Antimalarial Activity of Anacardium occidentale Leaf Extracts Against Plasmodium falciparum Transketolase (PfTK).

BACKGROUND: As per estimates by WHO in 2021 almost half of the world's population was at risk of malaria and > 0.6 million deaths were attributed to malaria. Therefore, the present study was aimed to explore the antimalarial activity of extracts derived from the leaves of the plant Anacardium occidentale L., which has been used traditionally for the treatment of malaria. Different extracts of A. occidentale leaves were prepared and tested for their inhibitory activity against recombinant P. falciparum transketolase (rPfTK) enzyme, in vitro. Further, growth inhibitory activity against cultivated blood stage P. falciparum parasites (3D7 strain), was studied using SYBR Green fluorescence-based in vitro assays. Acute toxicity of the hydro alcoholic extracts of leaves of A. occidentale (HELA) at different concentrations was evaluated on mice and Zebra fish embryos. HELA showed 75.45 ± 0.35% inhibitory activity against the recombinant PfTk and 99.31 ± 0.08% growth inhibition against intra-erythrocytic stages of P. falciparum at the maximum concentration (50 µg/ml) with IC50 of 4.17 ± 0.22 µg/ml. The toxicity test results showed that the heartbeat, somite formation, tail detachment and hatching of embryos were not affected when Zebra fish embryos were treated with 0.1 to 10 µg/ml of the extract. However, at higher concentrations of the extract, at 48 h (1000 µg/ml) and 96 h (100 µg/ml and 1000 µg/ml, respectively) there was no heartbeat in the fish embryos. In the acute oral toxicity tests performed on mice, the extract showed no toxicity up to 300 mg/kg body weight in mice. CONCLUSION: The hydro-alcoholic extract of leaves of A. occidentale L. showed potent antimalarial activity against blood stage P. falciparum. Based on the observed inhibitory activity on the transketolase enzyme of P. falciparum it is likely that this enzyme is the target for the development of bioactive molecules present in the plant extracts. The promising anti-malarial activity of purified compounds from leaves of A. occidentale needs to be further explored for development of new anti-malarial therapy.

Immunoreactivity of Brugia malayi Calreticulin and Its Domains with Sera of Different Categories of Bancroftian Filarial Patients.

PURPOSE: We identified calreticulin in human filaria Brugia malayi (BmCRT) that shares 97% homology with Wuchereria bancrofti calreticulin (WbCRT), but only 56% with human calreticulin. We found that BmCRT binds C1q and prevents complement-mediated parasite death; immunization with BmCRT leads to parasite death in a rodent model of the infection. BmCRT could, therefore, be a potential vaccine candidate. In the present study, we determined the levels of BmCRT-reactive IgG and its isotype in bancroftian filarial subjects. METHODS: Recombinant BmCRT (rBmCRT) was prepared, and the sera of endemic normal subjects (EN), microfilaraemics (Mf+) and chronic amicrofilaraemics (ChMf-) from a bancroftian filaria-endemic area and normal subjects from filaria-non-endemic area (NEN) were probed for IgG and its isotypes reacting with rBmCRT and its domains rN, rP and rC. RESULTS: rBmCRT and its rN domain-reactive IgG levels were high in EN and Mf+ groups; rC domain and rP domain showed moderate and very little reactivity, respectively. NEN sera were non-reactive. Moderate levels of rBmCRT-reactive IgG1, IgG3 and IgG4 in EN and Mf+ groups and low levels of IgG2 in Mf+ were found; IgG1 and IgG3 reactivity was found for rBmCRT and its rN domain only, while IgG4 reactivity was moderate for rN domain and low for rP and rC domains. While IgG reactivity was seen in all the endemic subjects, IgG isotype reactivity was found mostly in EN and Mf+ subjects. CONCLUSIONS: Moderate levels of rBmCRT (and its rN domain)-reactive IgG and its isotypes are present in bancroftian subjects. Preponderance of IgG1 and IgG3 isotypes which bind and activate complement has relevance to vaccine potential of BmCRT.

Immunization with Brugia malayi Calreticulin Protein Generates Robust Antiparasitic Immunity and Offers Protection during Experimental Lymphatic Filariasis.

