Aminoglycosides Suppress the Protein Folding Activity of the Molecular Chaperone HSC70: Implication of a Structure-Activity Relationship

Abstract

Background: The mechanism of aminoglycoside (AG)-induced nephrotoxicity has not been fully elucidated. We previously reported that gentamicin suppresses the functions of HSC70, a heat shock cognate protein, which is a cytosolic molecular chaperone in mammalian cells. However, the relationship between HSC70 dysfunction and nephrotoxicity has not been clarified yet. Methods: The effects of 11 AGs, including gentamicin, on the function, protein refolding and substrate binding of HSC70 were examined in vitro. The protein refolding activity of HSC70 was examined by enzymatic activity restoration of the denatured firefly luciferase. Substrate binding activity of HSC70 was examined by the binding of fluorescent-dye-labeled reduced carboxymethlylated α-lactalbumin determined by HSC70/substrate complex formation by native polyacrylamide gel electrophoresis. Results: Strong inhibitory activity of AGs on the protein refolding activity of HSC70 was observed for arbekacin, gentamicin, neomycin and sisomicin, but not spectinomycin, streptomycin, ribostamycin and paromomycin, and inhibition was weak for tobramycin, amikacin and kanamycin. Neomycin strongly suppressed the formation of the HSC70/substrate complex and had the lowest concentration that resulted in 50% inhibition (IC₅₀; in the order of 10^-4<smlcap>M</smlcap>). Arbekacin, gentamicin, sisomicin and tobramycin had IC₅₀ values in the order of 10^-3<smlcap>M</smlcap>. Amikacin, ribostamycin and paromomycin had no effective potencies. Conclusions: The inhibition efficacies of AGs for protein refolding of HSC70 were as follows: neomycin >> gentamicin, arbekacin, and sisomicin > tobramycin, amikacin and kanamycin > spectinomycin, streptomycin, ribostamycin and paromomycin. Amino groups and/or hydroxyl groups located at 2′, 3′, 4′, and 6′ of amino sugar residue II, as well as the number of amino sugar rings were closely associated with the inhibition efficacies of AGs. Inhibition efficacies of AGs towards the function of HSC70 correlated well with the nephrotoxicity of AGs deduced from the in vivo studies previously reported. Amino groups/hydroxyl groups and the number of amino sugar rings of AGs were shown to be important for inhibition efficacies. The results strongly suggest that the AG-induced dysfunction of HSC70 is involved in the nephrotoxicity.