AU2014200485A1 - Stabilized maml peptides and uses thereof - Google Patents

Abstract

Stably cross-linked a polypeptides related to human MAML are described. These cross-linked polypeptides contain at least two modified amino acids that together form an internal cross-link or tether that can help to stabilize the alpha-helical secondary structure that is thought to be important for binding of MAML peptides to the Notch transcription complex, a complex that includes ICN and CSL.

Description

WO 2008/061192 PCT/US2007/084838 Stabilized MAML Peptides and Uses Thereof BACKGROUND Notch receptors are transmembrane receptors that involved in a variety of important signaling pathways. Mutations in human NOTCH l are commonly found 5 in human T cell acute lymphoblastic leukemias (T-ALL) and it is thought that abnonnalities in Notch signaling are involved in other cancers. The Notch signaling pathway is complex. When an appropriate ligand binds to Notch a proteolytic event occurs which allows a portion of the Notch receptor called ICN to enter the cell nucleus where is interacts with CSL, a transcription 10 factor that binds DNA, and a protein that is a member of the Mastermind-like (MAML) family. The assembled complex can active transcription of certain genes. It is known that certain fragments of MAML (e.g., within amino acids 13-74 of human MAML-1) can act to interfere with Notch activation of transcription. SUMMARY 15 Described below are stably cross-linked a polypeptides related to human MAML. These cross-linked polypeptides contain at least two modified amino acids that together form an internal cross-link (also referred to as a tether) that can help to stabilize the alpha-helical secondary structure that is thought to be important for binding of MAML peptides to the Notch transcription complex, a complex that 20 includes ICN and CSL. It is thought that the constrained secondary structure can increase resistance of the polypeptide to proteolytic cleavage. Accordingly, a cross linked polypeptide described herein can have improved biological activity relative to a corresponding polypeptide that is not cross-linked. The cross-linked polypeptides described herein can be used therapeutically, e.g., to treat a variety of cancers in a 25 subject. Inhibitors of Notch function may be useful in reducing unwanted immune responses, undesirable angiogenesis, treatment of human T cell acute lymphoblastic leukemias, treatment of mucoepidermoid carcinomas, treatment of breast cancer, treatment of medulloblastoma, and treatment of pancreatic cancer, treatment of lung cancer, treatment of ovarian cancer, treatment of atherosclerosis (e.g., heart disease), 1 WO 2008/061192 PCT/US2007/084838 treatment of melanoma, treatment of colon cancer, and treatment of cancers that exhibit resistance to gamma secretase inhibitors. In one aspect, the invention features a modified polypeptide of Formula (I), H H 0 H 0 N [Xaaj,-N [Xaa], R [Xaa]y 5z,

R

3

R

2 I 5 - - z Formula (1) wherein; each R, and R 2 are independently H or a C, to CIO alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; 10 R 3 is alkyl, alkenyl, alkynyl; [R 4

-K-R

4 ],; each of which is substituted with 0 6 R;

R

4 is alkyl, alkenyl, or alkynyl;

R

5 is halo, alkyl, OR 6 , N(R 6

)

2 , SR6, SOR 6 , SO 2

R

6 , C0 2

R

6 , R 6 , a fluorescent moiety, or a radioisotope; 0 15 K is 0, S, SO, SO 2 , CO, CO 2 , CONR 6 , or

R

6 is H, alkyl, or a therapeutic agent; n is 3, 4 or 6; x is an integer from 2-10; w and y are independently an integer from 0-100 ( e.g., 1, 2 3, 4, 5, 6 or 7); 20 z is an integer from I-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and each Xaa is independently an amino acid, wherein the polypeptide comprises at least 8 (e.g., 8, 9, 10, 11, 12, 13 or more) contiguous amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6 or 7 except that: (a) within the 8 contiguous amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6 or 7 the side 25 chains of at least one pair of amino acids separated by 3, 4 or 6 amino acids is replaced by the linking group R 3 which connects the alpha carbons of the pair of amino acids as depicted in formula I; and (b) the alpha carbon of the first of the pair of amino acids is substituted with R, as depicted in formula I and the alpha carbon 2 WO 2008/061192 PCT/US2007/084838 of the second of the pair of amino acids is substituted with R 2 as depicted in formula I or a pharmaceutically acceptable salt thereof. SEQ ID NO:l is a sequence created from an alignment of human MAML-1, 2, and 3, starting at amino acid 19 of MAML-1 and extending to amino acid 61. 5 SEQ ID NOs:2 - 4 respectively are the amino acid sequences of MAML-I, 2 and 3 over this same region. SEQ ID NOs:5 - 7 respectively are the amino acid sequences of MAM L-1, 2 and 3 over a somewhat larger region. His, Ser2 Xaa 3 Xaa 4 Xaa 5 Glu 6 Arg 7 Lcus Arg 9 Xaa() Xaan 1 lle 12 Xaa 1 3 Xaa 1 4 CysIs 10 Argi 6 Xaa 1 , Hisls Hisig Xaa 2 o Xaa 2 Cys 22 Glu 23 Xaa 2 4 Arg 2 s Tyr 26 Xaa 27 Xaa 28 Xaa 29 Xaa 30 Xaa 3 , Glu 3 2 Xaa33 Xaa 34 Xaa 35 Xaa 36 GIu 37 Arg 3 8 Xaa 3 9 Xaa 4 0 Thr 4 1 Xaa 42 Xaa 43 Leu 44 Xaa 4 5 Xaa46 Xaa 47 (SEQ ID NO: 1), wherein Xaa 3 is Ala or Thr; Xaa 4 is VIa or lie; 15 Xaa 5 is Met or Val; Xaajo is Arg, Ala, or Gin Xaa 3 is Giu or Ala Xaa 14 is Leu, Val or Gly Xaa, 7 Arg or Gin 20 Xaa 20 is Ser, Leu or Val Xaa 21 is Thr, Ser or Asn Xaa 24 is Arg, Gly or Asn Xaa 2 7 is Glu or Gin Xaa 28 is Ala, Arg or Gin 25 Xaa 29 is Val, Gly or Ala Xaa 30 is Ser, Arg or Gin Xaa 3 1 Pro, Ala or Val Xaa 33 Arg, Ser or Gin Xaa 34 Leu or Ser 30 XaaSs is Glu or Asp Xaa 36 is Leu or Arg Xaa 3 ( is Gin Glu or Arg 3 WO 2008/061192 PCT/US2007/084838 Xaa4o is His, Ser or Asp Xaa 42 is Phe, Leu or Val Xaa 43 is Ala, Gin or Ser Xaa 4 5 is His, Lcu or Tyr 5 Xaa 4 6 is Gin or Ser Xaa 4 is Arg or Leu Within SEQ ID NO:1, the following pairs of amino acid can be cross-linked: 2/9, 6/13, 13/17, 17/20, 20/27, 20/24, 35/39, 39/46, and 39/43. The corresponding residues in SEQ ID NOs:2-8 can be cross-linked. 10 SEQ ID NO:2 (MAML-1; amino acids 19-62): VMER LRRRIELCRRHHSTCEARYEAVSPERLELERQHTFA LHQR 15 SEQ ID NO:3 (MAML-2): IVERLRARIAVCRHHI LSCEGRYERGRAESSDRERESTLQLLSL SEQ ID NO:4 (MAML-3): VVERLRQRIEGCRRH HVNCENRYQQAQVEQLELERRDTVSLYQR 20 SEQ ID NO:5 (MAML- 1; includes predicted domain for binding the transcription complex): HSAVMERLRRRIELCRRH HSTCEARYEAVSPERLELERQHTFALHQRCIQAK AKRAGKH 25 SEQ ID NO:6 (MAML-2; includes predicted domain for binding the transcription complex): HSAIVERLRARIAVCRQHHLSCEGRYERGRAESSDRERESTLQLLSLVQHGQ GARKAGKH 30 SEQ ID NO:7 (MAML-3; includes predicted domain for binding the transcription complex): 4 WO 2008/061192 PCT/US2007/084838 AVPKHSTVVERLRQRIEGCRRHHVNCENRYQQAQVEQLELERRDTVSLYQR TLEQRAKKS SEQ ID NO:8 (MAML-1 core) 5 ERLRRRIELCRRHHST SEQ ID NO:9 (MAML-2 core) ERLRARIAVCRQHHLSC 10 SEQ ID NO:10 (MAML-3 core) ERLRQRIEGCRRHHVN In some instances, the modified polypeptide binds a complex of ICN and CSL, e.g., ICN and CSL bound to DNA. 15 In some instances, each y is independently an integer between 3 and 15. In some instances each y is independently an integer between I and 15. In some instances, R, and R 2 are each independently H or Ci-C6 alkyl. In some instances, R, and R 2 are each independently CI-C 3 alkyl. In some instances, at least one of R I and R 2 are methyl. For example R I and 20 R 2 are both methyl. In some instances R 3 is alkyl (e.g., Cg alkyl) and x is 3. In some instances, R 3 is C 1 I alkyl and x is 6. In some instances, R 3 is alkenyl (e.g., C 8 alkenyl) and x is 3. In some instances x is 6 and R 3 is CI alkenyl. 25 In some instances, R 3 is a straight chain alkyl, alkenyl, or alkynyl. In some instances R 3 is -CH 2

-CH

2

-CH

2 -CH=CH-CH2-CH 2

-CH

2 -. In certain embodiments the two alpha, alpha disubstituted stereocenters (alpha carbons) are both in the R configuration or S configuration (e.g., i, i+4 cross link), or one stereocenter is R and the other is S (e.g., i, i+7 cross-link). Thus, where 30 Formula I is depicted as 5 WO 2008/061192 PCT/US2007/084838 H 0 H 0 [xaa NClXaa-N CK [Xaa],

R

3 R iz the C' and C" disubstituted stereocenters can both be in the R configuration or they can both be in the S configuration, for example when x is 3. When x is 6, the C' 5 disubstituted stereocenter is in the R configuration and the C" disubstituted stereocenter is in the S configuration. The R 3 double bond may be in the E or Z stereochemical configuration. In some instances R 3 is [R 4 -K-R4]n; and R 4 is a straight chain alkyl, alkenyl, or alkynyl. 10 In some instances, the polypeptide includes an amino acid sequence which is at least about 60% (70%, 80%, 85%, 90%, 95% or 98%) identical to the amino acid sequence of HSAVMERLRRRIELCRRHHSTCEARYEAVSPERLELERQHTFALHQRCIQAK AKR (SEQ ID NO:8). In some instances the modified polypeptide comprises at 15 least 8 contiguous amino acids of HSAVMERLRRRIELCRRHHSTCEARYEAVSPERLELERQHTFALHQRCIQAK AKR (SEQ ID NO:8). except that at least one pair of amino acids within the 8 (e.g., 8, 9, 10, 11, 12, 13 or more) contiguous amino acids are replaced by modified amino acids that can form an internal cross-link. 20 The tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., C 5 , CS or

C

1 alkyl or a C 5 , Cs or C 1 1 alkenyl, or C 5 , Cs or CI alkynyl). The tethered amino acid can be alpha disubstituted (e.g., C-C3 or methyl). [Xaa]y and [Xaa], are peptides that can independently comprise at least 1, 2 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more contiguous amino acids of a MAML 25 polypeptide and [Xaa]h is a peptide that can comprise 3 or 6 contiguous amino acids of acids of a MAML peptide. The polypeptide can comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 amino acids of a MAML polypeptide (e.g., human MAML-1, 2 or 3 or a consensus MAML polypeptide). The amino acids are contiguous except 6 WO 2008/061192 PCT/US2007/084838 that one or more pairs of amino acids separated by 3 or 6 amino acids are replaced by amino acid substitutes that form a cross-link, e.g., via R 3 . Thus, at least two amino acids can be replaced by tethered amino acids or tethered amino acid substitutes. Thus, where formula I is depicted as H 0 H 0 N , [Xaa]j-N, C, XaaJy.

R

3 5 z [Xaa]y, and [Xaa]y- can each comprise contiguous polypeptide sequences from the same or different MAML peptides. In some instances the polypeptide comprises an amino acid sequence 10 selected from SEQ ID NOs:8, 9 and 10 wherein: (a) the side chains of amino acids 8 and 12 are replaced by the linking group R 3 which connects the alpha carbons of amino acids 8 and 12 as depicted in formula I; and (b) the alpha carbon of amino acid 8 is substituted with R, as depicted in formula I and the alpha carbon of amino acid 12 is substituted with R 2 as depicted in formula 1. 15 The invention features cross-linked polypeptides comprising at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more) contiguous amino acids of a MAML polypeptide wherein the alpha carbons of two amino acids that are separated by three amino acids (or six amino acids) are linked via R 3 , one of the two alpha carbons is substituted by Ri and the other is 20 substituted by R 2 and each is linked via peptide bonds to additional amino acids. In some embodiments the polypeptide acts as dominant negative inhibitor of Notch. In some instances, the polypeptide also includes a fluorescent moiety or radioisotope. 25 In some instances, R, and R 2 are methyl; R 3 is C 8 alkyl, C 1 alkyl, C 8 alkenyl,

