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

Chagas disease, caused by Trypanosoma cruzi, 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 T. cruzi IMPDH (TcIMPDH) 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 T. foetus and T. brucei structures-within TcIMPDH. Recombinant TcIMPDH 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) and highest selectivity index (SI > 100), outperforming benznidazole (EC50 = 3.0 μM SI = 13.4) and other inhibitors. Our findings support TcIMPDH 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.