Leishmania donovani mitogen-activated protein kinases as a host-parasite interaction interface.

Leishmaniasis, a major globally re-emerging neglected tropical disease, has a restricted repertoire of chemotherapeutic options due to a narrow therapeutic index, drug resistance, or patient non-compliance due to toxicity. The disease is caused by the parasite Leishmania that resides in two different forms in two different environments: as sessile intracellular amastigotes within mammalian macrophages and as motile promastigotes in sandfly gut. As mitogen-activated protein kinases (MAPKs) play important roles in cellular differentiation and survival, we studied the expression of Leishmania donovani MAPKs (LdMAPKs). The homology studies by multiple sequence alignment show that excepting LdMAPK1 and LdMAPK2, all thirteen other LdMAPKs share homology with human ERK and p38 isoforms. Expression of LdMAPK4 and LdMAPK5 is less in avirulent promastigotes and amastigotes. Compared to miltefosine-sensitive L. donovani parasites, miltefosine-resistant parasites have higher LdMAPK1, LdMAPK3-5, LdMAPK7-11, LdMAPK13, and LdMAPK14 expression. IL-4-treatment of macrophages down-regulated LdMAPK11, in virulent amastigotes whereas up-regulated LdMAPK5, but down-regulated LdMAPK6, LdMAPK12-15, expression in avirulent amastigotes. IL-4 up-regulated LdMAPK1 expression in both virulent and avirulent amastigotes. IFN-γ-treatment down-regulated LdMAPK6, LdMAPK13, and LdMAPK15 in avirulent amastigotes but up-regulated in virulent amastigotes. This complex profile of LdMAPKs expression among virulent and avirulent parasites, drug-resistant parasites, and in amastigotes within IL-4 or IFN-γ-treated macrophages suggests that LdMAPKs are differentially controlled at the host-parasite interface regulating parasite survival and differentiation, and in the course of IL-4 or IFN-γ dominated immune response.

Insights into the ATP / GTP selectivity of a GTPase, adenylosuccinate synthetase from Leishmania donovani.

Many GTPases have been shown to utilize ATP too as the phosphoryl donor. Both GTP and ATP are important molecules in the cellular environments and play multiple and discrete functional role within the cells. In our present study, we showed that one of the purine metabolic enzymes Adenylosuccinate synthetase from Leishmania donovani (LdAdSS) which belongs to the BioD-superfamily of GTPases can also carry out the catalysis by hydrolysing ATP instead of its cognate substrate GTP albeit with less efficiency. Biochemical and biophysical studies indicated its ability to bind to ATP too but at a higher concentration of ATP compared to that of GTP. Sequence analysis and molecular dynamic simulations suggested that residues of the switch loop and the G4-G5 (593SAXD596) connected motif of LdAdSS plays a role in determining the nucleotide specificity. Though the crucial interaction between Asp596 and the nucleotide is broken when ATP is bound, interactions between the Ala594 and the adenine ring of ATP could still hold ATP in the GTP binding site. The results of the present study suggested that though LdAdSS is GTP specific, it still shows ATP hydrolysing activity.

Molecular explorations of the Leishmania donovani 6-phosphogluconolactonase enzyme, a key player in the pentose phosphate pathway.

The enzymes of the pentose phosphate pathway are vital to survival in kinetoplastids. The second step of the pentose phosphate pathway involves hydrolytic cleavage of 6-phosphogluconolactone to 6-phosphogluconic acid by 6- phosphogluconolactonase (6PGL). In the present study, Leishmania donovani 6PGL (Ld6PGL) was cloned and overexpressed in bacterial expression system. Comparative sequence analysis revealed the conserved sequence motifs, functionally and structurally important residues in 6PGL family. In silico amino acid substitution study and interacting partners of 6PGL were predicted. The Ld6PGL enzyme was found to be active in the assay and in the parasites. Specificity was confirmed by Western blot analysis. The ∼30 kDa protein was found to be a dimer in MALDI, glutaraldehyde crosslinking and size exclusion chromatography studies. Kinetic analysis and structural stability studies of Ld6PGL were performed with denaturants and at varied temperature. Computational 3D Structural modelling of Ld6PGL elucidates that it has a similar α/ß hydrolase fold structural topology as in other members of 6PGL family. The three loops are found in extended form when the structure is compared with the human 6PGL (Hs6PGL). Further, enzyme substrate binding mode and its mechanism were investigated using the molecular docking and molecular simulation studies. Interesting dynamics action of substrate 6-phosphogluconolactone was observed into active site during MD simulation. Interesting differences were observed between host and parasite enzyme which pointed towards its potential to be explored as an antileishmanial drug target. This study forms the basis for further analysis of the role of Ld6PGL in combating oxidative stress in Leishmania.

