Synthesis and structure-activity relationships of new quinolone-type molecules against Trypanosoma brucei.

Human African trypanosomiasis (HAT) or sleeping sickness is caused by two subspecies of Trypanosoma brucei , Trypanosoma brucei gambiense , and Trypanosoma brucei rhodesiense and is one of Africa's old plagues. It causes a huge number of infections and cases of death per year because, apart from limited access to health services, only inefficient chemotherapy is available. Since it was reported that quinolones such as ciprofloxacin show antitrypanosomal activity, a novel quinolone-type library was synthesized and tested. The biological evaluation illustrated that 4-quinolones with a benzylamide function in position 3 and cyclic or acyclic amines in position 7 exhibit high antitrypanosomal activity. Structure-activity relationships (SAR) are established to identify essential structural elements. This analysis led to lead structure 29, which exhibits promising in vitro activity against T. b. brucei (IC(50) = 47 nM) and T. b. rhodesiense (IC(50) = 9 nM) combined with low cytotoxicity against macrophages J774.1. Screening for morphological changes of trypanosomes treated with compounds 19 and 29 suggested differences in the morphology of mitochondria of treated cells compared to those of untreated cells. Segregation of the kinetoplast is hampered in trypanosomes treated with these compounds; however, topoisomerase II is probably not the main drug target.

Effect of dibutyltin(IV) on the ultrastructure of African Trypanosoma spp.

Diorganotins (R2SnX2) are compounds with a wide variety of biological properties. In an attempt to follow the morphological events and to characterize the toxic effects of diorganotins on in vitro cultured African Trypanosoma spp., the ultrastructural alterations induced on the parasites by dibutyltins (Bu2SnX2) were followed. The data obtained indicate that these compounds induced irreparable damage to the in vitro cultured bloodstream forms of the parasites. Transmission and scanning electron microscopy allowed observations on the perturbation of the kinetoplast, extensive cytoplasmic swellings, disconfiguration around the flagellar pocket and membrane disintegration. Fluorescence microscopy with 4,6-diamidine-2-phenylindole stain was also used to visualize the survival or degeneration of kDNA. Understanding the collateral cellular toxic effect of these compounds on the parasites may shed light on the possible mechanism by which they kill trypanosomes. Agarose gel electrophoresis resolution of isolated kDNAs revealed no fragmentation by these compounds following in vitro incubation at 37 degrees C. However, fragmentation was observed from the gel electrophoresis of kDNA isolated from in vitro cultured Bu2SnX2-exposed parasites. Transmission electron microscopy of the kDNAs revealed the same pattern as observed with gel electrophoresis. These results provide evidence for the possible involvement of the Bu2Sn moiety in the in vivo-induced fragmentation of trypanosomal kDNA and consequent trypanolysis. This observation also underlies the relevance of organometallics in the therapy of African trypanosomiasis.

In vitro trypanocidal activity of dibutyltin dichloride and its fatty acid derivatives.

Searching for new compounds against pathogenic trypanosomes has been substantially accelerated by the development of in vitro screening assays. In an attempt to explore the chemotherapeutic potential of organotin compounds and to broaden the search for newer trypanocides, fatty acid derivatives of dibutyltin dichloride were synthesized and their in vitro trypanocidal profiles studied on Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. A 24-h time course experiment was conducted with various concentrations of the compounds using a 24-well microtiter plate technique. The compounds tested were trypanocidal in a dose-dependent fashion: inhibiting survival and growth, resulting in irreversible morphological deformation and the eventual death of the parasites. The minimum inhibitory concentrations of the tested diorganotins are at low micromolar ranges: from 0.15-0.75 microM for T. b. brucei, T. b. gambiense and T. b. rhodesiense. These observations suggest that organotin has chemotherapeutic potential.

A proposed density-dependent model of long slender to short stumpy transformation in the African trypanosomes.

A simple arithmetic model is developed that is based upon the assumption: (1) that transformation of replicating long slender Trypanosoma brucei to nonreplicating short stumpy forms is parasite population density dependent; (2) that as the slender population increases there is a change in the external environment that triggers the slender to stumpy transformation; and (3) that stumpy forms of T. brucei do not induce the change in external environment that triggers slender to stumpy transformation or do so to a lesser extent than slender forms, thus preventing the proportion of stumpy forms in a population from reaching 100%. A simulation based on these assumptions shared many features with curves on numbers of long slender, intermediate, and short-stumpy forms of T. brucei during the first parasitemic wave of the 3 T. brucei subspecies in intact and immunosuppressed inbred mice.

Processing and transport of a lysosomal membrane glycoprotein is developmentally regulated in African trypanosomes.

We have used pulse-chase immunoprecipitations methods to study early post-translational processing of CBI-gp, a lysosomal membrane glycoprotein expressed by African trypanosomes, Rap67, a polyclonal antibody to CBI-gp, immunoprecipitated a 100-kDa glycoprotein, gp100, from both bloodstream forms (BF) and procyclic forms (PF) of Trypanosoma brucei gambiense immediately after a 5-min pulse with radiomethionine. N-Glycanase digestion released a 67-kDa core protein, p67, from gp100 of both life cycle forms V8 protease digestion of p67 from BF and PF yielded 13 identical methionyl peptides, suggesting that gp100 from both life cycle forms have very similar or identical p67 core molecules. In BF, gp 100 carried both endoglycosidase H (EndoH)-resistant and EndoH-sensitive, N-linked oligosaccharides immediately after labeling. In PF, all the N-linked sugars on gp100 were EndoH sensitive. In BF, gp100 chased progressively into slower migrating 150-180-kDa components that obtained the CBI epitope, traveled to the cell surface where they could be biotinylated, and were proteolytically processed. The increase in mass of gp100 during chase in BF resulted from an elongation of N-linked oligosaccharides. Maturation of gp100 into 150-180-kDa CBI-gp was inhibited if BF were chased in the presence of glucosidase inhibitors castanospermine or deoxynojirimycin. In PF, gp100 did not increase in mass, could not be biotinylated on the cell surface, and was not proetolyzed during extended chases. Cryoimmunoelectron microscopy revealed that the antigens detected by rap67 are abundant in lysosomes and endosomes in both BF and PF. Thus, BF and PF express very similar or identical lysosomal membrane glycoproteins but process and transport them in very different ways.

Trypanosoma brucei brucei and T. b. gambiense: stumpy bloodstream forms express more CB1 epitope in endosomes and lysosomes than slender forms.

CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.

Distribution of filipin-sterol complexes in the bloodstream form of Trypanosoma brucei gambiense.

The polyene antibiotic, filipin, was used as a probe for demonstrating sterols in the plasma membrane of Trypanosoma brucei gambiense. Three different regions of the continuous plasma membrane over the cell body proper, flagellar pocket, and flagellum, were compared as to density and distribution of the filipin-sterol complexes by freeze-fracture method. The density of the complexes was highest in the cell body membrane and lowest over the flagellar pocket. The filipin-sterol complexes in the cell body membrane were distributed homogeneously on both the protoplasmic and exoplasmic faces except in the zone of flagellar attachment. This junctional zone showed a linear, complex-poor region. In the flagellar membranes, a line of complex-poor regions was observed along the juncture of the flagellum to the cell body. At the neck of the flagellar pocket, the membranes of the flagellar pocket and flagellum were closely opposed, with few filipin-sterol complexes on either membrane. At the base of the flagellar shaft, the complexes were completely lacking on both faces. Based on these observations, the zones of juncture observed in T.b. gambiense seems to be similar in ultrastructure to mammalian cell junctions, such as tight, gap, septate junctions and desmosomes, which show a nearly complete absence of the filipin-sterol complexes.

Freeze-fracture study of the bloodstream form of Trypanosoma brucei gambiense.

The ultrastructure of Trypanosoma brucei gambiense was investigated by the freeze-fracture method. Three different regions of the continuous plasma membrane; cell body proper, flagellar pocket, and flagellum were compared in density and distribution of the intramembranous particles (IMP's). The IMP-density was highest in the flagellar pocket membrane and lowest in flagellum. Intra membranous particles of the cell body membrane were distributed uniformly on both the protoplasmic (P) and exoplasmic (E) faces. On the P face of the flagellar membrane, a single row of IMP-clusters was seen along the juncture of the flagellum to the cell body. Since the spacing of the IMP-clusters was almost equal to the spacing of the paired rivet structures observed in thin section, these clusters likely are related to the junction of flagellum and cell body. At the neck of the flagellar pocket, several linear arrays of IMP's were found on the P face of the flagellar membrane, while on the E face rows of depressions were seen. At the flagellar base, the clusters of IMP's were only seen on the P face. On the flagellar pocket membrane, particle-rich depressions and linear particle arrays were also found on the P face, while on the E face such special particle arrangements were not recognized. These particle-rich depressions may correspond to the sites of pinocytosis of the bloodstream forms which have been demonstrated in thin sections.

