RESUMEN
The immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO1) strengthens cancer immune escape, and inhibition of IDO1 by means of new chemotypes and mechanisms of action is considered a promising opportunity for IDO1 inhibitor discovery. IDO1 is a cofactor-binding, redox-sensitive protein, which calls for monitoring of IDO1 activity in its native cellular environment. We developed a new, robust fluorescence-based assay amenable to high throughput, which detects kynurenine in cells. Screening of a ca. 150 000-member compound library discovered unprecedented, potent IDO1 modulators with different mechanisms of action, including direct IDO1 inhibitors, regulators of IDO1 expression, and inhibitors of heme synthesis. Three IDO1-modulator chemotypes were identified that bind to apo-IDO1 and compete with the heme cofactor. Our new cell-based technology opens up novel opportunities for medicinal chemistry programs in immuno-oncology.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Línea Celular Tumoral , Cumarinas/química , Inhibidores Enzimáticos/química , Colorantes Fluorescentes/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/análisis , Estructura MolecularRESUMEN
Natural Killer (NK) cells are innate lymphocytes that are important for early immune reactions against viral infections and cancer. Their cytotoxic activity is mediated by the release of perforin and granzymes or by engaging death receptors on the surface of their target cells. Here we provide a protocol for the use of fluorescence localization reporters to measure the activity of granzyme B or caspase-8 activity inside living target cells. This method can be used to investigate how these two killing pathways are used by NK cells. By modifying the modular structure of the reporters, they can be adapted to study other cytotoxic effector cells or signaling pathways, where proteases play an important role.