Cellular effects of leishmanial tubulin inhibitors on L. donovani.

To aid our investigation of tubulin as an antileishmanial drug target, the effects of the mammalian antimicrotubule agents ansamitocin P3, taxol, and hemiasterlin on Leishmania donovani promastigotes were described. These drugs affected the assembly of purified leishmanial tubulin and inhibited the growth of L. donovani promastigotes at micromolar concentrations. When promastigotes were treated with these agents, mitotic partitioning of nuclear DNA and cytokinesis were usually inhibited. The spatial orientation of kinetoplasts was often disturbed, suggesting a role for microtubules in the segregation of these organelles during mitosis. Aberrant cell types produced in drug-treated cultures included parasites with one nucleus and two geometrically distinct kinetoplasts, parasites with multiple kinetoplasts, and cytoplasts containing a kinetoplast but no nucleus. A subset of unique cell types, parasites containing two nuclei, a spindle fiber, and two geometrically distinct kinetoplasts, were observed in hemiasterlin-treated cultures. Flow cytometric analysis of L. donovani promastigotes treated with these three drugs indicated a dramatic shift toward the G2 + M phase of the cell cycle, with some cells containing four times the amount of DNA present in G1. These results were used to evaluate the cellular effects of WR85915, an aromatic thiocyanate with in vitro antileishmanial and anti-tubulin activity, on L. donovani. Treatment of parasites with WR85915 did not produce the unusual cell types described above and did not cause the accumulation of parasites in G2 + M, suggesting that WR85915 acts on target(s) in Leishmania in addition to tubulin. These studies validate tubulin as a suitable antileishmanial drug target and provide criteria to assess the cellular mechanism of action of new candidate antileishmanial agents.

Analysis of stereoelectronic properties of camptothecin analogues in relation to biological activity.

Camptothecin and four of its 10,11-methylenedioxy analogues were examined for their activity against the pathogenic protozoan Leishmania donovani in vitro. The methylenedioxy analogues were 36- to 180-fold more potent than the parent camptothecin, possessing IC50 values ranging from 160 to 32 nM against the parasite. Our finding that the methylenedioxy camptothecins possess greater activity than camptothecin, which is also the case for other cell types and for the generation of cleavable complex in the presence of DNA and purified mammalian topoisomerase I, prompted us to examine the molecular features of camptothecin and methylenedioxy camptothecin analogues. A delocalization of positive potential was observed in the methylenedioxy camptothecin analogues, which could increase the affinity of these molecules for DNA. In addition, geometrical and electronic differences between the E ring of camptothecin and its methylenedioxy analogues were noted. One or both of these factors may contribute to the superior biological activity of the methylenedioxy camptothecin analogues.

Purification, characterization, and drug susceptibility of tubulin from Leishmania.

Past work suggests that tubulin from kinetoplastid parasites may present an excellent drug target. To explore this possibility, tubulin was purified on a milligram scale from Leishmania mexicana amazonensis promastigotes by sonication, DEAE-Sepharose chromatography, and one cycle of assembly-disassembly. Purified leishmanial tubulin is recognized by commercially available anti-tubulin antibodies and displays concentration dependent assembly in vitro. The vinca site agents vinblastine, maytansine, and rhizoxin bind to leishmanial tubulin as assessed by the quenching of intrinsic tubulin fluorescence and the alteration of the proteins reactivity with the sulfhydryl-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid). They also interfere with the assembly of leishmanial tubulin at low micromolar concentrations. Electrophilic compounds such as phenyl arsenoxide and 4-chloro-3,5-dinitro-alpha,alpha,alpha-trifluorotoluene (chloralin), which are of interest as traditional and experimental antiparasitic agents, respectively, inhibit the assembly of leishmanial tubulin in vitro as well. Colchicine-site agents and trifluralin, on the other hand, have little or no effect on leishmanial tubulin in these assays. Maytansine, taxol, and the electrophiles block the growth of Leishmania donovani amastigote-like forms in vitro at low ( <1 microM) concentrations, while colchicine site agents, trifluralin, vinblastine, and rhizoxin are at least two orders of magnitude less toxic to the parasite.

Synthesis and biological evaluation of 4-chloro-3, 5-dinitrobenzotrifluoride analogues as antileishmanial agents.

Desnitro analogues of 4-chloro-3,5-dinitrobenzotrifluoride (chloralin) (2), an in vitro microtubule inhibitor of several Leishmania species, have been synthesized from 2-halo-5-(trifluoromethyl)benzenesulfonyl chlorides 4 and 5. The analogues exhibited moderate to excellent activity when tested against Leishmania donovani amastigotes in vitro. Two representative compounds, 7f and 8, were tested against the Khartoum strain of L. donovani in a hamster model using chloralin (2) and Glucantime (one of the current therapeutics of choice in the treatment of Leishmania) as standards, the results of which will be discussed herein.

Detection and definition of canine intestinal carbohydrases, using a standardized method.

Intestinal mucosa and pancreas from purebred Beagle dogs were assayed for carbohydrase activity, using several methods of tissue treatment. The enzymes found and studied were alpha-amylase, sucrase, lactase, amyloglucosidase, cellobiase, maltase, and isomaltase. Experiments using polyacrylamide gel columns and heat inactivation showed the presence of an isozyme of maltase which degrades isomaltose. This activity had not been previously demonstrated in dogs. An optimal standard procedure is presented for the preparation and assay of canine digestive enzymes. A statistical analysis of variance of the results showed that the variance was primarily associated with differences among dogs and not by variance within the procedure. When the different extraction procedures were used, results indicated that the level of enzymes detected differed with the method of treatment.

Contamination of bacteriological media by Leptospira biflexa.

Contamination of media with a strain of Leptospira biflexa was traced to the deionized water supply. The leptospiral contaminant appeared in media sterilized by filtration through 0.45- and 0.22-mum pore size membrane filters.

Nucleic acid similarities among Pseudomonas pseudomallei, Pseudomonas multivorans, and Actinobacillus mallei.

Annealing experiments on membrane filters were carried out with deoxyribonucleic acids (DNA) from selected strains of the nomen-species of Pseudomonas, Actinobacillus, Chromobacterium, and Micrococcus, with the use of DNA of Pseudomonas pseudomallei and Actinobacillus mallei as reference materials. Under the usual conditions employed in these experiments, the results were not quantitatively reproducible. Incorporation of dimethylsulfoxide (DMSO) into the incubation medium greatly increased differences in comparative binding. DNA binding in agar matrices was examined in the presence and absence of DMSO at various incubation temperatures. It was found that the greatest specificity, stability, and total binding for DNA containing high amounts of guanine and cytosine occurred in the presence of DMSO. Under the most stringent annealing conditions permitted in agar, DNA species from P. pseudomallei and A. mallei in the presence of DMSO demonstrated interspecific relative bindings of 76 to 86% when compared to the homologous reactions. The thermal elution midpoints (E(m)) of these duplexed interspecific DNA species were quite close to the homologous E(m) values. The relative bindings of P. multivorans DNA types to either reference DNA ranged between 6 to 27%, and the E(m) values were 4 to 7 C less than those for the homologous reactions.