Identification of levothyroxine antichagasic activity through computer-aided drug repurposing

Cruzipain (Cz) is the major cysteine protease of the protozoan Trypanosoma cruzi, etiological agent of Chagas disease. A conformation-independent classifier capable of identifying Cz inhibitors was derived from a 163-compound dataset and later applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. 54 approved drugs were selected as candidates, 3 of which were acquired and tested on Cz and T. cruzi epimastigotes proliferation. Among them, levothyroxine, traditionally used in hormone replacement therapy in patients with hypothyroidism, showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.Facultad de Ciencias Exacta

Identification of levothyroxine antichagasic activity through computer-aided drug repurposing

Cruzipain (Cz) is the major cysteine protease of the protozoan Trypanosoma cruzi, etiological agent of Chagas disease. A conformation-independent classifier capable of identifying Cz inhibitors was derived from a 163-compound dataset and later applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. 54 approved drugs were selected as candidates, 3 of which were acquired and tested on Cz and T. cruzi epimastigotes proliferation. Among them, levothyroxine, traditionally used in hormone replacement therapy in patients with hypothyroidism, showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.Facultad de Ciencias Exacta

Flavins stimulate while analogs retard progression through <i>T</i>. <i>cruzi</i> life cycle.

<p>(A) <i>In vitro</i> metacyclogenesis assay scheme (for more details, see ‘<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005513#sec005" target="_blank">Material and methods</a>‘ section). Percentage of MT obtained in differentiation media (TAU3AAG) supplemented with (B) flavins (riboflavin: RF, FMN or FAD), or (C) chemical analogs (roseoflavin: RoF, lumiflavin: LF, or lumichrome: LC), at the indicated concentrations at 48 h. (D) <i>In vitro</i> infection assay scheme (for more details, see ‘<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005513#sec005" target="_blank">Material and methods</a>‘ section). Effect of 10 μM analogs on (E) cellular invasion at 48 h, expressed as percentage of H9C2 host cells infected with <i>T</i>. <i>cruzi</i> Y-GFP (∼400 cells counted) or (F) amastigote proliferation at 48 h, expressed as number of <i>T</i>. <i>cruzi</i> Y-GFP amastigotes per infected H9C2 host cell. Values are expressed as mean ± SD. Statistical analysis was performed by a Kruskal-Wallis non-parametric test followed by a post-hoc Dunn's multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.005).</p

<i>Tc</i>RibJ and <i>Tb</i>RibJ display flavin transport activity in <i>E</i>. <i>coli</i>.

<p>The <i>E</i>. <i>coli</i> ∆<i>ribB</i> strain was transformed with expression plasmids coding for either RibM, <i>Tc</i>RibJ, <i>Tb</i>RibJ, <i>Lmi</i>RibJ or <i>Tc</i>PAT12, or with an empty vector. Strains were (A) plated on LB agar (left: strain plating scheme) with the addition of 0, 5 or 750 μM riboflavin (RF) or (B) cultured at 37°C for 24 h in liquid LB supplemented with 0.02–100 μM FMN or FAD, or 0.02–20 μM riboflavin. (C) [³H]-riboflavin uptake (2 μM) was measured from 0 to 180 min in bacteria expressing RibM, <i>Tc</i>RibJ or <i>Tb</i>RibJ or containing an empty vector. (D) Displacement assays performed with 0.3 μM [³H]-riboflavin in the absence of competitors (Ctrl: control) or in the presence of 3–30 μM of unlabelled riboflavin; aliquots were sampled at 0 and 120 min after the addition of the radioactive material. Values are expressed as mean ± SD. Statistical analysis was performed by one way ANOVA test followed by a post-hoc Tukey's multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.005).</p

<i>Tc</i>RibJ functions as flavin transport <i>in vivo</i> in <i>T</i>. <i>cruzi</i>.

<p>Epimastigotes transfected with pRIBOTEX-GFP (wt) or pRIBOTEX-<i>Tc</i>RibJ (<i>Tc</i>RibJ) were incubated in fresh SDM-20-10% FBS in the presence of (A) 20 nM riboflavin (RF), FMN or FAD, or (B) roseoflavin (RoF), lumiflavin (LF), or lumichrome (LC), at the indicated concentrations. Parasites were counted daily using a hemocytometer chamber. <i>T</i>. <i>cruzi</i> proliferation (%) was calculated at the third culture round, where the control condition (20 nM flavin) was referenced as 100%. (C) [³H]-riboflavin (100 nM) uptake measurements in control and over-expressing <i>Tc</i>RibJ epimastigotes. Values are expressed as mean ± SD. (A-B) Statistical analysis was performed by a two-tailed unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.005).</p

Riboflavin apparent K_m and V_max uptake parameters measured in trypanosomatids.

<p>Riboflavin apparent K_m and V_max uptake parameters measured in trypanosomatids.</p