Topoisomerase I amino acid substitutions, Gly185Arg and Asp325Glu, confer camptothecin resistance in Leishmania donovani.
The antitumor compound camptothecin (CPT) is also recognized for its specific activity against Leishmania donovani topoisomerase I (Topo-I). In consequence, defining CPT resistance mechanisms represents an important strategic tool in the acquisition of a better understanding of its mode of action. In the present study, we selected a single highly resistant L. donovani strain termed LdRCPT.160 by stepwise exposure to CPT. Gene sequencing revealed two single nucleotide mutations in the LdRCPT.160 LdTOP1A gene, resulting in two amino acid substitutions (Gly185Arg and Asp325Glu) in the protein. Moreover, these two substitutions observed in the LdTOP1A protein were correlated with a decreased Topo-I DNA relaxation activity in these resistant parasites. Nevertheless, there was no change in the LdTOP1A gene expression level. Interestingly, transfection studies of the LdRCPT.160 LdTOP1A gene in its wild-type counterpart showed that it induced CPT resistance. Site-directed mutagenesis studies demonstrated that, despite a substantial level of resistance conferred by the Gly185Arg and Asp325Glu substitutions separately, both were essential to reach a high-resistance phenotype. Of interest, the amino acid substitutions observed in LdRCPT.160 LdTOP1A protein occurred near the amino acids previously predicted to interact with CPT, providing new insight into the mechanism of CPT molecular action.
Marquis
Jean-François
JF
Centre for the Study of Host Resistance and the Research Institute of McGill University Health Centre, Department of Experimental Medicine, McGill University, Lyman Duff Building, 3775 University St., Room 600, Montréal, Québec, Canada H3A 2B4.
Hardy
Isabelle
I
Olivier
Martin
M
D019943:Amino Acid Substitution; D000818:Animals; D002166:Camptothecin / Q000494:pharmacology*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics*; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007893:Leishmania donovani / Q000187:drug effects* / Q000201:enzymology; D021261:Parasitic Sensitivity Tests; D017422:Sequence Analysis, DNA
D004791:Enzyme Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin