant reduction in parasite load suggesting strong protective response. The molecule also augmented the CMI response as depicted by an increased lymphocyte proliferation, NO production, DTH responses and increased levels of IgG2 in immunized hamsters. The vaccinated hamsters exhibited a surge in IFN-γ, TNF-α, IL-12 and iNOS levels but down-regulation of IL-10 and IL-4. Thus, the results suggest the potentiality of the rLdDCP as a strong candidate vaccine.

Unraveling of interacting protein network of chaperonin TCP1 gamma subunit of Leishmania donovani.

T-complex polypeptide-1 (TCP1) is a group II chaperonin that folds various cellular proteins. About 10% of cytosolic proteins in yeast have been shown to flux through the TCP1 protein complex indicating that it interacts and folds a plethora of substrate proteins that perform essential functions. In Leishmania donovani, the gamma subunit of TCP1 (LdTCP1γ) has been shown to form a homo-oligomeric complex and exhibited ATP-dependent protein folding activity. LdTCP1γ is essential for the growth and infectivity of the parasite. The interacting partners of L. donovani TCP1γ, involved in many cellular processes, are far from being understood. In this study, we utilized co-immunoprecipitation assay coupled with liquid chromatography-mass spectrometry (LC-MS) to unravel protein-protein interaction (PPI) networks of LdTCP1γ in the L. donovani parasite. Label-free quantification (LFQ) proteomic analysis revealed 719 interacting partners of LdTCP1γ. String analysis showed that LdTCP1γ interacts with all subunits of TCP1 complex as well as other proteins belonging to pathways like metabolic process, ribosome, protein folding, sorting, and degradation. Trypanothione reductase, identified as one of the interacting partners, is refolded by LdTCP1γ. In addition, the differential expression of LdTCP1γ modulates the trypanothione reductase activity in L. donovani parasite. The study provides novel insight into the role of LdTCP1γ that will pave the way to better understand parasite biology by identifying the interacting partners of this chaperonin.

Immunological characterization of rLdTCP1γ for its prophylactic potential against visceral leishmaniasis in hamster model.

Visceral leishmaniasis (VL) is a chronic tropical disease responsible for devastating epidemics worldwide. Though current treatment relies on drugs, the emergence of resistance, toxic side-effects, and strenuous administration has led to an ineffective remedy. Hence, vaccination remains an alternative and desirable approach for VL control. Though extensive research on anti-leishmanial vaccine candidates has been carried out in past decades, presence of an effective molecule is still missing. In the present study, we have evaluated the immunogenicity and prophylactic potential of a recombinant T-complex protein-1 gamma subunit of L. donovani (rLdTCP1γ), against VL in hamster model. The antigen exhibited in vitro stimulation of lymphoproliferative and NO response in miltefosine and amphotericin B treated hamsters depicting its immunotherapeutic/immunogenic nature. Immunization with rLdTCP1γ revealed a strong protective response against experimental VL as indicated by reduced parasite load in the spleen of immunized group compared to infected control. The immunized animals gained body weight and exhibited significant reduction in the spleen and liver weight as compared to infected controls on days 60, 90, 120 post-challenge. A substantial augmentation of cell-mediated immune response as depicted by an increased lymphocyte proliferation, nitric oxide production, DTH responses and increased levels of IgG2 was observed in rLdTCP1γ immunized hamsters. The Th1 stimulatory potential, imparted by the antigen, was found to be intensified in the presence of adjuvant Bacillus Calmette-Guérin (BCG). The efficacy was further assisted by an upregulated mRNA transcript of Th1 induced cytokines (IL-12, IFN-γ and TNFα) and downregulation of IL-4 and IL-10. The results are thus suggestive of rLdTCP1γ having the potential of a strong vaccine candidate against VL.

Pathogenic role of mitogen activated protein kinases in protozoan parasites.

Protozoan parasites with complex life cycles have high mortality rates affecting billions of human lives. Available anti-parasitic drugs are inadequate due to variable efficacy, toxicity, poor patient compliance and drug-resistance. Hence, there is an urgent need for the development of safer and better chemotherapeutics. Mitogen Activated Protein Kinases (MAPKs) have drawn much attention as potential drug targets. This review summarizes unique structural and functional features of MAP kinases and their possible role in pathogenesis of obligate intracellular protozoan parasites namely, Leishmania, Trypanosoma, Plasmodium and Toxoplasma. It also provides an overview of available knowledge concerning the target proteins of parasite MAPKs and the need to understand and unravel unknown interaction network(s) of MAPK(s).

Comparative phosphoproteomic analysis unravels MAPK1 regulated phosphoproteins in Leishmania donovani.

Mitogen Activated Protein Kinase1 (MAPK1) of Leishmania donovani functions as key regulators of various cellular activities, which seem to be imperative for parasite survival, infectivity, drug resistance and post-translational modification of chaperones/co-chaperones. However, very less is known about LdMAPK1 target proteins. With recent advancements in proteomics, we aimed to identify phosphoproteins which were differentially expressed in LdMAPK1 overexpressing (Dd8++/++) and single replacement mutants (Dd8+/) as compared to wild type (Dd8+/+) parasites, utilizing LC-MS/MS approach. An in-depth label-free phospoproteomic analysis revealed that modulation of LdMAPK1 expression significantly modulates expression levels of miscellaneous phosphoproteins which may act as its targets/substrates. Out of 1974 quantified phosphoproteins in parasite, 140 were significantly differentially expressed in MAPK1 overexpressing and single replacement mutants. These differentially expressed phosphoproteins are majorly associated with metabolism, signal transduction, replication, transcription, translation, transporters and cytoskeleton/motor proteins, hence suggested that MAPK1 may act in concert to modulate global biological processes. The study further implicated possible role of LdMAPK1 in regulation and management of stress machinery in parasite through post translational modifications. Precisely, comparative phosphoproteomics study has elucidated significant role of LdMAPK1 in regulating various pathways contributing in parasite biology with relevance to future drug development. SIGNIFICANCE: MAPKinase1, the downstream kinase of MAPK signal transduction pathway, has drawn much attention as potential therapeutic drug target due to their indispensable role in survival and infectivity of Leishmania donovani. However, limited information is available about its downstream effector proteins/signaling networks. Utilizing label free LC-MS/MS analysis, phosphoproteome of LdMAPK1 over-expressing (Dd8++/++) and LdMAPK1 single replacement mutants (Dd8+/-) with wild type (Dd8+/+) parasites was compared and identified 140 LdMAPK1 modulated phosphoproteins, mainly involved in pathways like signal transduction, metabolism, transcriptional, translational, post-translational modification and regulation of heat shock proteins. Interestingly, LdMAPK1 interacts directly with only six phosphoproteins i.e. casein kinase, casein kinase II, HSP83/HSP90, LACK, protein kinase and serine/threonine protein kinase. Thus, the study elucidates significant role of LdMAPK1 in Leishmania biology which may drive drug-discovery efforts against visceral leishmaniasis.

Estrogens as regulator of hematopoietic stem cell, immune cells and bone biology.

Hematopoietic stem cells provide continuous supply of all the immune cells, through proliferation and differentiation decisions. These decisions are controlled by local bone marrow environment as well as by long-range signals for example endocrine system. Sex dependent differential immunological responses have been described under homeostasis and disease conditions. Females show higher longevity than male counterpart that seems to depend on major female sex hormone, estrogen. There are four estrogens - Estrone (E1), estradiol (E2), Estriol (E3) and Estetrol (E4) that spatially and temporarily present during different female reproductive phases. In this review, we discussed recent updates describing the effects of estrogen on HSC, immune cells and in bone biology. Estradiol (E2) being a major/abundant estrogen is extensively investigated, while effects of other estrogens E1, E3 and E4 are started to unravel recently. Furthermore, clinical effect of estrogen as hormone therapy is discussed in HSC and immune cells perspectives. The data presented in this review is compiled by searches of PubMed, database of American Cancer Society (ACS). We have included article from September 1994 to March 2020 as covering all article in chronological order is not fissile so we included relevant article with substantial information in this specific area of research by using the search term (alone or in combination) estrogen, hematopoietic stem cell, immune cells, gender difference, estrone, estriol, estetrol, therapeutic application, pregnancy, effect on bone.

Identification of potential anti-leishmanial agents using computational investigation and biological evaluation against trypanothione reductase.

Trypanothione reductase of Leishmania donovani is a flavin adenine dinucleotide containing homodimeric protein essential for parasite survival. The flavoenzyme utilizes nicotinamide adenine dinucleotide phosphate in the reaction to convert oxidized trypanothione to reduced trypanothione which is further used up by tryparedoxin/tryparedoxin peroxidase system to neutralize the reactive oxygen species generated by the macrophages. Some of the drugs previously reported against the disease include sodium stibogluconate, miltefosine and amphotericin B. However, due to the resistance and toxicity problem associated with these molecules, there is an urgent need to develop new drugs against L. donovani. Trypanothione reductase of L. donovani is one such essential target whose inhibition could lead to a decline in parasite growth. In this work, we have performed a computational studies using Maybridge library of chemical compounds to identify potential inhibitors of Trypanothione reductase of L. donovani. Structure-based virtual screening method in combination with molecular docking was employed to identify and prioritize 30 compounds which were further subjected to molecular dynamics simulation. Ten compounds which showed stable ligand root-mean-square deviation plot, c-alpha backbone and root-mean-square fluctuation were considered for trypanothione reductase inhibition assay and subsequent inhibition studies of parasite growth. Enzyme inhibition assay resulted in shortlisting of four compounds that were found to inhibit Trypanothione reductase of L. donovani. Subsequently, the anti-leishmanial screening highlighted one compound as the potential anti-leishmanial agent, with IC50 value of 15.2 µM, that can be further optimised with medicinal chemistry efforts to improve its activity. Communicated by Ramaswamy H. Sarma.

Efficient antileishmanial activity of amphotericin B and piperine entrapped in enteric coated guar gum nanoparticles.

Amphotericin B (AmB) exhibits potential antileishmanial activity, with only a little rate of recurrence. However, low bioavailability and severe nephrotoxicity are among the major shortcomings of AmB-based therapy. Various AmB nanoformulations have been developed, which to an extent, have reduced its toxicity and increased the drug efficacy. To further reduce the nonspecific tissue distribution and the cost of the treatment, the current AmB-based formulations require additional improvements. Combination of natural bioenhancers with AmB is expected to further increase its bioavailability. Therefore, we developed a nanoformulation of AmB and piperine (Pip), a plant alkaloid, known to enhance the bioavailability of various drugs, by entrapping them in guar gum, a macrophage targeting polymer. Owing to the ease of oral delivery, these nanoparticles (NPs) were coated with eudragit to make them suitable for oral administration. The formulated eudragit-coated AmB and Pip-loaded NPs (Eu-HDGG-AmB-Pip-NPs) exhibited controlled release of the loaded therapeutic agents and protected the drug from acidic pH. These NPs exhibited effective suppression of growth of both promastigotes and amastigotes of Leishmania donovani parasite under in vitro. In vivo evaluation of these NPs for therapeutic efficacy in golden hamster-L. donovani model demonstrated enhanced drug bioavailability, non-nephrotoxic nature, and potential antileishmanial activity with up to 96% inhibition of the parasite. Graphical abstract.

Guanylate Binding Proteins Restrict Leishmania donovani Growth in Nonphagocytic Cells Independent of Parasitophorous Vacuolar Targeting.

Interferon (IFN)-inducible guanylate binding proteins (GBPs) play important roles in host defense against many intracellular pathogens that reside within pathogen-containing vacuoles (PVs). For instance, members of the GBP family translocate to PVs occupied by the protozoan pathogen Toxoplasma and facilitate PV disruption and lytic parasite killing. While the GBP defense program targeting Toxoplasma has been studied in some detail, the role of GBPs in host defense to other protozoan pathogens is poorly characterized. Here, we report a critical role for both mouse and human GBPs in the cell-autonomous immune response against the vector-borne parasite Leishmania donovani Although L. donovani can infect both phagocytic and nonphagocytic cells, it predominantly replicates inside professional phagocytes. The underlying basis for this cell type tropism is unclear. Here, we demonstrate that GBPs restrict growth of L. donovani in both mouse and human nonphagocytic cells. GBP-mediated restriction of L. donovani replication occurs via a noncanonical pathway that operates independent of detectable translocation of GBPs to L. donovan-containing vacuoles (LCVs). Instead of promoting the lytic destruction of PVs, as reported for GBP-mediated killing of Toxoplasma in phagocytic cells, GBPs facilitate the delivery of L. donovani into autolysosomal-marker-positive compartments in mouse embryonic fibroblasts as well as the human epithelial cell line A549. Together our results show that GBPs control a novel cell-autonomous host defense program, which renders nonphagocytic cells nonpermissible for efficient Leishmania replication.IMPORTANCE The obligate intracellular parasite Leishmania causes the disease leishmaniasis, which is transmitted to mammalian hosts, including humans, via the sandfly vector. Following the bite-induced breach of the skin barrier, Leishmania is known to live and replicate predominantly inside professional phagocytes. Although Leishmania is also able to infect nonphagocytic cells, nonphagocytic cells support limited parasitic replication for unknown reasons. In this study, we show that nonphagocytic cells possess an intrinsic property to restrict Leishmania growth. Our study defines a novel role for a family of host defense proteins, the guanylate binding proteins (GBPs), in antileishmanial immunity. Mechanistically, our data indicate that GBPs facilitate the delivery of Leishmania into antimicrobial autolysosomes, thereby enhancing parasite clearance in nonphagocytic cells. We propose that this GBP-dependent host defense program makes nonphagocytic cells an inhospitable host cell type for Leishmania growth.

