A BODIPY-embedding miltefosine analog linked to cell-penetrating Tat(48-60) peptide favors intracellular delivery and visualization of the antiparasitic drug

Therapeutic application of many drugs is often hampered by poor or denied access to intracellular targets. A case in point is miltefosine (MT), an orally active antiparasitic drug, which becomes ineffective when parasites develop dysfunctional uptake systems. We report here the synthesis of a fluorescent BODIPY-embedding MT analogue with appropriate thiol functionalization allowing linkage to the cell-penetrating Tat(48-60) peptide through disulfide or thioether linkages. The resulting constructs are efficiently internalized into the otherwise MT-invulnerable R40 Leishmania strain, resulting in fast parasite killing, and hence successful avoidance of the resistance. In the disulfide-linked conjugate, an additional fluoro tag on the Tat moiety allows to monitor its reductive cleavage within the cytoplasm. Terminally differentiated cells such as peritoneal macrophages, impervious to MT unless infected by Leishmania, can uptake the drug in its Tat-conjugated form. The results afford proof-of-principle for using CPP vectors to avert drug resistance in parasites, and/or for tackling leishmaniasis by modulating macrophage uptake.     

Inhibition of human immunodeficiency virus type-1 (HIV-1) replication by immunor (IM28), a new analog of dehydroepiandrosterone

The inhibition of HIV-1 replication in vitro by Immunor 28 (IM28), an analog of dehydroepiandrosterone (DHEA), was monitored using the HIV-1 laboratory wild-type strain IIIB. Evaluation of the 50% inhibitory dose (IC50) revealed a decrease in HIV-1 replication giving an IC50 value around 22 microM. The toxicity of the drug has been determined also, in MT2 cells and PBMCs. 60 microM of IM28 provoked a 50% decrease in cell viability while DHEA caused the same decrease at 75 microM in MT2 cells. These values are 125 microM for IM28 in PBMCs and 135 microM for DHEA. Thus, DHEA is less toxic than IM28, but IM28 has a higher antiviral activity.     

Eribulin disrupts EB1-microtubule plus-tip complex formation

Eribulin mesylate is a synthetic analog of halichondrin B known to bind tubulin and microtubules, specifically at their protein rich plus-ends, thereby dampening microtubule (MT) dynamics, arresting cells in mitosis, and inducing apoptosis. The proteins which bind to the MT plus-end are known as microtubule plus-end tracking proteins (+TIPs) and have been shown to promote MT growth and stabilization. Eribulin's plus-end binding suggests it may compete for binding sites with known +TIP proteins such as End-binding 1 (EB1). To better understand the impact of eribulin plus-end binding in regard to the proteins which normally bind there, cells expressing GFP-EB1 were treated with various concentrations of eribulin. In a concentration dependent manner, GFP-EB1 became dissociated from the MT plus-ends following drug addition. Similar results were found with immuno-stained fixed cells. Cells treated with low concentrations of eribulin also showed decreased ability to migrate, suggesting the decrease in MT dynamics may have a downstream effect. Extended exposure of eribulin to cells leads to total depolymerization of the MT array. Taken together, these data show eribulin effectively disrupts EB1 +TIP complex formation, providing mechanistic insights into the impact of eribulin on MT dynamics.     

The inhibitory performance of flavonoid cyanidin-3-sambubiocide against H274Y mutation in H1N1 influenza virus

Oseltamivir (Tamiflu) is the most accepted antiviral drug that targets the neuraminidase (NA) protein to inhibit the viral release from the host cell. Few H1N1 influenza strains with the H274Y mutation creates drug resistance to oseltamivir. In this study, we report that flavonoid cyanidin-3-sambubiocide (C3S) compound acts as a potential inhibitor against H274Y mutation. The drug resistance mechanism and inhibitory activity of C3S and oseltamivir against wild-type (WT) and H274Y mutant-type (MT) have been studied and compared based on the results of molecular docking, molecular dynamics, and quantum chemical methods. Oseltamivir has been found less binding affinity with MT. C3S has more binding affinity with WT and MT proteins. From the dynamical study, the 150th loop of the MT protein has found more deformation than WT. A single H274Y mutation induces the conformational changes in the 150th loop which leads to produce more resistance to oseltamivir. The 150th cavity is more attractive target for C3S to stop the conformational changes in the MT, than 430th cavity of NA protein. The C3S is stabilized with MT by more number of hydrogen bonds than oseltamivir. The electrostatic interaction energy shows a stronger C3S binding with MT and this compound may be more effective against oseltamivir-resistant virus strains.     

