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Organometallic gold(III) complex [Au(Hdamp) (L14)]+ (L1 = SNS-donating thiosemicarbazone) as a candidate to new formulations against Chagas disease Carla Duque Lopes, Bruna Possato, Ana Paula Segantin Gaspari Giovanini, Ronaldo Junio de Oliveira, Ulrich Abram, José Paulo Aldério Almeida, Fernanda dos Reis Rocho, Andrei Leitao, Carlos A. Montanari, Pedro Ivo Silva Maia, João Santana Silva, Sergio de Albuquerque, and Zumira Carneiro ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00284 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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ACS Infectious Diseases

Organometallic gold(III) complex [Au(Hdamp)(L14)]+ (L1 = SNS-donating thiosemicarbazone) as a candidate to new formulations against Chagas disease

Carla Duque Lopes1,

5ǂ,

Bruna Possato1, Ana Paula S. Gaspari1, Ronaldo J.

Oliveira2, Ulrich Abram3, José P. A. Almeida6, Fernanda dos Reis Rocho4, Andrei Leitão4, Carlos A. Montanari4, Pedro V. S. Maia2, João S. da Silva5, Sérgio de Albuquerque1, Zumira A. Carneiro1,6*ǂ

1Department

of Clinical Toxicological and Bromatological Analysis School of

Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), 14040-903, Ribeirão Preto-SP, Brazil; 2Núcleo

de Desenvolvimento de Compostos Bioativos (NDCBio), Universidade Federal

do Triângulo Mineiro, 38025-470, Uberaba – MG, Brazil; 3Freie

Universität Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany;

4Grupo

de Estudos em Química Medicinal de Produtos Naturais–NEQUIMED-PN,

Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador Sancarlense 400, P.O. Box 780, 13560-960 São Carlos-SP, Brazil 5Departament

of Biochemistry and Immunology, School of Medicine, University of São

Paulo, 14049-900- Ribeirão Preto, São Paulo, Brazil; 6Present

adress: Centro Universitário Estácio de Ribeirão Preto, Abraão Issa Halach

Street, 980, 14096-160, Ribeirão Preto-SP, Brazil.

ǂ These authors contributed equally *Address correspondence to Zumira Carneiro, [email protected]

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Chagas disease remains a serious public health concern with unsatisfactory treatment outcomes due to strain-specific drug resistance and various side effects. To identify new therapeutic drugs against Trypanosoma cruzi, we evaluated both the in vitro and in vivo activity of the organometallic gold(III) complex [Au(III)(Hdamp)(L14)]Cl (L1 = SNSdonating thiosemicarbazone), was henceforth denoted 4-Cl. Our results demonstrated that 4-Cl was more effective than benznidazole (Bz) in eliminating both the extracellular trypomastigote and intracellular amastigote forms of the parasite without cytotoxic effects on mammalian cells. In in vivo assays, 4-Cl in PBS solution lose the protonation and become the 4-neutral. 4-neutral reduced parasitaemia and tissue parasitism in addition to protecting the liver and heart from tissue damage at 2.8mg/ kg/ day. All these changes resulted in the survival of 100% of the mice treated with the gold complex during the acute phase. Analyzing the surviving animals of the acute infection, the parasite load after 150 days of infection was equivalent to those treated with the standard dose of Bz without demonstrating the hepatotoxicity of the latter. In addition, we identified a modulation of interferon gamma (IFN-γ) levels that may be targeting the disease's positive outcome. To the best of our knowledge this is the first gold organometallic study that shows promise in an in vivo experimental model against Chagas disease.

Keywords: Gold (III) Complex, Thiosemicarbazones, Chagas disease, Trypanocidal activity, cruzain, IFN-γ production.


