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 <i>Trypanosoma cruzi</i>, 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 <i>T. cruzi</i> cultures. The assay detects as few as 3 × 10³ trypomastigotes/well, tracks linearly with the parasite load in a two-order range (3 × 10³-2 × 10⁵ 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 (IC₅₀) of six emerging antifungal compounds, not targeting CYP-51 and novel for <i>T. cruzi</i>. 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 (IC₅₀ = 1.9 μM) and the Gwt1 inhibitor Fosmanogepix (IC₅₀ = 7.2 μM) resulted in <i>bona fide</i> hits, inhibiting parasite replication with only low-to-moderate toxicity on Vero host cells, thus suggesting potential for repurposing to Chagas disease.