An insight into the mechanism of inhibition of unusual bi-subunit topoisomerase I from Leishmania donovani by 3,3′-di-indolylmethane, a novel DNA topoisomerase I poison with a strong binding affinity to the enzyme

DIM (3,3'-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.21 mu M. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73 x 10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOPILS (bi-subunit L. donovani topoisomerase 1). DIM stabilizes topoisomerase I-DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I-DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated 'cleavable complexes' in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOPI Delta 39LS (N-terminal deletion of amino acids I 39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 mu M respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1 Delta 39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.