New Insights Into the Mechanism of Action of the Cyclopalladated Complex – CP2 in Leishmania: Calcium Dysregulation, Mitochondrial Dysfunction and Cell Death

Background: Leishmaniasis is a parasitic disease that affects millions of people and lacks new molecules for treatment. The current treatment is associated with high toxicity, a difficult administration route, and the emergence of resistant strains. These disadvantages raise the necessity to develop novel compounds with effective leishmanicidal activity and elucidate their action mechanism. Methods: To provide new information about of mechanism of action of the binuclear cyclopalladated complex [Pd(dmba)(µ-N3)]2 (CP2) in Leishmania, we have utilized two-dimensional SDS-PAGE for proteomic analyses, analyses of the progression of the cell cycle by flow cytometry. The measurement of reactive oxygen species (ROS) and mitochondrial membrane potential by fluorescence, live-cell Fura-2 Ca2+ imaging, and a dual acridine orange/ethidium bromide (AO/EB) staining method for the cell death mechanism. In vivo leishmanicidal activity has been performed in a golden hamster model. Findings: Dysregulation of parasite Ca2+ levels coming mainly from mitochondria, increasing ROS production and collapsing the Leishmania mitochondrial membrane potential. The presence of CP2 promotes apoptotic-like features in promastigotes six hours post-CP2-treatment. Exposure of parasites for 24 h or 48 h to CP2 leads to necrosis or directs programmed cell death (PCD)-committed cells toward necrotic-like destruction of cells. Besides, CP2 leads to arrests of cell cycle progression at S-phase, even in the absence of ROS production, and increases the expression of stress-related proteins, including calreticulin, Heat shock 10 kDa protein 1 (Hsp10), Hsp70, GRP78/BiP, and protein disulfide-isomerase (PDI) and cell detoxification proteins (trypanothione reductase, peroxiredoxin, tryparedoxin peroxidase). These studies provide new insight into the mechanism of action of CP2. When Leishmania infantum-infected Golden hamsters were treated for 15 days with 1·5 mg/Kg/day, CP2 caused a reduction in the parasite load of both liver and spleen by ~50%, which is similar to the leishmanicidal activity of amphotericin B. Interpretation: Our finding established the ability of CP2 to cause oxidative stress with disruption of mitochondrial Ca 2+ homeostasis of Leishmania, leading to other effects observed downstream to CP2-treatment. This new insight into the cyclopalladated complex's mechanism of action may have wider therapeutic applications targeting the parasite's mitochondria since structural modification to improve the leishmanicidal effect of CP2 may offer an alternative therapeutic agent for this neglected disease. Funding Information: This work was supported by the Sao Paulo Research Foundation (FAPESP) grants: #2016/05345-4, #2016/177115, #2017/03552-5 and #2018/23015-7; Programa de Apoio ao Desenvolvimento Cientifico da Faculdade de Ciencias Farmaceuticas da UNESP (PADC); and Funding from the Thomas P. Infusino Endowment at Rutgers University. AMAV (#2016/19289-9 and #2019/21661-1) and SV (#2016/18191-5) were supported by FAPESP. This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - Brasil (CAPES) - Finance Code 001, IAPL, TGP, KBI, JMV. MARB, AVGN and MASG are recipienu of a Research Productivity Scholarship from the National Council for Research and Development (CNPq). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The Ethics Committee approved this study for Animal Experimentation of Sao Paulo State University (UNESP), the School of Pharmaceutical Sciences (CEUA/FCF/CAr, 18/2015 and 44/2015) in agreement with the guidelines of Sociedade Brasileira de Ciencia de Animais de Laboratorio (SBCAL) and Conselho Nacional de Controle da Experimentacao Animal (CONCEA).