(A) 18; (B) 16; (C) 20. Upper spectra: Absolute absorbance in the Soret band region (black line – no ligand, red line – plus ligand. Lower: Difference spectra upon titration with the ligands (0.5 μM steps). Insets: Titration curves.

(A) Ligands (1–6) found via HTS using FMP library of small organic molecules (20,000-compounds selected from ChemDiv, San Diego, CA); (B) inhibitors (7–11) found using web-database search for structural similarity to 5 (http://chemdiv.emolecules.com); (C) 9-like INDO-amide derivatives. Calculated from spectral responses apparent dissociation constants (μM) for the compounds not included in Table 1 are shown in brackets.

(A) Structural formulas of neutral lipids formed upon sterol biosynthesis (1-squalene; 2-squalene epoxide; 3-lanosterol; 4 - 24-methylenedihydrolanosterol; 5 – obtusifoliol; 6- ergosterol; 7- 24- methylergosterol) or up taken from the media (8-cholesterol). 14α-methyl group and formed by CYP51 Δ14–15 double bond are shown in red.
(B) TLC analysis of sterols extracted from TC epimastigotes. St, sterol standards; C, sterols in untreated cells; T, sterols in TC incubated for 24 hours with 50 μM 20.

(A) Dose-dependent inhibition of T. cruzi intracellular multiplication and infection in cardiomyocytes by TCCYP51 inhibitors. Trypomastigotes were pre-treated with several concentrations of TCCYP51 inhibitors or mock-treated, exposed to cardiomyocyte monolayers and T. cruzi multiplication was evaluated at 72 h by determining the number of T. cruzi/cell (left panel) and the percent of infection (right panel). The data represent the mean ± standard deviation (SD) of results from triplicate samples. SD did not exceed 10% of the mean. Differences in T. cruzi per cell and % infection between trypomastigotes-mock treated (DMSO) and trypomastigotes treated with each TCCYP51 inhibitor (5–50 μM) are p<0.05 determined by the Student t test. The two most potent TCCYP51 inhibitors (21 and 22) show antiparasitic activities at ≤ 10 μM concentrations) but release cardiomyocyte monolayers at higher concentrations.
(B). Microscopic observation of the inhibition of T. cruzi multiplication by 20 μM TCCYP51 inhibitors within cardiomyocytes at 72 hr. T. cruzi pre-treated with control DMSO showed high levels of parasite multiplication, whereas cells exposed to trypanosomes pre-incubated with the inhibitors showed a dramatic decrease in the number of intracellular parasites.