WO2004037978A2 - Methods of reducing the activity of and reducing the concentration of a mutant kit protein - Google Patents

Abstract

The present invention pertains to methods of reducing the activity of a mutant KIT protein and also to methods of reducing the concentration of a mutant KIT protein. The mutant KIT protein can be in a cell, or the mutant KIT protein can be in a host. The present inventive methods comprise administering to the cell or the host either an inhibitor of Hsp90, which binds to an ATP binding domain of Hsp90, or a compound comprising a macrocycle, such as 17-allylamino-17-demethoxygeldanamycin. The mutant KIT protein can comprise a mutation in the kinase domain or the juxtamembrane domain. The host can be afflicted with a disease that is characterized by a mutant KIT protein, such as mastocytosis, gastrointestinal stromal tumor, mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

Description

METHODS OF REDUCING THE ACTIVITY OF AND REDUCING THE CONCENTRATION OF A MUTANT KIT PROTEIN

FIELD OF THE INVENTION [0001] The present invention pertains to methods of reducing the activity of a mutant KIT protein and also to methods of reducing the concentration of a mutant KIT protein, and, more particularly, to a method of reducing the activity of a mutant KIT protein and a method of reducing the concentration of a mutant KIT protein with an inhibitor of Hsp90, wherein the inhibitor binds to an adenosine triphosphate (ATP) binding domain of Hsp90.

BACKGROUND OF THE INVENTION [0002] Proto-oncogene c-kit encodes the transmembrane type III tyrosine kinase, KIT protein (Yarden et al., EMBO J 6:3341-3351 (1987)), which is the receptor for stem cell factor (SCF) (Anderson et al., Cell, 63:235-243 (1990); Flanagan et al., Cell, 63:185:194 (1990); Martin et al., Cell, 63:203-211 (1990); Zsebo et al., Cell, 63:213-224 (1990)) . This subfamily of tyrosine kinases which also includes receptors for platelet-derived growth factor, macrophage colony-stimulating factor and flt3 ligand, are characterized by their five immunoglobulin-like extracellular domains and by an intracytoplasmic domain which contains an ATP binding domain and a phosphotransferase domain separated by an interkinase sequence (Yarden et al., EMBOJ., 6:3341-3351 (1987); Qiu et al., EMBOJ., 7:1003-1011 (1988)). The c-kit gene was mapped to the White spotting (W) locus in mice (Chabot et al., Nature 335:88-89 (1988); Geissler et al., Cell, 55:185-192 (1988)), which was first described in 1927 (Russell et al., Adv. Genet. 20:357-459 (1979)) and the SCF gene was mapped to the Steel locus (SI) (Anderson et al.(1990), supra; Huang, et al., Cell, 63:225-233 (1990)) in mice which was first described in 1956 (Russell et al.(1979), supra. Under normal circumstances SCF binds to KIT inducing homodimerization of the receptor leading to intrinsic kinase activity and resulting in autophosphorylation of tyrosine residues (Blechman et al., Cells, 11 Suppl 2:12-21 (1993)). KIT then becomes the docking site for various SH2 domain signaling molecules. The KIT receptor is expressed on melanocytes, mast cells, primitive hematopoietic cells, primordial germ cells, intraepithelial lymphocytes and interstitial cells of Cajal (Fleischman et al., Trends Genet. 9:285-290 (1993); Huizinga et al., Nature, 373 :347-349 (1995)) .

[0003] Activating mutations within c-kit, first described by Besmer et al. (Besmer et al, Nature, 320:415-421 (1986)) as the cellular homologue of viral oncogene v-kit isolated from the retrovirus Hardy-Zuckerman 4-feline sarcoma virus, were later described in various neoplastic diseases including mast cell disease and gastrointestinal stromal tumors (GIST) (Heinrich et al., J Clin Oncol, 20:1692-1703 (2002)). Activating mutations cause constitutive phosphorylation of the KIT protein independent of ligand binding (Moriyama et al., J Biol Chem., 271 :3347-3350 (1996)). In effect, important downstream signaling alterations occur, believed to contribute to abnormal proliferation and survival of these neoplastic cells. Examples of signaling pathways and proteins activated by KIT include the Ras-Raf-MAP kinase cascade, the phosphatidylinositol-3 -kinase- AKT cascade, JAK/STAT pathway and the Src family of kinases (Linnekin et al., Int. J. Biochem Cell Biol, 31:1053- 1074 (1999); Taylor et al., Hematol Oncol Clin North Am., 14:517-535 (2000)) . [0004] Mastocytosis, defined as a pathologic increase in the number of mast cells in tissue, is a very heterogeneous disease varying in clinical significance from skin involvement alone to systemic involvement with infiltration of the gastrointestinal tract, spleen and bone manow (Metcalfe et al., Leuk. Res, 25:577-582 (2001)). The childhood form is usually a self-limited cutaneous form not associated with mutated KIT protein, although exceptions have been published, and the sporadic adult systemic form of the disease is always associated with KIT activating mutations (Longley et al., Proc Natl Acad Sci U.S.A., 96:1609-1614 (1999)). Two types of mutations have been well described (Longley et al.(2001), supra. The first consist of mutations in codon 816 of c-kit which is a single residue substitution of valine for aspartic acid (Asp816Val) in the kinase domain of KIT (Nagata et al., Proc Natl Acad Sci., 92:10560-10564 (1995)). Mutations at this codon are present in essentially all cases of adult systemic mastocytosis (Longley et al.(2000), supra) and have been termed an enzymatic type pocket mutation (Ma et al., Blood, 99:1741- 1744 (2002)). The second type of mutation includes single residue substitutions and in- frame insertions or deletions in the intracellular juxtamembrane region of KIT protein (Ma et al., J Invest Dermatol, 112:165-170 (1999)). The juxtamembrane mutations are not found in all cases of adult mastocytosis although they are found consistently in other diseases such as gastrointestinal stromal tumors (Hirota et al., Science, 279:577-580 (1998)). Juxtamembrane type mutations have been termed regulatory type activating mutations. HMC-1 cells, a human mast cell line, contain both types of mutations (Furitsu et a\., JClin Invest, 92:1736-1744 (1993)). Subsequent to the development of the original HMC-1 cell line, a subclone was created and designated as HMC-1.1 which contains only the juxtamembrane mutation, valine to glycine (Val560Gly). Interestingly, HMC-1.1 cells are sensitive to specific tyrosine kinase inhibitors while the original HMC-1 cells (designated HMC-1.2) are relatively insensitive to these drugs (Ma et al. (2002), supra. [0005] Diseases and conditions other than mastocytosis are also characterized by a mutant KIT protein. Such diseases or conditions include gastrointestinal stromal tumors (GIST), mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer. (Longley et al. (2000), supra; Heinrich et al. (2002), supra) [0006] Geldanamycin is a benzoquinoid ansamycin antibiotic, which binds to heat shock protein 90 (Hsp90), and has been shown to decrease levels of various important kinases involved in proliferation and survival of malignant cells (Neckers, Trends Mol Med, 8:S55-S61 (2002); Schulte et al., JBiol Chem 270:24585-24588 (1995); Whitesell et al., Cancer Res, 52:1721-1728 (1992); Whitesell et al., Proc Natl Acad Sci USA, 91:8324-8328 (1994)). 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a chemical derivative of geldanamycin, which contains significant anti-tumor activity via the same mechanism but enjoys a favorable toxicity profile (Schulte et al., Cancer Chemother Pharmacol, 42:273- 279 (1998)). It is thought that Hsp90 takes on two configurations depending on its ATP binding status and each configuration associates with a distinct multi-chaperone complex. In general, one complex tends to stabilize client proteins while the other complex, in association with Hsp70 has been described to ubiquitinate client proteins, targeting them for degradation via the proteosome (Mimnaugh et al., JBiol Chem, 271 :22796-22801 (1996)). 17-AAG and other ansamycin antibiotics bind to the amino-terminus (N-terminus) of Hsp90 at the ATP binding domain (Stebbins et al., Cell 89:239-250 (1997)) effectively locking the chaperone into its ADP-dependent configuration causing the later complex to be favored and client proteins to be degraded (Schneider et al., Proc Natl Acad Sci USA, 93:14536- 14541 (1996)). Client proteins of Hsp90 that are sensitive to ansamycin antibiotics are typically mutated or chimeric proteins (Neckers (2002), supra) that may depend on Hsp90 to exist in their misfolded state. There is emerging evidence that client proteins with mutations in the kinase domain tend to be more sensitive to treatment with ansamycin antibiotics (Citri et al., EMBOJ 21 :2407-2417 (2002)). It has also been recently reported that flt-3, another type III tyrosine kinase receptor protein, is a client protein to Hsp90 (Minami et al., Leukemia 16:1535-1540 (2002)). Heretofor, the relationship between Hsp90 and the KIT protein was not known.

BRIEF SUMMARY OF THE INVENTION [0007] Applicants have now discovered that the KIT protein is a client protein of Hsp90, such that inhibition of Hsp90 leads to the degradation of KIT proteins, including mutants thereof. The present invention provides methods of reducing the activity of a mutant KIT protein and methods of reducing the concentration of a mutant KIT protein. In one embodiment, the mutant KIT protein is in a cell, and in another embodiment, the mutant KIT protein is in a host. The present inventive methods comprise administering to the cell or host an inhibitor of Hsp90 in an amount sufficient to reduce either the activity or the concentration of the mutant KIT protein, wherein the inhibitor binds to an ATP binding pocket of Hsp90. In alternative embodiments of the present inventive methods of reducing either the activity or concentration of a mutant KIT protein, a compound comprising a macrocycle of the following structure:

is administered to the cell or host in an amount sufficient to reduce either the activity or the concentration of the mutant KIT protein. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS [0008] Figure 1 demonstrates a listing of the sequences refened to herein.

DETAILED DESCRIPTION OF THE INVENTION [0009] The present invention provides a method of reducing the activity of a mutant KIT protein in a cell or in a host comprising the mutant KIT protein. In one embodiment, the method comprises administering to the cell or host an inhibitor of Hsp90 in an amount sufficient to reduce the activity of the mutant KIT protein, wherein the inhibitor binds to an ATP binding pocket of Hsp90. Through this method, the activity of the mutant KIT protein is reduced.

[0010] The present invention also provides a method of reducing the concentration of a mutant KIT protein in a cell or in a host comprising the mutant KIT protein. In one embodiment, the method comprises administering to the cell or host an inhibitor of Hsp90 in an amount sufficient to reduce the concentration of the mutant KIT protein, wherein the inhibitor binds to an ATP binding pocket of Hsp90. Through this method, the concentration of the mutant KIT protein is reduced.

[0011] The present invention also provides in other embodiments methods of reducing the activity or the concentration of a mutant KIT protein in a cell or in a host, which methods comprise administering to the cell or host a compound comprising a macrocycle as described herein in an amount sufficient to reduce the activity or concentration of the mutant KIT protein.

[0012] For purposes of the present invention, the phrase "inhibitor of Hsp90" refers to any chemical compound, natural or synthetic, that inhibits the function of Hsp90. As used herein, the term "inhibits," and words stemming therefrom, do not necessarily imply 100% or complete inhibition. Rather, there are varying degrees of inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this regard, inhibitors of Hsp90 can induce any level of Hsp90 inhibition. Desirably, the inhibitors of Hsp90 inhibit at least 10% of the function or activity of Hsp90 in the absence of any inhibitors of Hsp90. It is more prefened that the inhibitors of Hsp90 achieve a 50% inhibition. Most prefened, is that the inhibitors of Hsp90 inhibit 90% of the activity of Hsp90 in the absence of any inhibitors of Hsp90. The function of Hsp90, as generally known by one of ordinary skill in the art, is to stabilize a client protein, such that the client protein is not subject to degradation. Client proteins of Hsp90 include c-KIT, Flt-3 (Minami et al. (2002), supra), p53 (Blagosklonny et al., Proc. Natl. Acad. Sci. 93(16): 8379-8383 (1996)), Raf-1 (Schulte et al., Biochem. Biophys. Res. Comm. 239: 655-659 (1997)), Epidermal Growth Factor Receptor (EGFR), ErbB2 (Xu et al., J Biol. Chem. 276: 3702-3708 (2001)), AKT (Basso et al., J. Biol Chem. 277: 39858-39866 (2002)), BCR- ABL (Blagosklonny et al., Leukemia 15: 2537-1543 (2001)), SRC, Whitesell et al., Proc. Natl. Acad. Sci. USA 91: 8324-8328 (1994)), and HIFlα (Isaacs et al., JBiol Chem. Ill: 29936-39944 (2002)).

