Structural Basis of the Radicicol Resistance Displayed by a Fungal Hsp90

Abstract

Heat shock protein 90 (Hsp90) is a promising cancer drug target, as multiple oncogenic proteins are destabilized simultaneously when it loses its activity in tumor cells. Highly selective Hsp90 inhibitors, including the natural antibiotics geldanamycin (GdA) and radicicol (RAD), inactivate this essential molecular chaperone by occupying its nucleotide binding site. Often cancer drug therapy is compromised by the development of resistance, but a resistance to these Hsp90 inhibitors should not arise readily by mutation of those amino acids within Hsp90 that facilitate inhibitor binding, as these are required for the essential ATP binding/ATPase steps of the chaperone cycle and are tightly conserved. Despite this, the Hsp90 of a RAD-producing fungus is shown to possess an unusually low binding affinity for RAD but not GdA. Within its nucleotide binding site a normally conserved leucine is replaced by isoleucine, though the chaperone ATPase activity is not severely affected. Inserted into the Hsp90 of yeast, this conservative leucine to isoleucine substitution recreated this lowered affinity for RAD in vitro. It also generated a substantially enhanced resistance to RAD in vivo. Co-crystal structures reveal that the change to isoleucine is associated with a localized increase in the hydration of an Hsp90-bound RAD but not GdA. To the best of our knowledge, this is the first demonstration that it is possible for Hsp90 inhibitor resistance to arise by subtle alteration to the structure of Hsp90 itself.

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

“…There is no direct contact between Ser or Ala and radicicol. However, as described previously 16 , water molecules play a crucial role in…”

“…Single amino acid mutations in Hsp90 may increase resistance to radicicol through an increased affinity of mutated Hsp90 to cochaperone Aha1 44 and are not related to change in radicicol affinity. Other mutants, however, directly affect radicicol binding affinity at the active site 16 .…”

Hsp90 Inhibitors in Parasitic Nematodes: Prospects and Challenges

“…fuscoatra produces the N-terminal inhibitor RAD and provides an excellent example of an adaptation to survive self-toxicity [50]. Here a single amino acid residue (L34 to I) results in a RADresistant phenotype, although sensitivity to GA or ATPase activity is not significantly affected.…”

Recent Advances in Macrocyclic Hsp90 Inhibitors

“…57 Geldanamycin-based analogues compete with ATP/ADP for binding to the ATP-binding pocket of Hsp90. 58 The binding affinity (K d ) of geldanamycin (1) for the N-terminal domain of yeast Hsp90 is 1.2 mM, 59 and the crystal structure of geldanamycin bound to its N-terminal Hsp90 binding pocket has highlighted unique structural changes that occur at the binding interface. [59][60][61] Geldanamycin adopts a ''C-clamp'' conformation when bound to Hsp90, where the benzoquinone forms the top of the clamp and the aliphatic ansa ring, with cis-configured amide, forms the stem and base (Figure 2.3).…”