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. 2016 Oct 21;60(11):6828-6836.
doi: 10.1128/AAC.01129-16. Print 2016 Nov.

Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts

Gyongseon Yang  1   2 , Gahee Choi  1 , Joo Hwan No  3
Affiliations

Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts

Gyongseon Yang et al. Antimicrob Agents Chemother. .

Abstract

Leishmaniasis is a disease caused by pathogenic Leishmania parasites; current treatments are toxic and expensive, and drug resistance has emerged. While pentamidine, a diamidine-type compound, is one of the treatments, its antileishmanial mechanism of action has not been investigated in depth. Here we tested several diamidines, including pentamidine and its analog DB75, against Leishmania donovani and elucidated their antileishmanial mechanisms. We identified three promising new antileishmanial diamidine compounds with 50% effective concentrations (EC50s) of 3.2, 3.4, and 4.5 μM, while pentamidine and DB75 exhibited EC50s of 1.46 and 20 μM, respectively. The most potent antileishmanial inhibitor, compound 1, showed strong DNA binding properties, with a shift in the melting temperature (ΔTm) of 24.2°C, whereas pentamidine had a ΔTm value of 2.1°C, and DB75 had a ΔTm value of 7.7°C. Additionally, DB75 localized in L. donovani kinetoplast DNA (kDNA) and mitochondria but not in nuclear DNA (nDNA). For 2 new diamidines, strong localization signals were observed in kDNA at 1 μM, and at higher concentrations, the signals also appeared in nuclei. All tested diamidines showed selective and dose-dependent inhibition of kDNA, but not nDNA, replication, likely by inhibiting L. donovani topoisomerase IB. Overall, these results suggest that diamidine antileishmanial compounds exert activity by accumulating toward and blocking replication of parasite kDNA.

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Figures

FIG 1
FIG 1
Structures of compounds investigated in this work.
FIG 2
FIG 2
Dose-response curves of pentamidine (A); DB75 (B); and compounds 1 (C), 3 (D), and 5 (E) against L. donovani cell growth. Data represent means ± standard deviations for duplicate experiments.
FIG 3
FIG 3
Measured DNA binding properties with selected antileishmanial diamidines. (A to E) Thermal shift assay with fluorescently labeled dsDNA and diamidines. (F) Correlation between FRET- and DSC-based ΔTm values. *, data are from reference .
FIG 4
FIG 4
Fluorescence spectra of DB75 (A), compound 1 (B), and compound 3 (C) in the presence of dsDNA. A.U., arbitrary units.
FIG 5
FIG 5
Subcellular distribution of selected diamidines in L. donovani. The blue signal corresponds to the localizations of DB75 (A), compound 3 (B), and compound 5 (C) at 1 and 10 μM after 1, 4, and 20 h of incubation. The green signal (SYBR green) represents DNA, and the red signal (MitoTracker) represents mitochondria of parasites. Bar, 5 μm.
FIG 6
FIG 6
Effects of pentamidine; DB75; and compounds 1, 3, and 5 on nuclear and kinetoplast DNA replication in L. donovani. (A) Images of replication phenotypes after treatment with diamidines. The blue signal (DAPI) corresponds to nuclear and kinetoplast DNAs. The green signal (EdU) corresponds to newly synthesized DNA. (B to F) Quantification of populations with replication phenotypes after treatment with pentamidine (B); DB75 (C); and compounds 1 (D), 3 (E), and 5 (F) in dose-dependent manners. All images are representative. Data represent means ± standard deviations for duplicate experiments (B to F). Bar, 5 μm.
FIG 7
FIG 7
Inhibition of LdTOPΙ activity by diamidines using an in vitro plasmid DNA relaxation assay. (A) DMSO control (lane 1), LdTOPΙ control (lane 2), and treatments with pentamidine (lane 3 to 7) and DB75 (lane 8 to 12) in dose-dependent manners. (B) DMSO control (lane 1); LdTOPΙ control (lane 2); and treatments with compounds 1 (lane 3 to 6), 3 (lane 7 to 10), and 5 (lane 11 to 14) in dose-dependent manners. The positions of supercoiled (SC) and relaxed (R) DNAs are indicated at the left.
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This work was supported by the National Research Foundation of Korea (NRF-2014K1A4A7A01074645), a grant funded by the Korean Government Ministry of Science, Information/Communication Technology and Future Planning (MSIP), Gyeonggi-do, and the Korea Institute of Science and Technology Information (KISTI) (J.H.N.).

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