[0013] Any inhibitor of Hsp90 can be employed in the present inventive methods, provided that the inhibitor binds to an ATP binding pocket of Hsp90. It is generally known that Hsp90 comprises two ATP binding pockets or domains, one of which is found in the N- terminus of Hsp90 and can be comprised of amino acids 9 - 236 of SEQ ID NO: 1, which is the amino acid sequence of human Hsp90, and the other of which is found in the C-terminus of Hsp90 and can be comprised of amino acids 538-677 of SEQ ID NO: 1 or amino acids 601 - 728 of SEQ ID NO: 2, which is the amino acid sequence of chicken Hsp90. One of ordinary skill in the art will appreciate that binding of the inhibitor to either one of the ATP binding pockets of Hsp90 prevents the binding of a client protein to Hsp90, such that the client protein is subject to degradation.

[0014] Binding affinities and relative binding affinities, which quantify the degree to which an inhibitor of Hsp90 binds to Hsp90, can be measured by any suitable method known in the art. For example, competitive binding assays upon which the concentration at which a test inhibitor of Hsp90 not known to bind to an ATP binding domain of Hsp90 binds to a given percentage of an ATP binding domain of Hsp90 in the presence of a second Hsp90 inhibitor, which is known to bind to the ATP binding domain of Hsp90, can be ascertained. A prefened method of detennining the relative binding affinities is by comparing the concentration of the test inhibitor at which 50% of the ATP binding domain of Hsp90 is bound (otherwise know as the IC₅₀ concentration level) in a competitive binding assay to the IC₅₀ of another inhibitor of Hsp90, which is known to bind to the ATP binding domain of Hsp90. Such assays that test whether or not an inhibitor of Hsp90 binds to an ATP binding domain are described in Schulte et al., Cell Stress Chaperones 3: 100-108 (1998); Sharma et al., Oncogene 16: 2639-2645 (1998); and Schulte et al., Mol. Endocrin. 13: 1435-1448 (1999).

[0015] Many inhibitors of Hsp90 that bind to an ATP binding domain are known in the art, some of which are described in the following references: Agatsuma et al., Bioorg. Med. Chem. 10(11): 3445 (2002); Garbaccio c\ a\., JAm. Chem. Soc. 123(44): 10903-10908 (2001); Andrus et al., Org. Lett. 4(20): 3549-3552 (2002); Neckers, Trends Mol. Med. 8(4 Suppl): S55-S61 (2002); Gamier et al., J Biol. Chem. 277(14): 12208-12212 (2002); Soga et al., Cancer Chemother. Pharmacol. 48(6): 435-445 (2001); Neckers et al., Drug Resist. Updat. 2(3): 165-172 (1999); Shiotsu et al., Blood 96(6): 2284-2291 (2000); Marcu et al., J. Biol. Chem. 275(47) 37181-37186 (2000); An et al., Cell Growth Differ. 11(7): 355-360 (2000); Neckers et al., Invest. New Drugs 17(4): 361-373 (1999); Marcu et al., J Natl. Cancer Inst. 92(3): 242-248 (2000); Schulte et al., Mol. Endocrinol 13(9): 1435-1448 (1999); Soga et al., Cancer Res. 59(12): 2931-2938 (1999); Schulte et al., Cancer Chemother. Pharmacol. 42(4): 273-279 (1998); Schulte et al., Cell Stress Chaperones 3(2): 100-108 (1998); Mimnaugh et al., Biochem. 36(47): 14418-14429 (1997); Grenert et al., J. Biol. Chem. 272(38): 23843-23850 (1997); Schulte et al., Mol. Cell. Biol. 16(10): 5839- 5845 (1996); and Whitesell et al., Proc. Natl. Acad. Sci. USA 91(18): 8324-8328 (1994)). [0016] Compounds comprising a macrocycle of the following structure:

are known to inhibit Hsp90. Geldanamycin and derivatives thereof are examples of such compounds. Other inhibitors, which do not comprise the above macrocycle, are likewise suitable for use in the present inventive methods. Radicicol is an example of such inhibitors, which can be used in the practice of the present inventive methods. [0017] In a prefened embodiment, the compound comprising the above macrocycle is a compound of Formula I:

(Formula I) wherein n=0 or 1 and wherein, when n=l, each of R and R is hydrogen, and, when n=0, a double-bond exists between position 4 and position 5;

R³ is hydrogen or hydroxyl;

R⁴ is hydrogen, hydroxyl, or R⁷C(O)O-, wherein R⁷ is amino(C₁-C₈)alkyl or imino^-Cs^lkyl; wherein, when R³ is hydrogen, R⁴ is hydroxyl or R⁷C(O)O-, and when R³ is hydroxyl, R⁴ is hydrogen;

R⁵ is hydrogen or a group of the formula

wherein each of R⁸, R⁹, and R¹⁰ is independently selected from the group consisting of hydrogen, a halo, an azido, a nitro, a C C₈ alkyl, a C^Cs alkoxy, an aryl, a cyano, and an

1 ι 1 *5 ι *3 1 1 10 1 ^

NR R R , wherein each of R , R , and R is independently selected from the group consisting of hydrogen and a Cι-C₃ alkyl;

R⁶ is hydrogen, a methoxy, a Cι-C₈ alkylamino, a Cι-C₈ dialkylamino, an ,N'- dialkylaminodialkylamino-, an N,N'-dialkylaminoalkylamino, or an allylamino. [0018] The term "alkyl" includes saturated alkyl and unsaturated alkyl substituents. The term "saturated alkyl" means a straight-chain or branched-chain saturated alkyl containing, e.g., from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms. Examples of saturated alkyls include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, octyl, and the like. Saturated alkyl substituents can be unsubstituted or substituted, for example, with at least one substituent selected from the group consisting of a halogen, a nitro, an amino, a hydroxyl, an alkoxy, an aryloxy, an aralkoxy, an ester, an amide, a sulfhydryl, an alkyl sulfide, an aryl sulfide, an alkyl sulfoxide, an aryl sufoxide, an alkyl sulfonyl, an aryl sulfonyl, a keto, a thioketo, an alkyl, a cyano, and the like. The term "unsaturated alkyl" means an unsaturated alkyl (straight-chain or branched-chain), as defined herein, in which at least one single carbon-carbon bonds thereof is instead a multiple bond, for example, a double bond or a triple bond. Unsaturated alkyls include alkenyls and alkynyls, as well as substituents that have a combination of double and triple bonds. The term "alkenyl" means a straight-chain or branched-chain alkenyl having one or more double bonds. An alkenyl can contain, e.g., from 2 to about 8 carbon atoms, or from 2 to about 6 carbon atoms. Examples of alkenyls include vinyl, allyl, 1,4-butadienyl, isopropenyl, and the like. The term "alkynyl" means a straight-chain or branched-chain alkynyl having one or more triple bonds. An alkynyl can contain, e.g., from 2 to about 8 carbon atoms, or from 2 to about 6 carbon atoms. Examples of alkynyls include ethynyl, propynyl (propargyl), butynyl, and the like. Unsaturated alkyl substituents can be unsubstituted or substituted, for example, with at least one substituent selected from the group consisting of a halogen, a nitro, an amino, a hydroxyl, an alkoxy, an aryloxy, an aralkoxy, an ester, an amide, a sulfhydryl, an alkyl sulfide, an aryl sulfide, an alkyl sulfoxide, an aryl sufoxide, an alkyl sulfonyl, an aryl sulfonyl, a keto, a thioketo, an alkyl, a cyano and the like.

[0019] The term "alkoxy" means an alkyl, as defined herein, in which at least one hydrogen atom thereof is substituted with an oxygen atom. Examples of alkoxy substituents include, but are not limited to, methoxy, ethoxy, isopropoxy, 2-butenyloxy, and the like. [0020] The term "alkylamino" includes monoalkylamino and dialkylamino. The term "monoalkylamino" means an amino, which is substituted with an alkyl as defined herein. Examples of monoalkylamino substituents include, but are not limited to, methylamino, ethylamino, isopropylamino, 2-butenylamino, and the like. The term "dialkylamino" means an amino, which is substituted with two alkyls as defined herein, which alkyls can be the same or different. Examples of dialkylamino substituents include dimethylamino, diethylamino, ethylisopropylamino, diisopropylamino, di-2-butenylamino and the like.

[0021] The term "halo" includes halogens such as, e.g., fluoro (F), chloro (CI), bromo

(Br) and iodo (I).

[0022] The term "aryl" means an aromatic ring, as commonly understood in the art, and includes monocyclic and polycyclic aromatics. The aryl substituent preferably comprises 6-

14 carbon atoms in the carbocyclic skeleton thereof. Examples of aryl substituents include, but are not limited to, phenyl, naphthyl and the like, which are unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen, a saturated alkyl, an unsaturated alkyl, a hydroxyl, an alkoxy, an aryloxy, an aralkoxy, an ester, an amide, a sulfhydryl, an alkyl sulfide, an aryl sulfide, an alkyl sulfoxide, an aryl sulfoxide, an alkylsulfonyl, an arylsulfonyl, a keto, a thioketo, a cyano, a nitro, an amino, an alkylamino, a dialkylamino, and the like.

[0023] Inhibitors of Hsp90 useful in the present inventive methods can be in the form of a salt, which is preferably a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example p-toluenesulphonic acid.

[0024] The compound of Formula I can be geldanamycin:

(geldanamycin) or 17-N,N'-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG):

(DMAG).

[0025] In a more prefened embodiment, the compound used in the methods of the invention is 17-allylamino-17-demethoxygeldanamycin (17-AAG):

(17-AAG).

[0026] Methods of synthesizing inhibitors of Hsp90 are described herein as Example 1 and also in Agatsuma et al.(2002), supra; Garbaccio et al.(2001), supra; and Andrus et al. (2002), supra. [0027] Compounds that can be used in the present inventive methods, can be formed as a composition, such as a pharmaceutical composition. Pharmaceutical compositions containing the compound comprising the macrocycle described above or the inhibitor of Hsp90 can comprise more than one active ingredient, such as more than one compound comprising the macrocycle or more than one inhibitor of Hsp90. The pharmaceutical composition can alternatively comprise a compound comprising the macrocycle and/or an inhibitor of Hsp90 in combination with other pharmaceutically active agents or drugs. [0028] The carrier can be any suitable earner. Preferably, the canier is a :. ^'••; pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemicθ physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. It will be appreciated by one of skill in the art that, in addition to the following described pharmaceutical composition, the compounds and inhibitors of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0029] The pharmaceutically acceptable earners described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is prefened that the pharmaceutically acceptable canier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. [0030] The choice of canier will be determined in part by the particular compound comprising the macrocycle and/or the inhibitor of Hsp90, as well as by the particular method used to administer the compound and/or inhibitor. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present inventive methods. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering the compound comprising the macrocycle and/or the inhibitor of Hsp90 of the present invention are known, and, although more than one route can be used to administer a particular compound and/or inhibitor of Hsp90, a particular route can provide a more immediate and more effective response than another route.

[0031] Injectable formulations are among those formulations that are prefened in accordance with the present invention. The requirements for effective pharmaceutical earners for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). i [0032] Topical formulations are well-known to those of skill in the art. Sϋ^'<ϊh - formulations are particularly suitable in the context of the present invention fon application to the skin.

[0033] Formulations suitable for oral administration can consist of (a) liqui solutions, such as an effective amount of the inhibitor dissolved in diluents, such as water,! saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing^, a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol,- iand the polyethylene alcohols, either with or without the addition of a pharmaceuticallytaeceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin itype, \- containing, for example, surfactants, lubricants, and inert fillers, such as lactose,! sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia,, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium . stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistemng agents, preservatives j flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin; ipr "sucrose

; and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0034]. The compounds comprising the macrocycle or the inhibitors of Hsp90, alone or in combination with each other and/or with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa.

