Identification of Antifungal Agents AR-12 and Fosmanogepix as Anti- <i>Trypanosoma cruzi</i> Drugs through an Enhanced Fluorogenic β-Galactosidase Phenotypic Screening Assay

Phenotypic screening remains essential for identifying and characterizing bioactive compounds or their combinations against human parasitic pathogens. In the case of Trypanosoma cruzi, the etiological agent of Chagas disease, transgenic parasites expressing the reporter enzyme β-galactosidase have been extensively used to this end. Here, we replaced the traditional chromogenic substrate chlorophenol red-β- d -galactopyranoside (CPRG) with the fluorogenic 4-methylumbelliferyl-β- d -galactopyranoside (MUG) to derive a highly sensitive, continuous enzymatic assay to obtain a quantitative surrogate of parasite growth in T. cruzi cultures. The assay detects as few as 3 × 10 3 trypomastigotes/well, tracks linearly with the parasite load in a two-order range (3 × 10 3 –2 × 10 5 trypomastigotes/well), takes 1 h, and has a similar cost per assay as its colorimetric counterpart. To demonstrate its convenience and versatility, we used this assay to estimate the half-maximal inhibitory concentration (IC 50 ) of six emerging antifungal compounds, not targeting CYP-51 and novel for T. cruzi . Finally, the assay was adapted to a semiautomatic methodology and used to explore dual combinations of the active antifungals in the primary screening and with benznidazole. The multitarget compound AR-12 (IC 50 = 1.9 μM) and the Gwt1 inhibitor Fosmanogepix (IC 50 = 7.2 μM) resulted in bona fide hits, inhibiting parasite replication with only low-to-moderate toxicity on Vero host cells, thus suggesting potential for repurposing to Chagas disease.