WO2007128080A1 - An assay for modulators of apoptosis - Google Patents

Abstract

The present invention relates generally to an assay for molecules which modulate apoptosis, to molecules identified by the assay and to use of the molecules. More particularly, an assay is provided for screening for molecules that interact with and which antagonize or agonize the anti-apoptotic properties of a pro-survival molecule. Modulators of apoptosis identified by the subject assay and compositions comprising same and their use are also contemplated herein.

Description

An assay for modulators of apoptosis

FIELD

The present invention relates generally to an assay for molecules which modulate apoptosis, to molecules identified by the assay and to use of the molecules. More particularly, an assay is provided for screening for molecules that interact with and which antagonize or agonize the anti-apoptotic properties of a pro-survival molecule. Modulators of apoptosis identified by the subject assay and compositions comprising same and their use are also contemplated herein.

BACKGROUND

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Programmed cell death or apoptosis in higher organisms is controlled by a group of molecules involved in a series of overlapping, complex biochemical and physiological pathways. Within this group of molecules are pro-apoptotic molecules, which promote apoptosis and pro-survival molecules which help prevent apoptosis (Cory and Adams, Nature Review Cancer 2:647-656, 2002; Adams, Genes and Development 77:2481-2495, 2003; Daniel and Korsmeyer, Cell 116:205-219, 2004).

Apoptosis is an integral part of normal cell turnover but the ability to control apoptosis has potential efficacy in treating certain degenerative diseases. Equally, the ability to induce apoptosis provides an opportunity to promote apoptosis of cancer cells (Cory et al, Oncogene 22:8590, 2003).

One particular family of proteins involved in regulating and facilitation of apoptosis is the Bcl-2 family. This family has pro-apoptotic members such as Bak, Bim, Bad, Bid, Puma and Noxa and pro-survival members such as McI-I₅ BCI-X_L, Bcl-w and Al. Apoptosis is controlled at one level by the interaction between the pro-apoptotic and pro-survival Bcl-2 family proteins. All members of the Bcl-2 family of proteins contain a number of Bcl-2 homology (BH) domains. These domains are involved in interactions between the family members. In particular, the BH3 domain of pro-apoptotic proteins interact with a groove composed of the BHl, BH2 and BH3 domains of pro-survival (i.e. anti-apoptotic) proteins (Sattler et al, Science 275:983, 1997; Petros et al, Protein Science 9:2528, 2000; Liu et al, Immunity 19:341, 2003). Hence, this interaction is an important target for chemotherapeutic agents to promote cancer cell death (Oltersdorf et al, Nature 435:677, 2005).

Pro-survival Bcl-2 family protein, McI-I, or myeloid cell leukemia sequence-1, has been the focus of interest within the research community (Holleman et al, Blood, 27 September 2005; Mongini et al, J. Immunol l75(9):6H3-6l54, 2005; Balakrislinan et al, Clin Cancer Res 11(18):6745-6752, 2005). A murine form of McI-I has been expressed as a GST fusion using pGEX-6P3 (Amersham Biosciences, USA) [Day et al, J. Biol Chem 280:4738, 2005]. This fusion molecule contains an N-terminal truncation of 151 residues to remove a non-Bcl-2-like region of the molecule and a C-terminal truncation of 23 residues to remove the transmembrane anchor. This is referred to as a "mMcl-l^BLR-GST fusion". Such a construct leaves the Bcl-2-like region of murine McI-I in a form that can be expressed as a soluble protein.

Typically, the mMcl-l^BLR-GST fusion protein is expressed in E. coli BL21 (DE3) [Phillipps et al, J. Bacteriol 159:283, 1984; Studier and Moffatt, J. MoI Biol 189:113, 1986; available from Invitron Corporation, USA]. Protein is extracted from the bacteria and bound to giutathione-sepharose beads. The beads are washed with buffer to remove any proteins non-specifically bound to the beads. PreScission protease (Amersham Biosciences, USA) is then typically used to cleave the mMcl-l^BLR from the GST allowing it to be eluted from the beads.

Previous attempts to express the human form of McI-I have not been successful. There is a need, therefore, to develop an expression system for human McI-I so that the Bcl-2-like region and in particular the BH3 binding groove can be used as a target to identify antagonists and agonists of interaction between human McI-I and pro-apoptotic molecules.

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SUMMARY

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

All scientific citations, patents, patent applications and manufacturer's technical specifications referred to hereinafter are incorporated herein by reference in their entirety.

