WO2008066641A2

WO2008066641A2 - Methods for treating mitf associated diseases by the use of methionine aminopeptidase-2 inhibitors

Info

Publication number: WO2008066641A2
Application number: PCT/US2007/022923
Authority: WO
Inventors: Gerhard Hannig; Sylvie Bernier
Original assignee: Praecis Pharmaceuticals Incorporated
Priority date: 2006-11-06
Filing date: 2007-10-30
Publication date: 2008-06-05
Also published as: WO2008066641A3

Abstract

The instant invention provides methods and compositions for treating a subject suffering from an MITF associated disease, such as Microphthalmia, Waardenburg syndrome type 2a, Tietz syndrome, digenic ocular albinism, Sensorineural deafness, albinism, and osteoporosis. The invention also provides methods and compositions for modulating a function of MITF, inducing melanogenesis in a melanocyte or a melanoma cell, and sensitizing a melanoma cell to a chemotherapeutic agent.

Description

METHODS FOR TREATEVG MITF ASSOCIATED DISEASES BY THE USE OF METHIONINE AMINOPEPTIDASE-2 INHIBITORS

Related Applications This application claims priority to U.S. Provisional Application No. 60/857,063, filed November 6, 2006, the entire contents of which are incorporated herein by this reference.

Background of the Invention The Microphthalmia Associated Transcription Factor (MITF) is an integral transcriptional regulator that is required for the proper development of several cell lineages, including melanocytes, osteoclasts, retinal pigment epithelial (RPE) cells, mast cells, and natural killer cells (Steingrimsson et al, (1994) EMBO J. 15:6280-6289). The MITF gene was originally identified in a spotted mutant mouse, characterized by the loss of melanocytes, by the Hertwig group in 1942. The MITF gene was cloned in 1993 using a transgenic insertion into the MITF gene locus (Hodgkinson et al, (1993) Cell 74:395-404), and there are at least six isoforms of MITF designated by their unique 5' ends arising from multiple alternative promoter and initial exon usage (Shibahara et al, (2001) J Invest Dermatol. Symp. Proc. 6:99-104). MITF is a basic-helix-loop-helix-leucine-zipper (bHLHzip) protein, which plays a critical role in melanocyte differentiation through transcriptional regulation of several pigmentation enzymes, including Tyrpl (tyrosinase 1), Tyrp2, and Dct. Furthermore, it has been shown that MITF is involved in the M-CSF and RANKL signaling pathways that are critical for osteoclast proliferation, differentiation, and function. However, the cellular functions of MITF are wider than cell-differentiation and cell-fate pathways alone, since mature melanocytes and melanoma cells also require expression of this transcription factor. In humans, mutations in MITF have been linked to several disorders, including abnormal pigmentation and bone loss. MITF associated disorders include, but are not limited to, Waardenburg syndrome type 2a, Microphthalmia, Tietz syndrome, digenic ocular albinism, Sensorineural deafness, albinism and osteoporosis. There is no known cure for MITF associated diseases. Current treatment goals vary depending on the specific MITF associated disease and include symptomatic reduction of skin trauma, treatment of bacterial infection, early diagnosis and treatment of hearing defects, the reduction of pain and discomfort, the prevention of deformities and loss of joint function, and/or the suppression of inflammation. Accordingly, there is still a need for effective treatments for these diseases, e.g., methods for inducing melanogenesis and/or modulating the function of MITF within a subject.

Summary of the Invention

The present invention provides methods of treating a subject having an MITF associated disease. The methods include administering to a subject a MetAP-2 inhibitor in an amount effective to inhibit MITF function, thereby treating an MITF associated disease in a subject. The present invention is based, at least in part, on the discovery that Met-AP2 inhibitors potently inhibit the function of MITF.

In one aspect, the invention provides a method for treating an MITF associated disease in a subject, e.g., a mammal, e.g., a human, by administering to the subject a therapeutically effective amount of a methionine aminopeptidase 2 inhibitor, thereby treating an MITF associated disease in a subject. In one embodiment, the MITF associated disease is selected from the group consisting of Microphthalmia,

Waardenburg syndrome type 2a, Tietz syndrome, digenic ocular albinism, albinism, Sensorineural deafness, and osteoporosis.

In another aspect, the invention provides a method for modulating a function of MITF, by contacting MITF with an effective amount of a MetAP-2 inhibitor, thereby modulating the function of MITF. In one embodiment, the function of MITF that is modulated is the expression of MITF. In another embodiment, MITF is present within a human cell. In yet another embodiment, the human cell is present within a human subject. In yet another embodiment, the MITF function is selected from the group consisting of melanocyte differentiation, osteoclast proliferation, and melanogenesis. In one embodiment, the MITF function is the regulation of expression or activity of proteins regulated by MITF selected from the group consisting of melanin, mast cell protease 5 (MCP-5), MCP-6, c-kit, p75 nerve growth factor, granzyme B, tryptophan hydroxylase, cathepsin K, RbI, CDK2 , tyrosinase, tyrosinase related protein, and pink- eyed Pmel 17. In one embodiment, the MITF function is the regulation of the expression or activity of proteins that bind to and/or interact with MITF selected from the group consisting of TFEB, TFEC, TFE3, USF2, PKCI, PIAS3, PEBP2, MAZR, c- FOS, PU-I, LEF-I, GSK3β, and PAX-6. In another aspect, the invention provides a method for inducing melanogenesis in a melanocyte or a melanoma cell, by contacting the melanocyte with an amount of a MetAP-2 inhibitor effective to inhibit an MITF function, thereby inducing melanogenesis in the melanocyte or the melanoma cell. In yet another aspect, the invention provides a method for sensitizing a melanoma cell to a chemotherapeutic agent, by contacting a melanoma cell with an amount of a MetAP-2 inhibitor effective to inhibit an MITF function, thereby sensitizing a melanoma cell to a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is selected from the group consisting of dacarbazine (DITC), docetaxe, paclitaxel, temozolomide, carmustine (BCNU), lomustine (CCNU), fotemustine, cisplatin, carboplatin, vinblastine, and vindesine.

In one embodiment of the methods of the invention, the methionine aminopeptidase 2 inhibitor is a compound of Formula I,

wherein A is a Met-AP2 inhibitory core; W is O or NR₂; R₁ and R₂ are each, independently, hydrogen or alkyl; X is alkylene or substituted alkylene; n is 0 or 1; R₃ and R₄ are each, independently, hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; or R₃ and R₄, together with the carbon atom to which they are attached, form a carbocyclic or heterocyclic group; or R3 and R₄ together form an alkylene group; Z is -C(O)- or alkylene-C(O)-; and P is a peptide comprising from 1 to about 100 amino acid residues attached at its amino terminus to Z or a group OR5 or N(Re)R₇, wherein R₅, R₆ and R₇ are each, independently, hydrogen, alkyl, substituted alkyl, azacycloalkyl or substituted azacycloalkyl; or R₆ and R₇, together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic ring structure; or Z is -O-, -NR8-, alkylene-O- or alkylene-NRg-, where R₈ is hydrogen or alkyl; and P is hydrogen, alkyl or a peptide consisting of from 1 to about 100 amino acid residues attached at its carboxy terminus to Z; and a pharmaceutically acceptable salt or pro-drug thereof.

In another embodiment, the methionine aminopeptidase 2 inhibitor is a compound of Formula XV,

(XV)

wherein A is a MetAP-2 inhibitory core; W is O or NR; each R is, independently, hydrogen or alkyl; Z is -C(O)- or -alkylene-C(O)-; P is NHR, OR or a peptide consisting of one to about one hundred amino acid residues connected at the N-terminus to Z; Q is hydrogen, linear, branched or cyclic alkyl or aryl, provided that when P is - OR, Q is not hydrogen; or Z is -alkylene-O- or -alkylene-N(R)-; P is hydrogen or a peptide consisting of from one to about one hundred amino acid residues connected to Z at the carboxyl terminus; Q is hydrogen, linear, branched or cyclic alkyl or aryl, provided that when P is hydrogen, Q is not hydrogen; and pharmaceutically acceptable salts or a pro-drug thereof. In yet another embodiment, the methionine aminopeptidase 2 inhibitor is a compound of the formula

wherein W is O or NR; each R is, independently hydrogen or a Ci-C-j-alkyl; Q is hydrogen; linear, branched or cyclic Ci-Cβ-alkyl; or aryl; R₁ is hydroxy, CrC₄-alkoxy or halogen; Z is -C(O)- or Ci-C4-alkylene; P is NHR, OR, or a peptide comprising 1 to 100 amino acid residues attached to Z at the N-teπninus; or Z is alkylene-0 or alkylene- NR; and P is hydrogen or peptide comprising 1 to 100 amino acid residues attached to Z at the C-terminus; provided that when P is hydrogen, NHR or OR, Q is not hydrogen; and a pharmaceutically acceptable salt or a pro-drug thereof.

In a further embodiment, the methionine aminopeptidase 2 inhibitor is a compound comprising the structure

and a pharmaceutically acceptable salt or a pro-drug thereof.

In one embodiment, the methionine aminopeptidase 2 inhibitor is administered at a dosage range of about 0.1 to about 50 mg/kg, about 25 to about 35 mg/kg, about 10 to about 50 mg/kg, about 20 to about 40 mg/kg, about 30 to about 50 mg/kg, or about 30 to about 40 mg/kg. In another embodiment, the methionine aminopeptidase 2 inhibitor is administered at a dosage of about 30 mg/kg.

In another embodiment, the methionine aminopeptidase 2 inhibitor may be administered to the subject in a sustained-release formulation, e.g., a sustained-release formulation which provides sustained delivery of the methionine aminopeptidase 2 inhibitor to a subject for at least one, two, three, four or five weeks after the formulation is administered to the subject.

In another aspect, the present invention provides a method of treating an MITF associated disease in a subject, e.g., a human. The method includes administering to the subject a therapeutically effective amount of a methionine aminopeptidase 2 inhibitor comprising the structure (l-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-l-oxa- spiro[2.5]oct-6-yl ester, or a pharmaceutically acceptable salt thereof or a pro-drug thereof, thereby treating MITF associated disease in a subject. In another aspect, the invention provides a method of modulating a function of MITF, comprising contacting MITF with an effective amount of a methionine aminopeptidase 2 inhibitor comprising the structure (l-Carbamoyl-2-methyl-propyl)- carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3-methyl-but-2- enyl)-oxiranyl]-l-oxa-spiro[2.5]oct-6-yl ester, or a pharmaceutically acceptable salt or a pro-drug thereof, thereby modulating the function of MITF.

In yet another aspect, the instant invention provides a method of inducing melanogenesis in a melanocyte or a melanoma cell, comprising contacting the melanocyte with an an amount of a methionine aminopeptidase 2 inhibitor comprising the structure (l-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R )-5- methoxy-4-[(2R, 3R ) -2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-l-oxa-spiro[2.5]oct- 6-yl ester, or a pharmaceutically acceptable salt or a pro-drug thereof, effective to inhibit an MITF function, thereby inducing melanogenesis in the melanocyte.

In yet another aspect, the invention provides a method of sensitizing a melanoma cell to a chemotherapeutic agent, comprising contacting a melanoma cell with an amount of a methionine aminopeptidase 2 inhibitor comprising the structure (l-Carbamoyl-2- methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3- methyl-but-2-enyl)-oxiranyl]-l-oxa-spiro[2.5]oct-6-yl ester, or a pharmaceutically acceptable salt or a pro-drug thereof, effective to inhibit an MITF function, thereby sensitizing the melanoma cell to a chemotherapeutic agent.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

Figure 1 is a set of graphs demonstrating that MetAP-2 inhibitors inhibit the growth of the UACC-62, A375, and Ml 4 cell lines. The growth inhibition of cells in response to MetAP-2 inhibitors is linked to the amount of MetAP-2 enzyme inhibited in these cells, as determined by a MetAP-2 pharmacodynamic assay.

Figure2 is a graph demonstrating that MetAP-2 inhibitors inhibit the growth tumor cells in the mouse Ml 4 xenograft model. Figure 3 is a set of graphs demonstrating that MetAP-2 inhibitors inhibit the growth of M14 tumors in vivo. MetAP-2 levels detected in the white blood cell and tumor compartments 24 hours after the final dose of MetAP-2 inhibitor in the mouse Ml 4 xenograft model. Figure 4 depicts the results of an experiment demonstrating that melanogenesis is induced in vitro in response to exposure to MetAP-2 inhibitors.

Figure 5 depicts the results of an experiment demonstrating that the MetAP-2 inhibitor induced downstream regulation of MITF in vitro in M14 melanoma cells.

Figure 6 is a set of graphs demonstrating that targeted downregulation of MITF by small interfering RNA (siRNA) significantly inhibits the growth of melanoma cells and further sensitizes the cells to treatment with a MetAP-2 inhibitor or a chemo therapeutic agent.

Detailed Description of the Invention

The present invention provides methods of treating an MITF associated disease in a subject. The methods include administering to the subject a therapeutically effective amount of a methionine aminopeptidase 2 inhibitor, thereby treating an MITF associated disease in a subject. The present invention also provides methods for modulating a function of MITF, methods for inducing melanogenesis in a melanocyte, and methods for sensitizing a melanoma cell with an effective amount of a methionine aminopeptidase 2 inhibitor. The present invention is based, at least in part, on the discovery that MetAP-2 inhibitors potently inhibit the function of MITF. The present invention is also based, at least in part, on the discovery that MetAP-2 inhibitors induce melanogenesis in melanocytes.

The terms "Microphthalmia Associated Transcription Factor" and "MITF" are used interchangeably herein to refer to the well known transcriptional regulator. The human MITF is described in, for example, Hodgkinson et al, (1993) Cell 74:395-404, and all known iso forms of human MITF are intended to be encompassed by this term. The term "MITF associated disease," as used herein, is intended to include any disease, disorder or condition associated with (or caused by) the modulated, e.g., decreased or increased, expression or activity of MITF as compared to normal MITF expression or activity levels. This term includes diseases, disorders, or conditions in which melanogenesis mediated by MITF is impaired, in which mast cell development is inhibited, or in which the activity of NK cells is inhibited. MITF associated diseases include, but are not limited to, Microphthalmia, Waardenburg syndrome type 2a, Tietz syndrome, digenic ocular albinism, albinism, and Sensorineural deafness,. In one embodiment, this term does not include diseases such as cancer, e.g., melanoma or inflammatory diseases. In another embodiment, this term does not include diseases such as bone associated diseases, e.g., osteoporosis.

As used herein, the term "MITF function" or "function of MITF" is intended to include any function mediated by MITF. This term includes any action, process, signaling pathway, or activity which is affected or regulated by MITF. In one embodiment, this term is intended to include the expression of MITF. In another embodiment, general MITF functions include cell differentiation and pigmentation. In a preferred embodiment, MITF functions include melanocyte differentiation and melanogenesis. An MITF function, as used herein, also includes the regulation of the expresion or activity of other proteins which are regulated by MITF such as, for example, melanin, mast cell protease 5 (MCP-5) (see, e.g., Morii et al, (1997) Blood. 90:3057-3066), MCP-6 (see, e.g., Mom et al, (1996) Blood. 88:2488-2494), c-kit (see, e.g., Isozaki et al, (1994) Am. J. Pathol. 145:827-836), p75 nerve growth factor (see, e.g., Morii et al, 1997), granzyme B (see, e.g., Wo et al, (1998) Blood. 91:3210-3221), tryptophan hydroxylase (see, e.g., Ito et al, (\999) Blood. 93:1189-1196), cathepsin K (see, e.g., Motyckova et al, (2001) Proc. Natl Acad Sci. U.S.A. 98:5798-5803), RbI (see, e.g., Carreira et al, (2005) Nature 433(7027):764-769), CDK2 (see, e.g., Du et al, (2004) Cancer Cell 6(6): 565-576), tyrosinase, tyrosinase related protein, and pink-eyed Pmel 17 (see, e.g., Bentley et al, (1994) MoI Cell Biol 14:7996-8006). MITF function can be determined by monitoring the function or expression of proteins, or the underlying nucleic acids encoding proteins, which are regulated by MITF. The term "MITF function" also includes the regulation of the expression or activity of proteins which bind to, intract with and/or regulate MITF. For example, MITF can form heterodimers with TFEB, TFEC, TFE3, and USF2 (see, e.g., Beckmann et al, (1990) Genes Dev. 4:167-179; Carr and Sharp, (199O)M?/. Cell. Biol. 10:4384-4388;

Nechushtan ef tf/., (1997) Blood. 89:2999-3008; and Zhao et al, (1993) MoI. Cell Biol. 13:4505-4512), can associate with PKCI or PIAS3 to prevent MITF activation of target genes until the appropriate stimulus is encountered (see, e.g., Razin et al, (1999) J. Biol Chem. 274:34272-34276 and Levy et ai, (2002) J. Biol. Chem. 277:1962-1966), can interact with PEBP2 and MAZR to increase affect transcription of MCP-6 (see, e.g., Ogihara et al, (1999) Oncogene 8:4632-4639 and Morii et al, (2002) J. Biol. Chem. 277(10):8566-8571, respectively), and can bind to c-FOS and PU-I (see, e.g., Sato et al, (1999) Biochem BiophysRes. Commun. 254(2):384-387), LEF-I (see, e.g., Yasumoto et al, (2002) EMBOJ. 21(l l):2703-2714), and PAX-6 (see, e.g., Plangue et al, (200I) J Biol Chem. 276(31):29330-7). MITF function can be determined by monitoring the function or expression of proteins, or the underlying nucleic acids encoding proteins, which bind to and/or interact with the MITF protein. MITF function can also be determined by monitoring the function or expression of any proteins which post- translationally modify MITF or proteins which are post-translationally modified by MITF. For example, the Kit signaling pathway and the GSK3β molecule have been suggested to modulate MITF post-translationally (see, e.g., Wu et al, (2000) Gems Dev. 14:301-312). As used herein, the term "melanogenesis" is intended to refer to the formation of the melanin pigment by living cells. Melanogenesis also refers to a morphological feature of a differentiated melanocyte cell or a melanoma cell. Melanocytes utilize the enzyme tyrosinase in melanogenesis, e.g., the synthesis of the melanin pigment from tyrosine. The tyrosinase enzymes Trypl, Tryp2, and Dct (dopachrometautomerase) are transactivated by the transcription factor MITF.

As used herein, the term "contact" or "contacting" is intended to include both the direct and, preferably, the indirect contacting of MITF with a MetAP-2 inhibitor. For example, a cell containing MITF may be contacted with a MetAP-2 inhibitor.

As used interchangeably herein, the terms "methionine aminopeptidase 2 inhibitor" and "MetAP-2 inhibitor" are intended to include any compound which inhibits the activity of the methionine aminopeptidase 2 protein, the well known enzyme which cleaves the N-terminal methionine residue of newly synthesized proteins to produce the active form of the protein. In a preferred embodiment, MetAP-2 inhibitors useful in the methods of the invention include those inhibitors comprising a Fumagillin core, such as the ones described in sub-section I below.

The methods of the invention further include sensitizing a melanoma cell to a chemotherapeutic agent, comprising contacting a melanoma cell with an amount of a MetAP-2 inhibitor effective to inhibit an MITF function, thereby sensitizing the melanoma cell to a chemotherapeutic agent.

The term "chemotherapeutic agent" is well known in the art and includes dacarbazine (DITC), docetaxe, paclitaxel, temozolomide, carmustine (BCNU), lomustine (CCNU), fotemustine, cisplatin, carboplatin, vinblastine, and vindesine. Other well known chemotherapeutic agents may be found in Harrison's Principles of Internal Medicine, Thirteenth Edition, Eds. T.R. Harrison et al. McGraw-Hill N. Y., NY; and the Physicians Desk Reference 50th Edition 1997, Oradell New Jersey, Medical Economics Co., the complete contents of each of which are expressly incorporated herein by reference. The MetAP-2 inhibitor and the chemotherapeutic agent may be administered to the subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times).

As used herein, the term "subject" includes warm-blooded animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human.

As used herein, the term "administering" to a subject includes dispensing, delivering or applying a MetAP-2 inhibitor compound, e.g., a MetAP-2 inhibitor in a pharmaceutical formulation (as described herein), to a subject by any suitable route for delivery of the compound to the desired location in the subject, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.

As used herein, the term "effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to treat an MITF associated disease in a subject or sufficient to inhibit an MITF function. An effective amount of a MetAP-2 inhibitor, as defined herein, may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the MetAP-2 inhibitor to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the MetAP-2 inhibitor are outweighed by the therapeutically beneficial effects.

A therapeutically effective amount of a compound of the invention (i.e., an effective dosage) may range from about 0.001 to about 50 mg/kg body weight, preferably about 0.01 to about 35 mg/kg body weight, more preferably about 10 to about 40 mg/kg body weight, and even more preferably about 25 to about 35 mg/kg, about 27 to about 33 mg/kg, about 29 to about 31 mg/kg, or about 30 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present, if any. Moreover, treatment of a subject with a therapeutically effective amount of a compound of the invention can include a single treatment or, preferably, can include a series of treatments. In one example, a subject is treated with a compound of the invention in the range of between about 25 to about 35 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a compound used for treatment may increase or decrease over the course of a particular treatment.

I. Methionine Aminopeptidase 2 (MetAP-2) Inhibitors

Any methionine aminopeptidase 2 (MetAP-2) inhibitor capable of inhibiting the activity of the methionine aminopeptidase 2 protein may be used in the methods of the present invention. Such inhibitors are well known in the art and include those described in, for example, U.S. Patent No. 6,548,477 Bl; U.S. Patent No. 7,037,890; U.S. Patent No. 7,084,108; U.S. Patent No. 6,919,307; U.S. Patent No. 7,105,482; U.S. Patent No. 5,135,919; U.S. Patent No. 5,180,738; U.S. Patent No. 5,290,807; U.S. Patent No. 5,648,382; U.S. Patent No. 5,698,586; U.S. Patent No. 5,767,293; U.S. Patent No. 5,789,405, the contents of each of which are incorporated herein by reference. In a preferred embodiment, the MetAP-2 inhibitor is a compound of Formula I,

In Formula I, A is a MetAP-2 inhibitory core, W is O or NR₂, and R₁ and R₂ are each, independently, hydrogen or alkyl; X is alkylene or substituted alkylene, preferably linear d-Cβ-alkylene; n is 0 or 1; R₃ and R₄ are each, independently, hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or arylalkyl or substituted or unsubstituted heteroaryl or heteroalkyl. R3 and R₄ can also, together with the carbon atom to which they are attached, form a carbocyclic or heterocyclic group; or Ri and R₄ together can form an alkylene group; Z is -C(O)-, alkylene-C(O)- or alkylene; and P is a peptide comprising from 1 to about 100 amino acid residues attached at its amino terminus to Z or a group OR5 or N(R₆)R₇, wherein R5, R₆ and R₇ are each, independently, hydrogen, alkyl, substituted alkyl, azacycloalkyl or substituted azacyclo alkyl. R₆ and R₇ can also form, together with the nitrogen atom to which they are attached, a substituted or unsubstituted heterocyclic ring structure.

In another embodiment of the compounds of Formula I, W, X, n, Ri, R₃ and R₄ have the meanings given above for these variables; Z is -O-, -NRg-, alkylene-O- or alkylene-NRg-, where Rg is hydrogen or alkyl; and P is hydrogen, alkyl, preferably normal or branched Ci-C₄-alkyl or a peptide consisting of from 1 to about 100 amino acid residues attached at its carboxy terminus to Z.

In compounds of Formula I, when any of Ri-Rg is an alkyl group, preferred alkyl groups are substituted or unsubstituted normal, branched or cyclic Ci-C₆ alkyl groups. Particularly preferred alkyl groups are normal or branched C₁-C₄ alkyl groups. A substituted alkyl group includes at least one non-hydrogen substituent, such as an amino group, an alkylamino group or a dialkylamino group; a halogen, such as a fluoro, chloro, bromo or iodo substituent; or hydroxyl.

When at least one OfR₃ and R₄ is a substituted or unsubstituted aryl or heteroaryl group, preferred groups include substituted and unsubstituted phenyl, naphthyl, indolyl, imidazolyl and pyridyl. When at least one of R3 and R₄ is substituted or unsubstituted arylalkyl or heteroarylalkyl, preferred groups include substituted and unsubstituted benzyl, naphthylmethyl, indolylmethyl, imidazolylmethyl and pyridylmethyl groups. Preferred substituents on aryl, heteroaryl, arylalkyl and heteroarylalkyl groups are independently selected from the group consisting of amino, alkyl-substituted amino, halogens, such as fluoro, chloro, bromo and iodo; hydroxyl groups and alkyl groups, preferably normal or branched Ci-Cδ-alkyl groups, most preferably methyl groups. X is preferably linear d-C_ό-alkylene, more preferably d-C4-alkylene and most preferably methylene or ethylene. When Z is alkylene-C(O)-, alkylene-O- or alkylene- NR₈, the alkylene group is preferably linear Ci-Cβ-alkylene, more preferably C₁-C₄- alkylene and most preferably methylene or ethylene.

Re and R₇, in addition to alkyl, substituted alkyl or hydrogen, can each also independently be a substituted or unsubstituted azacycloalkyl group or a substituted or unsubstituted azacycloalkylalkyl group. Suitable substituted azacycloalkyl groups

include azacycloalkyl groups which have an N-alkyl substituent, preferably an N-C₁-C₄- alkyl substituent and more preferably an N-methyl substituent. Re and R₇ can also, together with the nitrogen atom to which they are attached, form a heterocyclic ring system, such as a substituted or unsubstituted five or six-membered aza- or diazacycloalkyl group. Preferably, the diazacyclo alkyl group includes an N-alkyl substituent, such as an N-C₁-C₄^IkVl substituent or, more preferably, an N-methyl substituent.

In particularly preferred embodiments, -N(Rs)R₇ is NH₂ or one of the groups shown below:

-N N v Preferably, the compounds of Formula I do not include compounds wherein Z is -O-, P is hydrogen, R₃ and R₄ are both hydrogen, n is 1 and X is methylene. Preferably, the compounds of Formula I further do not include compounds wherein Z is methylene- O-, R₃ and R₄ are both hydrogen, and n is 0.

In another embodiment, the MetAP-2 inhibitor is a compound of Formula XV,

(XV)

where A is a MetAP-2 inhibitory core and W is O or NR. In one embodiment, Z is - C(O)- or -alkylene-C(O)- and P is NHR, OR or a peptide consisting of one to about one hundred amino acid residues connected at the N-terminus to Z. In this embodiment, Q is hydrogen, linear, branched or cyclic alkyl or aryl, provided that when P is -OR, Q is not hydrogen. Z is preferably -C(O)- or C!-C₄-alkylene-C(O)-, and, more preferably, - C(O)- or C₁-C₂-alkylene-C(O)-. Q is preferably linear, branched or cyclic d-Cβ-alkyl, phenyl or naphthyl. More preferably, Q is isopropyl, phenyl or cyclohexyl.

In another embodiment, Z is -alkylene-O- or -alkylene-N(R)-, where alkylene is, preferably, Ci-Cβ-alkylene, more preferably d-C₄-alkylene and, most preferably, C₁-C₂- alkylene. P is hydrogen or a peptide consisting of from one to about one hundred amino acid residues connected to Z at the carboxyl terminus. In this embodiment, Q is hydrogen, linear, branched or cyclic alkyl or aryl, provided that when P is hydrogen, Q is not hydrogen. Q is preferably linear, branched or cyclic Ci-Cδ-alkyl , phenyl or naphthyl. More preferably, Q is isopropyl, phenyl or cyclohexyl.

In the compounds of Formula XV, each R is, independently, hydrogen or alkyl. In one embodiment, each R is, independently, hydrogen or linear, branched or cyclic C₁- Cβ-alkyl. Preferably, each R is, independently, hydrogen or linear or branched C₁-C₄- alkyl. More preferably, each R is, independently, hydrogen or methyl. In the most preferred embodiments, each R is hydrogen.

In Formulas I and XV, A is a MetAP-2 inhibitory core. As used herein, a "MetAP-2 inhibitory core" includes a moiety able to inhibit the activity of methionine aminopeptidase 2 (MetAP-2), e.g., the ability of MetAP-2 to cleave the N-terminal methionine residue of newly synthesized proteins to produce the active form of the protein. Preferred MetAP-2 inhibitory cores are Fumagillin derived structures.

Suitable MetAP-2 inhibitory cores include the cores of Formula II,

(H)

where R¹ is hydrogen or alkoxy, preferably C₁-C₄-alkoxy and more preferably, methoxy. R² is hydrogen or hydroxy; and R³ is hydrogen or alkyl, preferably d-C₄-alkyl and more preferably, hydrogen. D is linear or branched alkyl, preferably Ci-Cβ-alkyl; arylalkyl, preferably aryl-Cι-C₄-alkyl and more preferably phenyl-Ci-C₄-alkyl; or D is of the structure

where the dashed line represents a single bond or a double bond. "A" can also be a MetAP-2 inhibitory core of Formula III,

(up

Where R¹, R², R³ and D have the meanings given above for Formula II, and X is a leaving group, such as a halogen. Examples of suitable MetAP-2 inhibitory cores include, but are not limited to, the following.

(IV)

(VI)

(V)

(VII) (IX)

(VIII)₁₆ In each of Formulas IV-X, the indicated valence on the ring carbon is the point of attachment of the structural variable W, as set forth in Formulas I-XV. In Formula IX, p is an integer from 0 to 10, preferably 1-4. In Formulas IV, V and VI-IX, R₁ is hydrogen or C₁-C₄-alkoxy, preferably methoxy. In Formulas IV and V, the dashed line indicates that the bond can be a double bond or a single bond. In Formula V, X represents a leaving group, such as a thioalkoxy group, a thioaryloxy group, a halogen or a dialkylsulfinium group. In Formulas IV and V, R₂ is H, OH, amino, C₁-C4-alkylamino or di(d-C4-alkyl)amino), preferably H. In formulas in which the stereochemistry of a particular stereocenter is not indicated, that stereocenter can have either of the possible stereochemistries, consistent with the ability of the MetAP-2 inhibitor to inhibit the activity of MetAP-2.

In particularly preferred embodiments, A is the MetAP-2 inhibitory core of Formula X below.

(X)

As used herein, the terms "P" and "peptide" include compounds comprising from 1 to about 100 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues). In preferred embodiments, the peptide includes compounds comprising less than about 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acid residues, preferably about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, or 1-90 amino acid residues. The peptides may be natural or synthetically made. The amino acid residues are preferably α-amino acid residues. For example, the amino acid residues can be independently selected from among the twenty naturally occurring amino acid residues, the D-enantiomers of the twenty natural amino acid residues, and may also be non-natural amino acid residues (e.g. , norleucine, norvaline, phenylglycine, β-alanine, or a peptide mimetic such as 3-amino-methylbenzoic acid). In one embodiment, the amino acid residues are independently selected from residues of Formula XI, Formula XII, and Formula XIII.

XI xπ XIII

In Formula XI, X₁ is hydrogen, a side chain of one of the twenty naturally- occurring amino acid residues, a linear, branched or cyclic Ci-Cs-alkyl group, an aryl group, such as a phenyl or naphthyl group, an aryl-d-C-i-alkyl group, a heteroaryl group, such as a pyridyl, thienyl, pyrrolyl, or furyl group, or a heteroaryl-d-C-i-alkyl group; and X₂ is hydrogen a linear, branched or cyclic Ci-Cs-alkyl group, an aryl group, such as a phenyl or naphthyl group, an aryl-Ci-C4-alkyl group or a heteroaryl group as described above for X₁. Preferably, X₂ is hydrogen. In Formula XII, Y is methylene, oxygen, sulfur or NH, and a and b are each, independently, 0-4, provided that the sum of a and b is between 1 and 4. Formulas XI and XII encompass α-amino acid residues having either a D or an L stereochemistry at the alpha carbon atom. One or more of the amino acid residues can also be an amino acid residue other than an α-amino acid residue, such as a β-, γ- or ε-amino acid residue. Suitable examples of such amino acid residues are of Formula XIII, wherein q is an integer of from 2 to about 6, and each X₁ and X₂ independently have the meanings given above for these variables in Formula XI. In a preferred embodiment, the peptide used in the MetAP-2 inhibitors used in the methods of the invention may include a site-directed sequence in order to increase the specificity of binding of the MetAP-2 inhibitor to a cell surface of interest. As used herein, the term "site-directed sequence" is intended to include any amino acid sequence {e.g., comprised of natural or non natural amino acid residues) which serves to limit exposure of the MetAP-2 inhibitor to the periphery and/or which serves to direct the MetAP-2 inhibitor to a site of interest, e.g., a site of pigmentation loss. The peptide contained within the MetAP-2 inhibitors used in the methods of the invention may include a peptide cleavage site for an enzyme which is expressed at sites of pigment loss, allowing tissue-selective delivery of a cell-permeable active MetAP-2 inhibitor or fragment thereof (e. g. , a fragment containing the MetAP-2 inhibitory core of the MetAP-2 inhibitor). The peptide may also include a sequence which is a ligand for a cell surface receptor which is expressed at a site of pigment loss, thereby targeting MetAP-2 inhibitors to a cell surface of interest. However, the selection of a peptide sequence must be such that the active MetAP-2 inhibitor is available to be delivered to the cells in which MetAP-2 inhibition is desired. The peptide can be attached to the MetAP-2 inhibitory core at either its N- terminus or C-terminus. When the peptide is attached to the MetAP-2 inhibitory core at its C-terminus, the N-terminus of the peptide can be -NR₂R₃, where R₂ is hydrogen, alkyl or arylalkyl and R₃ is hydrogen, alkyl, arylalkyl or acyl. When the peptide is attached to the MetAP-2 inhibitory core at its N-terminus, the C-terminus can be - C(O)R₄, where R₄ is -OH, -O-alkyl, -O-arylalkyl, or -NR₂R₃, where R₂ is hydrogen, alkyl or arylalkyl and R₃ is hydrogen, alkyl, arylalkyl or acyl. In this embodiment, the C-terminal residue can also be present in a reduced form, such as the corresponding primary alcohol.

The methods of the present invention may also utilize pharmaceutically acceptable salts of the MetAP-2 inhibitors described herein. A "pharmaceutically acceptable salt" includes a salt that retains the desired biological activity of the parent MetAP-2 inhibitor and does not impart any undesired toxico logical effects. Examples of such salts are salts of acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosporic acid, nitric acid, and the like; acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, benzoic acid, pamoic acid, alginic acid, methanesulfonic acid, naphthalenesulfonic acid, and the like. Also included are salts of cations such as sodium, potassium, lithium, zinc, copper, barium, bismuth, calcium, and the like; or organic cations such as trialkylammonium. Combinations of the above salts are also useful.

The methods of the present invention may also use pro-drugs of the MetAP-2 inhibitors described herein. Preferred MetAP-2 inhibitors of Formula I

One set of particularly preferred MetAP-2 inhibitors to be used in the methods of the invention includes compounds in which A is the MetAP-2 inhibitory core of Formula X, W is O or NR₂, and the structure

is represented by the structures set forth below.

Preferred MetAP-2 inhibitors of Formula XV

A preferred subset of the MetAP-2 inhibitors of Formula XV to be used in the methods of the invention comprises Formula XIV shown below.

(XIV)

In one embodiment, W is O or NR. Z is -C(O) or -alkylene-C(O)-, preferably Cl-C4-alkylene-C(O)-. R is hydrogen or a d-C₄-alkyl. Q is hydrogen; linear, branched or cyclic Ci-Cβ-alkyl; or aryl. R₁ is hydroxy, d-C₄-alkoxy or halogen. P is NH₂, OR or a peptide attached to Z at its N-terminus and comprising from 1 to 100 amino acid residues independently selected from naturally occurring amino acid residues, D- enantiomers of the naturally occurring amino acid residues and non-natural amino acid residues. When Q is H, P is not NH2 or OR. In preferred embodiments, W is O or NH; Q is isopropyl; R₁ is methoxy; P comprises from 1 to 15 amino acid residues; and the dashed line present in Formula XIV represents a double bond. In particularly preferred embodiments, W is O, and P comprises 10 or fewer amino acid residues.

In another embodiment of the compounds of Formula XIV, W is O or NR. Z is alkylene-0 or alkylene-NR, preferably Cl-C4-alkylene-0 or Cl-C4-alkylene-NR-. R is hydrogen or a d-Gralkyl. Q is hydrogen; linear, branched or cyclic Ci-Cβ-alkyl; or aryl. Ri is hydroxy, Ci-C₄-alkoxy or halogen. P is hydrogen or a peptide attached to Z at its C-terminus and comprising from 1 to 100 amino acid residues independently selected from naturally occurring amino acid residues, D-enantiomers of the naturally occurring amino acid residues and non-natural amino acid residues. When Q is H, P is not H. In preferred embodiments, W is O or NH; Q is isopropyl; Ri is methoxy; P comprises from 1 to 15 amino acid residues; and the dashed line present in Formula XIV represents a double bond. In particularly preferred embodiments, W is O, and P comprises 10 or fewer amino acid residues or P is hydrogen. One set of particularly preferred MetAP-2 inhibitors for use in the methods of the invention is represented by the structures set forth below.

10

II, Methods of Treatment of MITF Associated Disease

The present invention provides a method of treating an MITF associated disease in a subject. The method includes administering to the subject a therapeutically effective amount of a MetAP-2 inhibitor, thereby treating an MITF associated disease in the subject.

The term "MITF associated disease," as used herein, is intended to include any disease, disorder or condition associated with (or caused by) the modulated, e.g., decreased or increased, expression or activity of MITF as compared to normal MITF expression or activity levels. This term includes diseases, disorders, or conditions in which melanogenesis mediated by MITF is impaired, in which mast cell development is inhibited, or in which the activity of NK cells is inhibited. MITF associated diseases include, but are not limited to, Microphthalmia, Waardenburg syndrome type 2a, Tietz syndrome, digenic ocular albinism, albinism, and Sensorineural deafness. In one embodiment, this term does not include diseases such as cancer, e.g., melanoma, or inflammatory diseases. In another embodiment, this term does not include diseases such as bone associated diseases, e.g., osteoporosis.

As used herein, the term "administering" to a subject includes dispensing, delivering or applying an MetAP-2 inhibitor, e.g., an MetAP-2 inhibitor in a pharmaceutical formulation (as described herein), to a subject by any suitable route for delivery of the compound to the desired location in the subject, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.

As used herein, the term "effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to treat an MITF associated disease in a subject or sufficient to inhibit an MITF function. An effective amount of a MetAP-2 inhibitor, as defined herein, may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the MetAP-2 inhibitor to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects {e.g., side effects) of the MetAP-2 inhibitor are outweighed by the therapeutically beneficial effects.

A therapeutically effective amount of a compound of the invention (i.e., an effective dosage) may range from about 0.001 to about 50 mg/kg body weight, preferably about 0.01 to about 35 mg/kg body weight, more preferably about 10 to about 40 mg/kg body weight, and even more preferably about 25 to about 35 mg/kg, about 27 to about 33 mg/kg, about 29 to about 31 mg/kg, or about 30 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present, if any. Moreover, treatment of a subject with a therapeutically effective amount of a compound of the invention can include a single treatment or, preferably, can include a series of treatments. In one example, a subject is treated with a compound of the invention in the range of between about 25 to about 35 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a compound used for treatment may increase or decrease over the course of a particular treatment. The present invention also provides methods for modulating the function of MITF. The methods include contacting MITF with an effective amount of a MetAP-2 inhibitor, thereby modulating the function of MITF.

As used herein, the term "MITF function" or "function of MITF" is intended to include any function mediated by MITF. This term includes any action, process, signaling pathway, or activity which is affected or regulated by MITF. In one embodiment, this term is intended to include the expression of MITF. In another embodiment, general MITF functions include cell differentiation and pigmentation. In a preferred embodiment, MITF functions include melanocyte differentiation, and melanogenesis. An MITF function, as used herein, also includes the regulation of the expresion or activity of other proteins which are regulated by MITF such as, for example, melanin, mast cell protease 5 (MCP-5) (see, e.g., Morii et al, (1997) Blood. 90:3057-3066), MCP-6 (see, e.g., Mom et al, (1996) Blood. 88:2488-2494), c-kit (see, e.g., Isozaki et al, (1994) Am. J. Pathol. 145:827-836), p75 nerve growth factor (see, e.g., Morii et al, 1997), granzyme B (see, e.g., Ito et al, (1998) Blood. 91 :3210-3221), tryptophan hydroxylase (see, e.g., Ito et al, (1999) Blood. 93:1189-1196), cathepsin K (see, e.g., Motyckova et al, (2001) Proc. Natl. Acad Sd. U.S.A. 98:5798-5803), RbI (see, e.g., Carreira et al, (2005) Nature 433(7027):764-769), CDK2 (see, e.g., Du et al, (2004) Cancer Cell 6(6):565-576), tyrosinase, tyrosinase related protein, and pink-eyed Pmel 17 (see, e.g., Bentley et al, (1994) MoI Cell. Biol. 14:7996-8006). MITF function can be determined by monitoring the function or expression of proteins, or the underlying nucleic acids encoding proteins, which are regulated by MITF. The term "MITF function" also includes the regulation of the expression or activity of proteins which bind to, intract with and/or regulate MITF. For example, MITF can form heterodimers with TFEB, TFEC, TFE3, and USF2 (see, e.g., Beckmann et al, (1990) Genes Dev. 4:167-179; Carr and Sharp, (199O)M?/. Cell. Biol. 10:4384-4388; Nechushtan ef α/., (1997) Blood 89:2999-3008; and Zhao et al, (1993) MoI. Cell. Biol. 13:4505-4512), can associate with PKCI or PIAS3 to prevent MITF activation of target genes until the appropriate stimulus is encountered (see, e.g., Razin et al, (1999) J. Biol. Chem. 274:34272-34276 and Levy et al, (2002) J. Biol. Chem. 277: 1962-1966), can interact with PEBP2 and MAZR to increase affect transcription of MCP-6 (see, e.g., Ogihara et al, (1999) Oncogene 8:4632-4639 and Morii et al, (2002) J. Biol. Chem. 277(10):8566-8571, respectively), and can bind to c-FOS and PU-I (see, e.g., Sato et al, (1999) Biochem Biophys Res. Commun. 254(2):384-387), LEF-I (see, e.g., Yasumoto et al., (2002) EMBO J. 21(l l):2703-2714), and PAX-6 (see, e.g., Plangue et al, (200I) J Biol Chem. 276(31):29330-7). MITF function can be determined by monitoring the function or expression of proteins, or the underlying nucleic acids encoding proteins, which bind to and/or interact with the MITF protein. MITF function can also be determined by monitoring the function or expression of any proteins which post- translationally modify MITF or proteins which are post-translationally modified by MITF. For example, the Kit signaling pathway and the GSK3β molecule have been suggested to modulate MITF post-translationally (see, e.g., Wu et al, (2000) Genes Dev. 14:301-312).

The present invention also provides methods of inducing melanogenesis in a melanocyte or a melanoma cell. These methods include contacting the melanocyte or the melanoma cell with an amount of a MetAP-2 inhibitor effective to inhibit an MITF function, thereby inducing melanogenesis in the melanocyte or a melanoma cell. As used herein, the term "melanogenesis" is intended to refer to the formation of the melanin pigment by living cells. Melanogenesis also refers to a morphological feature of a differentiated melanocyte cell or a melanoma cell. Melanocytes utilize the enzyme tyrosinase in melanogenesis, e.g., the synthesis of the melanin pigment from tyrosine. The tyrosinase enzymes Trypl, Tryp2, and Dct (dopachrometautomerase) are transactivated by the transcription factor MITF.

The present invention also provides methods for sensitizing a melanoma cell to a chemotherapeutic agent. The methods include contacting a melanoma cell with an amount of a MetAP-2 inhibitor effective to inhibit an MITF function, thereby sensitizing the melanoma cell to a chemotherapeutic agent. The term "chemotherapeutic agent" is well known in the art and includes dacarbazine (DITC), docetaxe, paclitaxel, temozolomide, carmustine (BCNU), lomustine (CCNU), fotemustine, cisplatin, carboplatin, vinblastine, and vindesine. Other well known chemotherapeutic agents may be found in Harrison's Principles of Internal Medicine, Thirteenth Edition, Eds. T.R. Harrison et al. McGraw-Hill N. Y., NY; and the Physicians Desk Reference 50th Edition 1997, Oradell New Jersey, Medical Economics Co., the complete contents of each of which are expressly incorporated herein by reference. The MetAP-2 inhibitor and the chemotherapeutic agent may be administered to the subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times).

As used herein, the term "sensitizing" or "sensitized" refers to rendering a cell, e.g., a melanoma cell, more sensitive or reactive to treatment, e.g., treatment with a chemotherapeutic agent. The term sensitizing may also refer to making a cell more readily or excessively affected by treatment, e.g., treatment with a chemotherapeutic agent. A cell may be sensitized in vitro, for example, in cell culture. In a preferred embodiment, a cell may be sensitized in vivo, for example, in a subject, e.g., a human subject.

III. Pharmaceutical Compositions

The MetAP-2 inhibitors to be used in the methods of the present invention are preferably administered to a subject using a pharmaceutically acceptable formulation. Such pharmaceutically acceptable formulations typically include one or more MetAP-2 inhibitors as well as a pharmaceutically acceptable carrier(s) and/or excipient(s). As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compounds of the invention, use thereof in the pharmaceutical compositions is contemplated.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble), or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions for injection. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the pharmaceutical composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol or sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the compound of the invention in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the MetAP-2 inhibitor into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the MetAP-2 inhibitor plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the MetAP-2 inhibitor can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also include an enteric coating. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the MetAP-2 inhibitor in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the MetAP-2 inhibitors are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the MetAP-2 inhibitors are formulated into ointments, salves, gels, or creams as generally known in the art. The pharmaceutical compositions of the invention can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the MetAP-2 inhibitors are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811, U.S. Patent No. 5,455,044, U.S. Patent No. 5,576,018 and U.S. Patent No. 4,883,666, the contents of all of which are incorporated herein by reference.

The MetAP-2 inhibitors can also be incorporated into pharmaceutical compositions which allow for the sustained delivery of the MetAP-2 inhibitors to a subject for a period of at least several weeks to a month or more. Such formulations are described in U.S. Patent No. 5,968,895; U.S. Patent No. 6,699,833 Bl; U.S. Patent No. 6,180,608 Bl; U.S. Publication No. US 2002-0176841 Al; U.S. Publication No.US 2005-0112087 Al; U.S. Publication No. US 2002-0086829 Al, the contents of each of which are incorporated herein by reference.

It is especially advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of one or more compounds of the invention calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are dictated by and directly dependent on the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such compounds for the treatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. MetAP-2 inhibitors which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the compounds of the invention lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compounds used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.

Such information can be used to more accurately determine useful doses in humans.

Levels in plasma may be measured, for example, by high performance liquid chromatography.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures are hereby incorporated by reference.

EXAMPLES

Materials and Methods for Examples 1-3 Reagents. The MetAP-2 inhibitor comprising the structure (l-Carbamoyl-2- methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3- methyl-but-2-enyl)-oxiranyl]-l-oxa-spiro[2.5]oct-6-yl ester was used in these experiments. For in vitro studies, a 10 mM stock solution in ethanol was prepared. For in vivo administration, the MetAP-2 inhibitor was dissolved in 11% 2-hydroxypropyl- beta-cyclodextran (HPCD) (Cargill Incorporation). Dacarbazine (DTIC) and melanin were obtained from Sigma. [³H]-thymidine was obtained from Amersham.

MetAP-2 pharmacodynamic assay. The MetAP-2 assay measures the amount of uninhibited MetAP-2 in cells or tissues which has not been derivitized by prior treatment with the MetAP-2 inhibitor (Bernier (2004) Proc. Natl. Acad Sci. USA lOl : 10768- 10773 and Bernier (2005) J. Cell. Biochem. 95: 1191-1203). Briefly, wbc from animals of each study group were pooled and cell lysates were prepared as previously described (Bernier (2004) Proc. Natl. Acad Sci. USA 101 : 10768-10773 and Bernier (2005) J. Cell. Biochem. 95: 1191-1203). 10 μg to 20 μg of wbc protein was incubated with a biotinylated analog of the MetAP-2 inhibitor which covalently binds to the catalytic site of MetAP-2. The biotinylated MetAP-2-inhibitor complex was captured on a plate with immobilized streptavidin (Pierce), and detected with the MetAP-2 antibody CM33 (0.5μg/ml), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody. The amount of uninhibited MetAP-2 was determined by measuring the absorption at 450 nm using a Labsystems Multiskan plate spectrophotometer. Human recombinant MetAP-2 (Mediomics), pre-bound to the biotinylated MetAP-2 inhibitor, was used to generate the standard curve. The detection limit of this assay was 0.47 ng MetAP-2 protein/mg wbc protein. Western Blot Analysis. After treatment, cells were washed once with phosphate- buffered saline (PBS), harvested in buffer A (50 mM Tris-HCl, pH 7.4, 1% Nonidet P- 40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 2 mM Na₃VO₄, 1 mM NaF, and complete protease cocktail inhibitor from Roche), solubilized for 30 minutes at 4⁰C, and centrifuged at 14,000 x g for 15 minutes. For Western blot analysis, 30 ug of cellular protein was used. The MetAP-2 polyclonal antibody used for detection was CM33 (Zymed). Trp-1 and Trp-2 polyclonal antibodies G-17 and G-15, respectively, were obtained from Santa Cruz.

The methods used in the Examples provided below are also generally described in Hannig et α/.,(2006) InternationalJournal ofOnclology 28:955-963, the entire contents of which are expressly incorporated herein by reference.

Example 1: The MetAP-2 inhibitor inhibits the growth of melanoma cells in vitro.

The MetAP-2 inhibitor used in the present studies is an orally available, irreversible MetAP-2 inhibitor of the fumagillin class of molecules disclosed in, for example, U.S. Patent Nos. 6,548,477 and 7,037,890. In order to investigate the activity of the MetAP-2 inhibitor on MITF function, several cell proliferation assays were performed. The human melanoma Ml 4 and B 16Fl O murine melanoma cells were obtained from the American Type Tissue Collection (ATCC). The cells were cultured in DMEM supplemented with fetal bovine serum (10% v/v), penicillin-streptomycin (100 units/ml and 100 ug/ml, respectively) and L-glutamine (2 mM). For proliferation assays, 1 x 10³ cells were seeded in 96-well plates (in triplicate) and incubated with varying concentrations of MetAP-2 inhibitor. After three days, 1 μCi/well [³H] -thymidine was added for the final 24 hours of incubation. Cell proliferation was determined by the amount of incorporated [³H]-thymidine, by using liquid scintillation counting.

The MetAP-2 inhibitor inhibited the growth of the B16F10 and M14 cell lines with GI50S of 0.2 and 2 nM, respectively (Figure 1). This growth inhibition was linked to the amount of MetAP-2 enzyme inhibited in these cells (Figure 1), as determined by a MetAP-2 pharmacodynamic assay described above, consistent with previous reports that show a link between MetAP-2 inhibitor-induced growth inhibition and the level of MetAP-2 inhibition.

Example 2: The MetAP-2 inhibitor inhibits the growth of melanoma cells in the mouse M14 xenograft model in vivo.

Male SCID mice (7 weeks of age) were purchased from Charles River Laboratories and randomized into groups of 10 animals each. 5 x 10⁶ M14 cells in PBS were injected subcutaneously above the right hind leg. Tumor growth was measured every 2-3 days using a CD-8" CS digital caliper (Mitutoyo Corporation, Japan), and tumor volumes were calculated following the equation: volume = (width x width) (length)/2. MetAP-2 inhibitor was formulated in 11% HPCD, and mice were dosed orally at 30 mg/kg, every other day (QOD). Treatment with MetAP-2 inhibitor or vehicle started at day 6 post tumor implantation and concluded at day 20. Mice were sacrificed 21 days post tumor implantation, tumors were removed, weighed, frozen in liquid nitrogen, and stored at -8O⁰C.

The MetAP-2 inhibitor inhibited the growth tumor cells when administered orally at 30 mg/kg, QOD (Figure 2). This growth inhibition was linked to the almost complete inhibition of MetAP-2 detected in the white blood cell and tumor compartments 24 hours after the final dose (Figure 3), as determined by a MetAP-2 pharmacodynamic assay described above. These results also indicate that MetAP-2 inhibitors inhibit the growth of melanoma cells in the mouse M14 xenograft model and that this growth inhibition is linked to the amount of MetAP-2 inhibited in those cells.

Example 3: The MetAP-2 inhibitor induces melanogenesis in vitro. To measure the amount of secreted melanin in response to a MetAP-2 inhibitor,

1 x 10⁵ cells from previously selected clones following exposure to either 0.1 μM or 1 μM of MetAP-2 inhibitor were seeded in 6-well plates in triplicate, and MetAP-2 inhibitor was added at different concentrations to the growth medium. After seven days, the amount of secreted melanin was determined by measuring the optical density at 492 nm, derived from a standard curve prepared with synthetic melanin dissolved in growth medium. To determine whether the MetAP-2 inhibitor induced melanogenesis was reversible, Bl 6F10 cells were seeded at 1 x 10⁵ cells in 6-well plates and incubated in either drug-free medium or MetAP-2 inhibitor-containing medium for seven days. The cells were split and seeded at 5 x 10⁴ cells/well in fresh medium containing either no drug or the MetAP-2 inhibitor for another seven day period. The growth medium was then collected and the amount of melanin was measured as described above.

Prolonged exposure of high concentrations of the MetAP-2 inhibitor to mouse Bl 6F10 melanoma cells in vitro did not result in resistance to this agent, but induced melanogenesis, a morphological feature of differentiated melanocytes (Figure 4). Figure 4 shows that melanogenesis was associated with the downregulation of MITF protein expression, an unexpected finding given the role of MITF as a positive regulator of this process. In contrast to B16F10 cells, prolonged exposure of human UACC-62, A375 and M14 cells to high concentrations of MetAP-2 inhibitor did not significantly induce melanogenesis, but moderate downregulation of MITF protein expression was detected (Figure 5). These data indicate that MITF plays a role as a downstream effector of MetAP-2 inhibitor-induced growth inhibition in mammalian cells.

Example 4: Targeted downregulation of MITF by small interfering RNA (siRNA) significantly inhibits the growth of melanoma cells and further sensitizes the cells to treatment with MetAP-2 inhibitors.

In order to study the role of MITF in the inhibition of cell growth in response to MetAP-2 inhibitors, the human melanoma cell lines UACC-62, A375, and M14 were transfected with MITF siRNA. MITF siRNA inhibited the growth of all three melanoma cell lines, albeit at different levels, and further sensitized these cells to treatment with MetAP-2 inhibitor or a chemotherapeutic agent (Figure 6).

Conclusion These results indicate that MetAP-2 inhibitors inhibit the growth of cells, e.g., human melanoma cells, in vitro and in vivo and that this growth inhibition is linked to the amount of MetAP-2 inhibited in these cells. Additionally, these results are the first to indicate that MITF plays a role as a downstream effector of MetAP-2 inhibitor induced growth inhibition in mammalian cells, e.g., melanoma cells.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more that routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWe claim:

1. A method for treating a subject having an MITF associated disease, comprising administering to a subject a MetAP-2 inhibitor in an amount effective to inhibit MITF function, thereby treating a subject having an MITF associated disease.