Lymphatic filariasis causes permanent and long-term disability worldwide. Lack of potent adulticidal drugs, the emergence of drug resistance, and the nonavailability of effective vaccines are the major drawbacks toward LF elimination. However, immunomodulatory proteins present in the parasite secretome are capable of providing good protection against LF and thus offer hope in designing new vaccines against LF. Here, we evaluated the immunogenicity and protective efficacy of B. malayi calreticulin protein (BmCRT) using in vitro and in vivo approaches. Stimulation with recombinant BmCRT (rBmCRT) significantly upregulated Th1 cytokine production in mouse splenocytes, mesenteric lymph nodes (mLNs), and splenic and peritoneal macrophages (PMΦs). Heightened NO release, ROS generation, increased lymphocyte proliferation, and increased antigen uptake were also observed after rBmCRT exposure. Mice immunized with rBmCRT responded with increased Th1 and Th2 cytokine secretion and exhibited highly elevated titers of anti-BmCRT specific IgG at day 14 and day 28 postimmunization while splenocytes and mLNs from immunized mice showed a robust recall response on restimulation with rBmCRT. Infective larvae (L3) challenge and protection studies undertaken in Mastomys coucha, a permissive model for LF, showed that rBmCRT-immunized animals mounted a robust humoral immune response as evident by elevated levels of total IgG, IgG1, IgG2a, IgG2b, and IgG3 in their serum even 150 days after L3 challenge, which led to significantly reduced microfilariae and worm burden in infected animals. BmCRT is highly immunogenic and generates robust antiparasitic immunity in immunized animals and should therefore be explored further as a putative vaccine candidate against LF.

Molecular characterization of recombinant arginase of Leishmania donovani.

Arginase catalyzes the first committed step in the biosynthesis of polyamines that enable cell growth and hence potential drug target for the treatment of leishmaniasis. The arginase from Leishmania donovani (LdARG) was cloned, overexpressed and characterized. Analysis of the deduced amino acid sequence of LdARG with homologous enzyme from other trypanosomatids arginases identified a non-conserved 12 residues long segment VWGLIERTFLSA from position 161-172. This counter segment in L. mexicana arginase exhibits a different conformation compared with human arginase I. The pH and temperature optima of LdARG were 9.0 and 37 °C, respectively. Biochemical studies revealed that the KM for the substrate L-arginine was 24.76 ±â€¯0.06 mM. Molecular modeling of LdARG studies revealed that the glutamic acid residue at position 288 plays a role in substrate binding. The importance of this glutamic acid residue was validated by constructing a mutant variant of LdARG (E288Q-LdARG) by replacing glutamic acid with glutamine through site-directed mutagenesis. The KM value of mutant variant for L-arginine was found to be 107 ±â€¯0.18 mM. The increase in KM value of E288Q-LdARG as compared to LdARG suggested that substrate binding was significantly affected which could be exploited further. Studies on biochemical and structural characterization of recombinant LdARG will help in evaluating this enzyme as a potential drug target for visceral leishmaniasis.

Metal binding study of calreticulin: An immunomodulatory protein of human filarial parasite Brugia malayi.

Calreticulin (CRT), a highly conserved ubiquitous eukaryotic protein with a molecular mass of 46 kDa, containing three domains (N, P, and C) is involved in promoting prolonged parasite-host relations. Brugia malayi Calreticulin (BmCRT) is involved in the establishment of parasite infection by suppression of C1q-mediated host immune response. Calcium plays important role in this immunomodulatory mechanism of BmCRT. In the present study binding of calcium with BmCRT region involved in this interaction was investigated and correlated with the accompanying changes in fluorescence, circular dichroism (CD) and UV absorption. The results obtained clearly indicated that BmCRT is a calcium binding protein and contains types two of Ca2+ binding sites, one high affinity Ca2+ binding site at P domain and another low affinity Ca2+ binding site at C domain. Zinc also binds to additional sites that do not have appreciable affinity for the calcium. These studies have provided new knowledge that allows us to describe how the structure of BmCRT responds to interactions with calcium and zinc which is different from human CRT and also discuss how this mechanism help to complex formation with host C1q.

Molecular cloning and characterization of protein disulfide isomerase of Brugia malayi, a human lymphatic filarial parasite.

