UMSBP2 is chromatin remodeler that functions in regulation of gene expression and suppression of antigenic variation in trypanosomes.

Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of kinetoplastids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and to play an essential role in chromosome end protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1. DNA decondensation is mediated via protein-protein interactions between TbUMSBP2 and these histones, independently of its previously described DNA binding activity. Silencing of the TbUMSBP2 gene resulted in a significant decrease in the disassembly of nucleosomes in T. brucei chromatin, a phenotype that could be reverted, by supplementing the knockdown cells with TbUMSBP2. Transcriptome analysis revealed that silencing of TbUMSBP2 affects the expression of multiple genes in T. brucei, with a most significant effect on the upregulation of the subtelomeric variant surface glycoproteins (VSG) genes, which mediate the antigenic variation in African trypanosomes. These observations suggest that UMSBP2 is a chromatin remodeling protein that functions in the regulation of gene expression and plays a role in the control of antigenic variation in T. brucei.

Fold-change of chromatin condensation in yeast is a conserved property.

During mitosis, chromatin is condensed and organized into mitotic chromosomes. Condensation is critical for genome stability and dynamics, yet the degree of condensation is significantly different between multicellular and single-cell eukaryotes. What is less clear is whether there is a minimum degree of chromosome condensation in unicellular eukaryotes. Here, we exploited two-photon microscopy to analyze chromatin condensation in live and fixed cells, enabling studies of some organisms that are not readily amenable to genetic modification. This includes the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, and Candida albicans, as well as a protist Trypanosoma brucei. We found that mitotic chromosomes in this range of species are condensed about 1.5-fold relative to interphase chromatin. In addition, we used two-photon microscopy to reveal that chromatin reorganization in interphase human hepatoma cells infected by the hepatitis C virus is decondensed compared to uninfected cells, which correlates with the previously reported viral-induced changes in chromatin dynamics. This work demonstrates the power of two-photon microscopy to analyze chromatin in a broad range of cell types and conditions, including non-model single-cell eukaryotes. We suggest that similar condensation levels are an evolutionarily conserved property in unicellular eukaryotes and important for proper chromosome segregation. Furthermore, this provides new insights into the process of chromatin condensation during mitosis in unicellular organisms as well as the response of human cells to viral infection.

Direct monitoring of the stepwise condensation of kinetoplast DNA networks.

Condensation and remodeling of nuclear genomes play an essential role in the regulation of gene expression and replication. Yet, our understanding of these processes and their regulatory role in other DNA-containing organelles, has been limited. This study focuses on the packaging of kinetoplast DNA (kDNA), the mitochondrial genome of kinetoplastids. Severe tropical diseases, affecting large human populations and livestock, are caused by pathogenic species of this group of protists. kDNA consists of several thousand DNA minicircles and several dozen DNA maxicircles that are linked topologically into a remarkable DNA network, which is condensed into a mitochondrial nucleoid. In vitro analyses implicated the replication protein UMSBP in the decondensation of kDNA, which enables the initiation of kDNA replication. Here, we monitored the condensation of kDNA, using fluorescence and atomic force microscopy. Analysis of condensation intermediates revealed that kDNA condensation proceeds via sequential hierarchical steps, where multiple interconnected local condensation foci are generated and further assemble into higher order condensation centers, leading to complete condensation of the network. This process is also affected by the maxicircles component of kDNA. The structure of condensing kDNA intermediates sheds light on the structural organization of the condensed kDNA network within the mitochondrial nucleoid.

Immune responses in liver and spleen against Plasmodium yoelii pre-erythrocytic stages in Swiss mice model.

Though the immunity to malaria has been associated with cellular immune responses, the exact function of the phenotypic cell population is still unclear. This study investigated the host immune responses elicited during the pre-erythrocytic stage, post-Plasmodium yoelii sporozoite infection in Swiss mice model. For this purpose, we analyzed the dynamics of different subsets of immune cells population and cytokine levels in the hepatic mononuclear and splenic cells population during pre-erythrocytic liver-stage infection. We observed a significant reduction in the effectors immune cells population including CD8+ T cell, F4/80+ macrophage and in plasmacytoid dendritic cells (CD11c+ B220+). Interestingly, substantial down-regulation was also noted in pro-inflammatory cytokines (i.e. IFN-γ, TNF-α, IL-12, IL-2, IL-17 and iNOS), while, up-regulation of anti-inflammatory cytokines (i.e. IL-10, IL-4 and TGF-ß) during asymptomatic pre-erythrocytic liver-stage infection. Collectively, this study demonstrated that during pre-erythrocytic development, Plasmodium yoelii sporozoite impaired the host activators of innate and adaptive immune responses by regulating the immune effector cells, gene expression and cytokines levels for the establishment of infection and subsequent development in the liver and spleen. The results in this study provided a better understanding of the events leading to malarial infection and will be helpful in supportive treatment and vaccine development strategy.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of Chiral Chloroquine Analogues as Antimalarial Agents.

In a focused exploration, we designed, synthesized, and biologically evaluated chiral conjugated new chloroquine (CQ) analogues with substituted piperazines as antimalarial agents. In vitro as well as in vivo studies revealed that compound 7c showed potent activity (in vitro 50% inhibitory concentration, 56.98 nM for strain 3D7 and 97.76 nM for strain K1; selectivity index in vivo [up to at a dose of 12.5 mg/kg of body weight], 3,510) as a new lead antimalarial agent. Other compounds (compounds 6b, 6d, 7d, 7h, 8c, 8d, 9a, and 9c) also showed moderate activity against a CQ-sensitive strain (3D7) and superior activity against a CQ-resistant strain (K1) of Plasmodium falciparum Furthermore, we carried out docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of all in-house data sets (168 molecules) of chiral CQ analogues to explain the structure-activity relationships (SAR). Our new findings specify the significance of the H-bond interaction with the side chain of heme for biological activity. In addition, the 3D-QSAR study against the 3D7 strain indicated the favorable and unfavorable sites of CQ analogues for incorporating steric, hydrophobic, and electropositive groups to improve the antimalarial activity.

Correlation between in vitro and in vivo antimalarial activity of compounds using CQ-sensitive and CQ-resistant strains of Plasmodium falciparum and CQ-resistant strain of P. yoelii.

Present efforts have been made to establish a correlation between in vitro and in vivo antimalarial activity using MIC, IC50 and IC90 values against CQ-sensitive (3D7) and CQ-resistant (K1) strains of Plasmodium falciparum and in vivo activity against Plasmodium yoelii. The method of discriminant function analysis (DFA) was applied to analyze the data. It was observed that in vitro IC90 values against both 3D7 and K1 strains (p < 0.001) have strong correlation with in vivo curative activity. The respective IC50 and IC90 values of compounds, which cured mice (i.e., animals did not show recrudescence of parasitemia even after 60 days posttreatment), ranged between 3 and 14 nM and 14 and 186 nM against 3D7 and between 9 and 65 nM and 24 and 359 nM against the K1 strain of P. falciparum. Whereas the IC50 and IC90 values of compounds which exhibited in vivo suppressive activity in mice ranged between 10 and 307 nm and 61 and >965 nM, respectively, against 3D7 and 75 and >806 nm and 241 and >1232 nM against the K1 strain of P. falciparum. The findings suggest that IC90 values against both 3D7 and K1 strains (p < 0.02) are the main contributors for the prediction of in vivo curative activity of a new molecule. Apart from this, a reasonable correlation between MIC and IC50 values of compounds has also been established.

Synthesis and Evaluation of Chirally Defined Side Chain Variants of 7-Chloro-4-Aminoquinoline To Overcome Drug Resistance in Malaria Chemotherapy.

A novel 4-aminoquinoline derivative [(S)-7-chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

Antimalarial activity of novel 4-aminoquinolines active against drug resistant strains.

In the present study we have synthesized a new class of 4-aminoquinolines and evaluated against Plasmodium falciparum in vitro (3D7-sensitive strain & K1-resistant strain) and Plasmodium yoelii in vivo (N-67 strain). Among the series, eleven compounds (5, 6, 7, 8, 9, 11, 12, 13, 14, 15 and 21) showed superior antimalarial activity against K1 strain as compared to CQ. In addition, all these analogues showed 100% suppression of parasitemia on day 4 in the in vivo mouse model against N-67 strain when administered orally. Further, biophysical studies suggest that this series of compounds act on heme polymerization target.

Repetitive live sporozoites inoculation under arteether chemoprophylaxis confers protection against subsequent sporozoite challenge in rodent malaria model.

Inoculation with live sporozoites under prophylactic antimalarial cover (CPS-immunization) represents an alternate approach to develop sterile, reproducible, and long-term protection against malaria. Here, we have employed arteether (ART), a semi synthetic derivative of artemisinin to explore its potential as a chemoprophylaxis candidate in CPS approach and systematically compared the protective potential of arteether with mefloquine, azithromycin and primaquine. Blood stage patency and quantitative RT-PCR of liver stage parasite load were monitored as primary key end-points for protection against malaria challenge infection. For this purpose, sequential exposures of Plasmodium yoelii sporozoites under prophylactic treatment with arteether (ART), mefloquine (MFQ), azithromycin (AZ) or primaquine (PQ) was conducted in experimental Swiss mice. Our results show that during the first three sequential exposures (1st, 2nd and 3rd challenge) no marked difference in the blood stage patency was observed between control and CPS-ART group. However, delayed patency was recorded following 4th sporozoite challenge and mice enjoyed sterile protection after 5th sporozoite challenge. A similar response was observed in CPS-MFQ group, whereas earlier protection was recorded in CPS-AZ group i.e., after 4th sprozoite challenge. However, mice under PQ cover did not show any protection/delay in patency even after five sequential sporozoite inoculations, possibly due to inhibition of liver stage development. Furthermore, protection acquired by CPS-immunization is stage-specific as the protected mice remained susceptible to challenge with blood stage parasites. In short, the present study demonstrates that sporozoite administration under ART, MFQ or AZ treatment confers strong protection against subsequent sporozoite infection and the acquired response is dependent on the presence of liver stage parasites.

Assessment of real-time method to detect liver parasite burden under different experimental conditions in mice infected with Plasmodium yoelii sporozoites.

Use of highly specific, sensitive and quantitative Real-Time PCR (qRT-PCR) based methods greatly facilitate the monitoring of experimental drug intervention and vaccination efficacy targeting liver stage malaria parasite. Here, in this study we have used qRT-PCR to detect the growing liver stage parasites following inoculation of Plasmodium yoelii sporozoite. Route of sporozoite administration and size of the sporozoite inoculums are two major determinants that affect the liver stage parasite load and therefore its detection and quantification. Thus, these factors need to be addressed to determine the accuracy of detection and quantification of Real-Time PCR method. Furthermore, applicability of quantitative RT-PCR system needs to be confirmed by analyzing the effect of different antimalarials on liver stage parasite burden. We have observed that parasite burden in mice infected via intravenous route was higher compared to that in subcutaneous, intradermal and intraperitoneal route infected mice. Moreover, this method detected liver stage parasite load with as low as 50 sporozoites. The inhibition studies with primaquine and atovaquone revealed inhibition of liver stage parasite and well correlated with patency and course of blood stage infection. This study characterized the simplicity, accuracy, and quantitative analysis of liver stage parasite development by real time PCR under different experimental conditions. Use of real time PCR method greatly improves the reproducibility and applicability to estimate the efficacy and potency of vaccine or drug candidates targeting liver stage parasite.

Host immune response is severely compromised during lethal Plasmodium vinckei infection.

Cytokines and immune effector cells play an important role in determining the outcome of infection with various intracellular pathogens, including protozoan parasites. However, their role during lethal and nonlethal malaria needs further validation. In the present study, we examined the role of cytokines and various immune effector cells during lethal and nonlethal malaria caused by Plasmodium vinckei in AKR mice. We show that lethal P. vinckei infection (PvAS) in AKR mice is characterized by increased parasite growth, decreased production of pro-inflammatory cytokines, and attenuated cell proliferation and nitric oxide (NO) synthesis resulting in increased parasitemia which ultimately leads to death of all animals by day 5 post infection. In contrast, AKR mice infected with lethal parasite (PvAR) showed elevated levels of pro-inflammatory cytokines, heightened cell proliferation, and NO synthesis leading to complete parasite clearance by day 22 post infection. Flow cytometric analysis performed on splenocytes from PvAS- and PvAR-infected mice shows that host immunity is severely compromised in PvAS-infected mice as was evident by decreased percentages of CD4(+) and CD8(+) T cells, B cells, plasma cells, dendritic cells (DCs), and macrophages (MΦs) which was in complete contrast to PvAR-infected animals which exhibited elevated numbers of all the cell types analyzed. Taken together, findings of the present study show that coordinated actions of pro-inflammatory cytokines and other immune effector cells are essential to control lethal malarial infection and their attenuation leads to increased parasite growth and, ultimately, death of animals.

Cloning, expression and functional characterization of heme detoxification protein (HDP) from the rodent malaria parasite Plasmodium vinckei.

Malaria parasite resides within the host red blood cells, where it degrades vast amount of haemoglobin. During haemoglobin degradation, toxic free heme is liberated which subsequently gets converted into hemozoin. This process is facilitated by action of various proteins viz. heme detoxification protein (HDP), and histidine rich proteins II and III (HRP II & III). Out of these, HDP is the most potent in hemozoin formation and plays indispensible role for parasite survival. Despite this, the detailed study of HDP from rodent and simian parasite has not been performed till date. Here, we have cloned and sequenced hdp gene from different malaria parasites Plasmodium vinckei, Plasmodium yoelii, Plasmodium knowlesi, and Plasmodium cynomolgi. Furthermore, HDP from P. vinckei (PvHDP) was over-expressed and purified for detailed characterization. The PvHDP is cytosolic, expressed throughout the intra erythrocytic stages and its expression is higher in late trophozoite and schizont stages of parasite. The PvHDP interacts with free heme (KD=89 nM) and efficiently converts heme into hemozoin in a time and concentration dependent manner. Moreover, PvHDP showed activity in acidic pH and over a broad range of temperature. Histidine modification of PvHDP using DEPC showed reduction in heme binding and hemozoin formation, thus emphasizing the importance of histidine residues in heme binding and subsequent hemozoin production. Furthermore, applicability of PvHDP to screen anti-plasmodial agents (targeting heme to hemozoin conversion) was also determined using chloroquine, and mefloquine as reference antimalarials. Results showed that these drugs inhibit heme polymerization effectively in a concentration dependent manner. In conclusion, our study identified and biochemically characterized HDP from rodent malaria parasite P. vinckei and this will help to develop a high throughput assay to evaluate new antimalarials targeting hemozoin pathway.

Synthesis of chiral chloroquine and its analogues as antimalarial agents.

In this investigation, we describe a new approach to chiral synthesis of chloroquine and its analogues. All tested compounds displayed potent activity against chloroquine sensitive as well as chloroquine resistant strains of Plasmodium falciparum in vitro and Plasmodium yoelii in vivo. Compounds S-13 b, S-13c, S-13 d and S-13 i displayed excellent in vitro antimalarial activity with an IC50 value of 56.82, 60.41, 21.82 and 7.94 nM, respectively, in the case of resistant strain. Furthermore, compounds S-13a, S-13c and S-13 d showed in vivo suppression of 100% parasitaemia on day 4 in the mouse model against Plasmodium yoelii when administered orally. These results underscore the application of synthetic methodology and need for further lead optimization.

Molecular cloning and biochemical characterization of iron superoxide dismutase from the rodent malaria parasite Plasmodium vinckei.

Plasmodium parasite utilizes superoxide dismutase family proteins to limit the toxicity of reactive oxygen species, such as produced through hemoglobin degradation. These proteins play an important role in parasite survival during intra-erythrocytic phase. We have identified, and biochemically characterized a putative iron dependent superoxide dismutase from rodent malaria parasite Plasmodium vinckei (PvSOD1). The recombinant PvSOD1 protein was purified to homogeneity through a combination of affinity and gel filtration chromatography. Crosslinking, Native-PAGE and FPLC gel filtration analyses documented that PvSOD1 exists as a dimer in solution, a common feature shared by other Fe-SODs. PvSOD1 is cytosolic in localization and its expression is comparatively higher during trophozoite as compared to that of ring and schizont stages. Enzymatic activity of recombinant PvSOD1 was validated using conventional zymogram analyses and xanthine-xanthine oxidase system. Under optimal conditions, PvSOD1 was highly active and catalyzed the dismutation of superoxide radicals. Furthermore, PvSOD1 showed activity over a broad range of pH and temperature. Inhibition studies suggested that PvSOD1 was inactivated by hydrogen peroxide, and peroxynitrite, but not by cyanide and azide. Since, PvSOD1 plays a central role in oxidative defense mechanism, therefore, characterization of PvSOD1 will be exploited in the screening of new superoxide dismutase inhibitors for their antimalarial activity.

Synthesis and antimalarial activity of 3,3-spiroanellated 5,6-disubstituted 1,2,4-trioxanes.

Novel 3,3-spiroanellated 5-aryl, 6-arylvinyl-substituted 1,2,4-trioxanes 19-34 have been synthesized and appraised for their antimalarial activity against multidrug-resistant Plasmodium yoelii nigeriensis in Swiss mice by oral route at doses ranging from 96 mg/kg × 4 days to 24 mg/kg × 4 days. The most active compound of the series (compound 25) provided 100% protection at 24 mg/kg × 4 days, and other 1,2,4-trioxanes 22, 26, 27, and 30 also showed promising activity. In this model, ß-arteether provided 100 and 20% protection at 48 mg/kg × 4 days and 24 mg/kg × 4 days, respectively, by oral route. Compound 25 displayed a similar in vitro pharmacokinetic profile to that of reference drug ß-arteether. The activity results demonstrated the importance of an aryl moiety at the C-5 position on the 1,2,4-trioxane pharmacophore.

Synthesis and insight into the structure-activity relationships of chalcones as antimalarial agents.

Licochalcone A (I), isolated from the roots of Chinese licorice, is the most promising antimalarial compound reported so far. In continuation of our drug discovery program, we isolated two similar chalcones, medicagenin (II) and munchiwarin (III), from Crotalaria medicagenia , which exhibited antimalarial activity against Plasmodium falciparum . A library of 88 chalcones were synthesized and evaluated for their in vitro antimalarial activity. Among these, 67, 68, 74, 77, and 78 exhibited good in vitro antimalarial activity against P. falciparum strains 3D7 and K1 with low cytotoxicity. These chalcones also showed reduction in parasitemia and increased survival time of Swiss mice infected with Plasmodium yoelii (strain N-67). Pharmacokinetic studies indicated that low oral bioavailability due to poor ADME properties. Molecular docking studies revealed the binding orientation of these inhibitors in active sites of falcipain-2 (FP-2) enzyme. Compounds 67, 68, and 78 showed modest inhibitory activity against the major hemoglobin degrading cysteine protease FP-2.

Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.

Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.

Antiplasmodial activity of novel keto-enamine chalcone-chloroquine based hybrid pharmacophores.

A series of novel keto-enamine chalcone-chloroquine based hybrids were synthesized following new methodology developed in our laboratory. The synthesized compounds were screened against chloroquine sensitive strain (3D7) of Plasmodium falciparum in an in vitro model. Some of the compounds were showing comparable antimalarial activity at par with chloroquine. Compounds with significant in vitro antimalarial activity were then evaluated for their in vivo efficacy in Swiss mice against Plasmodium yoelii (chloroquine resistant N-67 strain), wherein compounds 25 and 27 each showed an in vivo suppression of 99.9% parasitaemia on day 4. Biochemical studies reveal that inhibition of hemozoin formation is the primary mechanism of action of these analogues.