SEARCH FOR NEW INSECTICIDES AND ANTIPARASITICS THROUGH THE INHIBITION OF A SPECIFIC STEROL DESATURASE

PUCA, GERVASIO; ALONSO LEONARDO; A. NUSBLAT

Rosario

Congreso; LIX Annual Meeting SAIB 2023; 2023

The C7 sterol desaturase is a Rieske-type monooxygenase initially described in insects and nematodes.The enzyme is involved in the synthesis of steroid hormones and plays a crucial role in the embryonic andlarval development of the mentioned organisms. Orthologs have been found in other animals but not inmammals. Due to its role in the development of insects and nematodes of health and agriculturerelevance, and its absence in mammals, C7 sterol desaturase could be used as a target for thedevelopment of new, specific insecticides and antiparasitic agents. These compounds would act byinhibiting the enzyme, leading to the arrest of early development and subsequent death of the targetorganisms. However, the 3D structure and mechanism of action of the enzyme are unknown, making itscharacterization necessary for the rational development of inhibitors.The objectives of this study were to express and purify the C7 sterol desaturase for the subsequentdetermination of its conformational characteristics and to evaluate potential enzyme inhibitors. Toachieve this, the ciliated model organism Tetrahymena thermophila, which possesses an ortholog of C7sterol desaturase, was used.The enzyme was expressed in the ciliate fused to a tag for subsequent purification by affinitychromatography, for which a recombinant strain with this tag was generated. For the expression of theenzyme in E. coli, gene constructs were generated, optimizing codon usage frequency. Additionally, thesequence of the Maltose Binding Protein was added to the constructs to facilitate purification via affinitychromatography. As a result, the enzyme was successfully expressed in both systems and obtained withhigh purity.Furthermore, to evaluate potential compounds with insecticidal or antiparasitic activity, compounds wereselected based on similarity to the substrate and through an updated literature search in variousscientific and patent databases. The determination of in vivo inhibitory activity was carried out using T.thermophila. Ten compounds with potential inhibitory activity on the enzyme were evaluated, none ofwhich showed a significant difference in C7 sterol desaturase activity compared to the control.In conclusion, this study laid the groundwork for the development of new insecticides and antiparasiticagents targeting the inhibition of C7 sterol desaturase. An in vivo system for determining enzyme activityand inhibition was developed and optimized, and a recombinant protein production system wasgenerated. This advancements will allow for further testing of potential inhibitors, as well as thedetermination of the protein´s conformation and the prediction of the structure of the enzyme-substratecomplex for the rational identification of inhibitors