C

1 alkenyl, Cg alkynyl, or C 1 alkynyl; and x is 2, 3, or 6. In some instances, the polypeptide includes an PEG, tat protein, affinity label, a targeting moiety, and/or a biotin moiety. 7 WO 2008/061192 PCT/US2007/084838 In another aspect, the invention features a method of making a polypeptide of Formula (Ill), including providing a polypeptide of Formula (11); and H O H0 1 ,N [Xaa.-N [Xaajy R()2[Xaa]y 5 Formula (II) treating the compound of Formula (11) with a catalyst to promote a ring closing metathesis, thereby providing a compound of formula (111) H 0 H O I N [Xaa]-N [Xaa]w ( I [Xaa]y L- -- --- - ) Formula (III) 10 wherein each R, and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl; heteroarylalkyl; or heterocyclylalkyl; each n is independently an integer from 1-15; x is 2, 3, or 6 15 w and y are independently an integer from 0-100; z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and each Xaa is independently an amino acid; In some instances, the polypeptide binds to a complex of ICN and CSL. In some instances, the catalyst is a ruthenium catalyst. 20 In some instances, the method also includes providing a reducing or oxidizing agent subsequent to the ring closing metathesis. In some instances, the reducing agent is H 2 or the oxidizing agent is osmium tetroxide In some instances, the invention features a method of treating a subject 25 including administering to the subject any of the compounds described herein. In 8 WO 2008/061192 PCT/US2007/084838 some instances, the method also includes administering an additional therapeutic agent. In some instances, the invention features a method of treating cancer in a subject including administering to the subject any of the compounds described 5 herein. In some instances, the method also includes administering an additional therapeutic agent. In some instances, the invention features a library of the compounds described herein. Combinations of substituents and variables envisioned by this invention are 10 only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic administration to a subject or generation of reagents to study or discover a biological 15 pathway either in vitro or in vivo). The compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of 20 this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. 25 All crystal forms of the compounds described herein are expressly included in the present invention. The tenn "amino acid" refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D and L- isomers of the 20 common naturally occurring amino acids found in peptides 30 (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letter abbreviations)) as well as the naturally occurring and unnaturally occurring amino acids prepared by organic synthesis or other metabolic routes. The 9 WO 2008/061192 PCT/US2007/084838 table below provides the structures of the side chains for each of the 20 common naturally-occurring amino acids. In this table the "-" at right side of each structure is the bond to the alpha carbon. Single Three i Amiino acidLetter Letter Structure of side cham Alanine A Ala CH 3 Arginine R Arg I HN=C(NH 2

)-NH-(CH

2

)

3 Asparagine N Asn H 2

N-C(O)-CH

2 Aspartic acid D Asp HO(O)-CH 2 Cysteine C Cys __HS-CH.

2 Glutamine Q Gin H 2

N-C(O)-(CH

2

)

2 Glutamic acid E Glu HO(0)-(C--H 2

)

2 Glycine G Gly H

N=CH-NH-CH=C-CH

2 Histidine H His Isolcucine I Ile CH 3

-CH

2

-CH(CH

3

)

Leucine L Leu (CH 3

)

2

-CH-CH

2 ~~. ..- ...... Lysine K Lys H 2

N-(CH

2

)

4 Methionine M Met CH 3

-S-(CH

2

)

2 Phenylalanine F Phe Phenyl-CH,

-N-(CH

2

)

3

-CH

Proline PPro Serine S Ser HO-C 2 Threonine T Thr Cr1 3

-CH(OH)

Phenyl-NH-CH=C-CH 2 Tryptophan W rp Tyrosine Y Tyr 4-OH-Phenyl-CH Valine V Val CH 3

-CH(CH

2

)

5 A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide (without abolishing or substantially altering its activity. An "essential" amino acid residue is a residue that, when altered from the wild-typc sequence of the polypeptide, results in abolishing or substantially 10 abolishing the polypeptide activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families 10 WO 2008/061192 PCT/US2007/084838 of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), 5 nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a MAML polypeptide, for example, is preferably replaced with another amino acid 10 residue from the same side chain family. The symbol " "when used as part of a molecular structure refers to a single bond or a trans or cis double bond. The term "amino acid side chain" refers to a moiety attached to the a-carbon in an amino acids. For example, the amino acid side chain for alanine is methyl, the 15 amino acid side chain for phenylalanine is phenylmethyl, the amino acid side chain for cysteine is thiomethyl, the amino acid side chain for aspartate is carboxymethyl, the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Other non naturally occurring amino acid side chains are also included, for example, those that occur in nature (e.g., an amino acid metabolite) or those that are made synthetically 20 (e.g., an alpha di-substituted amino acid). The term polypeptide encompasses two or more naturally occurring or synthetic amino acids linked by a covalent bond (e.g., a amide bond). Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acids sequences (e.g., fragments of naturally occurring 25 proteins or synthetic polypeptide fragments). The term "halo" refers to any radical of fluorine, chlorine, bromine or iodine. The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, CI CIO indicates that the group may have from I to 10 (inclusive) carbon atoms in it. In 30 the absence of any numerical designation, "alkyl" is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it. The term "alkylene" refers to a divalent alkyl (i.e., -R-). 11 WO 2008/061192 PCT/US2007/084838 The term "alkenyl" refers to a hydrocarbon chain that may be a straight chain or branched chain having one or more carbon-carbon double bonds. The alkenyl moiety contains the indicated number of carbon atoms. For example, C 2 -CIo indicates that the group may have from 2 to 10 (inclusive) carbon atoms in it. The 5 term "lower alkenyl" refers to a C 2

-C

8 alkenyl chain. In the absence of any numerical designation, "alkenyl" is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it. The tern "alkynyl" refers to a hydrocarbon chain that may be a straight chain or branched chain having one or more carbon-carbon triple bonds. The alkynyl 10 moiety contains the indicated number of carbon atoms. For example, C 2 -CIO indicates that the group may have from 2 to 10 (inclusive) carbon atoms in it. The term "lower alkynyl" refers to a C 2