Lymphatic filariasis results in an altered lymphatic system and the abnormal enlargement of body parts, causing pain, serious disability and social stigma. Effective vaccines are still not available nowadays, drugs against the disease is required. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum which is involved in folding and chaperone activities in different biological systems. Here, we report the enzymatic characterization of a Brugia malayi Protein disulfide isomerase (BmPDI), which was expressed and purified from Escherichia coli BL21 (DE3). Western blotting analysis showed the recombinant BmPDI could be recognized by anti-BmPDI Rabbit serum. The rBmPDI exhibited an optimum activity at pH 8 and 40 °C. The enzyme was inhibited by aurin and PDI inhibitor. Recombinant BmPDI showed interaction with recombinant Brugia malayi calreticulin (rBmCRT). The three-dimensional model for BmPDI and BmCRT was generated by homology modelling. A total of 25 hydrogen bonds were found to be formed between two interfaces. There are 259 non-bonded contacts present in the BmPDI-BmCRT complex and 12 salt bridges were formed in the interaction.

Monitoring thermal and chemical unfolding of Brugia malayi calreticulin using fluorescence and Circular Dichroism spectroscopy.

Calreticulin of Brugia malayi (BmCRT) play very important role in host-parasite interaction. In previous study it was found that BmCRT is responsible for prevention of host classical complement pathway activation via its interaction with first component C1q of the human host. Therefore, BmCRT is an essential protein for parasite survival and an important drug target to fend filariasis. In the present study, we have carried out a systamatic biophysical characterization of BmCRT protein. Unfolding of BmCRT was found to be non-cooperative two-state process in the presence of both denaturant GdmCl and urea. The results also illustrated that protein lost its 50% activity at 1.5M GdmCl and 3M Urea. Partially unfolded and molten-globule like intermediate state was observed at 0.8 to 1.2M GdmCl while Urea unfolding showed intermediate state at 1.2 to 1.6M. Unfolding pathway monitored with the help of apolar quencher, favor above observations. All of these findings support the presence of detectable intermediate state during unfolding pathway of BmCRT. Furthermore, this study indicates that BmCRT is more stable toward temperature (Tm=65°C), pH and trypsin digestion. These differences in properties as compared to host can be fruitfully utilized for synthesis of compounds effective against the parasite.

Molecular cloning, purification and characterization of Brugia malayi phosphoglycerate kinase.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized. The SDS-PAGE analysis revealed native molecular weight of recombinant Brugia malayi 3-phosphoglycerate kinase (rBmPGK) to be ∼45 kDa. The enzyme was found sensitive to temperature and pH, it showed maximum activity at 25 °C and pH 8.5. The Km values for PGA and ATP were 1.77 and 0.967 mM, respectively. The PGK inhibitor, clorsulon and antifilarial drugs albendazole and ivermectin inhibited the enzyme. The specific inhibitor of PGK, clorsulon, competitively inhibited enzyme with Ki value 1.88 µM. Albendazole also inhibited PGK competitively with Ki value 35.39 µM. Further these inhibitory studies were confirmed by docking and molecular simulation of drugs with enzyme. Clorsulon interacted with substrate binding site with glutamine 37 as well as in hinge regions with aspartic acid 385 and valine 387 at ADP binding site. On the other hand albendazole interacted with asparagine 335 residues. These effects were in good association with binding interactions. Thus current study might help in designing and synthesis of effective inhibitors for this novel drug target and understanding their mode of interaction with the potent anthelmintic drugs.

Insights into the structure-function relationship of Brugia malayi thymidylate kinase (BmTMK).

Lymphatic filariasis is a debilitating disease caused by lymph dwelling nematodal parasites like Wuchereria bancrofti, Brugia malayi and Brugia timori. Thymidylate kinase of B. malayi is a key enzyme in the de novo and salvage pathways for thymidine 5'-triphosphate (dTTP) synthesis. Therefore, B. malayi thymidylate kinase (BmTMK) is an essential enzyme for DNA biosynthesis and an important drug target to rein in filariasis. In the present study, the structural and functional changes associated with recombinant BmTMK, in the presence of protein denaturant GdnHCl, urea and pH were studied. GdnHCl and urea induced unfolding of BmTMK is non-cooperative and influence the functional property of the enzyme much lower than their Cm values. The study delineate that BmTMK is more prone to ionic perturbation. The dimeric assembly of BmTMK is an absolute requirement for enzymatic acitivity and any subtle change in dimeric conformation due to denaturation leads to loss of enzymatic activity. The pH induced changes on structure and activity suggests that selective modification of active site microenvironment pertains to difference in activity profile. This study also envisages that chemical moieties which acts by modulating oligomeric assembly, could be used for better designing of inhibitors against BmTMK enzyme.

