Exploring <i>T. cruzi</i> IMPDH as a promising target through Chagas Box screening and AVN-944 inhibition

Chagas disease, caused by <i>Trypanosoma cruzi</i>, remains a leading cause of heart failure in Latin America, with current treatments limited to acute-phase efficacy, significant toxicity, and emerging resistance. Inosine monophosphate dehydrogenase (IMPDH) is an essential enzyme in guanine nucleotide salvage pathway and represents a promising alternative target. Here, we combined computational screening, biochemical and cell-based phenotypic assays that support <i>T. cruzi</i> IMPDH (<i>Tc</i>IMPDH) as a druggable target and identify repurposing opportunities among clinical-stage inhibitors. Using Tanimoto similarity scoring against the library of 222 Chagas Box compounds, we identified TCMDC-143376 as uniquely similar to the clinical IMPDH inhibitors merimepodib and AVN-944. Phylogenetic analysis and multiple sequence alignment confirmed conservation of both catalytic and allosteric residues-drawn from <i>T. foetus</i> and <i>T. brucei</i> structures-within <i>Tc</i>IMPDH. Recombinant <i>Tc</i>IMPDH kinetics revealed Michaelis constants of 155 µM for IMP and 292 µM for NAD⁺. Biochemical IC₅₀ assays showed submicromolar inhibition by AVN-944 (0.20 µM), (S)-Merimepodib (0.21 µM), and (R)-Merimepodib (0.37 µM). In H9c2 cardiomyoblasts infected with intracellular amastigotes, AVN-944 achieved the lowest EC₅₀ (0.4 µM), outperforming benznidazole (EC₅₀ = 3.0 µM) and other inhibitors. Our findings support <i>Tc</i>IMPDH as a promising alternative drug target for Chagas disease and position AVN-944 as a compelling candidate to evaluate this therapeutic strategy in animal models.