-C

8 alkynyl chain. In the absence of any numerical designation, "alkynyl" is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it. 15 The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term "arylalkyl" or the term "aralkyl" refers to alkyl substituted with an aryl. The tenn "arylalkoxy" refers to an alkoxy substituted with aryl. 20 The term "cycloalkyl" as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, 25 cyclohexenyl, cycloheptyl, and cyclooctyl. The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from 0, N, or S (e.g., carbon atoms and 1-3, 1-6, 30 or 1-9 heteroatoms of N, 0, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl., imidazolyl, 12 WO 2008/061192 PCT/US2007/084838 benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl. 5 The term "heterocyclyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from 0, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, 0, or S if monocyclic, bicyclic, or tricyclic, respectively), 10 wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like. The term "substituents" refers to a group "substituted" on an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of that group. 15 Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and 20 advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS FIG I depicts MAML polypeptides and locations for modification FIG 2 depicts a synthetic strategy for the generation of a-o'disubstituted 25 non-natural amino acids containing olefinic side chains. FIG 3 depicts certain MAML polypeptides used in studies described herein. FIG 4 depicts a stapled MAML polypeptide. FIG. 5 depicts a CD spectra of a stapled polypeptide. FIG 6 depicts stapled MAML polypeptides. 30 FIG 7 depicts the results of surface plasmon resonance showing that BioSAHNI I binds ICN in a dose dependent manner. 13 WO 2008/061192 PCT/US2007/084838 FIG 7 depicts the results of surface plasmon resonance showing that immobilized ICN associates with CSL in a dose-dependent manner. FIG. 9 depicts the results of studies examining the binding of immobilized stapled and unstapled SAHNI Ito ICNI. 5 FIG. 10 depicts the results of studies on cellular uptake of SAHN1, SAHN2 and SAHN6. FIG. 11 depicts the results of studies on cellular uptake of stapled and unstapled SAHNI1. FIG. 12 depicts the results of a study showing that stapled SAHN I1 reduces 10 expression of the CSL-responsive reporter in a dose-dependent manner. FIG. 13 depicts the results of a study showing that T-ALLI cells exposed to either SAlIN 11 -FITC or SAHN 1 -FITC exhibit reduced HES I expression relative to expression of a housekeeping gene. FIG. 14 depicts the results of a study showing that stapled SAHN2 reduces is the viability of MOLT4 cells. FIG. 15 depicts the results of a study showing that stapled SAHN1, but not unstapled SAHN 1, reduces the viability of ALL-SIL cells. FIG. 16 depicts the results of a study showing that stapled SAHN1, but not unstapled SAHNI, reduces the viability of KOPTK1 cells. 20 FIG. 17 depicts the results of a study showing that stapled SAHN 11 reduces the viability of MOLT4 cells. FIG. 18 depicts the results of a study showing that stapled SAHN 11 reduces the viability of TALL cells. FIG. 19 depicts the results of a study showing that SAHNI, but not SAHNI 25 D can bind to ICN l /CSL in T-ALL cellular lysates. FIG. 20 depicts the results of a study showing that SAHN I can compete off ICN 1 bound to MAML in T-ALL cellular lysates. FIG. 21 depicts the results of a study showing that SAHNI-D cannot compete off ICNI bound to MAML in T-ALL cellular lysates 30 FIG. 22 depicts the results of a study showing that SAHN1 and SAHNI 1 can cause a decrease in transcription from a CSL-responsive reporter in T-ALL cells. 14 WO 2008/061192 PCT/US2007/084838 FIG. 23 depicts the results of a study showing that SAHN1, but not SANNI D n cause a decrease in transcription from a CSL-responsive endogenous genes in T ALL cells. FIG. 24 depicts the results of a study showing that SAHNI can elicit an 5 apoptotic response in T-ALL cells. FIG. 25 depicts the results of a study showing that SAHN 1 decreases the viability of MOLT4 T-ALL cells. 10 DETAILED DESCRIPTION Described herein are internally cross-linked alpha helical domain polypeptides related to human MAML. The polypeptides include a tether between two non-natural amino acids, which tether significantly enhances the alpha helical 15 secondary structure of the polypeptide. Generally, the tether or cross-link (sometimes referred to as staple) extends across the length of one or two helical turns (i.e., about 3.4 or about 7 amino acids). Accordingly, amino acids positioned at i and i+3; i and i+4; or i and i+7 are ideal candidates for chemical modification and cross-linking. Thus, for example, where a peptide has the sequence ...Xaa 1 , 20 Xaa 2 , Xaa 3 , Xaa 4 , Xaas, Xaa 6 , Xaa 7 , Xaag, Xaae... (wherein ". ." indicates the optional presence of additional amino acids), cross-links between Xaa and Xaa 4 , or between Xaa 1 and Xaa 5 , or between Xaaj and Xaa 8 are useful as are cross-links between Xaa 2 and Xaa 5 , or between Xaa 2 and Xaa 6 , or between Xaa 2 and Xaag, etc. The polypeptides can include more than one crosslink within the polypeptide 25 sequence to either further stabilize the sequence or facilitate the stabilization of longer polypeptide stretches. If the polypeptides are too long to be readily synthesized in one part, independently synthesized cross-linked peptides can be conjoined by a technique called native chemical ligation (Bang, et al., J. Am. Chein Soc. 126:1377). 15 WO 2008/061192 PCT/US2007/084838 The novel cross-linked polypeptides are useful, for example, to mimic or study proteins or polypeptides having one or more alpha-helical domains. Analysis conserved residues among MAML-l, 2 and 3; analysis of the predicted interaction between MAML and Notch; and analysis of predicted alpha 5 helical regions led to the identification amino acids that might be replaced to provide a cross-link without significantly inhibiting binding to Notch. Thus, as shown in FIG 2 for MAML-1, residues that might be cross-linked are doubled underlined. Substitutions can be made at discrete locations, namely the "i, and i+4 positions" or the "i, and i+7 positions" shown for each phase (1 to 6) which facilitate cross 10 linking chemistry by placing reactive residues on the same face of the a-helix. Highly conserved amino acids among MAML polypeptides and those thought be important in protein-protein interactions based on X-ray crystallographic, are preferably not replaced. In FIG. 2 residues where changes are expected to be tolerated are single underlined. In certain circumstances, conserved amino acids can 15 be replaced by other amino acids (e.g., synthetic non-naturally occurring amino acids). ct,a-Disubstituted non-natural amino acids containing olefinic side chains of varying length can synthesized by known methods (Williams et al. 1991 J. Aim. C/em. Soc. 113:9276; Schafmeister et al. 2000J. Am. Chem Soc. 122:5891). FIG. 20 2 is a schematic depiction of the preparation of the non-natural amino acid (Fmoc S5) used in solid phase peptide synthesis (SPPS) of i linked to i+4 peptides (one turn of the alpha helix is stabilized). For peptides where an i linked to i+7 staple is used (two turns of the helix stabilized) one S5 amino acid is used and one R8 is used. R8 is synthesized using the same route, except that the starting chiral auxiliary 25 confers the R-alkyl-stereoisomer. Also, 8-iodooctene is used in place of 5 iodopentene. Inhibitors are synthesized on a solid support using solid-phase peptide synthesis (SPPS) on MBHA resin. Non-natural amino acids were synthesized by Moellering for incorporation into the final peptide product. Various internally cross-linked peptides (REM-GI to REM-G13, also called 30 SAHN I to SAHN 13, respectively) shown in FIG. 3 were produced (X is a modified amino acid forming a cross-link). The underlined portions indicate the extent of 16 WO 2008/061192 PCT/US2007/084838 each polypeptide, and the remainder of the MAML-1 sequence in each case is provided for context. FIG. 4 is a schematic drawing and detailed structural depiction of a modified polypeptide having the sequence of REM-GI (SAHN 1). As seen in the circular 5 dichromism spectra of FIG. 5, the modified polypeptide can be 96% alpha helical as compared to 40% when not cross-linked. FIG. 6 shows the detailed structure of versions of the modified, internally cross-linked polypeptide REM-GIl (SAHN 11) that include either biotin or FITC labels. Surface plasmon resonance was used to demonstrated that the biotin-labeled 10 cross-linker version of REM-GI I (BioSAHNI 1) binds ICN in a dose dependent manner (FIG. 7). Biochemical association between stapled peptides and the Notch complex was also investigated using surface plasmon resonance. These studies employed immobilized ICN protein (an anti-GST antibody and a GST-tagged, purified ICN 15 protein comprising the RAM and ANK domains). Other studies employed biotinylated stapled peptides and a streptavidin-functionalized sensor surface. We demonstrated that immobilized ICN associates with CSL in a dose dependent manner. The association exhibited a two-phase kinetic association, first with RAM binding and subsequently with a lower-affinity association with the ANK 20 domain (see FIG. 8). Non-specific binding to a reference surface with anti-GST antibody was only minimal. Binding of immobilized stapled and unstapled SAHN 11 to ICN I demonstrated that stapled (cross-linked) SAHN1 I binds ICN with greater affinity (Kd = 0.96 pM) than non-stapled SAHN 11 (Kd = 2.63 pM) (FIG. 9). 