Inhibition of phosphatidylcholine synthesis is not the primary pathway in hexadecylphosphocholine-induced apoptosis

The anticancer drug hexadecylphosphocholine (HePC), an alkyl-lysophospholipid analog (ALP), has been shown to induce apoptosis and inhibit the synthesis of phosphatidylcholine (PC) in a number of cell lines. We investigated whether inhibition of PC synthesis plays a major causative role in the induction of apoptosis by HePC. We therefore directly compared the apoptosis caused by HePC in CHO cells to the apoptotic process in CHO-MT58 cells, which contain a genetic defect in PC synthesis. HePC-provoked apoptosis was found to differ substantially from the apoptosis observed in MT58 cells, since it was (i) not accompanied by a large decrease in the amount of PC and diacylglycerol (DAG), (ii) not preceded by induction of the pro-apoptotic protein GADD153/CHOP, and (iii) not dependent on the synthesis of new proteins. Furthermore, lysoPC as well as lysophosphatidylethanolamine (lysoPE) could antagonize the apoptosis induced by HePC, whereas only lysoPC was able to rescue MT58 cells. HePC also induced a rapid externalisation of phosphatidylserine (PS). These observations suggest that inhibition of PC synthesis is not the primary pathway in HePC-induced apoptosis.     

Inhibition of Human Immunodeficiency Virus Type-1 (HIV-1) Replication by Immunor (Im28), a New Analog of Dehydroepiandrosterone

Abstract

The inhibition of' HIV-I replication in vitro by Immunor 28 (IM²⁸), an analog of dehydroepiandrosterone (DHEA), was monitored using the HIV- 1 laboratory wild-type strain IIIB. Evaluation of the 50% inhibitory dose (IC₅₀) revealed a decrease in HIV-1 replication giving an IC, value around 22 μM. The toxicity of the drug has been determined also, in MT2 cells and PBMCs. 60 μM of IM²⁸ provoked a 50% decrease in cell viability while DHEA caused the same decrease at 75 μM in MT2 cells. These values are 125 μM for IM²⁸ in PBMCs and 135 μM for DIIBA. Thus, DHEA is less toxic than IM²⁸, but IM²⁸ has a higher antiviral activity.     

Chemo-informatic design of antibiotic geldenamycin analogs to target stress proteins HSP90 of pathogenic protozoan parasites

Stress proteins HSP90 (Heat shock proteins) are essential molecular chaperones involved in signal transduction, cell cycle control, stress management, folding and degradation of proteins. HSP90 have been found in a variety of organisms including pathogens suggesting that they are ancient and conserved proteins. Here, using molecular modeling and docking protocols, antibiotic Geldenamycin and its analog are targeted to the HSP90 homolog proteins of pathogenic protozoans Plasmodium falciparum, Leishmania donovani, Trypanosoma brucei and Entamoeba Histolytica. The designed analogs of geldenamycin have shown drug like property with improved binding affinity to their targets. A decrease in insilico affinity of the analogs for the Human HSP90 target indicates that they can be used as potential drug candidates.     

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.     

Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa

Protozoa contain thymidylate synthase (TS) and dihydrofolate reductase (DHFR) on the same polypeptide. In the bifunctional protein, the DHFR domain is on the amino terminus, TS is on the carboxyl terminus, and the two domains are separated by a junction peptide of varying size depending on the source. The native protein is composed of a dimer of two such subunits and is 110-140 kDa. Most studies of the bifunctional TS-DHFR have been performed with the protein from anti-folate resistant strains of Leishmania major, which show amplification of the TS-DHFR gene and overproduction of the bifunctional protein. The Leishmania TS-DHFR has also been highly expressed in heterologous systems. There appears to be extensive communication among domains and channeling of the H2folate product of TS to DHFR. Anti-folates commonly used to treat microbial infections are poor inhibitors of L. major DHFR. However, selective inhibition of L. major vs. human DHFR does not appear difficult to achieve, and selective inhibitors are known. The TS-DHFR from Plasmodium falciparum has also been cloned and has recently been expressed in Escherichia coli, albeit in small amounts. Interestingly, pyrimethamine-resistant strains of P. falciparum all have a common point mutation in the DHFR coding sequence (Thr/Ser 108 to Asn), which causes decreased binding of the folate analog. It is suggested that if an appropriate inhibitor of the pyrimethamine-resistant P. falciparum DHFRs can be found, it may serve in combination with pyrimethamine as an antimalarial regimen with low propensity for the development of resistance. In the future, we project that we will have a detailed knowledge of the structure and function of TS-DHFRs, and have the essential tools necessary for a molecular-based approach to drug design.     

Elastokine-mediated up-regulation of MT1-MMP is triggered by nitric oxide in endothelial cells

Membrane-type I matrix metalloproteinase (MT1-MMP) has been previously reported to be up-regulated in human microvascular endothelial cell-1 line (HMEC) by elastin-derived peptides (elastokines). The aim of the present study was to identify the signaling pathways responsible for this effect. We showed that elastokines such as (VGVAPG)₃ peptide and kappa elastin induced nitric oxide (NO) production in a time-, concentration- and receptor-dependent manner as it could be abolished by lactose and a receptor-derived competitive peptide. As evidenced by the use of NO synthase inhibitors, elastokine-mediated up-regulation of MT1-MMP and pseudotube formation on Matrigel required NO production through activation of the PI₃-kinase/Akt/NO synthase and NO/cGMP/Erk1/2 pathways. Elastokines induced both PI₃-kinase p110γ sub-unit, Akt and Erk1/2 activation, as shown by a transient increase in phospho-Akt and phospho-Erk1/2, reaching a maximum after 5 and 15 min incubation, respectively. Inhibitors of PI₃-kinase and MEK1/2 suppressed elastokine-mediated MT1-MMP expression at both the mRNA and protein levels, and decreased the ability of elastokines to accelerate pseudotube formation. Besides, elastokines mediated a time- and concentration-dependent increase of cGMP, suggesting a link between NO and MT1-MMP expression. This was validated by the use of a guanylyl cyclase inhibitor, a NO donor and a cGMP analog. The guanylyl cyclase inhibitor abolished the stimulatory effect of elastokines on MT1-MMP expression. Inversely, the cGMP analog, mimicked the effect of both elastokines and NO donor in a concentration- and time-dependent manner. Overall, our results demonstrated that such elastokine properties through NO and MT1-MMP may be of importance in the context of tumour progression.     

Cloning,characterization and preliminary crystallographic analysis of Leishmania hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of nucleotides; therefore, this pathway is an attractive target for antiparasitic drug design. The hgprt gene was cloned from a Leishmania tarentolae genomic library and the sequence determined. The L. tarentolae hgprt gene contains a 633-nucleotide open reading frame that encodes a 23.4-kDa protein. A pairwise alignment of the different HGPRT's sequences revealed a 26%-53% sequence identity with the Leishmania sequences and 87% identity to the HGPRT of Leishmania donovani. A recombinant protein was expressed in Escherichia coli, purified to homogeneity and found to retain enzymatic activity. The steady-state kinetic parameters were determined for the recombinant enzyme and the enzyme is active as a homodimer in solution. Single crystals were obtained for the L. tarentolae HGPRT representing the first Leishmania HGPRT crystallized and initial crystallographic data were collected. The crystals obtained belong to the orthorhombic space group (P2(1)2(1)2(1)) with unit cell parameters a=58.104 A, b=85.443 A and c=87.598 A and diffract to a resolution of 2.3 A. The availability of the HGPRT enzyme from Leishmania and its crystallization suitable for X-ray diffraction data collection should provide the basis for a functional and structural analysis of this enzyme, which has been proposed as a potential target for rational drug design, in a Leishmania model system.     

Preparation and functional analysis of recombinant protein transduction domain-metallothionein fusion proteins

In order for proteins to be used as pharmaceuticals, delivery technologies need to be developed to overcome biochemical and anatomical barriers to protein drug transport, to protect proteins from systemic degradation, and to target the drug action to specific sites. Protein transduction domains (PTDs) are used for the non-specific transduction of bio-active cargo, such as proteins, genes, and particles, through cellular membranes to overcome biological barriers. Metallothionein (MT) is a low molecular weight intra-cellular protein that consists of 61 amino acids, including 20 cysteine residues, and is over-expressed under stressful conditions. Although MT has the potential to improve the viability of islet cells and cardiomyocytes by inhibiting diabetic-induced apoptosis and by removing reactive oxygen species (ROS), and thereby prevent or reduce diabetes and diabetic complications, all MT applications have been made for gene therapy or under induced over-expression of endogenous MT. To overcome the drawbacks of ineffective intra-cellular MT protein uptake, a human MT gene was cloned and fused with protein transduction domains (PTDs), such as HIV-1 Tat and undeca-arginine, in a bacterial expression vector to produce PTD–MT fusion proteins. The expression and purification of three types of proteins were optimized by adding Zn ions to maintain their stability and functionality mimicking intra-cellular stable conformation of MT as a Zn–MT cluster. The Zn–MT cluster showed better stability than MT in vitro. PTD–MT fusion proteins strongly protected Ins-1 beta cells against oxidative stress and apoptosis induced by glucolipotoxicity with or without hypoxia, and also protected H9c2 cardiomyocytes against hyperglycemia-induced apoptosis with or without hypoxia. PTD–MT recombinant fusion proteins may be useful protein therapeutics for the treatment or prevention of diabetes and diabetes-related complications.     

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability.     

Synthesis and biological evaluation of indolyl glyoxylamides as a new class of antileishmanial agents

A series of indolylglyoxylamide derivatives have been synthesized and evaluated in vitro against amastigote form of Leishmania donovani. Compound 8c has been identified as the most active analog of the series with IC₅₀ value of 5.17 μM and SI value of 31.48, and is several folds more potent than the standard drugs sodium stilbogluconate and pentamidine.     

