In silico toxicologic profile and in vivo trypanocidal activity of estafietin, a sesquiterpene lactone isolated from Stevia alpina Griseb.

Abstract Chagas disease is an infection caused by the protozoan Trypanosoma cruzi, affecting 6-8 million people worldwide. Only two drugs are available for its treatment, having a limited efficacy and adverse side-effects. Estafietin is a sesquiterpene lactone isolated from Stevia alpina with in vitro activity against T. cruzi and low cytotoxicity against mammalian cells. The aim of this work was to predict the toxicologic profile of estafietin by in silico methods and assess its in vivo activity on a murine model of Chagas disease. Estafietin showed low toxicity according to pkCSM web tool and passed the PAINS filter from PAINS-remover web server. The treatment of infected mice with 1 mg/Kg/day of estafietin for five consecutive days administrated by intraperitoneal route significatively decreased parasitemia levels and reduced inflammatory infiltrates and myocyte damage on muscle tissue. These results suggest that estafietin had effect both on acute and chronic stages of the infection. GRAPHICAL ABSTRACT


Introduction
Chagas disease is a parasitic disease caused by the protozoan Trypanosoma cruzi.It affects about 6-8 million people worldwide, mostly in rural areas of Latin America.In recent years, the disease has spread to other continents due to migration from endemic areas (World Health Organization 2022).
Nowadays, there are only two drugs available for Chagas disease treatment: the nitroheterocyclic compounds benznidazole and nifurtimox.Considering that both drugs have a limited cure efficacy (acute phase, 50-80%; chronic phase, 8-20%) and important side effects which can lead to treatment interruption (Sales Junior et al. 2017), the development of new drugs for the treatment of this parasitosis is needed.
Sesquiterpene lactones (STLs) are a group of secondary metabolites characteristic of the Asteraceae family.These natural compounds have shown a broad spectrum of biological activities, such as anticancer, anti-inflammatory, antifungal, anxiolytic, analgesic, and antiprotozoal activities (Moujir et al. 2020).Several STLs have displayed promising in vitro and in vivo activity against T. cruzi and other pathogenic protozoa (Boechat et al. 2018;Sanchez Alberti et al. 2018).
In a previous work, we reported the in vitro activity against T. cruzi and the cytotoxicity of four sesquiterpene lactones isolated from Argentinean species of Stevia and Mikania genus (Elso et al. 2020).Estafietin, a guaianolide isolated from Stevia alpina Griseb.(Asteraceae) with a high yield, displayed trypanocidal activity against bloodstream and intracellular forms of T. cruzi with low cytotoxicity against Vero cells.Moreover, this compound has shown a reduction in the activity of succinate dehydrogenase and affected the respiratory chain and the Krebs cycle in T. cruzi (Elso et al. 2022).The aim of this work was to perform the in silico toxicological prediction of estafietin and evaluate the in vivo activity of this STL on a murine model of Chagas disease.

Results and discussion
In silico technologies are useful tools for preliminary assessment of the toxicological profile of candidate molecules for subsequent preclinical studies (Valerio 2009).In this work, we used the pkCSM web tool to predict toxicological parameters of the STL estafietin before carrying out an in vivo assay on a murine model of Chagas disease infection.The same web tool was employed for the in silico toxicological analysis of other sesquiterpene lactones, including other guaianolides such as dehydrocostus lactone, cynaropicrin and artecanin (Oladele et al. 2021;Hajji et al. 2022;Hammud 2022).The results were compared with the reference compounds benznidazole and nifurtimox and the STLs artemisinin and arglabin, currently employed as antimalarial and antitumour drugs, respectively (Table S1).The acute oral toxicity in rats of estafietin, expressed as Ld 50 , was in the same range as arglabin, while benznidazole, nifurtimox and artemisinin showed a slightly higher value.In humans, the maximum tolerated dose of these compounds was in the same range, except for artemisinin, which showed significantly lower toxicity.The lowest adverse effect level (LOAEL) of estafietin can be compared with that of arglabin, being both values one-fold higher than nifurtimox and artemisinin.Estafietin, arglabin and artemisinin are not predicted to be mutagenic compounds (AMES toxicity negative) and are not predicted to exert cardiotoxicity (lack of hERG I and II inhibition), hepatotoxicity or skin sensitization while benznidazole and nifurtimox showed positive AMES toxicity and benznidazole showed hepatotoxicity according to the web tool employed.It is worth to mention that estafietin displayed a low cytotoxicity (240.4 µg/ml) when tested in vitro on Vero cells (Sülsen et al. 2019).Considering that PAINS compounds may not be acceptable for drug development as they offer unselective modes of action and false-positive results (Glaser and Holzgrabe 2016), estafietin was passed through a PAINS filter and this STL did not prove to be a PAINS compound.
The in vitro trypanocidal activity of estafietin on the clinical forms of T. cruzi was previously assayed by our group (Sülsen et al. 2019).despite the moderate in vitro activity of the STL against bloodstream trypomastigotes and intracellular amastigotes, a significant effect on parasitemia was observed when the compound was evaluated on a murine in vivo model of Chagas disease.Mice were infected with the K98 clone (TcI-dTU) as it does not present lethality on the acute phase of the infection, allowing the assessment of the treatment on the chronic stage (Batalla et al., 2013).
The pharmacological protocol (intraperitoneal administration of 1 mg/kg/day for five consecutive days) was established according to previous works of our group (Sülsen et al. 2011;2013;Laurella et al. 2017).The effect of estafietin on the acute stage was evaluated in terms of parasitemia reduction.The treatment with the STL exerted a significant parasitemia reduction in comparison with untreated control (AUC reduction of 80.6%), similar to that observed for the reference drug benznidazole (AUC reduction of 97.1%) (Figure S3).In line with these results, the sesquiterpene lactone eupatoriopicrin exerted a reduction in the AUC of 85.7%, when tested in the same murine model (Elso et al. 2020).It is known that trypanocidal activity of sesquiterpene lactones is strongly influenced by the presence of conjugated enone moieties and their relative position and orientation (Kimani et al. 2018).The significant in vivo trypanocidal activity of estafietin may be attributed not only to the presence of the α,β-unsaturated carbonyl system but also to the presence of an epoxide ring, which is able to covalently bind to nucleophilic centers from biological systems (Gomes et al. 2020).
Taking into account that T. cruzi chronic infection leads to inflammatory infiltrations and subsequent myocyte damage (Molina and Kierszenbaum 1988), samples of cardiac and skeletal muscles were dissected at 100 dpi and analyzed by optic microscopy in order to detect signs of inflammation and tissue damage.No significant differences were observed between samples from heart muscle of treated and control mice but when samples of skeletal muscle were analyzed, a significant reduction in inflammatory infiltrates and myocyte necrosis was observed in samples from mice treated with estafietin (Figure S4).Similar results were obtained with the STL eupatoriopicrin.This STL also reduced the inflammatory infiltrates and the damaged myocytes areas after 5-days treatment by intraperitoneal route (Elso et al. 2020).
Sesquiterpene lactones have demonstrated to be promising leads against Chagas disease as they showed encouraging results on in vivo models of T. cruzi infection.The STLs lychnopholide and goyazensolide showed high survival percentages and cure rates when administrated to mice infected with a benznidazole resistant strain of T. cruzi (de Mello et al. 2016;Milagre et al. 2020).Another STL worth to mention is tagitinin C, which not only reduced inflammatory infiltrates in muscle tissue but also potentiated the in vivo trypanocidal activity of benznidazole (Gonçalves-Santos et al. 2019).Other STLs assessed by our group also displayed significant activity on in vivo models of T. cruzi infection.The treatment of mice infected with a lethal dose of T. cruzi with the STL deoxymikanolide was able to produce a 1.5 fold reduction in parasitemia levels in comparison with untreated control and nearly 70% of the treated mice survived at the acute phase of the infection (Laurella et al. 2017).Similar results were obtained after treatment of infected mice with cumanin.This STL decreased the number of circulating parasites and exerted a significant reduction of mice mortality with regard to the untreated control group (Sülsen et al. 2013).In another work, psilostachyin C induced a significant reduction in parasitemia levels in comparison to the control group (Sülsen et al. 2011).
Our results suggest that the treatment of infected mice with the STL estafietin had effect not only in the acute phase reducing the parasite load, but also in the chronic stage of T. cruzi infection.A treatment administered orally along with parasitological tests of greater specificity and sensitivity to effectively demonstrate the potential use of estafietin as a lead against Chagas disease will be carried out shortly.

Conclusion
Estafietin has displayed trypanocidal activity on a murine model of Chagas disease, both in acute and chronic stages of the infection.The compound produced a significant decrease on parasitemia levels and reduced inflammatory infiltrates and myocyte damage in muscle tissue.Considering the in vivo activity of estafietin and the promising toxicological profile predicted, this natural compound could be considered for further preclinical studies.