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 ( Tc 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 T. foetus and T. brucei structures—within Tc IMPDH. Recombinant Tc IMPDH kinetics revealed Michaelis constants of 155 µM for IMP and 292 µM for NAD⁺. Biochemical IC₅ ₀ 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 EC₅ ₀ (0.4 µM) and highest selectivity index (SI > 100), outperforming benznidazole (EC₅ ₀ = 3.0 µM; SI = 13.4) and other inhibitors. Our findings support Tc 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.