TCP1γ Subunit Is Indispensable for Growth and Infectivity of Leishmania donovani.

T-complex protein-1 (TCP1) is a ubiquitous group II chaperonin and is known to fold various proteins, such as actin and tubulin. In Leishmania donovani, the γ subunit of TCP1 (LdTCP1γ) has been cloned and characterized. It forms a high-molecular-weight homo-oligomeric complex that performs ATP-dependent protein folding. In the present study, we evaluated the essentiality of the LdTCP1γ gene. Gene replacement studies indicated that LdTCP1γ is essential for parasite survival. The LdTCP1γ single-allele-replacement mutants exhibited slowed growth and decreased infectivity in mouse macrophages compared to the growth and infectivity of the wild-type parasites. Modulation of LdTCP1γ expression in promastigotes also modulated cell cycle progression. Suramin, an antitrypanosomal drug, not only inhibited the luciferase refolding activity of the recombinant LdTCP1γ (rLdTCP1γ) homo-oligomeric complex but also exhibited potential antileishmanial efficacy both in vitro and in vivo The interaction of suramin and LdTCP1γ was further validated by isothermal titration calorimetry. The study suggests LdTCP1γ as a potential drug target and also provides a framework for the development of a new class of drugs.

Leishmania donovani chaperonin TCP1γ subunit protects miltefosine induced oxidative damage.

T-complex protein-1 (TCP1) is a chaperonin protein known to fold various proteins like actin and tubulin. In Leishmania donovani only one subunit of TCP1 that is gamma subunit (LdTCP1γ) has been functionally characterized. It not only performs ATP dependent protein folding but is also essential for survival and virulence. The present work demonstrates that LdTCP1γ also has a role in miltefosine resistance. Overexpression of LdTCP1γ in L. donovani promastigotes results in decreased sensitivity of parasites towards miltefosine, while single-allele replacement mutants exhibited increased sensitivity as compared to wild-type promastigotes. This response was specific to miltefosine with no cross-resistance to other drugs. The LdTCP1γ-mediated drug resistance was directly related to miltefosine-induced apoptotic death of the parasite, as was evidenced by 2 to 3-fold decrease in cell death parameters in overexpressing cells and >2-fold increase in single-allele replacement mutants. Further, deciphering the mechanism revealed that resistance of overexpressing cells was associated with efficient ROS neutralization due to increased levels of thiols and upregulation of cytosolic tryparedoxin peroxidase (cTxnPx). Further, modulation of LdTCP1γ expression in parasite also modulates the levels of proinflammatory cytokine (TNF-α) and anti-inflammatory cytokine (IL-10) of the host macrophages. The study provides evidence for the involvement of a chaperonin protein LdTCP1γ in the protection against miltefosine induced oxidative damage and reveals the fundamental role of LdTCP1γ in parasite biology.

Antileishmanial assessment of isoxazole derivatives against L. donovani.

A chemical library comprising substituted 3-nitroisoxazoles and 3-aminoisoxazoles was prepared and screened for their antileishmanial activity against L. donovani. As compared to Miltefosine, the standard drug used in bioassays, several compounds displayed remarkably better inhibition of the promastigote and amastigote stages of parasites. The in vivo evaluation of a few compounds in a golden hamster model showed significant reduction of the parasite load post treatment via the intraperitoneal route by several compounds. The preliminary pharmacokinetic evaluation of a representative compound 4mf via the oral route, however, indicated high systemic clearance from the body.