NADP⁺ binding effects tryptophan accessibility, folding and stability of recombinant B. malayi G6PD.

Brugia malayi Glucose 6-phosphate dehydrogenase apoenzyme (BmG6PD) was expressed and purified by affinity chromatography to study the differences in kinetic properties of enzyme and the effect of the cofactor NADP(+) binding on enzyme stability. The presence of cofactor NADP(+) influenced the tertiary structure of enzyme due to significant differences in the tryptophan microenvironment. However, NADP(+) binding have no effect on secondary structure of the enzyme. Quenching with acrylamide indicated that two or more tryptophan residues became accessible upon cofactor binding. Unfolding and cross linking study of BmG6PD showed that NADP(+) stabilized the protein in presence of high concentration of urea/GdmCl. A homology model of BmG6PD constructed using human G6PD (PDB id: 2BH9) as a template indicated 34% α-helix, 19% ß-sheet and 47% random coil conformations in the predicted model of the enzyme. In the predicted model binding of NADP(+) to BmG6PD was less tight with the structural sites (-10.96 kJ/mol binding score) as compared with the coenzyme site (-15.47 kJ/mol binding score).

Designing, synthesis of selective and high-affinity chalcone-benzothiazole hybrids as Brugia malayi thymidylate kinase inhibitors: In vitro validation and docking studies.

In our continuing search for safe and efficacious antifilarials, a series of novel chalcone-benzothiazole hybrids have been synthesized and evaluated for their Brugia malayi thymidylate kinase (BmTMK) enzyme inhibition activity. Their selectivity towards BmTMK was studied and compared to the human TMK (HsTMK) by an in silico method. Out of seventeen derivatives, compounds 34 and 42 showed higher interactions with the BmTMK active site. MolDock docking model revealed the interactions of these two derivatives and the results corroborated well with their in vitro antifilarial activities. Our studies suggest that these hybrids are selective towards the BmTMK enzyme and may serve as potential therapeutic agents against filariasis.

Correction: Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis.

[This corrects the article DOI: 10.1371/journal.pntd.0003557.].

Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis.

BACKGROUND: The development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: LdSir2RP was successfully cloned, expressed and purified. The gene was present as a monomeric protein of ~45 kDa and further established by the crosslinking experiment. rLdSir2RP shown cytosolic localization in L. donovani and demonstrating NAD+-dependent deacetylase activity. Bioinformatic analysis also confirmed that LdSir2RP protein has NAD binding domain. The rLdSir2RP was further assessed for its cellular response by lymphoproliferative assay and cytokine ELISA in cured Leishmania patients and hamsters (Mesocricetus auratus) in comparison to soluble Leishmania antigen and it was observed to stimulate the production of IFN-γ, IL-12 and TNF-α significantly but not the IL-4 and IL-10. The naïve hamsters when vaccinated with rLdSir2RP alongwith BCG resisted the L. donovani challenge to the tune of ~75% and generated strong IL-12 and IFN-γ mediated Th1 type immune response thereof. The efficacy was further supported by remarkable increase in IgG2 antibody level which is indicative of Th1 type of protective response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of rLdSir2RP was done using computational approach. CONCLUSION/SIGNIFICANCE: The immunobiochemical characterization strongly suggest the potential of rLdSir2RP as vaccine candidate against VL and supports the concept of its being effective T-cell stimulatory antigen.

Antidyslipidemic Effect of Ocimum sanctum Leaf Extract in Streptozotocin Induced Diabetic Rats.

The antidyslipidemic activity of Ocimum sanctum leaf extract was studied in streptozotocin induced diabetic rats. In this model, there was significant increase in plasma markers of diabetic-dyslipidemia following diminution of lipid metabolizing enzymes. Oral administration of leaf extract (500 mg/kg b.w.p.o.) for 15 days resulted in significant decrease in diabetogenic and dyslipidemia parameters; namely blood glucose, glycosylated hemoglobin, lipid peroxide, free fatty acids, small dense low density lipoprotein, lipid and protein components of plasma lipoproteins, adipose and liver. The regulation of lipids was accompanied by stimulation of postheparin lipolytic activity, reactivation of lecithin cholesterol acyl transferase and hepatic lipoprotein lipase enzymes. The results of the present study demonstrated antidyslipidemic and antioxidant activities in leaf extract of O. sanctum which could be used in prevention of diabetic-dyslipidemia and related complications.