Long-term culture and cloning system for Trypanosoma brucei gambiense bloodstream forms in semi-defined medium in vitro.

A new semi-defined medium extremely useful for the long-term cultivation and cloning of Trypanosoma b. gambiense (Wellcome strain) bloodstream forms is described. Bloodstream forms could be continuously grown in 25 mM HEPES-buffered D-MEM supplemented with 10 microM bathocuproine sulfonate (BCS), 100 microM cysteine, and 20% heat-inactivated fetal calf serum at 37 degrees C in vitro. Under these culture conditions, T. b. gambiense bloodstream forms increased in number up to 2-3 x 10(6) trypanosomes/ml by day 3 after initiation of the culture. The trypanosomes maintained in this culture system for 200 days retained their infectivity for mice. Morphologically, they were long and slender, and a surface coat was evident on the cell surface and flagellar membrane. In vitro cloning with single bloodstream forms of T. b. gambiense could be achieved with high efficiency.

Trypanosoma brucei gambiense and T. b. rhodesiense: concanavalin A binding to the membrane and flagellar pocket of bloodstream and procyclic forms.

We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3-16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed greater than 95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.

Trypsin-stimulated transformation of Trypanosoma brucei gambiense bloodstream forms to procyclic forms in vitro.

The effect of trypsin treatment on the transformation of monomorphic Trypanosoma b. gambiense (Wellcome strain) bloodstream forms to procyclic forms was studied in HEPES-buffered RPMI 1640 medium supplemented with 20% inactivated fetal calf serum in the presence of GA-1 cells as feeder layers at 27 degrees C. In this system, 35%-40% of the bloodstream forms transformed to procyclic forms within 24 h, and over 95% of the trypanosomes changed into procyclic forms by day 3 after initiation of the culture. Established cultures of procyclic forms yielded up to 1.5-2 x 10(7) trypanosomes/ml. However, transformation of nontreated and inhibited trypsin-treated bloodstream forms were prolonged compared to trypsin-treated populations. In this experiment, the first procyclic forms could be detected on day 7 after initiation of the culture and transformation was complete within 15 days. The transformation of T. b. gambiense from bloodstream to procyclic forms required the living GA-1 cells as feeder layer cells, but established cultures of procyclic forms could be maintained in the culture medium without feeder cells for more than 300 days.

In vivo interaction between Trypanosoma gambiense and leucocytes in mice.

Naturally occurring phagocytosis of Trypanosoma gambiense by mouse eosinophils and neutrophils was reported. In vivo and in vitro experiments using monoclonal antibodies confirmed that the phagocytosis is triggered by G1 class antibodies against variable surface antigen. Ultrastructural observation revealed the mode of entry and the intracellular fate of T. gambiense: initial attachment, pseudopodia formation and complete invagination. This phagocytosis resulted in the killing of T. gambiense by mouse eosinophils and neutrophils, suggesting that eosinophils and neutrophils give at least partial protection against infection with T. gambiense in combination with the specific antibodies.

Possible conversion of axonemal microtubules to pellicular microtubules in Trypanosoma gambiense treated with vinblastine, colchicine plus concanavalin A.

The effects of vinblastine alone and in combination with vinblastine, colchicine and concanavalin A on microtubules of Trypanosoma gambiense cultured in vitro were studied ultrastructurally. Trypanosomes treated with vinblastine at 20 micrograms/ml, showed fusion of the extracellular flagellum with the plasma membrane of the parasite. As a result, the axoneme with the paraxial rod in the extracellular flagellum was taken into the cytoplasm. Although the axonemal and pellicular microtubules in T. gambiense differ in function and origin, the axonemal microtubules of the extracellular flagellum that was taken into the cytoplasm could be converted to pellicular microtubules by treatment with a combination of vinblastine (20 micrograms/ml), concanavalin A (10 micrograms/ml) and colchicine (100 micrograms/ml).

