Activator of Heat Shock Protein 90 ATPase 1 (herein: Aha1) is an activator of the ATPase-activity of Hsp90 and is able to stimulate the inherent activity of yeast Hsp90 by 12-fold and human Hsp90 by 50-fold (Panaretou, B., et al., Mol. Cell 2002, 10:1307-1318). Biochemical studies have shown that Aha1 binds to the middle region of Hsp90 (Panaretou et al. 2002, supra, Lotz, G. P., et al., J. Biol. Chem. 2003, 273; 17228-17235), and recent structural studies of the Aha1-Hsp90 core complex suggest that the co-chaperone promotes a conformational switch in the middle segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg380 and facilitates its interaction with ATP in the N-terminal nucleotide-binding domain (Meyer, P., et al., EMBO J. 2000, 23:511-519).
The molecular chaperons Heat shock protein 90 (Hsp90) is responsible for the in vivo activation or maturation of specific client proteins (Picard, D., Cell Mol. Life Sci. 2002, 59:1640-1648; Pearl, L. H., and Prodromou, C., Adv. Protein Chem. 2002, 59:157-185; Pratt, W. B., and Toft, D. O., Exp. Biol. Med. 2003, 228-111-133; Prodromou, C., and Pearl, L, H., Curr. Cancer Drug Targets 2003, 3:301-323). Crucial to such activation is the essential ATPase activity of Hsp90 (Panaretou, B., et al, EMBO J. 1998, 17:4829-4836), which drives a conformational cycle involving transient association of the N-terminal nucleotide-binding domains within the Hsp90 dimer (Prodromou, C., et al, EMBO J. 2000, 19:4383-4392).
As a molecular chaperone, HSP90 promotes the maturation and maintains the stability of a large number of conformationally labile client proteins, most of which are involved in biologic processes that are often deranged within tumor cells, such as signal transduction, cell-cycle progression and apoptosis. As a result, and in contrast to other molecular targeted therapeutics, inhibitors of HSP90 achieve promising anticancer activity through simultaneous disruption, of many oncogenic substrates within cancer cells (Whitesell L, and Dai C., Future Oncol. 2005; 1:529-540; WO 03/067262). Furthermore, HSP90 has been implicated in the degradation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), Mutations in the CFTR gene lead to defective folding and ubiquination of the protein as a consequence of HSP90 ATPase activity. Following ubiquitination, CFTR is degraded before it can reach its site of activity. Lack of active CFTR then leads to the development of cystic fibrosis in human subjects having such mutation. Therefore, the inhibition of HSP90 activity may be beneficial for subjects suffering from cancer or Cystic Fibrosis.
Hsp90 constitutes about 1-2% of total cellular protein (Pratt, W. B., Annu. Rev. Pharmacol Toxicol. 1997, 37:297-326), and the inhibition of such large amounts of protein by means of an antagonist or inhibitor would potentially require the introduction of excessive amounts of the inhibitor or antagonist into a cell. An alternative approach is the inhibition of activators of HSP90's ATPase activity, such as Anal, which are present in smaller amounts. By downregulating the amount of Aha1 present in the cell the activity of HSP90 may be lowered substantially.
Significant sequence homology exists between Homo sapiens (NM—01211.1), Mus musculus (NM—146036.1) and Pan troglodytes (XM—510094.1) Aha 1. A clear rattus norvegicus homologue of Aha 1 has not been identified; however, there is a Rattus norvegicus (XM—223680.3) gene which has been termed activator of heat shock protein. ATPase homolog 2 (Ahsa 2) on the basis of its sequence homology to yeast Ahsa 2. Its sequence is homologous to mus musculus RIKEN cDNA 1110064P04 gene (NM—172391.3), which is in turn similar in sequence to Aus musculus Aha 1 except for N-terminal truncation. A homo sapiens Ahsa 2 (NM—152392.1) has also been predicted, but sequence homology is limited. The functions of these latter three genes have not been sufficiently elucidated. However, there exists one region in which all of the above sequences are identical, and which may be used as the target for RNAi agents. It may be advantageous to inhibit the activity of more than one Aha gene.
Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi), WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
Despite significant advances in the field of RNAi and advances in the treatment of pathological processes mediated by HSP90, there remains a need for an agent that can selectively and efficiently attenuate HSP90 ATPase activity using the cell's own RNAi machinery. Such agent shall possess both high biological activity and in vivo stability, and shall effectively inhibit expression of a target Aha gene, such as Aha1, for use in treating pathological processes mediated directly or indirectly by Aha expression, e.g. Aha1 expression.
USPTO Backgrounds
USPTO Backgrounds