Mechanistic studies on Hsp90 inhibition by ansamycin derivatives.
Heat shock protein 90 (Hsp90) is a molecular chaperone that is required for the maturation and activation of a number of client proteins, many of which are involved in cancer development. The ansamycin family of natural products and their derivatives, such as geldanamycin (GA), are well-known inhibitors of the essential ATPase activity of Hsp90. Despite structural studies on the complexes of ansamycin derivatives with the ATPase domain of Hsp90, certain aspects of their inhibitory mechanism remain unresolved. For example, it is known that GA in solution exists in an extended conformation with a trans amide bond; however, it binds to Hsp90 in a significantly more compact conformation with a cis amide bond. GA and its derivatives have been shown to bind to Hsp90 with low micromolar affinity in vitro, in contrast to the low nanomolar anti-proliferative activity that these drugs exhibit in vivo. In addition, they show selectivity towards tumour cells. We have studied both the equilibrium binding, and the association and dissociation kinetics of GA derivative, 17-DMAG, and the fluorescently labelled analogue BDGA to both wild-type and mutant Hsp90. The mutants were made in order to test the hypothesis that conserved residues near the ATP-binding site may catalyse the trans-cis isomerisation of GA. Our results show that Hsp90 does not catalyse the trans-cis isomerisation of GA, and suggests that there is no isomerisation step before binding to Hsp90. Experiments with BDGA measured over a wide range of conditions, in the absence and in the presence of reducing agents, confirm recent studies that have suggested that the reduced dihydroquinone form of the drug binds to Hsp90 considerably more tightly than the non-reduced quinone species.
Onuoha
S C
SC
Chemistry Department, University of Cambridge, UK.
Mukund
S R
SR
Coulstock
E T
ET
Sengerovà
B
B
Shaw
J
J
McLaughlin
S H
SH
Jackson
S E
SE
D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001896:Boron Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002151:Calorimetry; D045744:Cell Line, Tumor; D005454:Fluorescence Polarization; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D047029:Lactams, Macrocyclic / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017828:Rifabutin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013816:Thermodynamics
C523490:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-geldanamycin; D016227:Benzoquinones; D001896:Boron Compounds; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C448659:17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; D017828:Rifabutin