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Background: In search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery., Methods: A new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm., Results: We have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 μΜ. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial kinases was subsequently undertaken to explain and rationalize the selectivity trend determined by the in vitro binding assays. Interestingly, the latter analysis showed that selectivity could be correlated with differences in kinase solvation thermo dynamics induced by minor sequence variations of the otherwise highly similar ATP binding pockets., Conclusions: In conclusion, 3'-bulky amino substituted 6-BIO derivatives, which demonstrate enhanced specificity towards LGSK-3, represent a new scaffold for targeted drug development to treat leishmaniasis.

Apoptosis is a regulated process of cell death originally described in multicelullar organisms contributing to their development and functionality. There is now increasing experimental evidence that a similar form of cell death is operative in unicellular eukaryotes, including trypanosomatids of the genera Trypanosoma and Leishmania. The determination of ancestral executors and regulators of 'apoptosis' in these protozoa belonging to the most primitive eukaryotes that appeared on earth 1.5 billion years ago, provide an exciting challenge in the understanding of the evolution of apoptosis-regulating processes. A review of the present knowledge of trypanosomatid apoptosis points to the fact that these dying protozoa acquire common apoptotic morphological features as metazoan cells, although they lack many of the molecules accepted today as canonical apoptosis mediators (Bcl-2 family members, caspases, TNF related family of receptors). Herein, we discuss how the knowledge of regulators and executors of trypanosomatid apoptosis may provide answers to the gaps concerning the origin of apoptosis. The aim of this addendum is to emphasize the need for classifying the ancestral death program and to discuss how this relates to the complex death programs in multicellular lineages, with the hope to stimulate further enquiry and research into this area.

Episomal expression of the major surface glycoprotein (gp63) sense and antisense mRNAs in Leishmania amazonensis was found previously to modulate the expression of this molecule as well as its infection of macrophages in vitro. Here, we evaluated the in vivo infectivity of these transfectants in BALB/c mice. Antisense downregulation of gp63 renders this parasite sensitive to complement-mediated lysis and less infective to mice, as indicated by a delay in lesion development and a significant reduction in lesion size and parasite loads at the site of inoculation and in the draining lymph nodes (DLNs). CD4+ cells at the site of inoculation decreased in number more rapidly and were 2-fold less numerous than those in controls by week 4. The number of IFN-gamma-positive cells was higher, while IL-10 positive cells were undetectable. In DLNs, CD4+ cells were higher in number, and the profile of cytokine-positive cells followed essentially the same patterns--found at the site of inoculation. These results suggest that the downregulation of gp63 increases extracellular lysis of the mutants by complement, in the in vivo environment, and reduces their infection of macrophages, resulting in a type 1 immune response seen at the site of inoculation and DLNs.

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.

The antigenic sequence Ac-IASRYDQL (gp63-SRYD) of the major surface glycoprotein of Leishmania, gp63, was covalently attached to the Lys-N epsilon H2 groups of a new sequential oligopeptide carrier (SOCn), namely, (Lys-Aib-Gly)n (n = 5.6), in order to obtain potent immunogens and site-specific antibodies. It was shown, using 1H-NMR spectroscopy, that the gp63-SRYD octapeptides bound to the SOCn retain their original structural profile outlined by an ionic interaction between R and D side chains and a type 1 beta-turn involving the QNH-->RCO hydrogen bonding. Also, the gp63-SRYD octapeptides linked to the carrier do not experience conformational restrictions, probably because of the favorable conformation of the SOCn. Immunizations of outbred rabbits with the peptide carriers designed resulted in high-titered antibody response to the gp63-SRYD octapeptide and the gp63 cognate protein. Thus, this chemically defined model may be used for incorporating "protective" Leishmania epitopes and ultimately for the design of a multivalent synthetic vaccine against leishmaniosis.