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Chagas disease is a neglected infection caused by a protozoan parasite named Trypanosoma cruzi (T. cruzi) which is transmitted by triatomine insect vectors and is endemic in Latin America. According to the World Health Organization (WHO), approximately eight million people are infected with T. cruzi worldwide1,2. In addition, the phenomena of globalization and immigration has also led to the appearance of several infectious cases in non-endemic areas increasing the overall impact of neglected disease2,3. Currently, only two drugs are available for the treatment of the Chagas disease: nifurtimox (NFX) and benznidazole (Bz)4,5. In some countries, NFX was discontinued due to serious side effects, such as neuropathy and anorexia, among others5,6. Bz which is only effective in the acute stage, with low cure infection but presents low cure rates mainly during the chronic phase of disease. Moreover, Bz may present undesirable systemic toxicity, such as rashes and gastrointestinal symptoms2,4,6,7. Therefore, the development of more efficacious and less toxic drugs is urgently needed. A potential strategy for the treatment of Chagas disease is the design of compounds that selectively inhibit essential enzymes for parasite survival inside the host cells8. In the case of T. cruzi, cruzain is an attractive drug target8–11. The structure of cruzain contains a cysteine protease domain, which plays an important role during the life cycle of the parasite, with including replication, metabolism, and evasion of host immune defence during the early events of macrophage infection9,10. Although many other potential drug targets exist in parasite metabolism8, cruzain is by far the most studied protease of T. cruzi due to its role as a virulence factor of the parasite10–12. Thus, compounds that inhibit the biological function of cruzain, such as some thiosemicarbazones, may be an effective alternative for pharmacological treatment of Chagas disease13. In this context, transition metal complexes that have thiosemicarbazones as ligands have been developed and tested against various forms of T. cruzi14–18.Gold(III) complexes have received increased



attention due to their biological properties, such as anti-cancer19 and antiparasitic effects20. In a recent paper, our research group identified complexes of the general formula [Au(III)(Hdamp)(L1)]Cl (Figure 1) that have high stability in aqueous solution and antiparasitic activity against the Tulahuen LacZ strain. Among these compounds, 4Cl, [Au(III)(Hdamp)(R1R2L14)]Cl (R1 = R2 = Methyl) demonstrated low cytotoxicity in spleen cells, leading to a selectivity index (SI) of approximately 3021, which indicated that this compound is a promising candidate for the development of trypanocidal drugs.

Figure 1. Organometallic gold(III) complexes containing hybrid SNS-donating [Au(III)(Hdamp)(L1)]Cl thiosemicarbazone ligands (Hdamp = 14 dimethylammoniummethylphenyl) . Adapted with permission from Maia et al. Organometallic Gold (III) Complexes with Hybrid SNS-Donating Thiosemicarbazone Ligands: Cytotoxicity and Anti-Trypanosoma cruzi Activity. 46 (8), 2559–2571 Copyright, 2017/ Dalton Trans. Camb. Engl, DOI 10.1039/c6dt04307k. Clinical manifestations associated with the T. cruzi infection are dependent on the intricate equilibrium between the parasite and the host immune response. The production of interferon gamma (IFN-γ) generated for adaptive immune responses is important for inhibition of parasite proliferation22,23. Intense production of IFN-γ activates the CD4 T lymphocytes with Type 1 Helper T (Th1) profile and cytotoxic CD8 T lymphocytes, which efficiently eliminate the parasite24. T-Helper 17 (Th17) cells have the potential to


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become antigen-specific CD8 T cells against the parasite and show greater contributions than the subpopulation of CD4 T cells which are producers of IFN-γ25. Consequently, if cytotoxic and cytokine secretion functions of Th17 cells are decreased, the elimination of the parasite is hampered26. Therefore, the modulation of immune responses is fundamental for a good prognosis of Chagas disease. In the present study, the in vitro and in vivo 4-Cl trypanocidal activities were evaluated against the Y (group TcII) strain, partially resistant to Bz, to assess the use of this compound as a candidate for the development of organometallics to treatment of Chagas Disease.