TDP1 knockout Leishmania donovani accumulate topoisomerase 1-linked DNA damage and are hypersensitive to clinically used antileishmanial drugs.

Leishmania donovani, a unicellular protozoan parasite, causes a wide range of human diseases including fatal visceral leishmaniasis. Tyrosyl DNA-phosphodiesterase 1 (TDP1) hydrolyzes the phosphodiester bond between DNA 3'-end and a tyrosyl moiety of trapped topoisomerase I-DNA covalent complexes (Top1cc). We have previously shown Leishmania harbors a TDP1 gene (LdTDP1), however, the biological role of TDP1 remains largely unknown. In the present study, we have generated TDP1 knockout L. donovani (LdTDP1-/- ) promastigotes and have shown that LdTDP1-/- parasites are deficient in 3'-phosphodiesterase activities and were hypersensitive to Top1-poison like camptothecin (CPT), DNA alkylation agent like methyl methanesulfonate, and oxidative DNA lesions generated by hydrogen peroxide but were not sensitive to etoposide. We also detected elevated levels of CPT-induced reactive oxygen species triggering cell cycle arrest and cell death in LdTDP1-/- promastigotes. LdTDP1-/- promastigotes accumulate a significant change in the membrane morphology with the accumulation of membrane pores, which is associated with oxidative stress and lipid peroxidation. To our surprise, we detected that LdTDP1-/- parasites were hypersensitive to antileishmanial drugs like amphotericin B and miltefosine, which could be rescued by complementation of wild-type TDP1 gene in the LdTDP1-/- parasites. Notably, multidrug-resistant L. donovani clinical isolates showed a marked reduction in TDP1 expression and were sensitive to Top1 poisons. Taken together, our study provides a new role of LdTDP1 in protecting L. donovani parasites from oxidative stress-induced DNA damage and resistance to amphotericin B and miltefosine.

An immunoprophylactic evaluation of Ld-ODC derived HLA-A0201 restricted peptides against visceral leishmaniasis.

Five (5) HLA-A 0201 restricted epitopes of ornithine decarboxylase derived from Leishmania donovani (Ld-ODC) were examined by reverse vaccinology to develop prophylactics against visceral leishmaniasis (VL). These consensus epitopes comprising (P1: RLMPSAHAI, P2: LLDQYQIHL, P3: GLYHSFNCI, P4: AVLEVLSAL and P5: RLPASPAAL) were observed and presented by diverse HLA alleles screened by immune-informatics tools. These epitopes were also observed for strong stability for appropriate immune response in in silico screening and molecular dynamics. Top five selected epitopes filtered from population coverage analysis and TAP binding affinity were identified and evaluated against treated cases of VL subjects. Experiments were run individually with synthetic peptides or as the cocktail of peptides. A major population of CD8+ T cells were predominantly IFN-γ producers but not the IL-10 cytokines and shown with granzyme-B activity. Therefore, it can be concluded that the screened HLA-A0201 restricted epitope hotspots derived from Leishmania ODC can trigger CD8+ T cells, which can skew other immune cells functions toward protection. However, a detailed analysis can explore its potentiality as a vaccine candidate.Communicated by Ramaswamy H. Sarma.

Identification of potential antileishmanial 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-ones, in vitro metabolic stability, cytotoxicity and molecular modeling studies.

We report the synthesis and in vitro evaluation of 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-one derivatives against Leishmania donovani. Amongst the compound library synthesized, molecules 3d, 3f, 3h, 3i, 3l, and 3m demonstrated substantial dose-dependent killing of the promastigotes. Their IC50 values range from 55.0 to 77.0 µg/ml, with 3m (IC50 55.75 µg/ml) being equipotent with amphotericin B (IC50 50.0 µg/ml, used as standard). The most active compound 3m, is metabolically stable in rat liver microsomes. Furthermore, the molecules are highly specific against leishmania as shown by their weak antibacterial and antifungal activity. In vitro cytotoxicity studies show the compounds lack any cytotoxicity. Furthermore, molecular modeling studies show plausibility of binding to Leishmania donovani topoisomerase 1 (LdTop1). Structure activity relationships reveal bulky substitutions on the pyridone nitrogen are well-tolerated, and such compounds have better binding affinity. Intramolecular hydrogen bonds confer some rigidity to the molecules, rendering a degree of planarity akin to topotecan. Taken together, we emphasis the merits of molecules possessing the 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-one skeleton as potential antileishmanial agents warranting further investigation.

Mevalonate kinase of Leishmania donovani protects parasite against oxidative stress by modulating ergosterol biosynthesis.

Leishmaniasis comprises of a wide variety of diseases, caused by protozoan parasite belonging to the genus Leishmania. Leishmania parasites undergo different types of stress during their lifetime and have developed strategies to overcome this damage. Identifying the mechanistic approach used by the parasite in dealing with the stress is of immense importance for unfolding the survival strategy adopted by the parasite. Mevalonate kinase (MVK) is an important regulatory factor in the mevalonate pathway in both bacteria and eukaryotes. In this study, we explored the role of Leishmania donovani mevalonate kinase (LdMVK) in parasite survival under stress condition. Hydrogen peroxide (H2O2) and menadione, the two known oxidants were used to carry out the experiments. The MVK expression was found to be up regulated ∼2.1 fold and ∼2.3 fold under oxidative stress condition and under the effect of anti-Leishmania drug, AmBisome respectively. The cell viability declined under the effect of MVK inhibitor viz: vanadyl sulfate (VS). The level of intracellular ROS was also found to be increased under the effect of MVK inhibitor. To confirm the findings, LdMVK over expression (LdMVK OE) and LdMVK knockdown (LdMVK KD) parasites were generated. The level of ergosterol, an important component of plasma membrane in L. donovani, was observed and found to be reduced by nearly 60 % in LdMVK KD parasite and increased by nearly 30 % in LdMVK OE parasites as compared to wild type. However, the ergosterol content was found to be elevated under oxidative stress. Furthermore, LdMVK was also found to be associated with maintaining the plasma membrane integrity and also in preventing the peroxidation of cellular lipids when exposed to oxidative stress. The above data clearly suggests that MVK has a vital role in protecting the parasite from oxidative stress. These findings may also explore the contribution of LdMVK in drug unresponsiveness which may help in future rational drug designing for leishmaniasis.

Functional characterization of the LdNAGD gene in Leishmania donovani.

The N-acetyl glucosamine catabolic pathway has been well established as a critically essential pathway for the survival and pathogenesis of several intracellular pathogens. The intracellular form of Leishmania donovani resides inside the parasitophorous vacuole of macrophages. Recent studies have shown that amino sugars, such as N-acetyl glucosamine, are released from the turnover of host macromolecules, such as glycosaminoglycans, glycoproteins, and proteoglycans, inside the parasitophorous vacuole. Three enzymes, hexokinase (Hxk), N-acetyl glucosamine-6-phosphate deacetylase (NAGD) and glucosamine-6-phosphate deaminase (GND), are sequentially involved in the catabolism of GlcNAc. The Leishmania donovani genome encodes all enzymes of the GlcNAc catabolic pathway. Here, we investigated the role of the GlcNAc catabolic pathway in the proliferation and survival of L. donovani by characterizing the NAGD gene of this pathway. Recombinant LdNAGD displayed deacetylation activity and was localized inside the glycosomes. LdNAGD gene deletion impaired GlcNAc catabolism and was indispensable for the viability of L. donovani in media containing GlcNAc as the sole carbon source. Furthermore, these Δnagd cells showed attenuated virulence in THP-1 cells and a significantly reduced proliferation rate compared to wild type (WT) cells inside THP-1 cells. Our data suggested that LdNAGD is important for the intracellular proliferation of L. donovani and may represent a potential drug target.

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits.

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 µM; LRL-TP-94: 2.9 µM; and LRL-TP-101: 35.3 µM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 µM in extracellular promastigote assay and 34.0, 53.7, and 11.4 µM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

Ile209 of Leishmania donovani xanthine phosphoribosyltransferase plays a key role in determining its purine base specificity.

Xanthine phosphoribosyltransferase (XPRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are purine salvaging enzymes of Leishmania donovani with distinct 6-oxopurine specificities. LdXPRT phosphoribosylates xanthine, hypoxanthine, and guanine, with preference toward xanthine, whereas LdHGPRT phosphoribosylates only hypoxanthine and guanine. In our study, LdXPRT was used as a model to understand these purine base specificities. Mutating I209 to V, the conserved residue found in HGPRTs, reduced the affinity of LdXPRT for xanthine, converting it to an HGXPRT-like enzyme. The Y208F mutation in the active site indicated that aromatic residue interactions with the purine ring are limited to pi-pi binding forces and do not impart purine base specificity. Deleting the unique motif (L55-Y82) of LdXPRT affected enzyme activity. Our studies established I209 as a key residue determining the 6-oxopurine specificity of LdXPRT.

Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP-PKA signaling axis for its intra-macrophage survival during infection.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

Repurposing of FDA-approved drugs as inhibitors of sterol C-24 methyltransferase of Leishmania donovani to fight against leishmaniasis.

Leishmaniasis is a vector-borne disease caused by around 20 species of Leishmania. The main clinical forms of leishmaniasis are cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). VL is caused by Leishmania infantum in Central and South America, Mediterranean Basin, Middle East, and by L. donovani in Asia and Africa. Sterol C-24 methyltransferase (LdSMT) of L. donovani is a transferase enzyme of the sterol biosynthesis pathway. This pathway is one of the major targets for drug developments in Leishmania. Due to insufficient evidence about the exact function of SMT inside the cell and the uniqueness of the SMT enzyme in the Leishmania parasites made it a significant target for an effective drug development approach. We performed virtual screening of the Food and Drug Administration (FDA)-approved drug library against LdSMT and found simeprevir, an antiviral drug on top in the binding score. It showed a significant binding affinity with LdSMT. The binding was supported by hydrogen bonds and several other interactions. Simeprevir inhibited L. donovani growth of promastigotes with 50% inhibitory concentration (IC50 ) of 51.49 ± 5.87 µM. Further studies showed that simeprevir induced ROS generation in 44.7% of parasites at 125-µM concentration. Here, we for the first time reported simeprevir as an antileishmanial lead molecule using a drug repurposing approach.

Multiple unbiased approaches identify oxidosqualene cyclase as the molecular target of a promising anti-leishmanial.

Phenotypic screening identified a benzothiophene compound with activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Using multiple orthogonal approaches, oxidosqualene cyclase (OSC), a key enzyme of sterol biosynthesis, was identified as the target of this racemic compound and its enantiomers. Whole genome sequencing and screening of a genome-wide overexpression library confirmed that OSC gene amplification is associated with resistance to compound 1. Introduction of an ectopic copy of the OSC gene into wild-type cells reduced susceptibility to these compounds confirming the role of this enzyme in resistance. Biochemical analyses demonstrated the accumulation of the substrate of OSC and depletion of its product in compound (S)-1-treated-promastigotes and cell-free membrane preparations, respectively. Thermal proteome profiling confirmed that compound (S)-1 binds directly to OSC. Finally, modeling and docking studies identified key interactions between compound (S)-1 and the LdOSC active site. Strategies to improve the potency for this promising anti-leishmanial are proposed.

The HIV - 1 protease inhibitor Amprenavir targets Leishmania donovani topoisomerase I and induces oxidative stress-mediated programmed cell death.

The global prevalence of HIV is a major challenge for the control of visceral leishmaniasis. Although the effectiveness and usefulness of amprenavir (APV) are well studied in anti-retroviral regimens, very little is known on HIV/VL-co-infected patients. In the present study, we report for the first time the protective efficacy of APV against visceral leishmaniasis by inhibition of DNA Topoisomerase I (LdTOP1LS) and APV-induced downstream pathway in programmed cell death (PCD). During the early phase of activation, reactive oxygen species (ROS) is increased inside the cells, which causes subsequent elevation of lipid peroxidation. Endogenous ROS formation and lipid peroxidation cause eventual depolarization of mitochondrial membrane potential (ΔΨm). Furthermore, the release of cytochrome c and activation of CED3/CPP32 group of proteases lead to the formation of oxidative DNA lesions followed by DNA fragmentation. The promising in vitro and ex vivo results promoted to substantiate further by in vivo animal experiment, which showed a significant reduction of splenic and hepatic parasites burden compared to infected controls. Interestingly, APV selectively targets LdTOPILS and does not inhibit the catalytic activity of human topoisomerase I (hTopI). Moreover, based on the cytotoxicity test APV is not toxic for host macrophage cells, which is correlated with non-responsiveness of inhibition of catalytic activity of hTopI. Taken together, this study provides the opportunity for discovering and evaluating newer potential molecular therapeutic targets for drug designing. The present study might be exploited in future as important therapeutics, which will be useful for treatment of VL as well as HIV-VL co-infection.

Cloning, purification, and homology modeling of Histone deacetylase in Leishmania donovani.

Neglected diseases, such as leishmaniasis, are still a major health problem in poor countries. To date, there is a severe lack of effective, safe, and affordable treatment for leishmaniasis. Currently, there are very limited chemotherapeutic options, and the development of vaccines is still underway. Hence, novel therapeutic strategies need to be developed against leishmanial parasites. Histone deacetylases (HDACs), silent regulators of many critical pathways, have been validated as potential therapeutic targets in cancer and several parasitic diseases. In the present work, we have isolated and characterized biologically active Zn2+-dependent HDAC protein from leishmania that can be studied further as a potential anti-leishmanial drug target to develop new therapies against neglected diseases. The nucleotide sequence of the HDAC gene with no intervening sequence was amplified, cloned in a pET-28a vector, and later transformed into the BL21(DE3) competent E. coli bacterial cells. After transformation, the cells were cultured and induced with 0.6 mM of IPTG to express histidine-tagged HDAC protein (LD_HDAC), which was later purified using nickel affinity chromatography. The approximate protein size confirmed with the help of 10% SDS-PAGE was ~48.0 kDa. The enzymatic assay using the purified protein confirmed it as biologically active. A three dimensional structure of LD_HDAC was modeled using the crystal structure of HDAC2 protein of Homo sapiens (PDB ID: 6G3O). This protein can be utilized for the screening of Leishmania-specific HDAC inhibitors.

Identification of Selective Inhibitors of LdDHFR Enzyme Using Pharmacoinformatic Methods.

Dihydrofolate reductase (DHFR) is a well-known enzyme of the folate metabolic pathway and it is a validated drug target for leishmaniasis. However, only a few leads are reported against Leishmania donovani DHFR (LdDHFR), and thus, there is a need to identify new inhibitors. In this article, pharmacoinformatic tools such as molecular docking, virtual screening, absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling, and molecular dynamics (MD) simulations were utilized to identify potential LdDHFR inhibitors. Initially, a natural DHFR substrate (dihydrofolate), a classical DHFR inhibitor (methotrexate), and a potent LdDHFR inhibitor, that is, "5-(3-(octyloxy)benzyl)pyrimidine-2,4-diamine" (LEAD) were docked in the active site of the LdDHFR and MD simulated to understand the binding mode characteristics of the substrates/inhibitors in the LdDHFR. The shape of the LEAD molecule was used as a query for shape-based virtual screening, while the three-dimensional structure of LdDHFR was utilized for docking-based virtual screening. In silico ADMET factors were also considered during virtual screening. These two screening processes yielded 25 suitable hits, which were further validated for their selectivity toward LdDHFR using molecular docking and prime molecular mechanics/generalized born surface area analysis in the human DHFR (HsDHFR). Best six hits, which were selective and energetically favorable for the LdDHFR, were chosen for MD simulations. The MD analysis showed that four of the hits exhibited very good binding affinity for LdDHFR with respect to HsDHFR, and two hits were found to be more selective than the reported potent LdDHFR inhibitor. The present study thus identifies hits that can be further designed and modified as potent LdDHFR inhibitors.

Development of quinoline-based hybrid as inhibitor of methionine aminopeptidase 1 from Leishmania donovani.

Methionine aminopeptidase 1 (MetAP1) is a target for drug discovery against many adversaries and a potential antileishmanial target for its role in N-terminal methionine processing. As an effort towards new inhibitor discovery against methionine aminopeptidase 1 from Leishmania donovani (LdMetAP1), we have synthesized a series of quinoline-based hybrids, that is (Z)-5-((Z)-benzylidine)-2-(quinolin-3-ylimino)thiazolidin-4-ones (QYT-4a-i) whose in vitro screening led to the discovery of a novel inhibitor molecule (QYT-4h) against LdMetAP1. The compound QYT-4h showed nearly 20-fold less potency for human MetAP1 and had drug-like features. Time-course kinetic assays suggested QYT-4h acting through a competitive mode by binding to the metal-activated catalytic site. Notably, QYT-4h was most potent against the physiologically relevant Mn(II) and Fe(II) supplemented forms of LdMetAP1 and less potent against Co(II) supplemented form. Surface plasmon resonance and fluorescence spectroscopy demonstrated high affinity of QYT-4h for LdMetAP1. Through molecular modelling and docking studies, we found QYT-4h binding at the LdMetAP1 catalytic pocket occupying both the catalytic and substrate binding sites mostly with hydrogen bonding and hydrophobic interactions which provide structural basis for its promising potency. These results demonstrate the feasibility of employing small-molecule inhibitors for selective targeting of LdMetAP1 which may find use to effectively eliminate leishmaniasis.