The successful system in long-term cultivation of Trypanosoma gambiense bloodstream forms.

Rat astroglioma cell line (GA-1) was extremely useful for long-term in vitro cultivation of Trypanosoma gambiense blood-stream forms. Parasites could be continuously grown at 37 degrees C for more than 200 days in the culture system, consisted of HEPES-buffered RPMI 1640 (pH 7.2, 300 milliosmole/kg) supplemented with 20% inactivated fetal calf serum in the presence of GA-1 cells. Parasites cultured for more than 200 days still retained not only their virulence for mice but also their original antigenic type. Morphologically, they resembled host infected bloodstream forms by way of having a subterminal kinetoplast and surface coat. The best growth rate of trypanosomes was obtained with 1 X 10(6) GA-1 cells/25 cm2 culture flask. Under this culture condition trypomastigote form populations increased in number up to 7-8 X 10(6) trypanosomes/ml by day 3 after initiation of the culture. The population doubling time in this culture system within the first 24 hours was almost the same as in mice. Most of the cultured trypanosomes were in suspension, but 15-20% of the parasites adhered to the surface of GA-1 cells. The culture system was also shown to be useful for cloning of T. gambiense which is important for separation of mutants.

Electron-microscopic localization of Trypanosoma brucei gambiense transmitted by Glossina morsitans centralis in Microtus montanus.

In Microtus montanus infected with T. b. gambiense, electron microscopic examination of lymph nodes, spleen, liver, heart, choroid plexus and brain demonstrated extravascular populations of trypanosomes distributed throughout interstitial spaces, accompanied by a moderate cellular infiltration of plasma cells. The trypanosomes exhibited numerous profiles; some were dividing, others were in different stages of lysis, or phagocytosed. Penetration of trypanosomes into hepatocytes was observed. The present investigation indicated that trypanosomes migrated to the brain parenchyma from the Virchow-Robin spaces but could not confirm whether the parasites reached the Virchow-Robin spaces by traversing the ependymal cells lining the choroid plexus or by migrating through the endothelial cells of the cerebral blood vessels.

The kinetoplast of DNA of Trypanosoma gambiense: comparison with the kDNA of Trypanosoma equiperdum.

The molecular components of the kinetoplast DNA (kDNA) network of Trypanosoma gambiense have been studied and compared with those of the very closely related species T. equiperdum, previously studied in detail. The kDNA of T. gambiense contains about 80 maxicircles of 20 kilobase pairs and 4000 minicircles of 1 kilobase pairs. The restriction cleavage sites of 7 restriction endonucleases have been mapped on the T. gambiense maxicircle. The majority of these sites were also found in T. equiperdum maxicircles; however their relative positions which are different do not allow us to conclude to relatedness of maps. Maxicircles of the Cairn or of the rolling circle type have been observed and thought to be replicative intermediates. Experiments on renaturation kinetics and hybridization after blotting transfer, show that T. gambiense and T. equiperdum maxicircles have base sequences in common. The T. gambiense minicircles are heterogeneous in base sequence, in contrast to the T. equiperdum minicircles which are homogeneous. The minicircles of the two species have also common base sequences.

Transmission and scanning electron microscopic studies of the micronemata of Trypanosoma gambiense.

The structure of micronemata arising from the surface of the bloodstream form of Trypanosoma gambiense was studied by electron microscopy. In order to produce micronemata, trypanosomes were incubated in either 1) phosphate buffered saline supplemented with glucose (PBSG), 2) immune mouse serum or 3) PBSG after passage through a DEAE-cellulose column. Electron microscopic examination of the parasite revealed the presence of thread-like micronemata arising from the anterior end and from the flagellar pocket regardless of the incubation conditions. Negative staining revealed a distinct peripheral fringe layer with nodular protrusions covering the entire surface of the micronema. The distribution and number of intramembrane particles (IMP) on the P and E faces of the micronema were similar to those of the flagellum of T. gambiense, indicating a close relationship between the membrane structure of the micronema and the flagellum. Micronemata became fragmented and adhered to each other after incubation of the parasite in the media for 12 h. Since micronemata tend to have the characteristics of adhesiveness and fragmentation, fragments of these structures might adhere to various host organs. Dispersal of potential antigenic material might be responsible, in part, for the induction of the host immune response.