25 Immunoprecipitation studies using MOLT4 cell lysates, ALL-SIL cell lysates and KOPTK1 cell lysates found that biotin labeled SAHNI can be used to pull down ICN. A reverse immunoprecipitation assay using FITC labeled SAHN I found that SAHN I can be used to pull down ICN in MOLT4 cell lysates. Automated quantitative immunofluorescence was used to determine the 30 intracellular distribution of fluorophore-labeled stapled alpha helices. Cells were incubated with FITC-conjugated peptides SAHN 1, SAHN2, or SAHN6; or control. At 16 hours measurements of cellular fluorescence were taken using epifluorescence 17 WO 2008/061192 PCT/US2007/084838 microscopy. The results of these studies are shown in FIG. 10 in which each circle represents an individual cell and each column represents a treatment condition. Both SAl-IN I and SAHN2 exhibited significant intracellular passage, whereas SAHN6 did not. 5 Automated quantitative immunofluorescence was also used to determine the intracellular distribution of fluorophorc-labeled stapled alpha helices. Cells were incubated with FITC-conjugated peptides SAHN 1, or unstapled SAHN 1. As shown in FIG. 11, stapling of the peptide did not, in this instance, appear to impact intracellular passage. 10 Epifluorescence microscopy demonstrated that SAN 11 exhibits intracellular distribution. Confocal microscopy analysis suggested that both stapled and unstapled SAHNI peptides appear to distribute to the intracellular compartment through endocytosis. MOLT4 cells transfected with a CSL-responsive reporter were used to test 15 whether stapled SAHN I 1 can interfere with Notch-mediated activation of transcription. As can be seen in FIG. 12, stapled SAHN I I reduced expression of the CSL-responsive reporter in a dose-dependent manner. HESI is Notch responsive gene. As shown in FIG. 13, T-ALLI cells exposed to either SAHNI l-FITC or SAHNI -FITC exhibit reduced HESI expression 20 relative to expression of a housekeeping gene (beta-actin). As shown in FIG. 14, stapled SAHN2 reduced the viability of MOLT4 cells. As shown in FIG. 15, stapled SAHN1, but not unstapled SAHN 1, reduced the viability of ALL-SIL cells. As shown in FIG. 16, stapled SAHNI, but not unstapled SAHN1, reduced the viability of KOPTKI cells. As shown in FIG. 17, stapled 25 SAHN I I reduced the viability of MOLT4 cells. As shown in FIG. 18, stapled SAHN I 1 reduced the viability of TALL] cells. Immobilized SAHN I was used to measure the apparent Kd for a pre assembled ICN-CSL complex by surface plasmon resonance. The result of this analysis revealed an apparent Kd of 98 nM. (I DON'T THINK THERE IS ANY 30 NEED TO SHOW SLIDE 8) A damaged variant of SAHN I was created by changing the Glu indicated by * in the SAHN1 depicted in FIG. 6 to an Arg and changing the Arg indicated by + 18 WO 2008/061192 PCT/US2007/084838 in the SAHN 1 depicted in FIG. 6 to Glu. This damaged variant, which has the same net charge as SANN1, is referred to as SAHNI-D. Immobilized SAHN I -D was used to measure the apparent Kd for a pre assembled ICN-CSL complex by surface plasmon resonance. The result of this 5 analysis revealed an apparent Kd of 1.40 gM. (I DON'T THINK THERE IS ANY NEED TO SHOW SLIDE 10) Immunoprecipitation studies using T-ALL (KOPTKI) cell lysates demonstrated that SAHN 1 and SAHN 11, but not SAHNI-D, can pull down both ICN and CSL. The results of the analysis are shown in FIG. 19. 10 A study using T-ALL cellular lysates found that SAHNI can compete away ICNI that is bound to immunoprecipitated MAML. The results of this analysis are shown in FIG 20. A study using T-ALL cellular lysates found that SAHNI -D cannot effectively compete away ICNI that is bound to immunoprecipitated MAML. The 15 results of this analysis are shown in FIG 21. T-ALL cells (MOLT4) harboring a beta-lactamase gene under the control of a CSL responsive promoter was used to study the effect of SAHN I and SAHN 11 on Notch complex mediated transcription. This study found that both SAHN I and SAHN II decreased transcription and that the decrease was similar in magnitude to 20 that caused by an RNAi directed against Notch and an RNAi directed against lactamase. The results of this analysis are shown in FIG. 22. A study in T-ALL cells (MOLT4) found that SAHN1, but not SAHNI-D, decrease expression of HES I and HEY 1, both Notch-driven genes, in a dose dependent manner. The results of this analysis are show in FIG. 23. 25 A study in gamma secretase resistant T-ALL cells (MOLT4) found that SAHN 1, but not SAHN1-D induces an apoptotic response after 24 or 48 hours. The results of this analysis are show in FIG. 24. A study in T-ALL cells (KOPTK1) found that SAHNI decreased cell viability (IC 5 o = 8 pM). SAHNI-D had little effect on cell viability. The results of 30 this analysis are show in FIG. 25. Additional studies found that SAHN1, but not SAHN1-1), is cytotoxic to neoplastic murine lymphocytes derived from transgenic mice harboring the 19 WO 2008/061192 PCT/US2007/084838 clinically relevant human Notch mutations (a L to P change at position 1601 and a PEST domain mutation). Polypeptides 5 In some instances, the hydrocarbon tethers (i.e., cross links) described herein can be further manipulated. In one instance, a double bond of a hydrocarbon alkenyl tether, (e.g., as synthesized using a ruthenium-catalyzed ring closing metathesis (RCM)) can be oxidized (e.g., via epoxidation or dihydroxylation) to provide one of compounds below. H H H 0 H 0 \N L[Xaa] 3 -N N [Xaa]3-N 10 0 HO OH Either the epoxide moiety or one of the free hydroxyl moieties can be further functionalized. For example, the epoxide can be treated with a nucleophile, which provides additional functionality that can be used, for example, to attach a tag (e.g., a radioisotope or fluorescent tag). The tag can be used to help direct the compound 15 to a desired location in the body or track the location of the compound in the body. Alternatively, an additional therapeutic agent can be chemically attached to the functionalized tether (e.g., an anti-cancer agent such as rapamycin, vinblastine, taxol, etc.). Such derivitization can alternatively be achieved by synthetic manipulation of the amino or carboxy terminus of the polypeptide or via the amino 20 acid side chain. Other agents can be attached to the functionalized tether, e.g., an agent that facilitates entry of the polypeptide into cells. While hydrocarbon tethers have been described, other tethers are also envisioned. For example, the tether can include one or more of an ether, thioether, ester, amine, or amide moiety. In some cases, a naturally occurring amino acid side 25 chain can be incorporated into the tether. For example, a tether can be coupled with a functional group such as the hydroxyl in serine, the thiol in cysteine, the primary amine in lysine, the acid in aspartate or glutamate, or the amide in asparagine or glutamine. Accordingly, it is possible to create a tether using naturally occurring amino acids rather than using a tether that is made by coupling two non-naturally 20 WO 2008/061192 PCT/US2007/084838 occurring amino acids. It is also possible to use a single non-naturally occurring amino acid together with a naturally occurring amino acid. It is further envisioned that the length of the tether can be varied. For instance, a shorter length of tether can be used where it is desirable to provide a 5 relatively high degree of constraint on the secondary alpha-helical structure, whereas, in some instances, it is desirable to provide less constraint on the secondary alpha-helical structure, and thus a longer tether may be desired. Additionally, while examples of tethers spanning from amino acids i to i+3, i to i+4; and i to i+7 have been described in order to provide a tether that is primarily 10 on a single face of the alpha helix, the tethers can be synthesized to span any combinations of numbers of amino acids. In some instances, alpha disubstituted amino acids are used in the polypeptide to improve the stability of the alpha helical secondary structure. However, alpha disubstituted amino acids are not required, and instances using 15 mono-alpha substituents (e.g., in the tethered amino acids) are also envisioned. As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the described herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry 20 transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. 25 Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. The peptides of this invention can be made by chemical synthesis methods, which arc well known to the ordinarily skilled artisan. See, for example, Fields et al., 30 Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence, peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the ce-NH 2 protected 21 WO 2008/061192 PCT/US2007/084838 by either t-Boc or F-moc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 43 1. One manner of making of the peptides described herein is using solid phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked 5 polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups. 10 Longer peptides could be made by conjoining individual synthetic peptides using native chemical ligation. Alternatively, the longer synthetic peptides can be synthesized by well known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse 15 translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed. Next, a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The 20 peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods. The peptides can be made in a high-throughput, combinatorial fashion, e.g., using a high-throughput multiple channel combinatorial synthesizer available from 25 Advanced Chcmtech. In the modified polypeptides one or more conventional peptide bonds replaced by an a different bond that may increase the stability of the polypeptide in the body. Peptide bonds can be replaced by: a retro-inverso bonds (C(O)-NH); a reduced aide bond (NH-CH 2 ); a thiomethylene bond (S-CI 2 or CH 2 -S); an oxornethylene bond (0-CH 2 or CHO 2 -); an ethylene bond (CH 2

-CH

2 ); a thioamide bond 30 (C(S)-NH); a trans-olefine bond (CH=CH); an fluoro substituted trans-olefine bond (CF=CH); a ketomethylene bond (C(O)-CHR) or CHR-C(O) wherein R is HI or CH4 3 ; and a fluoro-ketoinethylene bond (C(O)-CFR or CFR-C(O) wherein R is H or F or CH1 3 . 22 WO 2008/061192 PCT/US2007/084838 The polypeptides can be further modified by: acetylation, amidation, biotinylation, cinnamoylation, farnesylation, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfurylation. The polypeptides of the invention may also be conjugated to, for 5 example, polyethylene glycol (PEG); alkyl groups (e.g., CI-C20 straight or branched alkyl groups); fatty acid radicals; and combinations thereof. Methods of Treatment The present invention provides for both prophylactic and therapeutic 10 methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant (e.g., excessive) Notch activity. This is because the polypeptides are expected to act as dominant negative inhibitors of Notch activity. As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of 15 a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and 20 antisense oligonucleotides. The polypeptides of the invention can be used to treat, prevent, and/or diagnose cancers and neoplastic conditions. As used herein, the terms "cancer", "hyperproliferative" and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly 25 proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or 30 organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor 23 WO 2008/061192 PCT/US2007/084838 growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders. The compounds (i.e., 5 polypeptides) can act as novel therapeutic agents for controlling breast cancer, T cell cancers and B cell cancer. The polypeptides may also be useful for treating mucoepidermoid carcinoma and medulloblastoma. Examples of proliferative disorders include hematopoictic neoplastic disorders. As used herein, the term "hematopoictic neoplastic disorders" includes 10 diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Exemplary disorders include: acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promycloid leukemia (APML), acute myelogenous 15 leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), multiple mylenoma, hairy cell leukemia (HLL) and 20 Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease. 25 Examples of cellular proliferative and/or differentiative disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ 30 (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, 24 WO 2008/061192 PCT/US2007/084838 medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma. 5 Pharmaceutical Compositions and Routes of Administration As used herein, the compounds of this invention, including the compounds of formulae described herein, are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A "pharmaceutically acceptable derivative or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester, or other 10 derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention. Particularly favored derivatives and prodrugs arc those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be 15 more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group which enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. 20 The compounds of this invention may be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, 25 alter metabolism and alter rate of excretion. Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoatc, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, 30 glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, 25 WO 2008/061192 PCT/US2007/084838 succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl) 4 + salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or 5 oil-soluble or dispersible products may be obtained by such quaternization. The compounds of the formulae described herein can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or. by inhalation, with a 10 dosage ranging from about 0.001 to about 100 mg/kg of body weight, or according to the requirements of the particular drug. The methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about I to about 6 times per day or 15 alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such 20 preparations contain from about 20% to about 80% active compound. Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug 25 combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician. Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if 30 necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is 26 WO 2008/061192 PCT/US2007/084838 retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms. Pharmaceutical compositions of this invention comprise a compound of the formulae described herein or a pharmaceutically acceptable salt thereof; an 5 additional agent including for example, morphine or codeine; and any phannaceutically acceptable carrier, adjuvant or vehicle. Alternate compositions of this invention comprise a compound of the formulae described herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle. The compositions delineated herein include the compounds of 10 the formulae delineated herein, as well as additional therapeutic agents if present, in amounts effective for achieving a modulation of disease or disease symptoms. The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is 15 nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying 20 drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, 25 such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat. Cyclodextrins such as c-, p-, and 30 y-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein. 27 WO 2008/061192 PCT/US2007/084838 The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection. The pharmaceutical compositions of this invention may 5 contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, 10 intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using 15 suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butancdiol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic 20 sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in 25 their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or 30 bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. 28 WO 2008/061192 PCT/US2007/084838 The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose 5 and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening 10 and/or flavoring and/or coloring agents may be added. The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and 15 therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols. The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as 20 solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. When the compositions of this invention comprise a combination of a compound of the formulae described herein and one or more additional therapeutic 25 or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about I to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents may be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents may be 30 part of a single dosage form, mixed together with the compounds of this invention in a single composition. 29 WO 2008/061192 PCT/US2007/084838 Screeninp Assays The invention provides methods (also referred to herein as "screening assays") for identifying polypeptides which modulate the activity of one or more Notch complexes. 5 The binding affinity of polypeptides to Notch can measured using the methods described herein, for example, by using a titration binding assay. Notch complex lacking MAML (i.e., a complex of ICN and CSL) be exposed to varying concentrations of a candidate compound (i.e., polypeptide) (e.g., I nM, 10 nM, 100 nM, I pM, 10 pM, 100 iM, 1 mM, and 10 mM) and binding can be measured using 10 surface plasmon resonance to determine the Kd for binding. Candidate compounds could also be screened for biological activity in vivo, for example, by measuring expression of a Notch responsive reporter in a suitable cell, e.g., in MOLT-4 cells. Cell permeability screening assays in which fluorescently labeled candidate compounds are applied to intact cells, which arc then assayed for cellular 15 fluorescence by microscopy or high-throughput cellular fluorescence detection can also be used. The assays described herein can be performed with individual candidate compounds or can be performed with a plurality of candidate compounds. Where the assays are performed with a plurality of candidate compounds, the assays can be 20 performed using mixtures of candidate compounds or can be run in parallel reactions with each reaction having a single candidate compound. The test compounds or agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art. 25 Other applications A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. 30