Mitochondrial damage contribute to epigallocatechin-3-gallate induced death in Leishmania amazonensis

Epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, has been reported to have antiproliferative effects on Trypanosoma cruzi however, the mechanism of protozoan action of EGCG has not been studied. In the present study, we demonstrate the mechanism for the antileishmanial activity of EGCG against Leishmania amazonensis promastigotes. Incubation with EGCG significantly inhibited L. amazonensis promastigote proliferation in a time- and dose-dependent manner. The IC(50) for EGCG at 120h was 0.063mM. Ultrastructural alterations of the mitochondria were observed in promastigote treated with EGCG, being the organelle injury reinforced by the decrease in rhodamine 123 fluorescence. The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. These data suggest mitochondrial collapse as a part of the EGCG mechanism of action and demonstrate the leishmanicidal effect of EGCG.     

Increased metallothionein gene expression in 5-aza-2'-deoxycytidine-induced resistance to cadmium cytotoxicity

The pyrimidine analog, 5-azacytidine (AZA-CR), has been shown to increase the expression of the metallothionein (MT) gene and to induce tolerance to cadmium toxicity. Since incorporation into DNA of AZA-CR appears to be required for this effect, the deoxynucleoside of AZA-CR should also be effective. Therefore, this study was undertaken to assess the effect of 5-aza-2'-deoxycytidine (AZA-CdR) pretreatment on cadmium-induced cytotoxicity and MT expression in cultured cells. TRL 1215 cells in log phase of growth were exposed to AZA-CdR (0.4, 0.8, 4.0, 8.0 microM) followed 48 h later by the addition of cadmium (10 microM). MT concentrations were measured 24 h after the addition of cadmium. AZA-CdR alone caused modest, dose-related increases in MT levels (2.3-fold maximum), while cadmium alone resulted in a 9.5-fold increase. Pretreatment with AZA-CdR in combination with cadmium caused a 19--24-fold increase in cellular MT at all doses of AZA-CdR. Addition of the DNA synthesis inhibitor, hydroxyurea (HU), to the incubation medium during AZA-CdR exposure prevented the enhancing effect of the analog on cadmium induction of MT accumulation. Time course studies revealed that AZA-CdR pretreatment reduced the time required for cadmium to induce MT levels from 4--8 h to 0--2 h. AZA-CdR pretreated cells placed in suspension with cadmium (125 microM) showed a marked reduction in cadmium-induced cytotoxicity as reflected by reduced glutamic-oxaloacetic transaminase (GOT) loss. Uptake studies showed that AZA-CdR pretreatment had no effect on cadmium transport during the initial phases of exposure, indicating that an alteration in the toxicokinetics of the metal did not account for the reduction in toxicity. AZA-CdR did, however, cause hypomethylation of the MT-I gene. These results suggest that AZA-CdR pretreatment induces tolerance to cadmium toxicity by increasing the genetic expression of MT possibly through hypomethylation of the MT gene.     

The NAD+ metabolism of Leishmania, notably the enzyme nicotinamidase involved in NAD+ salvage, offers prospects for development of anti-parasite chemotherapy

NAD+ plays multiple, essential roles in the cell. As a cofactor in many redox reactions it is key in the cellular energy metabolism and as a substrate it participates in many reactions leading to a variety of covalent modifications of enzymes with major roles in regulation of expression and metabolism. Cells may have the ability to produce this metabolite either via alternative de novo synthesis pathways and/or by different salvage pathways. In this issue of Molecular Microbiology, Gazanion et al. (2011) demonstrate that Leishmania species can only rely on the salvage of NAD+ building blocks. One of the enzymes involved, nicotinamidase, is absent from human cells. The enzyme is important for growth of Leishmania infantum and essential for establishing an infection. The crystal structure of the parasite protein has been solved and shows prospects for design of inhibitors to be used as leads for development of new drugs. Indeed, NAD+ metabolism is currently being considered as a promising drug target in various diseases and the vulnerability of Leishmania for interference of this metabolism has been proved in previous work by the same group, by showing that administration of NAD+ precursors has detrimental effect on the pathogenic, amastigote stage of this parasite.     

LD1 amplifications in Leishmania

The genome of Leishmania is quite plastic. Chromosomal rearrangements and DNA amplifications are common events in all the species of the genus. Gene amplification occurs both as a mechanism of drug resistance and in the absence of drug pressure. The best known spontaneous amplification in Leishmania is the so-called LD1 family of amplicons. In the past few years there have been great advances in our knowledge of LD1 elements; here, Manuel Segovia and Ginés Ortiz review all the available data.     

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.