RESULTS AND DISCUSSION The gold complex acts directly on T. cruzi parasites. The organometallic gold(III) complex 4-Cl has interesting characteristics from both chemical and biological points of view14. The synthesis is quite simple, occurring at room temperature in a short period of time, and has an excellent yield (almost 90%). The compound is easily crystallised, which leads to a high purity. Therefore, although it is a gold compound, the costs for preparation do not limit its use. Furthermore, the ESI+ MS spectrum shows an exclusive [M]+ complex ion peak with no evidence for the formation of gold(I) compounds, a common finding for gold(III) thiosemicarbazone derivatives14,28,29. Concerning 4-Cl stability, through observation of UV-vis-NIR absorption spectra (Supplementary Figure 1), it was possible to verify that incubation in DMSO and RPMI (10% of SBF) during 72 hours could not change significantly the complex structure. Although stable in the aforementioned media, in PBS, a great decrease in 416 nm band could lead one erroneously to believe there was the complex consumption during the stability experiment. Actually, after careful observation and analysis through mass spectrometry (ESI+MS), the decrease is due to precipitation, after complex deprotonation. The crystalline structure of this neutral species



(4-neutral) had already been explored by our group14 (Figure 2). In aqueous solution or in the presence of a reducing agent, such as glutathione, for 24 h at 37 °C, the compound also did not show changes in retention time on HPLC14.

Figure 2. Organometallic gold(III) complexes. (A) 4-Cl and (B) 4-neutral

These characteristics are interesting for a drug to be delivered along areas of difficult access and high ambient temperature typically found in tropical countries in which Chagas Disease is inserted.2 Then, we initially assessed the effectiveness of 4-Cl against the T. cruzi Y strain, which is partially resistant to Bz treatment and more virulent than the Tulahuen strain. The trypomastigotes of the Y strain were incubated with serial dilutions of 4-Cl or Bz for 24 h, and live parasites were counted by colorimetric analyses. The 4-Cl treatment was highly efficient compared to Bz treatment, reaching an IC50Try (concentration needed to kill 50% of the parasites) of 0.3±0.06 µM, whereas the IC50Try of Bz was 0.84±0.29 µM. These results indicate that 4-Cl is almost three times more effective than Bz in killing the trypomastigote forms of T. cruzi (Figure 3A). To determine the effects of 4-Cl on intracellular amastigotes of the T. cruzi Y strain, BMMs (Bone Marrow Macrophages) were differentiated and infected with the trypomastigote forms for


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16 h. The extracellular parasites were removed by extensive washes and incubated with low concentrations of 4-Cl or Bz for 24 h (Figure 3B). The trypanocidal capacity of Bztreatment decreased with subsequent concentrations (IC50 (Bz) = 1.84 ± 0.5µM). In 3.2 µM, the treatment eliminated, approximately, 58% of intracellular parasites. In contrast, at the same concentration, the 4-Cl-treatment killed 75% of the intracellular amastigotes of T. cruzi and demonstrated a high trypanocidal ability in all concentration tested (IC50(4-Cl) = 0.64 ± 0.1µM). Interestingly, 4-Cl exhibits capacity of inhibit 50% of parasite replication at 780 nM, which suggested that 4-Cl is four times more potency than Bz (Figure 3B). Together, these data indicate that 4-Cl is a more potent agent against amastigote forms of T. cruzi.

Figure 3. In vitro trypanocidal activity of 4-Cl against the trypomastigote and amastigote forms of the Y strain of T. cruzi. (A) Percentage of trypanocidal activity of 4-Cl and Bz against the Y strain of T. cruzi analysed by quantifying viable parasites 24 h post-treatment. (B) Macrophages derived from bone marrow were infected with the trypomastigote form of the Y strain of T. cruzi. After 24 h of infection, the extracellular



parasites were removed, and treated with 4-Cl or Bz. Statistical difference was considered when *p

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