Claims (52)

1. A modified polypcptide of Formula (1), 0 0 [Xaa]N [Xa [Xaay 5 Z Formula (I) Wherein: each R, and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; 10 each R 3 is independently alkyl, alkenyl, alkynyl; [R 4 -K-R 4 ],,; each of which is substituted with 0-6 R5; each R 4 is independently alkyl, alkenyl, or alkynyl; each R 5 is independently is halo, alkyl, OR 6 , N(R 6 ) 2 , SR 6 , SOR 6 , SO 2 R 6 , CO 2 R 6 , R 6 , a fluorescent moiety, or a radioisotope; 0 15 each K is independently 0, S, SO, SO 2 , CO, C0 2 , CONR 6 , or each R is independently H, alkyl, or a therapeutic agent; n is an integer from 1-4; x is 3, 4 or 6; y and w are independently integers from 0-100; 20 z is an integer from 1-10; and each Xaa is independently an amino acid; wherein the modified polypeptide comprises at least 8 contiguous amino acids of a MAML polypeptide except that: (a) within the 8 contiguous amino acids the side chains of at least one pair of amino acids separated by 3, 4 or 6 amino acids 25 is replaced by the linking group R 3 which connects the alpha carbons of the pair of amino acids as depicted in Formula I and (b) the alpha carbon of the first of the pair of amino acids is substituted with R, as depicted in formula I and the alpha carbon 31 WO 2008/061192 PCT/US2007/084838 of the second of the pair of amino acids is substituted with R 2 as depicted in Formula I and wherein and N or C can be substituted by PEG, spermine, or a carbohydrate.

2. The modified polypeptide of claim 1, wherein the MAML 5 polypeptide comprises any of SEQ ID NOs: 1 to 7.

3. The polypeptide of claim 1, wherein the modified polypeptide binds a complex comprising CSL and ICN. 10 4. The modified polypeptide of claim 1, wherein the MAML polypeptide ins selected from human MAML-I, MAML-2 and MAML-3.

5. The polypeptide of claim 1, wherein the polypeptide reduces the viability of at least one of MOLT4 cells, ALL-SIL T-ALL cells, KOPTKI T-ALL 15 cells and TALL I cells.

6. The modified polypeptide of claim 1, wherein x is 3.

7. The modified polypeptide of claim 1, wherein x is 4. 20

8. The modified polypeptide of claim 1, wherein x is 6.

9. The modified polypeptide of claim 1, wherein x is 2 or 6; R 3 is an alkenyl containing a single double bond, and both R, and R 2 are H. 25

10. The modified polypeptide of claim 1, wherein each y is independently an integer between 3 and 15.

11. The modified polypeptide of claim 1, wherein R, and R 2 are each 30 independently H or CI-C 6 alkyl. 32 WO 2008/061192 PCT/US2007/084838

12. The modified polypeptide of claim 1, wherein R, and R 2 are each independently C-C 3 alkyl.

13. The modified polypeptide of claim 11, wherein at least one of R, and 5 R 2 are methyl.

14. The modified polypeptide of claim 12, wherein Ri and R 2 are methyl.

15. The modified polypeptide of claim 1, wherein R3 is alkyl. 10

16. The modified polypeptide of claim 14, wherein x is 3.

17. The modified polypeptide of claim 15, wherein R 3 is Cs alkyl. 15 18. The modified polypeptide of claim 14, wherein x is 6.

19. The modified polypeptide of claim 17, wherein R 3 is C, 1 alkyl.

20. The modified polypeptide of claim 1, wherein R 3 is alkenyl. 20

21. The modified polypeptide of claim 18, wherein x is 3.

22. The modified polypeptide of claim 20, wherein R 3 is C 8 alkenyl. 25 23. The modified polypeptide of claim 19, wherein x is 6.

24. The modified polypeptide of claim 19, wherein R 3 is C 1 alkenyl.

25. The modified polypeptide of claim 1, wherein R 3 is a straight chain 30 alkyl, alkenyl, or alkynyl. 33 WO 2008/061192 PCT/US2007/084838

26. The modified polypeptide of claim 1, wherein R 3 is [R 4 -K-R 4 ]; and R4 is a straight chain alkyl, alkenyl, or alkynyl.

27. The modified polypeptide of claim 1, wherein the polypeptide 5 comprises at least 8 contiguous amino acids of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 except that within the 8 contiguous amino the side chains of at least one pair of amino acids separated by 3 or 6 amino acids is replaced the linking group R 3 as depicted in Formula I which connects the alpha carbons of the pair of amino acids and the alpha carbon of the first of the pair of amino acids is substituted with R I as 10 depicted in formula I and the alpha carbon of the second of the pair of amino acids is substituted with R 2 as depicted in Formula 1.

28. The modified polypeptide of claim I wherein [Xaa]. comprises an amino acid sequence selected from: AVMERL, RLRRRI, LCR, LCRRHH, TCE, 15 RYE, TCEARY CEARYE, LERQHT, HTF, and HTFALH.

29. The modified polypeptide of claim 15, wherein at least one of R, or R 2 is alkyl. 20 30. The modified polypeptide of claim I1, wherein each R, and R 2 is independently H or Cr-C 3 alkyl.

31. The modified polypeptide of claim 1, wherein R I and R 2 are methyl. 25 32. The modified polypeptide of claim 1, wherein x is 3 or 6 and z is 1.

33. The modified polypeptide of claim 1, wherein R 3 is Cs or C 1 alkyl or alkenyl. 30 37. The modified polypeptide of claim 1, further comprising a copolymer of lactic and glycolic acid 34 WO 2008/061192 PCT/US2007/084838

38. The modified polypeptide of claim 1, further comprising a PEG.

39. The modified polypeptide of claim 1, further comprising an targeting moiety. 5 40. The modified polypeptide of claim 1, further comprising a biotin moiety.

41. A method of making a polypeptide of Formula (111), comprising providing a polypeptide of Fonnula (11); and H 0 H O [Xaa] N [Xaa],-N [Xaa y R1, (),R2 10 z Formula (II) treating the compound of Formula (II) with a catalyst to promote a ring closing metathesis, thereby providing a compound of Formula (III) H O H O N L[Xaa],-N I [[Xaa ] Xaa,- [Xaa~y R 1 ( ), ( 2 - Z 15 Formula (III) wherein each R, and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl; heteroarylalkyl; or heterocyclylalkyl; each n is independently an integer from 1-15; 20 xis3,4,or6 each y is independently an integer from 0-100; z is an integer from 1-3; and each Xaa is independently an amino acid; and wherein the polypeptide comprises an alpha helical structure in aqueous 25 solution. 35 WO 2008/061192 PCT/US2007/084838

42. The method of claim 41, wherein the polypeptide binds to a complex comprising ICN and CSL. 5 43. The method of claim 41, wherein the catalyst is a ruthenium catalyst.

44. The method of claim 41, further comprising providing a reducing agent or oxidizing agent subsequent to the ring closing metathesis. 10 45. The method of claim 44, wherein the reducing agent is H 2 or oxidizing agent is osmium tetroxide

46. A method of treating a subject comprising administering to the subject a compound of claim 1. 15

47. The method of claim 46, further comprising administering an additional therapeutic agent.

48. A method of treating cancer in a subject comprising administering to 20 the subject a compound of claim 1.

49. The method of claim 48, further comprising administering an additional therapeutic agent. 25 50. A library of compounds of claim 1, formula (I).

51. A compound having the formula 30 36 WO 2008/061192 PCT/US2007/084838 R1 0 R 3 O R 5 O R7 0 R 9 0 R11 O Rz, I N Y)N I N Y)'N , N )UN rN y ,N N-- N N,,NTR 0 R R4 0 0 R8 R 1 i R12 5 Ri is -CH 2 CH 2 COOH [E] or -CH 2 COOH [D] or R, and R 8 together are R.; R 2 is -CH 2 CH(CH 3 )2 [L] or -CH 2 CH 2 CH 2 N(H)C(NH)NH 2 [R]; 10 R 3 is -CH 2 CH 2 COOH [E] R4 is -CHCH 2 CH 2 N(H)C(NH)NH 2 [R]; RS is -CH 2 CH 2 C(O)NH 2 [Q], -CH 2 CH 2 COOH [E], or 15 CH 2 CH 2 CH 2 N(H)C(NH)NH 2 [R); or R 5 and R 9 together are Ry; or R5 and R 12 together are Rx; CH 2 R 6 is -CH 2 OH [S], NH [H] or -CH 2 COOH [D]; 20 R 7 is -C(OH)CH 3 [T]; Rs is benzyl [F], -CH 2 CH(CH 3 ) 2 [L], -CH(CH 3 ) 2 [V] or R and Rg together are R,; 25 R 9 is selected from: -CH 3 [A] or -CH 2 CH 2 C(O)NH 2 [Q] and -CH 2 OH [S]; or RS and R, together are Ry; Rio is -CH 2 CH(CH 3 ) 2 [L]; 37 WO 2008/061192 PCT/US2007/084838 ^^AP vyvP CH 2 NH OH R 1 1 is [H], -CH 2 CH(CH 3 ) 2 [L] and R 12 is -CH 2 CH 2 C(O)NH 2 [Q] and -CH 2 OH [S]; or R 5 and R12 together are R., 5 provided that when R 1 and Rg together are Rx, R 5 and R 9 are not together Ry and R 5 and R 12 arc not together R,; further provided that when R 5 and R 9 together are Ry, R, and Rg are not together R, and R 5 and R 12 are not together R,; further provided that when R 5 and R 1 2 are together R,, R 5 and R, are not 10 together Ry and RI and R 8 are not together R,; R. and Ry alkyl, alkenyl, alkynyl; [Ri-K-Rai],,; each of which is substituted with 0-6 R, 2 ; Ri is alkyl, alkenyl, or alkynyl; R.2 is halo, alkyl, ORO 3 , N(R, 3 ) 2 , SR. 3 , SOR. 3 , SO 2 R. 3 , CO 2 Rx 3 , RO 3 , a 15 fluorescent moiety, or a radioisotope; 0 K is 0, S, SO, SO 2 , CO, CO 2 , CONRO 3 , or RO 3 is H, alkyl or a therapeutic agent; and Rz and R, are independently: H, hydroxyl, an amino acid, 2 to 10 amino acids linked by peptide bonds; tat; and PEG. 20

52. The polypeptide of claim 51, wherein each y is independently an integer between 3 and 15.

53. The polypeptide of claim 51, wherein R, and R 2 are each 25 independently H or C-C 6 alkyl. 54 The polypeptide of claim 51, wherein R, and R 2 are each independently C-C 3 alkyl. 38 WO 2008/061192 PCT/US2007/084838

55. The polypeptide of claim 51, wherein at least one of R, and R 2 are methyl. 5 56. The polypeptide of claim 51, wherein R, and R 2 are methyl.

57. The polypeptide of claim 51, wherein R 3 is alkyl.

58. The polypeptide of claim 51, wherein x is 3. 10

59. The polypeptide of claim 51, wherein R 3 is C 8 alkyl.

60. The polypeptide of claim 51, wherein x is 6. 15 61. The polypeptide of claim 51, wherein R 3 is C 1 , alkyl.

62. The polypeptide of claim 51, wherein R 3 is alkenyl.

63. The polypeptide of claim 51, wherein R 3 is C 8 alkenyl. 20

64. The polypeptide of claim 51, wherein R 3 is C1, alkenyl.

65. The polypeptide of claim 51, wherein R 3 is a straight chain alkyl, alkenyl, or alkynyl. 25

66. The polypeptide of claim 51, wherein R 3 is [R 4 -K-R 4 ]; and R4 is a straight chain alkyl, alkenyl, or alkynyl. 66. The polypeptide of claim 51, wherein x is 3 and z is 1. 30

67. The polypeptide of claim 51, wherein the polypeptide is transported through the cell membrane. 39 WO 2008/061192 PCT/US2007/084838

68. The polypeptide of claim I wherein polypeptide comprises an amino acid sequence selected from SEQ ID NOs:8, 9 and 10 wherein: (a) the side chains of amino acids 8 and 12 are replaced by the linking group R 3 which connects the alpha 5 carbons of amino acids 8 and 12 as depicted in formula 1; and (b) the alpha carbon of amino acid 8 is substituted with Ri as depicted in formula I and the alpha carbon of amino acid 12 is substituted with R 2 as depicted in formula 1. 40