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.
Werbovetz
K A
KA
Department of Parasitology, Walter Reed Army Medical Institute of Research, Washington, DC 20307, USA. cpt_karl_werbovetz@wrsmtp-ccmail.army.mil
Brendle
J J
JJ
Sackett
D L
DL
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D001152:Arsenicals / Q000494:pharmacology; D001665:Binding Sites; D004137:Dinitrochlorobenzene / Q000031:analogs & derivatives / Q000494:pharmacology; D007891:Leishmania / Q000737:chemistry* / Q000187:drug effects; D007893:Leishmania donovani / Q000737:chemistry / Q000187:drug effects; D007894:Leishmania mexicana / Q000737:chemistry / Q000187:drug effects; D008453:Maytansine / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D015800:Protozoan Proteins / Q000302:isolation & purification*; D051381:Rats; D013045:Species Specificity; D014274:Trifluralin / Q000494:pharmacology; D014404:Tubulin / Q000187:drug effects* / Q000302:isolation & purification* / Q000648:ultrastructure
D000981:Antiprotozoal Agents; D001152:Arsenicals; D004137:Dinitrochlorobenzene; D015800:Protozoan Proteins; D014404:Tubulin; C029341:oxophenylarsine; D008453:Maytansine; C027133:3,5-dinitro-4-chloro-alpha,alpha,alpha-trifluorotoluene; D014274:Trifluralin; D017239:Paclitaxel