Elsevier

Biochemical Pharmacology

Volume 85, Issue 10, 15 May 2013, Pages 1433-1440
Biochemical Pharmacology

Gimatecan and other camptothecin derivatives poison Leishmania DNA-topoisomerase IB leading to a strong leishmanicidal effect

https://doi.org/10.1016/j.bcp.2013.02.024Get rights and content

Abstract

The aim of this work is the in vitro and ex vivo assessment of the leishmanicidal activity of camptothecin and three analogues used in cancer therapy: topotecan (Hycantim®), gimatecan (ST1481) and the pro-drug irinotecan (Camptosar®) as well as its active metabolite SN-38 against Leishmania infantum. The activity of camptothecin and its derivatives was studied on extracellular L. infantum infrared-emitting promastigotes and on an ex vivo murine model of infected splenocytes with L. infantum fluorescent amastigotes. In situ formation of SDS/KCl precipitable DNA–protein complexes in Leishmania promastigotes indicated that these drugs are DNA topoisomerase IB poisons. The inhibitory potency of camptothecin derivatives on recombinant L. infantum topoisomerase IB was assessed in vitro showing that gimatecan is the most active compound preventing the relaxation of supercoiled DNA at submicromolar concentrations. Cleavage equilibrium assays in Leishmania topoisomerase IB show that gimatecan changes the equilibrium towards cleavage at much lower concentrations than the other camptothecin derivatives and that this effect persists over time. Gimatecan and camptothecin were the most powerful compounds preventing cell growth of free-living L. infantum promastigotes within the same concentration range. All these compounds killed L. infantum splenocyte-infecting amastigotes within the nanomolar range. The amastigote form showed higher sensitivity to topoisomerase IB poisons (with high therapeutic selectivity indexes) than free-living promastigotes. All the compounds assayed poisoned L. infantum DNA topoisomerase IB leading to a strong leishmanicidal effect. Camptothecin derivatives are suitable for reducing the parasitic burden of ex vivo infected splenocytes. The selectivity index of gimatecan makes it a promising drug against this neglected disease.

Graphical abstract

In this article, we characterized the leishmanicidal profile of several camptothecin derivatives. Gimatecan was the most powerful DNA TopIB poison, killing infecting-amastigotes in the nanomolar range.

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Introduction

Visceral leishmaniasis in Europe is a disease caused by the protozoan pathogen Leishmania infantum that affects all countries of the Mediterranean region. The disease has a low prevalence in humans – the definitive host – but not in dogs, which act as reservoirs of the parasite. The risk of human disease significantly increased in immunosuppressed individuals mainly linked to HIV, but due to highly active antiretroviral therapies its presence has dramatically decreased [1]. Current pharmacopoeia against leishmaniasis includes old-fashioned pentavalent antimonium derivatives, as well as amphotericin B, paromomycin and alkylphospholipids [2]. Most of them have many undesirable side effects or require parental administration and long-term treatments, which can make treatment difficult to adhere to [3].

Searching for differentiable targets between the host and pathogen is a recognized strategy for designing new drugs. Type IB DNA topoisomerases (TopIB) were indicated as putative targets in proliferative processes when their mechanism of action was originally shown [4]. Since then, numerous compounds have shown antiproliferative effects: (i) by interfering with the catalytic properties of enzymes (TopIB inhibitors) or (ii) by stabilizing the enzyme–DNA complex – a transient step of all Top activities – that can be hindered in time by many compounds (TopIB poisons). TopIB poisons prevent the religation step and produces single DNA breaks that interfere with the replication fork of the dividing cells [5]. An amazing result found in 2003 was that L. infantum TopIB (LiTopIB) was a heterodimeric enzyme encoded by two genes that were placed on different chromosomes [6]. This characteristic is only shared by certain phylogenetically close microorganisms such as Trypanosoma cruzi and T. brucei [7]. Despite these differences, most of the domains related to the enzymatic activity are conserved between both subunits, which are interconnected by two polypeptide extensions that play the role of a putative linker. This region is not needed for TopIB activity, but it contributes to DNA binding and camptothecin inhibition, theoretically by slowing down the religation step of the nicking-closing reaction [8], [9], [10].

