RESUMEN
Cathepsin B plays key roles in tumor progression with its overexpression being associated with invasive and metastatic phenotypes and is a primary target of protease activated antibody-directed prodrug therapy. It therefore represents a potential therapeutic and diagnostic target and effort has been made to develop fluorescent probes to report on Cathepsin B activity in cells and animal models of cancer. We have designed, synthesized, and thoroughly evaluated four novel "turn on" probes that employ a lysosomotropic dansylcadaverine dye to report on Cathepsin B activity. Enzyme activity assays using a recombinant human enzyme and cancer cell lysates coupled with confocal microscopy experiments demonstrated that one of the probes, derivatized with the self-immolative prodrug linker p-aminobenzyl alcohol, can selectively report on Cathepsin B in biological samples including live cells.
Asunto(s)
Cadaverina/análogos & derivados , Catepsina B/análisis , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Neoplasias/diagnóstico por imagen , Compuestos de Aminobifenilo/química , Cadaverina/síntesis química , Cadaverina/metabolismo , Catepsina B/metabolismo , Catepsina L/análisis , Catepsina L/metabolismo , Línea Celular Tumoral , Humanos , Hidrólisis , Cinética , Microscopía Confocal , Estructura Molecular , Imagen Óptica , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Cathepsin B (CTB) is a cysteine protease believed to be an important therapeutic target or biomarker for several diseases including aggressive cancer, arthritis, and parasitic infections. The development of probes capable of assessing CTB activity in cell lysates, living cells, and animal models of disease are needed to understand its role in disease progression. However, discovering probes selective to cathepsin B over other cysteine cathepsins is a significant challenge due to overlap of preferred substrates and binding site homology in this family of proteases. Herein we report the synthesis and detailed evaluation of two prodrug-inspired fluorogenic peptides designed to be efficient and selective substrate-based probes for CTB. Through cell lysate and cell assays, a promising lead candidate was identified that is efficiently processed and has high specificity for CTB over other cysteine cathepsins. This work represents a key step toward the design of rapid release prodrugs or substrate-based molecular imaging probes specific to CTB.