Hypolipidemic Activity of Cassia tora Seeds in Hyperlipidemic Rats.

The hypolipidemic activity of Cassia tora (Chakvat, Chakunda) (Family: Caesalpiniaceae) seeds extract have been studied in two models of hyperlipidemia in rats. In an acute model, hyperlipidemia was induced by injecting a single dose of Triton WR-1339 (400 mg/kg, b.w.) intraperitonially in rats. Feeding with C. tora seed extract at the dose of 500 mg/kg, b.w. exerted significant lipid lowering effect as assessed by the reversal of plasma levels of total cholesterol, phospholipids, triglyceride and reactivation of post heparin lipolytic activity. In the chronic model, hyperlipidemia was induced by feeding with cholesterol rich-HFD in rats. The treatment with seeds extract of C. tora (500 mg/kg, b.w.) simultaneously for 15 days also caused lowering of lipid levels in plasma and liver following reactivation of plasma post heparin lipolytic activity and hepatic lipoprotein lipase activity in animals. The hypolipidemic activity of C. tora seeds was compared with a standard drug guggulipid (200 mg/kg, b.w.) in both models.

Arg-265: a critical residue of L.donovani cytosolic SHMT in maintaining the binding of THF and catalysis.

Serine hydroxymethyltransferase belongs to the class of pyridoxal-5-phosphate enzymes along with aspartate aminotransferase. To explore the function of residue(s) involved in binding of the carboxylate group of Tetrahydrofolic acid (THF) to L. donovani cytosolic serine hydroxymethyltransferase (LdcSHMT), the gene was cloned in pET-28(a) vector, overexpressed and purified to homogeneity. With the help of docking results of THF to the active site of protein, the key residues involved in interaction were identified. In an attempt to unravel the function of Arg265 residue involved in binding of the carboxylate group of THF, Arg-265 was mutated to Ala by site-directed mutagenesis. The Arg265Ala-LdcSHMT showed increased Km value (threefold) and decreased kcat/Km value (threefold) for H4-folate as compared with wild type enzyme. The wild and mutant enzymes exhibited similar Km and kcat/Km values for L-allo-threonine. Unlike the wild type enzyme, mutant failed to form characteristic quinonoid intermediate and was unable to carry out the exchange of α-proton from glycine in the presence of Tetrahydrofolate. These results suggested that Arg265 residue is required for the binding of Tetrahydrofolate and may be the base that abstracts α-proton from glycine, leading to formation of quinonoid intermediate in cytosolic SHMT of L. donovani.

Identification of novel PTP1B inhibitors by pharmacophore based virtual screening, scaffold hopping and docking.

Design and synthesis of protein tyrosine phosphatases-1B (PTP1B) inhibitors are important for the drugs targeted to treat diabetes and obesity. The pharmacophore modeling, docking and scaffold hopping techniques have been applied to discover the novel PTP1B inhibitors. The ten prioritized compounds (115-119, 120-121, 127, 130-131) from the library of 86 compounds were synthesized and found positive in the micro molar range for PTP1B in-vitro inhibitory assays as compared to Suramin (IC50 9.5 µM). Among these five active compounds (115-119) were tested in STZ-s induced diabetic rat model and the most active compound 115 in this test, was further tested in C57BL/KsJ-db/db mice where it significantly improved OGTT along with the fasting and random blood glucose level. The treatment by the compound 115 significantly improved the insulin resistance and insulin signaling by restoring the insulin level and normalizing the serum lipid profile. Compound 115 also augmented the insulin action by modulating the expression of genes involved in insulin signaling like IRS 1-2, PI3K, PTPN1, Akt2, AMPK and PPAR-α. Western blot analysis of both skeletal muscle and liver demonstrated that proteins and intermediate enzymes of insulin signaling were also increased as compared to control group. The compound 115 was also investigated for anti-adipogenic effect on 3T3L-1 cells. The compound 115 inhibited MDI induced lipid accumulation in a dose-dependent manner. The oral bioavailability of compound 115 was ∼10.29% after 30 mg/kg oral dosing assessed in rat.

In silico and in vitro studies on the protein-protein interactions between Brugia malayi immunomodulatory protein calreticulin and human C1q.

Filarial parasites modulate effective immune response of their host by releasing a variety of immunomodulatory molecules, which help in the long persistence of the parasite within the host. The present study was aimed to characterize an immunomodulatory protein of Brugia malayi and its interaction with the host immune component at the structural and functional level. Our findings showed that Brugia malayi Calreticulin (BmCRT) is responsible for the prevention of classical complement pathway activation via its interaction with the first component C1q of the human host. This was confirmed by inhibition of C1q dependent lysis of immunoglobulin-sensitized Red Blood Cells (S-RBCs). This is possibly the first report which predicts CRT-C1q interaction on the structural content of proteins to explain how BmCRT inhibits this pathway. The molecular docking of BmCRT-C1q complex indicated that C1qB chain (IgG/M and CRP binding sites on C1q) played a major role in the interaction with conserved and non-conserved regions of N and P domain of BmCRT. Out of 37 amino acids of BmCRT involved in the interaction, nine amino acids (Pro(126), Glu(132), His(147), Arg(151), His(153), Met(154), Lys(156), Ala(196) and Lys(212)) are absent in human CRT. Both ELISA and in silico analysis showed the significant role of Ca(+2) in BmCRT-HuC1q complex formation and deactivation of C1r2-C1s2. Molecular dynamics studies of BmCRT-HuC1q complex showed a deviation from ∼ 0.4 nm to ∼ 1.0 nm. CD analyses indicated that BmCRT is composed of 49.6% α helix, 9.6% ß sheet and 43.6% random coil. These findings provided valuable information on the architecture and chemistry of BmCRT-C1q interaction and supported the hypothesis that BmCRT binds with huC1q at their targets (IgG/M, CRP) binding sites. This interaction enables the parasite to interfere with the initial stage of host complement activation, which might be helpful in parasites establishment. These results might be utilized for help in blocking the C1q/CRT interaction and preventing parasite infection.

Molecular cloning and characterization of Brugia malayi thymidylate kinase.

Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of deoxythymidine triphosphate, which is an essential component for DNA synthesis. The gene encoding thymidylate kinase of Brugia malayi was amplified by PCR and expressed in Escherichia coli. The native molecular weight of recombinant B. malayi thymidylate kinase (rBmTMK) was estimated to be ∼52kDa by gel filtration chromatography, suggesting a homodimeric structure. rBmTMK activity required divalent cation and Mg(2+) was found to be the most effective cation. The enzyme was sensitive to pH and temperature, it showed maximum activity at pH 7.4 and 37°C. The Km values for dTMP and ATP were 17 and 66µM, respectively. The turnover number kcat was found to be 38.09s(-1), a value indicating the higher catalytic efficiency of the filarial enzyme. The nucleoside analogues 5-bromo-2'-deoxyuridine (5-BrdU), 5-chloro-2'-deoxyuridine (5-CldU) and 3'-azido-3'-deoxythymidine (AZT) showed specific inhibitory effect on the enzyme activity and these effects were in good association with binding interactions and the scoring functions as compared to human TMK. Differences in kinetic properties and structural differences in the substrate binding site of BmTMK model with respect to human TMK can serve as basis for designing specific inhibitors against parasitic enzyme.

Behavior of Plasmodium falciparum purine nucleoside phosphorylase in macromolecular crowded environment.

Biochemical and biophysical properties of enzymes have been studied in dilute buffer system, which are far from the crowded physiological condition of cell. We report the enzyme kinetics and refolding of Plasmodium falciparum purine nucleoside phosphorylase under crowded conditions. Enzyme catalytic efficiency was inversely affected in the presence of polyethylene glycols and Dextran whereas it was increased in the presence of osmolytes. We detected a non-linear relationship between Km and increasing macromolecular crowding agents. At low concentrations of PEGs and Dextran, we observed decreased substrate binding whereas higher concentrations of PEGs and Dextran favored substrate binding. The presence of sucrose decreased the Km values. We detected decrease in Kcat value in the presence of PEGs and Dextran, whereas osmolytes increased the Kcat values. Thermal resistance of enzyme was increased in the presence of crowding agents. Intrinsic and extrinsic fluorescence analysis indicated change around active site loop region having single tryptophan residue. Preferential exclusions of polyols favor the catalytic mechanism of the enzyme. Urea denatured enzyme showed fast refolding when diluted and rate of refolding was not affected by the presence of crowding agents. It is important to draw together significant knowledge about modulation of inherent properties of this enzyme in crowded environment which will be helpful in better understanding of this drug-target enzyme and in further inhibitor design.