The bisbenzimidazole compound Hoechst 33342 (Ho342) has been identified as a specific Topoisomerase-I (Topo-I) inhibitor in mammalian cells. More recently, we have reported the ability of Ho342 to target L. donovani Topo-I, leading to parasite growth inhibition in vitro by mechanisms involving DNA breakage and apoptosis-like phenomenon. As the Ho342 lead molecule (2,5′-Bi-1H-benzimidazole) can be used as a starting structure for derivative compounds more effective against Leishmania, defining the Ho342 resistance mechanism(s) in Leishmania represents an important strategic tool. In the present study, we selected resistant parasites to Ho342 (LdRHo.300). While we observed an increase of the Topo-I gene expression correlated by a higher Topo-I DNA relaxation activity, the Topo-I genes (LdTOP1A and LdTOP1B) sequencing did not reveal any mutation for the resistant parasites. Moreover, our results on Ho342 cellular accumulation suggested the presence of a potential energy-dependent Ho342 transporter in the wild-type parasite, and that an alteration of this transporter has occurred in LdRHo.300, leading to an altered drug accumulation. Collectively, Ho342 resistance characterization provided results supporting that the resistance developed by LdRHo.300 involves complex mechanisms, most likely dominated by an altered drug accumulation, providing new insight in the Ho342 resistance mechanisms.

This study reports that inhibition of Leishmania Topo-I with the minor groove-binding ligands (MGBLs) Hoechst 33342 (Ho342) blocks parasite growth in culture by mechanisms involving DNA breakage. While Ho342 inhibited the replication of several species of Leishmania in a dose- and time-dependent manner, Ho258 was not effective. Cytofluorometric analysis suggested that superior effectiveness of Ho342 over Ho258 was attributed to Leishmania parasites being more permeable toward Ho342. This observation was supported by the ability of both Ho342 and Ho258 to block the relaxation of supercoiled pBR322 DNA by Leishmania Topo-I. The Ho342 specificity toward L. donovani Topo-I was reinforced by the observation that increased Topo-I gene expression and Topo-I activity in Leishmania was paralleled by augmented resistance for this compound. Furthermore, the capacity of NaCl treatment to reverse MGBL-mediated DNA break suggests that Ho342 targetted Topo-I. Moreover, we observed that Ho342-inducible arrest of Leishmania growth was accompanied by G1 arrest and induction of cell death that closely resembles apoptosis. Taken together, our results suggest that MGBL compounds show promise as Topo-I inhibitors against Leishmania infection.