[0035] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compounds comprising the macrocycle or the inhibitors of Hsp90 can be administered in a physiologically acceptable diluent in a pharmaceutical canier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3- dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending! agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or ^ carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. [0036] Oils, which can be used in parenteral formulations include pefroleuπ}, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean^ sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in;parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0037] Suitable soaps for use in parenteral formulations include fatty alkali/metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyLpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sύlfόnates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and . polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-b-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salti=.,<and (e) mixtures thereof.

[0038] The parenteral formulations will typically contain from about 0.5%.to about 25% by weight of the active ingredient in solution. Preservatives and buffers may beiused. In order to minimize or eliminate i itation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0039] Additionally, the compounds comprising the macrocycle and/or inhibitors of Hsp90, or compositions comprising such compounds and/or inhibitors of Hsp90, can be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such earners as are known in the art to be appropriate. [0040] One of ordinary skill in the art will readily appreciate that the compounds comprising the macromolecule and the inhibitors of Hsp90 of the present inventive methods can be modified in any number of ways, such that the therapeutic efficacy of the inhibitor is increased through the modification. For instance, the compound or inhibitor could be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds or inhibitors to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 111 (1995), and U.S. Patent No. 5,087,616. The term "targeting moiety" as used herein, refers to any molecule or agent that specifically recognizes and binds to a cell-surface receptor, such that the targeting moiety directs the delivery of the compound or inhibitor to a population of cells on which surface the receptor is expressed. Targeting moieties include, but are not limited to, antibodies, or fragments thereof, peptides, hormones, growth factors, cytokines, and any other naturally- or non- naturally-existing ligands, which bind to cell surface receptors. The term "linker" as used herein, refers to any agent or molecule that bridges the compound or inhibitor to the targeting moiety. One of ordinary skill in the art recognizes that sites on the compounds or inhibitors, which are not necessary for the function of the compound or inhibitor, are ideal sites for attaching a linker and/or a targeting moiety, provided that the linker and/or targeting moiety, once attached to the compound or inhibitor, do(es) not interfere with the function of the compound or inhibitor, i.e., the ability to reduce the activity or concentration of a mutant KIT protein.

[0041] Alternatively, the compounds comprising the macrocycle or the inhibitors of Hsp90 of the present invention can be modified into a depot form, such that the manner in which the compound or inhibitor of Hsp90 is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms of compounds or inhibitors can be, for example, an implantable composition comprising the compound or inhibitor and a porous material, such as a polymer, wherein the compound or inhibitor is encapsulated by or diffused throughout the porous material. The depot is then implanted into the desired location within the body and the compound or inhibitor is released from the implant at a predetermined rate by diffusing through the porous material.

[0042] Preferably, the compound comprising the macrocycle or the inhibitor of Hsp90 is administered to the cell in vitro. As used herein, the term "in vitro" means that the cell is not in a living organism. It is also prefened that the compound comprising the macrocycle or the inhibitor of Hsp90 is administered to the cell in vivo. As used herein, the term "in vivo" means that the cell is a part of a living organism or is the living organism. The inhibitor of Hsp90 can alternatively be administered to the host ex vivo, wherein the inhibitor of Hsp90 is administered to the cell in vitro and the cells are subsequently administered to the host.

[0043] Furthermore, the present inventive methods can comprise the administration of the compound or inhibitor of Hsp90, in the presence or absence of an agent that enhances its efficacy, or the methods can further comprise the administration of other suitable components, such as bleomycin, taxanes, analogues thereof, anthracyclines, and kinase inhibitors.

[0044] For purposes of all of the present inventive methods, the amount of dose of the compound or inhibitor administered should be sufficient to effect a therapeutic response in the animal over a reasonable time frame. Particularly,. the dose of the compound or inhibitor of Hsp90 should be sufficient to reduce the activity or concentration of a mutant KIT protein in a cell within about 1-2 hours, if ήot3-4 hours, fro the.time of administration. The dose will be determined by the efficacy of the particular compound or inhibitor and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated. Many assays for determining an administered dose are known in the art. For purposes of the present invention, an assay, which comprises comparing the extent to which the activity or concentration of a mutant KIT protein is reduced in a cell upon administration of a given dose of a compound or an inhibitor of Hsp90 to a mammal among a set of mammals that are each given a different dose of the compound or inhibitor, could be used to determine a starting dose to be administered to a mammal. The extent to which the activity or concentration of a mutant KIT protein is reduced upon administration of a certain dose can be assayed as described herein as Example 2.

[0045] The dose also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular compound or inhibitor. Ultimately, the attending physician will decide the dosage of the compound or inhibitor of the present invention with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inhibitor to be administered, route of administration, and the severity of the condition being treated.

[0046] By way of example, and not in limitation of the present invention, the dose of the compound comprising the macrocycle or the inhibitor of Hsp90 administered to a cell comprising a mutant KIT protein will typically be from about 250 nM to the maximum tolerable dose, which is cunently known to be about 2700 nM (80 mg/m²) (Munster et al., Proceeding of the American Society of Clinical Oncology, Abstract #327 (2001)). Based on preliminary results of the on-going Phase I clinical trials of 17-AAG, however, the maximum tolerable dose of 17-AAG appears to be even higher. For example, 17-AAG has been administered to and tolerated by humans at doses of 308 mg/m²/week once a week for three weeks.

[0047] The phrase "activity of a mutant KIT protein" as used herein, refers to the enzymatic activity of the mutant KIT protein, which is a protein tyrosine kinase. The activity of a KIT protein, including some but not all mutants thereof, are known in the art to conelate with the state of tyrosine phosphorylation of the KIT protein (Furitsu et al., J. Clin. Invest. 92(4): 1736-1744 (1993); Serve et al., J. Biol. Chem. 269(8): 6026-6030 (1994); and Thommes et al., Biochem. J. 341(Pt 1): 211-216 (1999). Therefore, a KIT protein that is not phosphorylated on tyrosine, is usually recognized to be inactive, whereas a tyrosyl- phosphorylated KIT means that this enzyme is activated or is actively phosphorylating tyrosyl residues on either itself or other proteins. Suitable methods of testing the activity of a protein tyrosine kinase, such as a mutant KIT protein, include in vitro kinase assays and Western blotting. These methods are described in references, such as Sambrook et al., supra; Hong et al., J. Biol. Chem. 277(35):31703-31714 (2002) and Example 2 below.) [0048] The phrase "concentration of a mutant KIT protein" as used herein, refers to the expression level of the mutant KIT protein. Suitable methods of measuring the concentration of proteins, such as KIT, in a cell are known in the art, and include, but are not limited to Western blotting, which is described herein as Example 2. [0049] As used herein, the term "reduce," and words stemming therefrom, do not necessarily imply a complete reduction. Rather, there are varying degrees of reduction of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this regard, the activity or concentration of a mutant KIT protein in a cell can be reduced to any level through the present inventive methods. Preferably, the activity or concentration of a mutant KIT protein is reduced by at least 10%. It is more prefened that the activity or concentration of a mutant KIT protein is reduced by 50%. Most prefened, is that the activity or concentration of a mutant KIT protein is reduced by 90% or greater. [0050] The term "mutant" as used herein encompasses any form of a protein that comprises at least one mutation, such that the amino acid sequence of the mutant protein differs by at least one amino acid from that of the wild-type protein. Such mutations include, for example, insertions, deletions, and substitutions of at least one amino acid. Many methods employed to make mutant proteins are known in the art, including, for example, site-directed mutagenesis. See, for example, Sambrook et al. (1989), supra. [0051] For purposes of the present inventive methods, the mutant KIT protein comprises at least one mutation in any part of the mutant KIT protein. For example, the mutant KIT protein can have a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein. The kinase domain of a human KIT protein comprises amino acids 721 - 840 of SEQ ID NO: 3, which is the amino acid sequence of human KIT amino acid sequence, whereas the juxtamembrane domain of a human KIT comprises amino acids 544 - 580 of the human KIT amino acid sequence (SEQ ID NO: 3). The mutant KIT protein of the present inventive methods can comprise an amino acid substitution mutation. For instance, the mutant KIT protein can have a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, or a substitution of glycine for valine amino acid position 560. Although these mutations are defined in terms of the human KIT amino acid sequence, one of ordinary skill in the art will appreciate that the present inventive methods can be used for other analogous mutant KIT protein, wherein the analogous sites within the amino acid sequences of other species are substituted similarly. [0052] With respect to the present inventive methods, the cell can be a cell from any tissue of any living system, provided that a mutant KIT is expressed in the cell. The cell can be a cell that expresses endogeneously a mutant KIT or it can be a cell, which is manipulated to express a mutant KIT protein. Many methods of manipulating a cell to express a mutant KIT protein are known in the art (see, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Such methods include, but are not limited to transfection, transformation, infection, and transduction.

[0053] hi a prefened embodiment of the present invention, the cell comprising a mutant KIT protein is in a host. It will also be understood by one skilled in the art that the methods of the present invention can be used to treat hosts including mammals that are affected by mutant KIT proteins The benefits of the invention, that is, reduction of the activity or concentration of a mutant KIT protein, that can be observed and realized at the cellular level are also observable and realized in the host. The host can be any host, including for example, bacteria, yeast, fungi, plants, and mammals. Preferably, the host is a mammal. For purposes of the present invention, mammals include, but are not limited to, the order Rodentia, such as mice, and the order Logomorpha, such as rabbits. It is prefened that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more prefened that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most prefened that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially prefened mammal is the human. Treatment of the host in accordance with the present invention will result in a reduction of the activity and/or the concentration of the mutant KIT protein in the host. [0054] In a prefened embodiment of the present inventive methods, the host is afflicted with a disease that is characterized by a mutant KIT protein. The term "disease that is characterized by a mutant KIT protein" as used herein, refers to any abnormal condition or state that conelates with a mutant KIT protein. Such diseases include, but are not limited to, mastocytosis, gastrointestinal stromal tumor, mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

EXAMPLES [0055] Abbreviations

[0056] For convenience, the following abbreviations are used herein: N-terminal, amino-terminal; ATP, adenosine friphosphate; SCF, stem cell factor; W, White spotting; SI, Steel locus; GIST, gastrointestinal tumors; Hsp90, Heat shock protein 90; Hsp70; Heat shock protein 70; 17-AAG, 17-allylamino-17-demethoxygeldanamycin; N-terminus, amino- terminus; HLB, hydrophile-lipophile balance, IC50, inhibitory concentration 50; HRP, horseradish peroxidase; DMAP, 4-dimethylaminopyridine; EDTA, ethylenediamminetetraacetic acid; MTT, methylthiazol-tertzolium; BCA, bicinchoninic acid; SDS, sodium dodecyl sulfate; NC, nitrocellulose; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PARP, poly (ADP-ribose) polymerase; fluorescein isothiocyanate (FITC); ED50, effective dose, also known as the LD50; EGFR, Epidermal Growth Factor Receptor; and DMAG, 17-N,N'-dimethylaminoethylamino-17- demethoxygeldanamycin.

[0057] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0058] Example 1

[0059] This example demonstrates methods of synthesizing various inhibitors of Hsp90. [0060] Preparation of Geldanamycinglycinate Hydrochloride [0061] A mixture of geldanamycin (6 g, 10.7 mmol) (NCI Lot Nos. 3059-25-1 and 2047-72-1), l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-^-toluenesulfonate (5.3 g, 12.5 mmol) (Aldrich, Lot No. AF02307AZ), 4-dimethylaminopyridine (DMAP) (160 mg, 1.3 mmol) (Lancaster, Lot. No. P00472) and N-(tert-butoxycarbonyl) glycine (1.6 g, 9.13 mmol) (Aldrich, Lot No. MX08309EW) was stined at room temperature overnight (ca. 18 hours). Two such reactions were canied out simultaneously. Both of these reaction mixtures were then filtered through the same celite pad. The resulting filtrate was washed with 5% NaHCO₃ (35 mL X 2) (J.T. Baker, Lot No. F24728) and water (35 mL X 3), dried using MgSO₄ (J.T. Baker, Lot No. F43145) and concentrated. The resulting crude product was triturated with water (100 mL X 3) and redissolved in ether (200 mL) (EM Science, Lot No. 32202). The ether solution was washed with water (70 mL X 4), dried using MgSO and evaporated to give a solid (6 g), which was stined in water (150 mL) at room temperature for 4 hours. The solid was collected and air-dried to give 3.8 g of product (Geldanamycin N-(tert-butoxycarbonyl) glycinate). This product was further purified by silica gel column chromatography using 5% MeOH/CH₂Cl₂ as eluent (2.4 g) to form an intermediate compound of Geldanamycin N-(tert-butoxycarbonyl) glycinate. [0062] Trifluoroacetic acid (7.5 mL) (Aldrich, Lot No. PZ06421KZ) was added to a clear solution of the intermediate compound described above (1.4 g, 1.95 mmol) in dry methylene chloride (15 mL) (Burdick & Jackson, Lot No. BE512) at room temperature. The resulting clear red solution was stined at room temperature for 30 minutes, evaporated and pumped (0.1 mmHg) to dryness. While still at room temperature, the residue was triturated with ether (30 mL) and filtered to give a yellow powder (1.2 g), which was subsequently dissolved in methylene chloride (30 mL). The resulting solution was then washed with 5% NaHCO₃ (10 mL X 2) and with brine (10 mL X 1), dried using MgSO₄ and filtered. The pH of the solution was then adjusted to ca. 3 by the addition of methanolic HCl. After adjusting the pH, the filtrate was evaporated and pumped (0.1 mmHg) to dryness. The remaining residue was dissolved in methylene chloride (2 mL) and subsequently diluted with dilute ethereal HCl (50 mL) at room temperature. This mixture was stined for 30 minutes and filtered to give prepurified product. [0063] To purify the product, the salt (1 g, 1.53 mmol) was redissolved in methylene chloride (50 mL). The solution was then washed with 1% NaHCO₃ (25 mL X 1) and water (25 mL X 2), dried using MgSO and filtered. The filtrate was adjusted to pH 3 with dilute methanolic HCl to produce a red solution, which was subsequently evaporated and pumped (0.1 mmHg) to dryness. The residue was then dissolved in methylene chloride (2 mL), seeded and diluted with dilute ethereal HCl (30 mL). The mixture was stined at room temperature for 30 minutes and filtered to produce 4.8 g of purified Geldanamycinglycinate hydrochloride. The water-solubility of the purified product was greater than 3 mg/ml. [0064] Preparation of 17-Demethoxy-17-allylamino geldanamycin- 11-aminoacetate hydrochloride

[0065] A mixture of geldanamycinglycinate hydrochloride (900 mg, 1.38 mol) (NCI Supplies, Lot No. ML-04-02) and allylamine (1.04 mL, 13.8 mmol) (Acros Organics, Lot No. 81223/1) in methylene chloride (36 mL) (J.T. Baker, Lot No. J51613) was stined at room temperature for 18 hours. The reaction mixture was evaporated in an aspirator to give a purple gummy residue (1.0 g). A second run (800 mg) was canied out in a similar manner and gave 910 mg of residue. The residues were combined and subsequently purified by silica gel chromatography (36 g) eluting with methylene chloride (100 mL) followed by methylene chloride-methanol (195:5, 200 mL; 97:3, 200 mL; 193:7, 200 mL; 96:4, 200 mL; 95:5, 200 mL, 94:6, 200 mL). The product-containing fractions were combined and concentrated in an aspirator to dryness. The residue (1 g) was dissolved in methylene chloride (10 mL) and the solution was filtered through a celite pad (Celite Corp., Lot No. 96316). To the stined filtrate was added 1 mL of cold 1.2 M hydrogen chloride-ethanol solution, which was prepared by passing hydrogen chloride gas (Matheson, Lot No. T410240) into ethanol, followed by diethyl ether (100 mL) (J.T. Baker, Lot No. K42582). The resulting mixture was refrigerated for 1 hour and filtered. The solid was washed with diethyl ether (3 X 20 mL) and dried at 64 °C/0.1 mmHg for 5 hours to give 830 mg (47%) of target compound as a purple solid. The water solubility of the purified product was greater than 5 mg/mL.

[0066] Preparation of 17-Demethoxy-17-allylamino geldanamycin- 11-T3- (dimethylaminoVpropionate hydrochloride

[0067] A mixture of methyl 3-(dimethylamino) propionate (5 g, 38.1 mmol) (Aldrich, Lot No. 10016AN) in 3 N hydrochloric acid (30 mL) (Chempure, Lot No. M152KLTS) was heated on a steam bath for 1 hour with stining and then concentrated in an aspirator to a volume of about 8 mL. The resulting mixture was cooled to room temperature and quenched with acetone (30 mL) (Mallinckrodt, Lot No. 2440KTML). The solid was collected by filtration, washed with acetone (2 X 10 mL) and air-dried to give 4.7 g of 3- (dimethylamino) propionic acid hydrochloride as a white crystalline solid (80%), mp 188- 192 °C.

[0068] A suspension of 3-(dimethylamino) propionic acid hydrochloride (4.7 g, 30.6 mmol) in thionyl chloride (20 mL) (Fluka, Lot No. 340130/11194) was heated to reflux in a steam bath for 30 minutes to form a clear yellow solution. The excess thionyl chloride was removed by distillation to give a light brown residue. The residue was then triturated with anhydrous ether (3 X 10 mL) (Fisher Scientific, Lot No. 96732415) under nitrogen and dried in a nitrogen stream to give 5.0 g of 3-(dimethylamino) propionic acid chloride as a light brown solid. This material was taken on directly to the next step without further purification.

[0069] A solution of 17-demethoxy-17-allylamino geldanamycin (1 g, 1.7 mmol) (Ash Stevens Inc., Lot No. BK-10-303) in methylene chloride (20 mL) was added to 3- (dimethylamino) propionic acid chloride (0.4 g) with stining at room temperature. The reaction mixture was stined for 1 hour and concentrated in an aspirator to give a purple gummy residue, which was chromatographed over silica gel (35 g). The column was eluted with methylene chloride (100 mL) followed by methylene chloride-methanol (97:3, 200 mL; 96:4, 100 mL; 95:5, 100 mL; 94:6, 100 mL; 93:7, 100 mL; 92:8, 100 mL). The product-containing fractions were combined and concentrated in an aspirator to give a residue which was rechromatographed over silica gel (27 g) using methylene chloride- methanol (97:3, 100 mL; 193:7, 200 mL; 96:4, 200 mL; 95:5, 100 mL; 94:6, 200 mL; 93:7, 200 mL) as eluant. The product-containing fractions were combined and concentrated in an aspirator to give a residue, which was dissolved in a mixture of methylene chloride (3 mL) and diethyl either (5 mL). To the stined solution, cooled in an ice bath, was added cold 1.2 M hydrogen chloride-ethanol solution (0.8 mL) followed by diethyl ether (15 mL). The mixture was refrigerated for 1 hour and filtered. The solid was washed with diethyl ether (2 X 10 mL) and air-dried to give 600 mg of target compound. A second run (1 g) was carried out in the same manner and gave 580 mg of target compound. The combined product from both runs (1.18 g) was dissolved in methylene chloride (10 mL) and filtered through a celite pad (Celite Corp., Lot No., 96316). The filtrate was diluted with diethyl ether (60 mL) with stining, then refrigerated for 1 hour. The solid was collected by filtration, washed with diethyl ether (3 X 8 mL), and dried at 64 °C/0.1 mmHg for 4 hours to give 960 mg (39%) of pure target compound as a purple solid. The water solubility of the purified product was greater than 3 mg/mL.

[0070] Preparation of 17-Demethoxy-17-allylamino geldanamycin- 11-|^"3- (dimethylamino)-propionate^"| hydrochloride

[0071] A mixture of methyl 3-(dimethylamino) propionate (5 g, 38.1 mmol) (Aldrich, Lot No. 10016AN) in 3 N hydrochloric acid (30 mL) (Chempure, Lot No. M152KLTS) was heated on a steam bath for 1 hour with stining and then concentrated in an aspirator to a volume of about 8 L. The resulting mixture was cooled to room temperature and quenched with acetone (30 mL) (Mallinckrodt, Lot No. 2440KTML). The solid was collected by filtration, washed with acetone (2 X 10 mL) and air-dried to give 4.7 g of 3- (dimethylamino) propionic acid hydrochloride as a white crystalline solid (80%), mp 188- 192 °C.

[0072] A suspension of 3-(dimethylamino) propionic acid hydrochloride (4.7 g, 30.6 mmol) in thionyl chloride (20 mL) (Fluka, Lot No. 340130/11194) was heated to reflux in a steam bath for 30 minutes to form a clear yellow solution. The excess thionyl chloride was removed by distillation to give a light brown residue. The residue was then triturated with anhydrous ether (3 X 10 mL) (Fisher Scientific, Lot No. 96732415) under nitrogen and dried in a nitrogen stream to give 5.0 g of 3-(dimethylamino) propionic acid chloride as a light brown solid. This material was taken on directly to the next step without further purification.

[0073] A solution of 17-demethoxy-17-allylamino geldanamycin (1 g, 1.7 mmol) (Ash Stevens Inc., Lot No. BK-10-303) in methylene chloride (20 mL) was added to 3- (dimethylamino) propionic acid chloride (0.4 g) with stining at room temperature. The reaction mixture was stined for 1 hour and concentrated in an aspirator to give a purple gummy residue, which was chromatographed over silica gel (35 g). The column was eluted with methylene chloride (100 mL) followed by methylene chloride-methanol (97:3, 200 mL; 96:4, 100 mL; 95:5, 100 mL; 94:6, 100 mL; 93:7, 100 mL; 92:8, 100 mL). The product-containing fractions were combined and concentrated in an aspirator to give a residue which was rechromatographed over silica gel (27 g) using methylene chloride- methanol (97:3, 100 mL; 193:7, 200 mL; 96:4, 200 mL; 95:5, 100 mL; 94:6, 200 mL; 93:7, 200 mL) as eluant. The product-containing fractions were combined and concentrated in an aspirator to give a residue, which was dissolved in a mixture of methylene chloride (3 mL) and diethyl either (5 mL). To the stined solution, cooled in an ice bath, was added cold 1.2 M hydrogen chloride-ethanol solution (0.8 mL) followed by diethyl ether (15 mL). The mixture was refrigerated for 1 hour and filtered. The solid was washed with diethyl ether (2 X 10 mL) and air-dried to give 600 mg of target compound. A second run (1 g) was canied out in the same manner and gave 580 mg of target compound. The combined product from both runs (1.18 g) was dissolved in methylene chloride (10 mL) and filtered through a celite pad (Celite Corp., Lot No., 96316). The filtrate was diluted with diethyl ether (60 mL) with stining, then refrigerated for 1 hour. The solid was collected by filtration, washed with diethyl ether (3 X 8 mL), and dried at 64 °C/0.1 mmHg for 4 hours to give 960 mg (39%) of pure target compound as a purple solid. The water solubility of the purified product was greater than 3 mg/mL.

[0074] Preparation of 1 l-(4-aminobutyrate -geldanamycin hydrochloride [0075] 4-aminobutyric acid (44.0 g, 0.43 mol) was added to a solution of sodium carbonate (22.0 g, 0.21 mol) and water (160 ml). The resulting clear solution was diluted with dioxane (160 ml) and di-tert-butyldicarbonate (110 g, 0.50 mol) was added to the dilution. The mixture was stined at room temperature overnight (approximately 18 hours), after which the solvent was evaporated. Water (660 ml) was added to the residue and the mixture was extracted three times with ethyl acetate (400 ml aliquots). The pH of the aqueous layer was adjusted from a pH of 9 to pH of 3 by the addition of solid sodium bisulfate monohydrate (59.0 g, 0.43 mol), and the pH-adjusted solution was extracted three times with ether (450 ml aliquots). The combined ether extract was washed with water (200 ml), dried using MgSO₄, and concentrated. The residue (87.9 g) was dissolved iri ether (100 ml). The solution was diluted with petroleum ether (50 ml), seeded with product from a previous preparation and cooled in an ice-water bath. After the product crystallized, more petroleum ether (250 ml) was added and the mixture was stined at 0 °C for 1 hour. The white solid was collected by filtration to give 71.9 g of 4-(tert-butoxycarbonyl)- aminobutyric acid (mp 50-52 °C).

[0076] A mixture of geldanamycin (10.0 g, 17.8 mmol), 1,3-diisopropylcarbodiimide (8.0 g, 63.4 mmol), DMAP (0.8 g, 6.5 mmol), and 4-(tert-butoxycarbonyl) aminobutyric acid (9.4 g, 46.2 mmol) in dry methylene chloride (300 ml) was stined at room temperature for 24 hours. Two such reactions were carried out simultaneously. Both of the reaction mixtures were filtered through the same celite pad. The filtrate was washed twice with aqueous 5% NaHCO₃ (100 ml aliquots) and with water (100 ml), then dried using MgSO and concentrated. The residue was dissolved in the mimmum volume of refluxing ethanol (1 L). The solution was cooled to room temperature and stined for 3 hours. The yellowish orange powder was collected by filtration, washed twice with ethanol (30 ml aliquots), three times with arid petroleum ether (50 ml aliquots), and air-dried to give 13.6 g of pure 11-(4- (tert-butoxycarbonyl) aminobutyrate-geldanamycin (mp 235 °C). Additional geldanamycin (70.0 g) was processed in this manner to give an additional 43.7 g of the intermediate product.

[0077] The unreacted geldanamycin was recovered according to the following process. The mother liquor recovered from the production of the 1 l-(4-(tert-butoxycarbonyl) aminobutyrate-geldanamycin was concentrated to a small volume (100 ml) and the mixture was filtered. The solid was washed twice with ethanol (10 ml aliquots), three times with petroleum ether (20 ml aliquots), and air-dried to yield unreacted geldanamycin (24.4 g) contaminated with 1 l-(4-(tert-butoxycarbonyl) aminobutyrate-geldanamycin. All of the recovered geldanamycin was recycled to give additional intermediate (13.9 g, mp 235 °C), with acceptable elemental analysis.

[0078] Thus, a total of 90.0 g of fresh geldanamycin and 24.4 g of recovered material was processed to give 71.2 g of pure 1 l-(4-(tert-butoxycarbonyl) aminobutyrate- geldanamycin.

[0079] Trifluoroacetic acid (50 ml) was added to a clear solution of 1 l-(4-(tert- butoxycarbonyl) aminobutyrate-geldanamycin (10.0 g, 13.4 mmol) in dry methylene chloride (150 ml) at room temperature. The red solution was stined at room temperature for 30 minutes, then evaporated and pumped (0.3 mmHg) to dryness. [0080] An identical scale reaction was canied out simultaneously. Each residue was triturated with ether (60 ml) at room temperature, then diluted with petroleum ether (120 ml). Each mixture was stined at room temperature for 30 mm and filtered through the same filter paper. The combined solid was dissolved in methylene chloride (300 ml). The solution was washed twice with 5% NaHCO₃ (aq.) (100 ml aliquots), once with water (100 ml), dried with MgSO , and filtered through a celite pad. The filtrate was acidified with methanolic HCl. The solution was evaporated and pumped dry to give 20.1 g of crude 11- (4-aminobutyrate)-geldanamycin hydrochloride.

[0081] In the same manner, additional 1 l-(4-(tert-butoxycarbonyl) aminobutyrate- geldanamycin (50.0 g total in several batches) was deprotected and converted to 50.3 g of crude 1 l-(4-aminobutyrate)-geldanamycin hydrochloride. The combined crude product (70.4 g) was dissolved in methylene chloride (500 ml) and the solution was filtered through a celite pad. The filtrate was concentrated to a volume of about 200 ml, then diluted with ether (600 ml) and stined at room temperature for 1 hour and filtered. The orange, powdery solid (57.2 g) was suspended in hot ethanol (100 ml). The partial solution was diluted slowly with petroleum ether (800 ml). The mixture was stined at room temperature for 1 hour. The yellow powder was collected by filtration and dried at 25 °C and at 0.1 mmHg for 10 hours to give 54.2 g of pure 1 l-(4-aminobutyrate)-geldanamycin hydrochloride. The water solubility of the purified product was about 3 mg/ml. [0082] Preparation of 17-Demethoxy-17-allylamino geldanamycin- 11-[4- (dimethylamino -butyrateT

[0083] A suspension of 4-(dimethylamino) butyric acid hydrochloride (3 g, 17.9 mmol) (Aldrich, Lot No. 09128AR) in thionyl chloride (20 mL) (Fluka, Lot No. 340130/1 1194) was heated at reflux on a stream bath for 30 min to form a clear yellow solution. The excess thionyl chloride was removed by distillation to give a light brown residue. This material was triturated with anhydrous ether (10 mL X 3) (Fisher Scientific, Lot No. 967324-15) under a nitrogen atmosphere and dried in a nitrogen stream to give 3.3 g of acid chloride as a light brown solid.

[0084] To a solution of 17-demethoxy-17-allylamino geldanamycin (500 mg, 0.85 mmol) (Ash Stevens Inc., Lot No. BK-10-303) in methylene chloride (8 mL) (J.T. Baker, Lot No. K33619) was added pyridine (0.1 mL) (J.T. Baker, Lot No. E13618) and thionyl chloride (300 mg). The reaction mixture was stined at room temperature for 18 hours and then partitioned between methylene chloride (50 mL) and a 0.2 M sodium hydroxide solution (30 mL) (Aldrich, Lot No. M152KPCX). The organic phase was separated, washed successively with water (20 mL X 2) and brine (20 mL), dried over anhydrous MgSO (EM Science, Lot No. 33146334), and filtered. The residue was purified by silica gel chromatography (20 g) eluting with methylene chloride (50 mL) followed by methylene chloride-methanol (97:3, 200 mL; 96:4, 200 mL); 95:5, 100 mL; 94:6, 100 mL; 93:7, 200 mL; 92:8, 200 mL) (Fisher Scientific, Lot No. 966097). The fractions containing product were combined and concentrated in an aspirator to dryness. The solution was filtered through a celite pad, and the filtrate was diluted with hexanes (30 mL) (Fisher Scientific, Lot No. 963814) and refrigerated for 3 hours. The solid was then collected by filtration and washed with hexanes (10 mL X 2), then air-dried to give 260 mg of target compound as a purple crystalline solid. In a similar manner, additional target compound was prepared in two batches and added to the first batch. The combined solids were then dried at 64 °C/0.1 mmHg for 5 hours to give 760 mg (32%) of 17-Demethoxy-17-allylamino geldanamycin- 1 l-[4-(dimethylamino)-butyrate] as a purple crystalline solid. [0085] Preparation of 17-Dimethylaminoethylamino-17-demethoχygeldanamvcin [0086] A mixture of geldanamycin (1.5g, 2.68 mmol) (NCI, Lot No. 3059-25-1) and N,N-dimethylethylenediamine (1.5 mL, 13.7 mmol) (Aldrich, Lot No. 04216AL) in dry methylene chloride (30 mL) (Burdick & Jackson, Lot No. BH516) was stined at room temperature for 1 hour and subsequently poured into ice water (50 mL). After the organic layer and the aqueous layer became visually separated, the aqueous layer was extracted with methylene chloride (2 X 10 mL). The combined methylene chloride solution was washed with water (2 X 10 mL), dried with MgSO₄ (J.T. Baker, Lot No. E01098) and evaporated in an aspirator. The remaining residue was purified by silica gel chromatography (30 g) (EM Science, Lot No. 325TA509634) eluting with 10% methanol (Chempure, Lot No. M178KMDH) in methylene chloride. The fractions containing product were combined and concentrated in an aspirator. The residue was then dissolved in hot methylene chloride (3 mL), cooled to room temperature and diluted with hexane (60 mL) (Chempure, Lot No. M148KMRC). This mixture was then stined at room temperature for 30 minutes. A purple powder formed and was collected by filtration, washed with hexane (2 X 10 mL) and dried at 55 °C/0.1 mmHg for 6 hours to give pure target compound. Approximately 1.59 g of 17- Dimethylaminoethylamino-17-demethoxygeldanamycin (96%) was isolated. [0087] Preparation of (17-Dimethoxy- 17- [^" r2-(dimethylamino ethyll aminol geldanamycin hydrochloride

[0088] A mixture of geldanamycin (15 g, 26.75 mmol) (SAIC-Frederick, Lot No. 3155- 28-1) and N,N-dimethyl-ethylenediamine (15 mL, 37.83 mmol) (Aldrich, Lot No. HN04216AL) in dry methylene chloride (300 mL) (Mallinckrodt, Lot No. 4881KVJP) was stined at room temperature for 1 hour and subsequently poured into ice water (500 mL). After the organic layer and aqueous layer became visually separated, the aqueous layer was extracted with methylene chloride (2 X 100 mL). The combined methylene chloride solution was washed with water (2 X 100 mL), dried with MgSO (Spectrum, Lot No. HJ302), and evaporated in a diaphragm pump. The remaining residue (16.5 g, 26.75 mmol) was dissolved in methylene chloride (130 mL). Dissolved hydrogen chloride (1.1 eq, 29.43 mmol) (Matheson, Lot No. T410242) in dioxane (Aldrich, Lot No. 16619CQ) was added to the solution resulting in immediate precipitation. After stining for 30 minutes, the solids were collected, rinsed with methylene chloride (50 mL), and air-dried for 18 hours to give the crude hydrochloride salt (17.2 g). A solution of the crude product (17.2 g) in aqueous alcohol (50% v/v, 200 mL) (Aaper Alcohol, Lot. No. 97A28UARAT) was heated to reflux, filtered, and allowed to cool for 30 minutes. After cooling on ice for an additional 30 minutes, the solids were collected, washed with cold anhydrous alcohol (50 mL), and air- dried for 1 hour. The product was dried further at 70 °C/0.1 mmHg for 17 hours to give pure target compound. Approximately 13.4 g of 17-Demethoxy-17- [[2(dimethylamino)ethyl] amino] geldanamycin hydrochloride (82%) was isolated as a violet crystalline solid.

[0089] Preparation of 17-Dimethylaminopropylamino-17-demethoxy geldanamycin [0090] A mixture of geldanamycin (1.5 g, 2.68 mmol) (NCI, Lot No. 3059-25-1) and 3- dimethylaminopropylamine (1.5 mL, 11.9 mmol) (Aldrich, Lot No. 08103CF) in dry methylene chloride (30 mL) (Burdick & Jackson, Lot No. BH516) was stined at room temperature for 30 minutes and subsequently poured into ice water (50 mL). After the organic layer and the aqueous layer became visually separated, the aqueous layer was extracted with methylene chloride (2 X 100 mL). The combined methylene chloride solution was washed with water (2 X 100 mL), dried with MgSO (J.T. Baker, Lot No. E01098) and evaporated in an aspirator. The remaining residue was purified by silica gel chromatography (30 g) eluting with 10% methanol in methylene chloride. The fractions containing product were combined and concentrated in an aspirator. The residue was then dissolved in hot methylene chloride (3 mL), cooled to room temperature and diluted with hexane (60 mL) (Chempure, Lot No. M148KMRC). The resulting mixture was stined at room temperature for 30 minutes. A purple powder formed and was collected by filtration, washed with hexane (2 X 10 mL) and dried at 55 °C/0.1 mmHg for 5 hours to give pure target compound. Approximately 1.64 g of 17-Dimethylaminopropylamino-17- demethoxygeldanamycin (97%) was isolated.

[0091] The cells and antibodies used in Examples 2-5 are as follows. [0092] Original HMC-1 cells (HMC-1.2) were provided by Joseph Butterfield (Mayo Clinic, Rochester, MN) and the subclone, HMC-1.1 cells, were provided by Dean Metcalfe (National Institutes of Health, Bethesda, MD). HMC-1 cells derived from a human patient with mast cell leukemia and the original cells (HMC-1.2) contain the two mutations, Asp816Val and Val560Gly (Furitsu et al., JClin Invest 92:1736-1744 (1998)). The subclone, HMC-1.1, contains the single mutation Val560Gly (Ma et al., Blood 99:1741- 1744 (2002)). Cells were maintained in Iscove's medium with 25 mM Hepes and L- Glutamine (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA) and 1.2 mM alphathioglycerol (Sigma, St. Louis, MO) and grown at 37C in 5% CO2. Anti-KIT antibody (M-14) for immunoprecipitation and western analysis was purchased from Santa Cruz (Santa Cruz, CA). Mouse anti-phosphotyrosine antibody (Ab-4) was purchased from Oncogene, rat anti-Hsp90 antibody (SPA-840) from Stressgen (San Diego, CA), goat anti-Hsp70 (K-20) antibody from Santa Cruz (Santa Cruz, CA), rabbit anti-phospho-AKT antibody and rabbit anti-AKT from Cell Signaling Technology (Beverly, MA), mouse anti-raf-1 antibody (E-10) from Santa Cruz (Santa Cruz, CA), rabbit anti-parp antibody from Upstate USA, and mouse anti-tubulin antibody from Sigma (St. Louis, MO).

[0093] Example 2

[0094] This example demonstrates a method of reducing the concentration of a mutant KIT protein and a method of reducing the activity of a mutant KIT protein, both of which comprise the administration of an inhibitor of Hsp90.

[0095] HMC-1.1 or HMC-1.2 cells (7.5 x 10⁶) were incubated with 0 nM, 100 nM, 500 nM, or 1 μM 17-AAG for 15 hours and were subsequently centrifuged at 800 rpm x g for 5 minute. The pellet was washed once with cold phosphate-buffered saline pH 7.0 (PBS), re- centrifuged and resuspended in TNSEV buffer (50 mM Tris pH 7.5, 2 mM EDTA, 100 mM NaCl, 2 mM Sodium Ortho vanadate, 1% NP-40) supplemented with CompleteTM protease inhibitors (Roche Molecular Biochemicals Indianapolis, IN). After sitting on ice for 10 minutes, cells lysates were clarified by centrifuging at 13,000 rpm x g at 4°C for 20 minutes. The protein concentration was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Clarified cell lysate was resuspended in SDS buffer (80 mM Tris-HCL pH 7.5, 2% SDS, 10% glycerol, 100 mM dithiothreitol and 0.0005% bromphenol blue). For each experiment, equal amounts of total protein were separated on 10% SDS-PAGE gel, transfened onto nitrocellulose paper (NC paper) by electroblotting and analyzed using horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ) to the primary antibody in conjunction with western blot chemoluminescence (Pierce, Rockford, IL). NC paper was also probed with anti-tubulin antibody to confirm equal loading of total protein. [0096] As seen in Tables 1 and 2, the levels of KIT protein decreased upon the administration of 50Q nM 17-AAG or 1 μM 17-AAG, which are doses that have been shown by ongoing Phase I clinical trials of 17-AAG to be tolerable in humans for up to 8 hours of exposure. A sharp decrease in the phosphorylated form of KIT, which is representative of KIT in its activated form, was also observed upon treatment with 500 nM 17-AAG. In contrast, the level of tubulin protein remained the same, regardless of the concentration of 17-AAG administered to the cells. [0097] Table 1. Dose response of HMC-1.1 cells to 17-AAG after exposed for 15 hours

[0099] This example demonstrated dose-dependent decreases in KIT protein and activated KIT protein in both HMC-1.1 and HMC-1.2 cells following treatment with 17- AAG at doses that have been shown to be tolerable by humans.

[00100] Example 3

[00101] This example demonstrates time-dependent decreases in the concentration of KIT and other cellular proteins upon the administration of an Hsp90 inhibitor.

[00102] HMC-1.1 and HMC-1.2 cells (7.5 x 10⁶) were exposed to 500 nM 17-AAG for 0,

1, 2, 3, 4, 8, 12, or 24 hours. The cells were harvested and analyzed by Western blotting as described in Example 2.

[00103] As seen in Tables 3 and 4, KIT protein decreased as early as 2 hours following treatment and continued to decrease over 24 hours. The level of KIT protein, which was phosphorylated on tyrosine, was also reduced after 1 hour of treatment. Interestingly, KIT was not phosphorylated upon treatment with 17-AAG for 12 hours or 24 hours in HMC-1.2 cells, but had a small degree of phosphorylation at these time point in HMC-1.1 cells.

Furthermore, while the level of AKT protein in HMC-1.2 cells remained essentially the same throughout the timecourse, the level of phosphorylated AKT in these cells decreased after 2 hours of treatment with 17-AAG. In HMC-1.1 cells, the levels of both AKT and phosphorylated AKT remained essentially the same. The level of Raf decreased gradually in HMC-1.2 cells, while that in HMC-1.1 cells remained basically unchanged. The level of tubulin also did not change throughout the timecourse experiment.

[00104] Table 3. Quantified levels of various proteins. HMC-1.2 cells exposed to 500nM of 17-AAG for increasing number of hours. Values shown as relative levels to control (defined as sample density divided by control density measured on densitometer)

[00105] Table 4. Quantified levels of various proteins. HMC-1.1 cells exposed to 500nM of 17-AAG for increasing number of hours. Values shown as relative levels of protein to control (defined as sample density divided by control density measured on densitometer)

[00106] This example demonstrated that KIT, as well as phosphorylated KIT and phosphorylated AKT, decreases upon longer incubations with 17-AAG. This example further demonstrated that proliferation and survival signaling pathways downstream to the KIT protein, in which AKT, phosphorylated AKT, and Raf-1 participate, are downregulated following treatment with 17-AAG.

[00107] Example 4

[00108] This example demonstrates that Hsp90 and Hsp70 are associated with mutant KIT protein in cells.

[00109] Co-immunoprecipitation was performed as described previously (Xu et al., J Biol Chem 276:3702-3708 (2001)) with the following minor changes. Briefly, cells (1.875 x 10⁷ were grown to a concentration of 10⁶/cc and washed as described in Example 1. Cells were then resuspended in TMNSV lysis buffer (50 mM Tris-HCL pH 7.5, 20 mM Na₂MoO₄, 0.09% Nonidet P-40, 150 mM NaCl and 2 mM sodium orthovanadate) supplemented with CompleteTM protease inhibitors. 1.5 mg of lysate protein was incubated with 5 μg of anti-KIT antibody for 15 hours at 4C. Protein A-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ) were added and rotated at 4C for 3 hours. For a negative control, protein A-sepharose beads were incubated with 1.5 mg of lysate protein in the absence of anti-KIT antibody. The beads were washed with TMNSV buffer 3 times then re-suspended in 1 x SDS buffer. Immunoprecipitated protein was boiled for 5 minutes, and then separated on 10% SDS-PAGE gel. Protein transfer and probing was done as described in Example 2.

[00110] As seen in Table 5, HMC-1.1 cells treated with 500 nM 17-AAG for 1 hour caused Hsp-90 to dissociate, but resulted in an increased Hsp70 association. KIT protein containing the 816 mutation (HMC-1.2 cells) by this method, however, did not show an association between Hsp90 but does not preclude an association in vivo. Antibody recognition of the Asp816Val mutated protein, a more unstable Asp816Val protein or a less stable interaction with Hsp90, which can be effected by detergents used in lysis buffer, could all have been reasons why the association with Hsp90 was not observed.

[00111] Table 5

[00112] This example demonstrates that the Asp816Val mutant of KIT is also a client protein of Hsp90.

[00113] Example 5

[00114] This example demonstrates that the administration of an inhibitor of Hsp90 prevents downstream signaling in cells containing a mutant form of KIT, showing that the activity of KIT is reduced upon the administration of the Hsp90 inhibitor. [00115] Cell viability was determined on four consecutive days in the presence or absence of 17-AAG using mefhylthiazol-tetrzolium (MTT) as described previously (Park et al., Hum Gene Ther 10:889-898 (1999)). Cells were suspended in medium on day -1, at a concentration of 7.5 x 10⁵ cells/cc, in a 96 well plate. On day 0, 17-AAG was added at various concentrations in sample plates. Starting on day 0 (day 0 plate used as control), MTT was added to medium each day and incubated for 4 hours at 37C. Cells were then spun down at 2000 rpm, medium was removed and 120 ul of DMSO was added to wells. Plates were shaken for 30 minutes at 1500 rpm and absorbance was measured at 560nm using the ELx808iu Ultra Microplate Reader (BIO-TEK Instruments, Winooski, VT). Samples were done in sextant at each concentration and the mean absorbance was used to determine the relative cell viability (relative growth). Relative growth is defined as sample absorbance divided by control absorbance.

[00116] Viable cells will enzymatically convert soluble MTT into the insoluble compound, formazan, which is then easily quantified after dissolved in DMSO by measuring light absorbance at 560 nm. Using this method we showed that 17-AAG causes cytotoxicity in HMC- 1.2 cells within 24 hours at concentrations similar to those resulting in molecular changes, while causing cytostasis in HMC-1.1 cells (Tables 6 and 7). There is a clear cytotoxic effect on HMC- 1.2 cells at doses of 250 nM and greater even with a relatively short exposure time of 1 day. HMC-1.1 cells appear to be relatively resistant to a cytotoxic effect though growth is clearly inhibited at similar concentrations of 250 nM and greater. The effective dose (ED50), also know as the LD50, of 17-AAG was calculated using values obtained by MTT assay after HMC-1.2 cells were exposed to drug for 24, 48 and 72 hours by method previously described (Chou et al., Adv Enzyme Regul 22:27-55 (1984)) and found to be 251 nM, 195 nM and 114 nM respectively.

[00117] Table 6. Relative Growth of HMC-1.1 cells (relative growth defined as absorbance of samples divided by absorbance of control on day 0).

[00119] This example demonstrated that 17-AAG-mediated cytotoxicity in HMC- 1.2 cells and 17-AAG cytostasis in HMC-1.1 cells. [00120] The following cells and antibodies were used for Examples 6-10. [00121] HMC-1 cells (Furitsu et al., J Clin Invest., 92:1736-1744, (1993)), derived from a patient with mast cell leukemia, were provided by Joseph Butterfield (Mayo Clinic, Rochester, MN). HMC-1.2 cells contain the two mutations Asp816Val and Val560Gly and HMC-1.1 cells contain a single mutation, Val560Gly (Ma et al., Blood, 99:1741-1744, (2002)). They were maintained in Iscove's medium with 25 mM Hepes and L-Glutamine (hivitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gemini Bio- Products, Clabasas, CA) and 1.2 mM alphathioglycerol (Sigma, St. Louis, MO) and grown at 37°C in 5% CO₂. Cos-7 cells were obtained from American Type Culture Collection (Rockville, MD) and were grown in DMEM medium (BioSource International, Camarillo, CA) supplemented with glutamine and 10% fetal bovine serum (Gemini Bio-Products, Clabasas, CA) and grown at 37°C in 5% CO₂. For Western blot analysis, anti-KIT antibody C-19 (Santa Cruz Biotechnologies, Santa Cruz, CA) was used as the primary antibody to detect total KIT and anti-phosphoKIT (Tyr719) (Cell Signaling Technology, Beverly, MA) was used to detect phosphorylated KIT. KIT protein immunoprecipitation efficiency was increased significantly by combining several primary antibodies. Thus, anti-KIT antibodies M-14, H-300, C-19, Ab81 (Santa Cruz) and PC34 (Oncogene Research, San Diego, CA) were used for immunoprecipitation. Rat anti-Hsp90 antibody (SPA-840) was purchased from Stressgen Biotechnology (San Diego, CA), goat anti-Hsp70 (K-20) antibody (Santa Cruz), rabbit anti-phospho-AKT antibody, rabbit anti-AKT, rabbit anti-STAT3 and rabbit anti-phophoSTAT3 (Tyr705) from Cell Signaling Technology (Beverly, MA), and mouse anti-tubulin antibody from Sigma.

[00122] Example 6

[00123] This example demonstrates that mutated KIT protein and KIT activity decrease in HMC-1.1 and HMC- 1.2 cells following treatment with 17-AAG. [00124] Cells grown to a concentration of approximately 1 x 10 (Blechman, EMBOJ., 7:1003-1011, (1988)) cells/ml, in the presence or absence of 17-AAG, were spun down at 800 rpm for 5 min. The pellet was washed once with cold phosphate-buffered saline (PBS), pH 7.0, re-centrifuged and resuspended in TNSEV buffer (50 mM Tris, pH 7.5, 2 mM EDTA, 100 mM NaCl, 2 mM sodium orthovanadate, 1% NP-40) supplemented with CompleteTM protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). After being placed on ice for 10 min, cell lysates were clarified by centrifuging at 13,000 rpm at 4°C for 20 min. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce Chemical Co., Rockford, IL). Equal aliquots of clarified cell lysate were heated at 100°C for 5 min in SDS buffer (80 mM Tris-HCL pH 7.5, 2% SDS, 10% glycerol, 100 mM dithiothreitol and 0.0005% bromphenol blue). Equal amounts of total protein were separated by 10% SDS-PAGE, transfened onto nitrocellulose paper by electroblotting and analyzed using horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ) in conjunction with Western blot chemiluminescence (Pierce Chemical Co.). Blots were probed with anti-tubulin antibody to confirm equal loading of total protein whenever necessary. [00125] Minor changes were made to a previously described method for co- immunoprecipitation (Xu et al., JBiol. Chem., 276: 3702-3708, (2001)). Cells were grown and washed as described above. They were then resuspended in TMNSV lysis buffer (50 mM Tris-HCL, pH 7.5, 20 mM Na₂MoO₄, 0.09% Nonidet P-40, 150 mM NaCl and 2 mM sodium orthovanadate) supplemented with CompleteTM protease inhibitors (Roche). Three milligrams of lysate protein was incubated with 3 μg of each anti-KIT antibody used for immunoprecipitation for 2 h at 4°C. Protein G-Sepharose beads (Invitrogen, Carlsbad, CA) were added and rotated at 4°C for 2 h. As a negative control, Protein G-Sepharose beads were incubated with 3 mg of lysate protein in the absence of anti-KIT antibody. The beads were washed three times with TMNSV buffer and then re-suspended in SDS sample buffer. Immunoprecipitated protein was boiled for 5 min, and then separated by 10% SDS-PAGE. Protein transfer and probing were done as described above.

[00126] To determine the effects of 17-AAG on KIT protein in malignant human mast cells, HMC-1 cell lines were treated for 8 hours with increasing concentrations of 17-AAG and levels of total KIT protein and phosphorylated KIT protein were measured by Western blot analysis. There was a dose-dependent decrease in total KIT protein and KIT activity (as measured by KIT autophosphorylation) in both cell lines with a more profound effect at concentrations greater than 500 nM 17-AAG. This is relevant as concentrations of 1 μM can be achieved in vivo in humans as shown by preliminary data from phase I clinical trials (Munster et al., Proceeding American Society of Clinical Oncology, 327, (2001) (Generic)). [00127] This example demonstrated that an inhibitor of Hsp90 inhibits the activity of a mutated KIT protein and reduces the protein concentration of a mutated KIT protein.

[00128] Example 7

[00129] This example demonstrates that proliferation and survival signaling pathways downstream of KIT protein are down-regulated following treatment with 17-AAG. [00130] To determine whether 17-AAG affects downstream signaling pathways, levels of total AKT, activated AKT (phosphorylated AKT), STAT3 and activated STAT3 (phosphorylated STAT3) were analyzed. Activation of both pathways appears to be necessary for factor-independent growth and proliferation of Asp816Val KIT mutated mast cells and MO7e cells (Ning et al., Blood, 97: 3559-3567, (2001); and Ning et al., Oncogene, 20: 4528-4536, (2001)), and both signaling pathways play an important role in protecting cells from apoptosis (Catlett-Falcone et al., Immunity, 10: 105-115, (1999); and Datta et al., Genes Dev., 13: 2905-2927, (1999)). AKT has previously been shown to be degraded and de-activated by hsp90 inhibitors in breast cancer cell lines over-expressing HER-2 (Shulte et al., J Biol. Chem., 270: 24585-24588: (1995); Fujita et al., J. Biol. Chem, 277: 10346- 10353, (2002); Munster et al., Cancer Res., 62:3132-3137, (2002); and Basso et al., Oncogene, 62: 3132-3137, (2002)). Both phospho-AKT and phospho-STAT3 were rapidly down-regulated in human mast cell lines after treatment with 17-AAG. Inhibition of AKT and STAT3 is similar in both cell lines and conelates well with 17-AAG effects on KIT. In these HMC cell lines, loss of AKT and STAT3 activity precedes the more gradual decrease in their total levels. Unlike AKT, and supported by our data, STAT3 is not known to be a client protein of hsp90. Nevertheless, STAT3 is rapidly de-phosphorylated following exposure of mast cells to 17-AAG. Therefore, it is likely that the decreased activity of AKT and STAT3 is, at least in part, secondary to inhibition of KIT, rather than to a direct effect of 17-AAG.

[00131] This example demonstrated the down-regulation of proliferation and survival signaling pathways upon administration of an inhibitor of Hsp90.

[00132] Example 8

[00133] This example demonstrates that 17-AAG causes apoptosis in HMC-1.1 cells and HMC- 1.2 cells.

[00134] Apoptosis was determined by propidium iodide uptake. Cells grown to a concentration of 1x10 cells/ml (Qui et al., EMBO , 1: 1003-1011, (1998)) were incubated in the presence of no drug, 1 μM 17-AAG or 1 μM Imatinib for 24 hours. Cells (5 10; Xsebo et al., Cell, 63: 213-224, 1990) cells were washed with buffer solution (5 mM Hepes, pH 7.4, 75 mM NaCl, 1.25 mM CaCl₂, 0.5 mM MgCl₂, 2% BSA) and then resuspended in 500 ml of the same buffer. Three hundred nanograms of propidium iodide (Oncogene Research, San Diego, Ca) were added, and samples were analyzed immediately by flow cytometry using an argon ion laser tuned to 488 nm (Becton Dickinson FACSCalibur, San Jose, CA) to quantify propidium iodide flourescence. Cells (10,000) were counted in each experiment and data are expressed as dot plots.

[00135] Analysis of apoptosis was performed by flow cytometry. Viable cells exclude propidium iodide and, therefore, appear in the lower half of the dot plots. Late apoptotic cells cannot exclude propidium iodide and appear in the upper half of these plots. In control HMC-1.1 and HMC- 1.2 cells, 3% and 2% of the total population, respectively, exhibited spontaneous apoptosis. Treatment of cells with 1 μM 17-AAG for 24 hours increased the percentage of apoptotic cells to 21% and 14% for HMC-1.1 cells and HMC-1.2 cells, respectively. In contrast, treatment with 1 μM Imatinib for 24 hours caused apoptosis only in HMC-1.1 cells and not in HMC-1.2 cells. It has previously been shown that imatinib is ineffective in the treatment of HMC-1.2 cells because they harbor a kinase domain mutation in KIT (Ma et al., Blood, 99: 1741-1744, (2002)). The data shown here further document the sensitivity of both HMC-1.1 and HMC-1.2 cells to 17-AAG, the latter of which is not sensitive to imatinib.

[00136] This example demonstrated that an inhibitor of Hsp90 caused apoptosis in mast cells.

[00137] Example 9

[00138] This example demonstrates that patient neoplastic mast cells treated ex vivo are sensitive to 17-AAG.

[00139] Bone manow aspirates were obtained from 4 patients (2 males, 2 females; age range 45-57) with indolent systemic mastocytosis after informed consent. The Asp816Val c-kit mutation was verified in all patients. Bone manow mononuclear cells were isolated on a Ficoll-Hypaque (1.077) density gradient and cultured in Stem-Pro serum-free medium (Invitrogen, Carlsbad, CA) supplemented with 100 ng/ml recombinant human SCF (Peprotech, Rocky Hill, NJ) for 7-9 days as described (Akin et al., Experimental Hematology, 31 :686-692, (2003) (Generic)). 17-AAG was added to the cultures at concentrations of 0.1-1 μM. At the end of the culture period, total cell counts and mast cell percentages were determined. Mast cells were detected by their characteristic appearance in flow cytometric analysis of the bone manow sample stained with phycoerythrin-conjugated anti-human CD117 (BD Biosciences, San Jose, CA) as described (Akin et al., Experimental Hematology, 31:686-692, (2003) (Generic)).

[00140] Because 17-AAG showed cytotoxicity against HMC1.2 cells carrying the D816V c-kit mutation, whether or not the drug exhibited a similar effect on human bone manow mast cells isolated from patients with mastocytosis was examined. Bone manow mononuclear cells from 4 patients with indolent systemic mastocytosis were isolated by density gradient centrifugation, and were each incubated with 0.1-1 μM 17-AAG for 7-9 days. A dose-dependent reduction in mast cell percentage, determined by flow cytometry, was observed. A significant reduction (p<0.05) in mast cell percentage was observed at concentrations greater than 500 nM 17-AAG. Consistent with a previous report (Akin et al., Experimental Hematology, 31 :686-692, (2003) (Generic)), imatinib did not cause a significant reduction in mast cell percentages using this assay.

[00141] This example demonstrated that neoplastic mast cells are sensitive to an inhibitor ofHsp90. [00142] Example 10

[00143] This example demonstrates that KIT transiently transfected into Cos-7 cells is downregulated following treatment with 17-AAG.

[00144] Cos-7 cells were grown to -60% confluence in 6-well plates. Transfections were performed using the Fugene 6 Reagent (Roche) and manufacturer's guidelines were followed. PcDNA3 plasmid vectors coding for either wild-type or Asp816Val mutated KIT were provided by Dr. Gunnar Nilsson (University of Uppsala, Sweden) (Taylor et al. Blood, 1195-1199, (2001)). The Fugene 6 reagent to DNA ratio was 2:1. Plasmid DNA (1.5 μg) was used in each well and transfection proceeded overnight. Medium was then exchanged for fresh medium. SCF (Peprotech, Rocky Hill, NJ) was added (to appropriate wells) at a concentration of 100 ng/ml 1 h prior to addition of 1 μM 17-AAG (to appropriate wells). Cells were lysed with TNSEV buffer, and Western blot analysis was performed as described above.

[00145] There are cunently no widely available mast cell lines that contain either wild- type KIT or only the Asp816Val mutation, which is the most common KIT mutation in human mast cell disease. To test whether 17-AAG was effective on KIT harboring this mutation alone, as well as wild-type KIT, Cos-7 cells were transfected with plasmid coding for these proteins. Transfected cells were treated with 1 μM 17-AAG and incubated for up to 24 hours. Consistent with our earlier data, KIT activity is downregulated before the total protein is degraded. Within 4 to 8 hours of drug exposure, Asp816Val KIT activity is significantly reduced, while total levels of KIT are only modestly affected. By 24 hours, phosphorylated KIT was undetectable, while total KIT protein was present by Western analysis, though at a reduced level. Both wild-type and kinase domain mutated KIT were sensitive to 17-AAG, suggesting that the effect of 17-AAG on mutated KIT was a relative effect.

[00146] This example demonstrated that mutated KIT is down-regulated by an inhibitor ofHsp90.

[00147] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00148] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

Prefened embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those prefened embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A method of reducing the activity of a mutant KIT protein in a cell comprising the mutant KIT protein, which method comprises administering to the cell an inhibitor of Hsp90 in an amount sufficient to reduce the activity of the mutant KIT protein, wherein the inhibitor binds to an adenosine friphosphate (ATP) binding pocket of Hsp90.

2. The method of claim 1 , wherein the mutant KIT protein comprises a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein.

3. The method of claim 2, wherein, when the mutant KIT protein is a human mutant KIT protein, the mutation is selected from the group consisting of a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, and a substitution of glycine for valine amino acid position 560.

4. The method of claim 1, wherein the inhibitor of Hsp90 is administered to the cell in vitro.

5. The method of claim 1, wherein the inhibitor of Hsp90 is administered to the cell in vivo.

6. The method of claim 1 , wherein the cell is in a host.

7. The method of claim 6, wherein the host is a mammal.

8. The method of claim 7, wherein the mammal is a human.

9. The method of claim 6, wherein the host is afflicted with a disease that is characterized by a mutant KIT protein.

10. The method of claim 9, wherein the disease is selected from the group consisting of mastocytosis, gastrointestinal stromal tumor (GIST), mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

11. A method of reducing the activity of a mutant KIT protein in a cell comprising the mutant KIT protein, which method comprises administering to the cell a compound comprising a macrocycle of the following structure:

in an amount sufficient to reduce the activity of the mutant KIT protein.

12. The method of claim 11 , wherein the compound is a compound of Formula I:

(Formula I) wherein n=0 or 1 and wherein, when n=l, each of R and R is hydrogen, and, when n=0, a double-bond exists between position 4 and position 5; R³ is hydrogen or hydroxyl;

R⁴ is hydrogen, hydroxyl, or R⁷C(O)O-, wherein R⁷ is amino(CrC₈)alkyl or imino(C₁-C₈)alkyl; wherein, when R³ is hydrogen, R⁴ is hydroxyl or R⁷C(O)O-, and when R³ is hydroxyl, R⁴ is hydrogen;

R⁵ is hydrogen or a group of the formula:

wherein each of R⁸, R⁹, and R¹⁰ is independently selected from the group consisting of hydrogen, a halo, an azido, a nitro, a C_!-C₈ alkyl, a Cι-C₈ alkoxy, an aryl, a cyano, and an NR^HR¹ R¹³, wherein each of R¹¹, R¹², and R¹³ is independently selected from the group consisting of hydrogen and a -C₃ alkyl;

R⁶ is hydrogen, a methoxy, a -Cs alkylamino, a -Cs dialkylamino, an N,N'- dialkylaminodialkylamino-, an N,N'-dialkylaminoalkylamino, or an allylamino; or salts thereof.

