Dados do Resumo

Título

Drug repositioning in oncology: a study on Idebenone

Introdução

Triple-negative breast cancer (TNBC) is one of the subtypes with the worst prognosis and still lacks a specific treatment. The enzyme glutaminase C (GAC) was previously identified as an oncological therapeutic target and is essential for the growth of TNBC cells. The compound Idebenone was identified in a high-throughput screening campaign targeting GAC. The inhibitory behavior of this molecule was then studied using cell lines and biochemically through purified enzymes.

Objetivo

Our objective is drug repurposing, which consists of discovering new therapeutic uses for an already existing or investigational drug, different from its original indication. In this strategy, there is a reduction in overall development costs and time.

Métodos

The cellular impact of idebenone treatment was evaluated based on the inhibition of cell growth in different TNBC and non-TNBC cell lines. The level of glutamine consumption, an amino acid metabolized by GAC, was quantified in a hybrid model assay using cell culture byproducts and biochemical analyses. The mode of enzyme inhibition and kinetic constant values were accessed through a double reciprocal assay for all enzymes in the coupled reaction (glutamate dehydrogenase - GDH). The ability to alter enzymatic thermal stability and prevent the formation of characteristic GAC filaments or the formation of protein aggregates was analyzed by Thermal shift and Dynamic Light Scattering (DLS) assays, respectively. IC50 values of IDB were also defined for all isoforms of GAC, GDH, and for one TN and non-TN cell lines. All assays were performed in comparison with a previously established compound that inhibits GAC with high specificity.

Resultados

The IDB’s behavior was comparable to the treatment with CB-839 in TN cells, reducing the concentration of glutamine in the cellular medium and, consequently, the amount of glutamate excreted. The IC50 of IDB on TN MDA-MB-231 and non-TN SKBR3 was 15.2µM and 23.7µM, respectively. While the IC50 of IDB on the coupled reaction GAC+GDH is 194µM, it is lower at 67.7µM in the reaction with GDH alone, showing that the compound is capable of inhibiting both targets. IDB inhibits GAC+GDH in a non-competitive manner, and GDH in an uncompetitive manner. IDB led to a significant decrease in the temperature variation, suggesting that it causes a loss in enzyme stability. At 500 mM NaCl, IDB and CB-839 did not lead to protein aggregation. IDB disrupted the active oligomeric structures of glutaminase induced by Pi. For the isoforms, the IC50 of KGA was 47.1µM, close to that obtained for GAC, while for GLS2 it was 48.3µM indicating that the inhibitor acts on all glutaminases with similar potencies.

Conclusões

Our assays allowed us to conclude that idebenone inhibited breast cancer cells, exhibiting activity against the GAC isoform, KGA, and the isoenzyme GLS2. In addition, idebenone also inhibited the enzyme GDH. Since we have confirmed that idebenone does not have a nonspecific action of NADH oxidation, a component of the enzymatic reaction, and did not lead to GAC aggregation, there is a chance that it acts on these enzymes through some specific mechanism.

Financiador do resumo

FAPESP #2016/09077-4, 21/13736-1, #2014/15968-3, #2015/25832-4, and #2019/16351-3
CAPES and LNBio

Palavras Chave

drug repositioning; glutaminase c; idebenone