Differential induction of Leishmania donovani bi-subunit topoisomerase I–DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I

‘LeishMan’ topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani

The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme

Mechanistic Studies of the Unusual Properties Inherent to Topoisomerase I of Leishmania donovani

It is a unicellular, protozoan parasite, which exists as motile promastigotes in the sandfly gut and is transmitted into the human host where it transforms into sessile amastigotes that manifests a dreadful clinical symptom called Leishmaniasis. It was discovered at the turn of the 19th century, viz. by Cunningham, Leishman, Donovan, Borovsky, Wright and Vianna (Gardener et. al. 1977). But the name Leishmania donovani was given by Ross in 1903

Mutational Studies Reveal Lysine 352 on the Large Subunit is Indispensable for Catalytic Activity of bi-subunit Topoisomerase I from Leishmania Donovani

From the vanadate complex crystal structure of Leishmania donovani topoisomerase I, several amino acid residues have been implicated to be involved in the catalytic reaction. Although several predictions and propositions have been made, the exact role of these amino acids has not yet been clearly demonstrated in vitro. Among these residues, lysine 352 and arginine 314 stand as potential candidates for playing the role of a general acid during the cleavage step. In this study,we have characterized the role of lysine 352 on the large subunit, by site-directed mutagenesis and have tried to identify the general acid that can protonate the 5�-O atom of the leaving strand. Studies with the mutant enzymes reveal that, relaxation activity was severely affected when Lys352was mutated to arginine or alanine (K352R or K352A). Mutation of Arg314 to Lys (R314K) has very little effect on the relaxation activity.Detailed study reveals that, both cleavage and religation steps are severely affected in case of K352R and K352A and the cleavage religation equilibrium is shifted towards the cleavage. On the contrary, the R314K mutant exhibits only a slightly slower rate of cleavage compared to wild-type enzyme. Cleavage assays with an oligonucleotide containing 5�-bridging phosphorothiolate indicate that Lys352 acts as a general acid in the cleavage step. Altogether, this study establishes the indispensable role of lysine 352 in the catalytic reaction of L. donovani topoisomerase I

Impact of Linker Groups on Self-Assembly, Gene Transfection, Antibacterial Activity, and In Vitro Cytotoxicity of Cationic Bolaamphiphiles

Cationic bolaamphiphiles have gained significant attention in various research fields, including materials science, drug delivery, and gene therapy, due to their unique properties and potential applications. The objective of the current research is to develop more effective cationic bolaamphiphiles. Thus, we have designed and synthesized two cationic bolaamphiphiles (−(CH2)12(2,3-dihydroxy-N,N-dimethyl-N-(3-ureidopropyl)propan-1-aminium chloride))2 (C12(DDUPPAC)2)) and (−(CH2)12(N-(3-(carbamoyloxy)propyl)-2,3-dihydroxy-N,N-dimethylpropan-1-aminium chloride)2 (C12(CPDDPAC)2) containing urea and urethane linkages, respectively. We have investigated their self-assembly properties in water using several techniques, including surface tension, electrical conductivity, fluorescence probe, calorimetry, dynamic light scattering, and atomic force microscopy. Their biological applications, e.g., in vitro gene transfection, antibacterial activity, and cytotoxicity, were studied. Both bolaamphiphiles were observed to produce aggregates larger than spherical micelles above a relatively low critical aggregation concentration (cac). The calorimetric experiments suggested the thermodynamically favorable spontaneous aggregation of both bolaforms in water. The results of interaction studies led to the conclusion that C12(CPDDPAC)2 binds DNA with a greater affinity than C12(DDUPPAC)2. Also, C12(CPDDPAC)2 is found to act as a more efficient gene transfection vector than C12(DDUPPAC)2 in 264.7 cell lines. The in vitro cytotoxicity assay using MTT, however, revealed that neither of the bolaamphiphiles was toxic, even at higher quantities. Additionally, both bolaforms show beneficial antibacterial activity

Mitochondria-Dependent Reactive Oxygen Species-Mediated Programmed Cell Death Induced by 3,3�-Diindolylmethane through Inhibition of F0F1-ATP Synthase in Unicellular Protozoan Parasite Leishmania donovan

Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3�-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (��m). Because ��m is the driving force for mito mitochondrial ATP synthesis, loss of ��m results in depletion of cellular ATP level. The loss of ��m causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of ��m leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets

Betulinic Acid, a Catalytic Inhibitor of Topoisomerase I, Inhibits Reactive Oxygen Species–Mediated Apoptotic Topoisomerase I–DNA Cleavable Complex Formation in Prostate Cancer Cells but Does Not Affect the Process of Cell Death

The ubiquitious enzyme topoisomerase I can be targeted by drugs which turn these enzymes into cellular poisons and subsequently induce cell death. Drugs like staurosporine, which do not target topoisomerase I directly, can also lead to stabilization of topoisomerase I–DNAcleavable complexes by an indirect process of reactive oxygen species (ROS) generation and subsequent oxidative DNAdamage. In this study, we show that betulinic acid, a catalytic inhibitor of topoisomerases, inhibits the formation of apoptotic topoisomerase I–DNAclea vable complexes in prostate cancer cells induced by drugs like camptothecin, staurosporine, and etoposide. Although events like ROS generation, oxidative DNA damage, and DNAfragmentation were observed after betulinic acid treatment, there is no topoisomerase I–DNAclea vable complex formation, which is a key step in ROS-induced apoptotic processes. We have shown that betulinic acid interacts with cellular topoisomerase I and prohibits its interaction with the oxidatively damaged DNA. Using oligonucleotide containing 8-oxoguanosine modification, we have shown that betulinic acid inhibits its cleavage by topoisomerase I in vitro. Whereas silencing of topoisomerase I gene by small interfering RNAreduces cell death in the case of staurosporine and camptothecin, it cannot substantially reduce betulinic acid– induced cell death. Thus, our study provides evidence that betulinic acid inhibits formation of apoptotic topoisomerase I–DNAcomplexes and prevents the cellular topoisomerase I from directly participating in the apoptotic proces

Analysis of drug induced covalent topoisomerase I–DNA complex formation in promastigotes by KCl-SDS precipitation assay

<p><b>Copyright information:</b></p><p>Taken from "Differential induction of bi-subunit topoisomerase I–DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I"</p><p>Nucleic Acids Research 2006;34(4):1121-1132.</p><p>Published online 18 Feb 2006</p><p>PMCID:PMC1373691.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Exponentially growing promastigotes (5 × 10 cells/ml) were labeled with [H]thymidine at 22°C for 24 h and then treated with different concentrations of drugs as indicated. Parts of the labeled cells were treated with DHBA (150 µM) for 10 min before the addition of different concentration of baicalein as indicated. SDS-K precipitable complex were measured as described in Materials and Methods. Experiments were performed three times and representative data from one set of experiments are expressed as means ± SD. Variations among different set of experiments wer

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 μM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73×10−9 M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual 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 non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1�39LS (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 μM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1�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