13. The method of claim 12, wherein the compound of Formula I is 17- allylamino- 17-demethoxygeldanamycin (17-AAG) :

(17-AAG).

14. The method of claim 12, wherein the compound of Formula I is geldanamycin:

(geldanamycin).

15. The method of claim 12, wherein the compound of Formula I is 17-N,N'- dimethylaminoethylamino- 17-demethoxygeldanamycin (DMAG) :

(DMAG).

16. The method of claim 11 , wherein the mutant KIT protein comprises a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein.

17. The method of claim 16, wherein, when the mutant KIT protein is a human mutant KIT protein, the mutation is selected from the group consisting of a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, and a substitution of glycine for valine amino acid position 560.

18. The method of claim 11 , wherein the compound is administered to the cell in vitro.

19. The method of claim 11 , wherein the compound is administered to the cell in vivo.

20. The method of claim 11 , wherein the cell is in a host.

21. The method of claim 20, wherein the host is a mammal.

22. The method of claim 21 , wherein the mammal is a human.

23. The method of claim 20, wherein the host is afflicted with a disease that is characterized by a mutant KIT protein.

24. The method of claim 23, wherein the disease is selected from the group consisting of mastocytosis, GIST, mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

25. A method of reducing the concentration of a mutant KIT protein in a cell comprising the mutant KIT protein, which method comprises administering to the cell an inhibitor of Hsp90 in an amount sufficient to reduce the concentration of Hsp90, wherein the inhibitor binds to an ATP binding pocket of Hsp90.

26. The method of claim 25, wherein the mutant KIT protein comprises a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein.

27. The method of claim 26, wherein, when the mutant KIT protein is a human mutant KIT protein, the mutation is selected from the group consisting of a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, and a substitution of glycine for valine amino acid position 560.

28. The method of claim 25, wherein the inhibitor of Hsp90 is administered to the cell in vitro.

29. The method of claim 25, wherein the inhibitor of Hsp90 is administered to the cell in vivo.

30. The method of claim 25, wherein the cell is in a host.

31. The method of claim 30, wherein the host is a mammal.

32. The method of claim 31 , wherein the mammal is a human.

33. The method of claim 30, wherein the host is afflicted with a disease that is characterized by a mutant KIT protein.

34. The method of claim 33, wherein the disease is selected from the group consisting of mastocytosis, gastrointestinal stromal tumor (GIST), mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

35. A method of reducing the concentration of a mutant KIT protein in a cell comprising the mutant KIT protein, which method comprises administering to the cell a compound comprising a macrocycle of the following structure:

in an amount sufficient to reduce the concentration of the mutant KIT protein.

36. The method of claim 35, wherein the compound is a compound of Formula I, wherein n=0 or 1 and wherein, when n=l, each of R and R is hydrogen, and, when n=0, a double-bond exists between position 4 and position 5;

R³ is hydrogen or hydroxyl;

R⁴ is hydrogen, hydroxyl, or R⁷C(O)O-, wherein R⁷ is amino(C₁-C₈)alkyl or imino(C₁-C₈)alkyl; wherein, when R³ is hydrogen, R⁴ is hydroxyl or R⁷C(O)O-, and when R³ is hydroxyl, R⁴ is hydrogen;

R⁵ is hydrogen or a group of the formula:

wherein each of R⁸, R , and R , 1^ι0^υ is independently selected from the group consisting of hydrogen, a halo, an azido, a nitro, a -Cs alkyl, a -Cs alkoxy, an aryl, a cyano, and an NR^πR¹²R¹³, wherein each of R¹¹, R¹², and R¹³ is independently selected from the group consisting of hydrogen and a Ci-Cs alkyl;

R⁶ is hydrogen, a methoxy, a d-C₈ alkylamino, a -Cs dialkylamino, an N,N'- dialkylaminodialkylamino, an N_jN'-dialkylaminoalkylamino, or an allylamino; or salts thereof.

37. The method of claim 36, wherein the compound of Formula I is 17-AAG.

38. The method of claim 36, wherein the compound of Formula I is geldanamycin.

39. The method of claim 36, wherein the compound of Formula I is DMAG.

40. The method of claim 35, wherein the mutant KIT protein comprises a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein.

41. The method of claim 40, wherein, when the mutant KIT protein is a mutant human KIT protein, the mutant human KIT protein has a mutation selected from the group consisting of a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, and a substitution of glycine for valine amino acid position 560.

42. The method of claim 35, wherein the compound is administered to the cell in vitro.

43. The method of claim 35, wherein the compound is administered to the cell in vivo.

44. The method of claim 35, wherein the cell is in a host.

45. The method of claim 44, wherein the host is a mammal.

46. The method of claim 45, wherein the mammal is a human.

47. The method of claim 35, wherein the host is afflicted with a disease that is characterized by a mutant KIT protein.

48. The method of claim 47, wherein the disease is selected from the group consisting of mastocytosis, GIST, mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

49. A method of reducing the activity of a mutant KIT protein in a host comprising the mutant KIT protein, which method comprises administering to the host an inhibitor of Hsp90 in an amount sufficient to reduce the activity of the mutant KIT protein, wherein the inhibitor binds to an ATP binding pocket of Hsp90.

50. The method of claim 49, wherein the mutant KIT protein comprises a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein.

51. The method of claim 50, wherein, when the mutant KIT protein is a human mutant KIT protein, the mutation is selected from the group consisting of a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, and a substitution of glycine for valine amino acid position 560.

52. The method of claim 49, wherein the inhibitor of Hsp90 is administered to the cell in vitro.

53. The method of claim 49, wherein the inhibitor of Hsp90 is administered to the cell in vivo.

54. The method of claim 49, wherein the host is a mammal.

55. The method of claim 54, wherein the mammal is a human.

56. The method of claim 49, wherein the host is afflicted with a disease that is characterized by a mutant KIT protein.

57. The method of claim 56, wherein the disease is selected from the group consisting of mastocytosis, GIST, mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

58. A method of reducing the activity of a mutant KIT protein in a host comprising the mutant KIT protein, which method comprises administering to the host a compound comprising a macrocycle of the following structure:

in an amount sufficient to reduce the activity of the mutant KIT protein.

59. The method of claim 58, wherein the compound is a compound of Formula I,

1 "? wherein n=0 or 1 and wherein, when n=l, each of R and R is hydrogen, and, when n=0, a double-bond exists between position 4 and position 5;

R³ is hydrogen or hydroxyl;

R⁴ is hydrogen, hydroxyl, or R⁷C(O)O-, wherein R⁷ is amino(C₁-C₈)alkyl or imino(Cι-C8)alkyl; wherein, when R³ is hydrogen, R⁴ is hydroxyl or R⁷C(O)O-, and when R³ is hydroxyl, R⁴ is hydrogen;

R⁵ is hydrogen or a group of the formula

wherein each of R , R , and R , 10 is independently selected from the group consisting of hydrogen, a halo, an azido, a nitro, a -Cs alkyl, a -Cs alkoxy, an aryl, a cyano, and an NR^UR¹²R¹³, wherein each of R¹¹, R¹², and R¹³ is independently selected from the group consisting of hydrogen and a C1-C3 alkyl;

R⁶ is hydrogen, a methoxy, a d-Cg alkylamino, a Q-Cg dialkylamino, an N,N'- dialkylaminodialkylamino-, an N,N'-dialkylaminoalkylamino, or an allylamino; or salts thereof.

60. The method of claim 59, wherein the compound of Formula I is 17-AAG.

61. The method of claim 59, wherein the compound of Formula I is geldanamycin.

62. The method of claim 59, wherein the compound of Formula I is DMAG.

63. The method of claim 58, wherein the mutant KIT protein comprises a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein.

64. The method of claim 63, wherein, when the mutant KIT protein is a human mutant KIT protein, the mutation is selected from the group consisting of a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, and a substitution of glycine for valine amino acid position 560.

65. The method of claim 58, wherein the host is a mammal.

66. The method of claim 65, wherein the mammal is a human.

67. The method of claim 58, wherein the host is afflicted with a disease that is characterized by a mutant KIT protein.

68. The method of claim 67, wherein the disease is selected from the group consisting of mastocytosis, GIST, mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

69. A method of reducing the concentration of a mutant KIT protein in a host comprising the mutant KIT protein, which method comprises administering to the host an inhibitor of Hsp90 in an amount sufficient to reduce the concentration of Hsp90, wherein the inhibitor binds to an ATP binding pocket of Hsp90.

70. The method of claim 69, wherein the mutant KIT protein comprises a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein.

71. The method of claim 70, wherein, when the mutant KIT protein is a human mutant KIT protein, the mutation is selected from the group consisting of a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, and a substitution of glycine for valine amino acid position 560.

72. The method of claim 69, wherein the inhibitor of Hsp90 is administered to the cell in vitro.

73. The method of claim 69, wherein the inhibitor of Hsp90 is administered to the cell in vivo.

74. The method of claim 69, wherein the host is a mammal.

75. The method of claim 74, wherein the mammal is a human.

76. The method of claim 69, wherein the host is afflicted with a disease that is characterized by a mutant KIT protein.

77. The method of claim 76, wherein the disease is selected from the group consisting of mastocytosis, GIST, mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.

78. A method of reducing the concentration of a mutant KIT protein in a host comprising the mutant KIT protein, which method comprises administering to the host a compound comprising a macrocycle of the following structure:

in an amount sufficient to reduce the concentration of the mutant KIT protein.

79. The method of claim 78, wherein the compound is a compound of Formula I, wherein n=0 or 1 and wherein, when n=l, each of R and R is hydrogen, and, when n=0, a double-bond exists between position 4 and position 5;

R³ is hydrogen or hydroxyl;

R⁴ is hydrogen, hydroxyl, or R⁷C(O)O-, wherein R⁷ is amino(C₁-C₈)alkyl or imino(Cι-C₈)alkyl; wherein, when R³ is hydrogen, R⁴ is hydroxyl or R⁷C(O)O-, and when R³ is hydroxyl, R⁴ is hydrogen;

R⁵ is hydrogen or a group of the formula

wherein each of R⁸, R⁹, and R¹⁰ is independently selected from the group consisting of hydrogen, a halo, an azido, a nitro, a d-C₈ alkyl, a d-C₈ alkoxy, an aryl, a cyano, and an

1 1 1 ι 1 1 19 1 ^

NR R R , wherein each of R , R , and R is independently selected from the group consisting of hydrogen and a d-d alkyl;

R⁶ is hydrogen, a methoxy, a d-C₈ alkylamino, a d-C₈ dialkylamino, an N,N'- dialkylaminodialkylamino, an N;N'-dialkylaminoalkylamino, or an allylamino; or salts thereof.

80. The method of claim 79, wherein the compound of Formula I is 17-AAG.

81. The method of claim 79, wherein the compound of Formula I is geldanamycin.

82. The method of claim 79, wherein the compound of Formula I is DMAG.

83. The method of claim 78, wherein the mutant KIT protein comprises a mutation in the kinase domain of the mutant KIT protein or the juxtamembrane domain of the mutant KIT protein.

84. The method of claim 83, wherein, when the mutant KIT protein is a mutant human KIT protein, the mutant human KIT protein has a mutation selected from the group consisting of a substitution of valine for aspartic acid at amino acid position 816, a substitution of tyrosine for aspartic acid at amino acid position 816, and a substitution of glycine for valine amino acid position 560.

85. The method of claim 78, wherein the host is a mammal.

86. The method of claim 85, wherein the mammal is a human.

87. The method of claim 78, wherein the host is afflicted with a disease that is characterized by a mutant KIT protein.

88. The method of claim 87, wherein the disease is selected from the group consisting of mastocytosis, GIST, mast cell leukemia, myelogenous leukemia, lymphoma, and testicular cancer.