Abstract
Heat shock proteins (HSPs) are involved in a number of cellular processes, including cell cycle, growth, and survival, apoptosis, stress responses, angiogenesis, and oncogenesis. Among the characterized HSPs, the molecular chaperone HSP90 has emerged as an exciting molecular target for cancer therapy since its discovery as the target protein of the antibiotic geldanamycin. The stress-inducible HSP70, which is upregulated in many cancers, contributing to tumor cell survival and resistance to therapy, has important roles as a housekeeper in the cell, assisting in the correct folding, trafficking, and degradation of many proteins. 2-Phenylethynesulfonamide (PES) physically interacts with HSP70 and disrupts the association between HSP70 and several of its cofactors and client proteins, leading to cancer cell death that is selectively mediated through caspase-independent mechanisms involving increased protein aggregation, impairment of lysosomal functions, and inhibition of autophagy. Mammalian HSP60 has several functions in the cell, including apoptosis, an immune-regulatory function, and cell spreading. HSP60 is a mitochondrial protein that is essential for the folding and assembly of newly imported proteins in the mitochondria. Epolactaene/ETB covalently binds to HSP60, inhibiting its chaperone activity. Molecular chaperone inhibitors are significantly valuable not only as tools to reveal the unknown cellular functions of molecular chaperones, but also as lead compounds for drug discovery. Thus, high-throughput screening systems are necessary for the discovery of more effective inhibitors. Here, we describe the methodology for 4 characteristic types of high-throughput screening systems for inhibitors of molecular chaperones, mainly HSP90 and HSP70: the colorimetric method, the fluorescence polarization method, the chemical array method, and the AlphaScreen® method.
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