In this study, we have surveyed gp63 expression in sinefungin-(SF)-resistant and wild-type Leishmania promastigotes. Documentation of gp63 expression in Leishmania promastigotes was carried out by Western blotting, purification of the protein and assessment of gp63 protease activity. We demonstrated a 3-4-fold and 1.5-2-fold increase of gp63 protein in SF-resistant Leishmania donovani and Leishmania tropica promastigotes compared to wild-type, respectively. Northern blot analysis showed that the increase in the amount of gp63 protein in SF-resistant compared to wild-type parasites was concomitant with an increase in gp63 mRNA. No extrachromosomal DNA was identified by alkaline lysis of isolated DNA samples and Southern blot analysis. Treatment of SF-resistant and wild-type L. donovani promastigotes with cycloheximide resulted in an increase of the steady state levels of gp63 mRNA in the SF-resistant parasites to approximately fivefold that of the wild type. After treating parasites with actinomycin D, estimated gp63 mRNA t1/2 in the wild type was 40 min and increased to 83 min in SF-resistant promastigotes. Therefore, the overexpression of gp63 may be mediated, at least in part, by post-transcriptional stabilization of a gp63 transcript by a protein factor. Down regulation of the latter factor may account for the observed increase in gp63 expression in SF-resistant promastigotes. Attempts to correlate gp63 expression with promastigote virulence suggested that the observed increase in gp63 expression did not result in a significant change in the virulence of SF-resistant compared to wild-type L. donovani promastigotes.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.

In a previous report, we have presented several lines of evidence, derived from widely different methodologies, suggesting that Leishmania has specific receptors for transferrin with a Kd similar to the mammalian transferrin receptor. This paper describes the identification, purification, and biochemical characterization of Leishmania transferrin receptor. The Leishmania transferrin receptor, detected on intact parasites by immunoperoxidase staining, was first identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis, using 125I-transferrin, as a 70-kDa protein. It has been isolated initially from Leishmania infantum promastigotes using affinity chromatography on a transferrin-Sepharose column and, subsequently, from Leishmania major promastigotes. The use of polyclonal antisera to the purified 70-kDa Leishmania transferrin receptor and to the purified rat transferrin receptor showed that the two receptors are antigenically distinct. The 70-kDa Leishmania transferrin receptor was subsequently characterized as an integral membrane glycoprotein. The monomeric state of the Leishmania transferrin receptor was demonstrated by gel filtration of purified receptor complexed with 125I-transferrin. Thus, the Leishmania transferrin receptor, unlike the mammalian receptor, is not a disulfide-linked dimer but a single 70-kDa polypeptide.

The major surface glycoprotein of Leishmania, gp63, is a membrane-bound metalloprotease. Contradictory data supporting a neutral or acidic nature of this enzyme have been presented. Seven strains of Old and New World Leishmania, including Leishmania donovani complex (Leishmania infantum and L. donovani), Leishmania major, Leishmania tropica and Leishmania mexicana amazonensis were used for the purification and comparative study of gp63. The protein was extracted from promastigotes by phase separation in Triton X-114 and purified by anion exchange chromatography. In agreement with previous reports, all purified gp63 were found to be structurally and immunologically related. Both membrane-bound gp63, on the surface of promastigotes, and the purified proteases had optimal activity at neutral to alkaline pH on azocasein, whereas their activity was optimal at acidic to neutral pH against 125I-insulin B-chain. The IC50 concentrations of 1,10-phenathroline against the two substrates, at the optimal pH, were comparable, suggesting that both activities measured were associated with gp63 rather than another contaminating enzyme. This was further supported by the comparable enrichment values, estimated from the specific activity of the enzyme during purification, using both assays. These results explain the earlier apparent discrepancies and suggest that the optimum pH of gp63 is substrate-dependent and not related to species differences or to the different purification procedures applied.

Ten monoclonal antibodies (MAbs) produced against isolated Leishmania infantum membranes were used as probes of L. infantum membrane antigens. Western blots of L. infantum membranes, sodium dodecyl sulfate solubilized and heated at 100 degrees C before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that all 10 MAbs recognized a band at 58 kilodaltons (kDa). However, when solubilized membranes were not heated, 2 of the 10 MAbs recognized, in addition to the 58-kDa band, bands of higher molecular weight. Limited digestion of heated or nonheated membranes showed that both groups of MAbs (i.e., not capable or capable of binding to the high-molecular-weight bands) recognized the same proteolytic digests. Hydrophilic forms of the above proteins, possessing proteolytic activity, were detected and isolated by gel filtration. Protein staining of the isolated monomer analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing and heating conditions, revealed incomplete reduction of the 58-kDa protein. The reduced form of the 58-kDa protein migrated at 63 to 65 kDa and was not recognized by the MAbs. These results suggest the existence of a monomeric and an oligomeric form of the 58-kDa antigen. The observed inhibition of Leishmania promastigote-macrophage binding caused by MAbs representative of the two groups (capable of oligomeric and/or monomeric antigen recognition) suggest that the 58-kDa monomer and oligomer play an important role in promastigote-macrophage interaction. We suggest that the 58-kDa L. infantum antigen is the major surface Leishmania antigen (p63) identified by others.