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 1.2 microM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73x10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). 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 LdTOP1Delta39LS (N-terminal deletion of amino acids 1-39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 muM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Delta39LS 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.
Roy
Amit
A
Department of Molecular Parasitology, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata-700032, India.
Das
Benu Brata
BB
Ganguly
Agneyo
A
Bose Dasgupta
Somdeb
S
Khalkho
Neeta V M
NV
Pal
Churala
C
Dey
Sumit
S
Giri
Venkatachalam Sesha
VS
Jaisankar
Parasuraman
P
Dey
Sanjit
S
Majumder
Hemanta K
HK
D000818:Animals; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000633:toxicity*; D006801:Humans; D007211:Indoles / Q000633:toxicity*; D007893:Leishmania donovani / Q000187:drug effects / Q000201:enzymology*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D059004:Topoisomerase I Inhibitors
C512018:1,1-bis(3'-indolyl)methane; D004791:Enzyme Inhibitors; D007211:Indoles; D021122:Protein Subunits; D015800:Protozoan Proteins; D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin