Advancing Trypanosoma cruzi N-Myristoyltransferase as a Drug Target for Chagas Disease through In Silico Discovery and Biochemical Evaluation

Diana C. Gonzalez-Garcia¹Angel Torres¹Alan Talevi²Lucas N. Alberca²Miguel A. Beltran¹Frida Lara¹Marina da Silva Ferreira¹Priscila Silva Grijó Farani¹Igor C Almeida¹Rosa Amelia Maldonado^1*

• ¹The University of Texas at El Paso, El Paso, United States
• ²Universidad Nacional de la Plata Facultad de Ciencias Exactas, La Plata, Argentina

N-myristoylation is a crucial lipid modification that governs protein localization, intracellular trafficking, and function in eukaryotic cells. The enzyme N-myristoyltransferase (NMT), which catalyzes this modification, has emerged as an attractive drug target for parasitic diseases. In this study, we performed a comprehensive biochemical and antiparasitic evaluation of Trypanosoma cruzi NMT (TcNMT), utilizing novel "in silico–identified inhibitors" to assess its potential as a therapeutic agent for Chagas disease. Recombinant TcNMT was successfully cloned, expressed, and purified. Enzymatic activity was confirmed using a fluorescence-based assay, which demonstrated high affinity for peptide substrates. Four candidate inhibitors were assessed for their ability to inhibit TcNMT, their cytotoxicity in host cells, and their antiparasitic activity in T. cruzi–infected human cardiomyocytes. Among the candidates, QUINE emerged as the most favorable drug lead. Although it exhibited moderate antiproliferative activity against the parasite, it showed remarkably low toxicity in human cardiomyocytes, resulting in a high selectivity index (SI=28.11), indicative of a wide therapeutic window. In contrast, the reference inhibitor DDD85646 demonstrated higher antiparasitic potency but a substantially lower selectivity index (SI=4.67), reflecting greater host toxicity. Proteomic analysis of T. cruzi myristoylated proteins from amastigotes and trypomastigotes treated with DDD85646 revealed stage-specific downregulation, including ARF GTPases and enzymes involved in lipid metabolism. These findings suggest widespread disruption of vesicular trafficking, signal transduction, and membrane biosynthesis pathways. Notably, host cell proteomes remained largely unaffected, underscoring the selective mechanism of NMT inhibition in the parasite.