Camptothecin and derivatives, known as TopIB poisons, develop their function by binding in a specific and reversible manner to the transient DNA–enzyme complexes [11]. These drugs effectively target the TopIB–DNA binary complex, while they do not bind to the enzyme alone and display a weak affinity for DNA in the absence of the enzyme [12]. The presence of cleavage complexes generates collisions with the replication fork, causing DNA breaks by converting transient complexes to permanent strand damage and consequently making these compounds powerful anticancer drugs. Camptothecin in fact shows a remarkable antiproliferative potential in vitro against a wide range of tumor cells in the submicromolar range [13], [14] and it has been the first compound described as a specific inhibitor of eukaryotic cell TopIB with no effect on the bacterial TopIA and a well-defined mechanism of action. The development of camptothecin derivatives against several types of cancer has resulted in two water-soluble compounds currently used in clinical practice: topotecan (Hycantim®) and the pro-drug irinotecan (Camptosar®). In addition a third compound, gimatecan (ST1481) is an orphan drug that is being studied in clinical phase II against astrocytoma, glioblastoma and oligodendroglial tumors [15].

TopIB is essential for DNA replication, recombination and repair mechanisms. Most organisms are unable to survive in the absence of this enzyme [16]. Previous works have unsuccessfully tried to create Leishmania [17] or T. brucei [18] strains lacking the small monomer or both protomers, respectively. For these reasons and because of distinct structural differences between human and leishmanial TopIB, this protein is considered a valuable target for chemotherapy [19].

This paper explains the effect of these camptothecin derivatives on an ex vivo murine model of infected splenocytes with L. infantum. Furthermore, the in vitro effect of these compounds reveals for the first time their ability to trap TopIB–DNA covalent complexes on Leishmania parasites, thus preventing the religation step at micromolar concentrations.

Section snippets

Reagents and culture media

Pyrococcus furiosus (Pfu), klenow polymerases and restriction enzymes were acquired from Roche (Roche Farma SA, Spain) and GE Healthcare (Spain). T4 DNA ligase was obtained from Stratagene (La Jolla, CA, USA). Cell culture media, camptothecin, irinotecan and SN-38 were purchased from Sigma (Sigma–Aldrich, Spain). Topotecan (Hycantim®) was obtained from GlaxoSmithKline (UK). Gimatecan and camptothecin-N-oxide were kind gifts of Sigma Tau to Alessandro Desideri. Primers for PCR amplification were

Leishmanicidal effect of camptothecin and analogues

We have analyzed the effect of the specific TopIB inhibitors outlined in Fig. 1 on the proliferation rate of L. infantum-IFR1.4 promastigotes and amastigotes. Table 1 shows the IC50 values obtained from dose–response plots, based on the percentage of infrared fluorescence signal at 708 nm, obtained for free-living promastigotes and amastigote-infected splenocytes, exposed for 48 h to several concentrations of the tested compounds in comparison to untreated controls. The IC50 value obtained with

Discussion

One of the most problematic aspects of leishmaniases is the fast emergence of resistant strains against conventional drugs, which hinders the treatment and consequently, constant development of new therapeutic resources is required. In the present study, we have shown that gimatecan is a powerful growth inhibitor of L. infantum promastigotes that is even more lethal for its intracellular form (amastigote) (Table 1). In addition, gimatecan is also shown to be a potent LiTopIB-poison.

In vitro

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This research was supported by Ministerio de Ciencia y Tecnología (grants AGL2009–11935/GAN and AGL2010–16078/GAN), Instituto de Salud Carlos III (grants PI09/0448, PI12/00104 and the Network of Tropical Diseases RICET) and Junta de Castilla y León (grant Gr238) and AIRC (grant no. 10121). RAV, CFP and ECA are pre-doctoral fellows granted by Instituto de Salud Carlos III, JCyL-ESF and University of León, respectively. CP was supported by a Juan de la Cierva programme (JCI-2009-05444) of the

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