2. The method of claim 1, wherein said MITF associated disease is selected from the group consisting of Microphthalmia, Waardenburg syndrome type 2a, Tietz syndrome, digenic ocular albinism, Sensorineural deafness, and albinism.

3. The method of claim 1, wherein said subject is a mammal.

4. The method of claim 3, wherein said mammal is a human.

5. A method for modulating a function of MITF, comprising contacting MITF with an effective amount of a MetAP-2 inhibitor, thereby modulating the function of MITF.

6. The method of claim 5, wherein said MITF is present within a human cell.

7. The method of claim 6, wherein said human cell is present within a human subject.

8. The method of claim 5, wherein said MITF function is selected from the group consisting of melanocyte differentiation and melanogenesis.

9. The method of claim 5, wherein said MITF function is the regulation of expression or activity of proteins regulated by MITF selected from the group consisting of melanin, mast cell protease 5 (MCP-5), MCP-6, c-kit, p75 nerve growth factor, granzyme B, tryptophan hydroxylase, cathepsin K, RbI, CDK2 , tyrosinase, tyrosinase related protein, and pink-eyed Pmel 17.

10. The method of claim 5, wherein said MITF function is the regulation of the expression or activity of proteins that bind to and/or interact with MITF selected from the group consisting of TFEB, TFEC, TFE3, USF2, PKCI, PIAS3, PEBP2, MAZR, c- FOS, PU-I, LEF-I, GSK3β, and PAX-6.

11. A method for inducing melanogenesis in a melanocyte or a melanoma cell, comprising contacting the melanocyte or the melanoma cell with an amount of a MetAP- 2 inhibitor effective to inhibit an MITF function, thereby inducing melanogenesis in the melanocyte or the melanoma cell.

12. A method for sensitizing a melanoma cell to a chemotherapeutic agent, comprising contacting a melanoma cell with an amount of a MetAP-2 inhibitor effective to inhibit an MITF function, thereby sensitizing a melanoma cell or the melanoma cell to a chemotherapeutic agent.

13. The method of claim 12, wherein said chemotherapeutic agent is selected from the group consisting of dacarbazine (DITC), docetaxe, paclitaxel, temozolomide, carmustine (BCNU), lomustine (CCNU), fotemustine, cisplatin, carboplatin, vinblastine, and vindesine.

14. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is a compound of Formula I,

wherein

A is a Met-AP2 inhibitory core;

W is O or NR₂;

R₁ and R₂ are each, independently, hydrogen or alkyl; X is alkylene or substituted alkylene; n is O or 1;

R₃ and R₄ are each, independently, hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; or R₃ and R₄, together with the carbon atom to which they are attached, form a carbo cyclic or heterocyclic group; or R3 and R₄ together form an alkylene group;

Z is -C(O)- or alkylene-C(O)-; and

P is a peptide comprising from 1 to about 100 amino acid residues attached at its amino terminus to Z or a group OR₅ or N(Rg)R?, wherein R5, Rδ and R₇ are each, independently, hydrogen, alkyl, substituted alkyl, azacycloalkyl or substituted azacycloalkyl; or Rg and R₇, together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic ring structure; or Z is -O-, -NRg-, alkylene-O- or alkylene-NRg-, where Rg is hydrogen or alkyl; and

P is hydrogen, alkyl or a peptide consisting of from 1 to about 100 amino acid residues attached at its carboxy terminus to Z; or a pharmaceutically acceptable salt or a pro-drug thereof.

15. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is a compound of Formula XV,

(XV)

wherein

A is a MetAP-2 inhibitory core;

W is O or NR; each R is, independently, hydrogen or alkyl; Z is -C(O)- or -alkylene-C(O)-;

P is NHR, OR or a peptide consisting of one to about one hundred amino acid residues connected at the N-terminus to Z; Q is hydrogen, linear, branched or cyclic alkyl or aryl, provided that when P is -OR, Q is not hydrogen; or

Z is -alkylene-O- or -alkylene-N(R)-;

P is hydrogen or a peptide consisting of from one to about one hundred amino acid residues connected to Z at the carboxyl terminus;

Q is hydrogen, linear, branched or cyclic alkyl or aryl, provided that when P is hydrogen, Q is not hydrogen; or a pharmaceutically acceptable salt or a pro-drug thereof.

16. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is a compound of the formula

wherein

W is O or NR; each R is, independently hydrogen or a Ci-C4-alkyl;

Q is hydrogen; linear, branched or cyclic d-Cβ-alkyl; or aryl;

R₁ is hydroxy, C₁-C₄-alkoxy or halogen; Z is -C(O)- or d^-alkylene;

P is NHR, OR, or a peptide comprising 1 to 100 amino acid residues attached to Z at the

N-terminus; or

Z is alkylene-0 or alkylene-NR; and P is hydrogen or peptide comprising 1 to 100 amino acid residues attached to Z at the C- terminus; provided that when P is hydrogen, NHR or OR, Q is not hydrogen; or a pharmaceutically acceptable salt or a pro-drug thereof.

17. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is a compound comprising the structure

or a pharmaceutically acceptable salt or a pro-drug thereof.

18. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is a compound comprising the structure (l-Carbamoyl-2- methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3- methyl-but-2-enyl)-oxiranyl]-l-oxa-spiro[2.5]oct-6-yl ester, or a pharmaceutically acceptable salt or a pro-drug thereof.

19. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is administered at a dosage range of about 0.1 to about 50 mg/kg.

20. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is administered at a dosage range of about 25 to about 35 mg/kg.

21. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is administered at a dosage of about 30 mg/kg.

22. The method of any one of claims 1, 5, 11 or 12, wherein said methionine aminopeptidase 2 inhibitor is administered in a sustained-release formulation.

23. The method of claim 22, wherein said sustained-release formulation provides sustained delivery of the methionine aminopeptidase 2 inhibitor to a subject for at least one week after the formulation is administered to the subject.

24. The method of claim 22, wherein said sustained-release formulation provides sustained delivery of the methionine aminopeptidase 2 inhibitor to a subject for at least two weeks after the formulation is administered to the subject.

25. The method of claim 22, wherein said sustained-release formulation provides sustained delivery of the methionine aminopeptidase 2 inhibitor to a subject for at least three weeks after the formulation is administered to the subject.

26. A method of treating an MITF associated disease in a subject, comprising administering to the subject a therapeutically effective amount of a methionine aminopeptidase 2 inhibitor comprising the structure (l-Carbamoyl-2-methyl-propyl)- carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3-methyl-but-2- enyl)-oxiranyl]-l-oxa-spiro[2.5]oct-6-yl ester, or a pharmaceutically acceptable salt or a pro-drug thereof, thereby treating the MITF associated disease in a subject.

27. A method of modulating a function of MITF, comprising contacting MITF with an effective amount of a methionine aminopeptidase 2 inhibitor comprising the structure

(l-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-l-oxa-spiro[2.5]oct-6-yl ester, or a pharmaceutically acceptable salt or a pro-drug thereof, thereby modulating the function of MITF.

28. A method of inducing melanogenesis in a melanocyte or a melanoma cell, comprising contacting the melanocyte or the melanoma cell with an an amount of a methionine aminopeptidase 2 inhibitor comprising the structure (l-Carbamoyl-2-methyl- propyl)-carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3-methyl- but-2-enyl)-oxiranyl]-l-oxa-spiro[2.5]oct-6-yl ester, or a pharmaceutically acceptable salt or a pro-drug thereof, effective to inhibit an MITF function, thereby inducing melanogenesis in the melanocyte or the melanoma cell.

29. A method of sensitizing a melanoma cell to a chemotherapeutic agent, comprising contacting a melanoma cell with an amount of a methionine aminopeptidase 2 inhibitor comprising the structure (l-Carbamoyl-2-methyl-propyl)-carbamic acid-(3R, 4S, 5S, 6R )-5-methoxy-4-[(2R, 3R ) -2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-l-oxa- spiro[2.5]oct-6-yl ester, or a pharmaceutically acceptable salt or a pro-drug thereof, effective to inhibit an MITF function, thereby sensitizing the melanoma cell to a chemotherapeutic agent.