Cynaroside inhibits Leishmania donovani UDP-galactopyranose mutase and induces reactive oxygen species to exert antileishmanial response.

Cynaroside, a flavonoid, has been shown to have antibacterial, antifungal and anticancer activities. Here, we evaluated its antileishmanial properties and its mechanism of action through different in silico and in vitro assays. Cynaroside exhibited antileishmanial activity in time- and dose-dependent manner with 50% of inhibitory concentration (IC50) value of 49.49 ± 3.515 µM in vitro. It inhibited the growth of parasite significantly at only 20 µM concentration when used in combination with miltefosine, a standard drug which has very high toxicity. It also inhibited the intra-macrophagic parasite significantly at low doses when used in combination with miltefosine. It showed less toxicity than the existing antileishmanial drug, miltefosine at similar doses. Propidium iodide staining showed that cynaroside inhibited the parasites in G0/G1 phase of cell cycle. 2,7-dichloro dihydro fluorescein diacetate (H2DCFDA) staining showed cynaroside induced antileishmanial activity through reactive oxygen species (ROS) generation in parasites. Molecular-docking studies with key drug targets of Leishmania donovani showed significant inhibition. Out of these targets, cynaroside showed strongest affinity with uridine diphosphate (UDP)-galactopyranose mutase with -10.4 kcal/mol which was further validated by molecular dynamics (MD) simulation. The bioactivity, ADMET (absorption, distribution, metabolism, excretion and toxicity) properties, Organisation for Economic Co-operation and Development (OECD) chemical classification and toxicity risk prediction showed cynaroside as an enzyme inhibitor having sufficient solubility and non-toxic properties. In conclusion, cynaroside may be used alone or in combination with existing drug, miltefosine to control leishmaniasis with less cytotoxicity.

Targeting the Trypanothione Reductase of Tissue-Residing Leishmania in Hosts' Reticuloendothelial System: A Flexible Water-Soluble Ferrocenylquinoline-Based Preclinical Drug Candidate.

Since inception, the magic bullets developed against leishmaniasis traveled a certain path and then dropped down due to either toxicity or the emergence of resistance. The route of administration is also an important concern. We developed a series of water-soluble ferrocenylquinoline derivatives, targeting Leishmania donovani, among which CQFC1 showed the highest efficacy even in comparison to other drugs, in use or used, both in oral and intramuscular routes. It did not induce any toxicity to splenocytes and on hematopoiesis, induced protective cytokines, and did not hamper the drug-metabolizing enzymes in hosts. It acts through the reduction and the inhibition of parasites' survival enzyme trypanothione reductase of replicating amastigotes in hosts' reticuloendothelial tissues. Unlike conventional drugs, this molecule did not induce the resistance-conferring genes in laboratory-maintained resistant L. donovani lines. Experimentally, this easily bioavailable preclinical drug candidate overcame all of the limitations causing the discontinuation of the other conventional antileishmanial drugs.

Crystal structure of the GDP-bound GTPase domain of Rab5a from Leishmania donovani.

Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 Šresolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows the canonical GTPase fold, with a six-stranded central mixed ß-sheet surrounded by five α-helices. The positions of the Switch I and Switch II loops confirm an open conformation, as expected in the absence of the γ-phosphate. However, in comparison to other GTP-bound and GDP-bound homologous proteins, the Switch I region traces a unique disposition in LdRab5a. One magnesium ion is bound to the protein at the GTP-binding site. Molecular-dynamics simulations indicate that the GDP-bound structure exhibits higher stability than the apo structure. The GDP-bound LdRab5a structure presented here will aid in efforts to unravel its interactions with its regulators, including the guanine nucleotide-exchange factor, and will lay the foundation for a structure-based search for specific inhibitors.