An assay for modulators of apoptosis is provided. In particular, a chimeric molecule is generated comprising the Bcl-2-like region (BLR) of human McI-I fused at its N-terminal end to a portion of murine McI-I. This molecule is readily expressed in prokaryotic or eukaryotic cells as a fusion with a carrier molecule such as but not limited to glutathione- S- transferase (GST) fusion molecule.

One particular human chimeric molecule is referred herein as "m/h McI- 1^BLR " and comprises amino acids 152 to 189 from murine McI-I and amino acids 209 to 327 from human McI-I . When fused to GST, the construct is referred to as "m/h Mcl-1^BLR-GST". This designation should not be construed as limiting the location of the GST portion of the fusion molecule. The GST portion may be at the N-terminal or C-terminal end portion of the fusion molecule. Having the GST at the N-terminal portion has some advantage in ensuring that it is fully translated prior to the remainder of the fusion molecule. The chimeric molecule is a target for screening for molecules which bind to the BH3 binding groove of human McI-I which may then inhibit its anti-apoptotic properties or may promote its pro-survival properties.

In one embodiment, the chimeric molecule is immobilized to a solid support and contacted with potential BH3 domain ligands. The molecules identified either promote apoptosis by inhibiting the pro-survival properties of McI-I or inhibit apoptosis by acting as agonists of

McI-I interaction with the pro-apoptotic molecules. The assay may be conducted with the McI-I immobilized to beads or free in solution.

The identification of pro-apoptotic molecules enables the development of anti-cancer agents whereas anti-apoptotic molecules are useful in treating degenerative and necrotic disease conditions.

Accordingly, a chimeric molecule is provided comprising the BLR from a human pro- survival protein of the Bcl-2 family of proteins and a portion of a non-human homolog thereof fused to the N-terminal region of the human protein wherein said chimeric molecule is produced in a cell in which a nucleic acid molecule encoding said chimeric molecule is expressed.

Generally, the chimeric molecule further comprises a carrier protein which is removable through use of a protease.

Hence, another aspect is directed to a fusion protein comprising a C-terminal portion comprising the Bcl-2-like region (BLR) from a human pro-survival Bcl-2 protein and an N-terminal portion comprising a non-human homolog of the human pro-survival protein fused at its C-terminal end to a portion to the human pro-survival Bcl-2 protein said fusion protein further comprising a carrier protein fused to the N-terminal portion of the non- human homolog or the C-terminal protion of the human pro-survival Bcl-2 protein.

The human pro-survival Bcl-2 protein is, in one embodiment, McI-I and the non-human homolog is murine McI-I or another non-human form of McI-I.

Hence, a chimeric molecule is provided comprising a Bcl-2-like region (BLR) from human pro-survival protein McI-I of the Bcl-2 family of proteins and a portion of a non-human homolog thereof fused to the N-terminal region of McI-I wherein said chimeric molecule is capable of being produced in a cell in which a nucleic acid molecule encoding said chimeric molecule is expressed. More particularly, a chimeric molecule is provided comprising the BLR of human McI-I or its functional equivalent fused at its N-terminal end region to a portion of murine McI-I or its functional equivalent wherein the chimeric molecule is produced in a cell in which a nucleic acid molecule encoding said chimeric molecule is expressed.

In another aspect, a fusion protein is provided comprising a C-terminal portion comprising a Bcl-2 homology region of human McI-I and an N-terminal portion comprising murine McI-I or a portion thereof, said fusion protein further comprising a carrier protein fused to the N-terminal portion of murine McI-I or the C-terminal portion of human McI-I .

In a particular embodiment, the carrier protein is GST or a portion thereof.

A particular embodiment, therefore, is directed to a fusion protein useful for screening for modulators of apoptosis of cells, said fusion protein comprising an N-terminal region comprising GST fused to amino acids 152 to 189 of murine McI-I which is fused at its C- terminal end to amino acids of 209 to 327 of human McI-I .

Conveniently, the GST end portion or other carrier is useful for immobilizing to a solid support. The C-terminal region of the fusion protein comprises the human-derived BH3 binding groove comprised of the BHl, BH2, and BH3 domains to which the BH3 domain of pro-apoptotic proteins interact. In addition, a protease such as PreScission protease (GE Biosciences) or HRV-3C protease is used to cleave the fusion protein away from GST. The fusion molecule may then be used free in solution.

The fusion protein is useful inter alia for screening for molecules which interact with the human BLR of McI-I. Such molecules may interrupt pro-apoptotic molecules interacting with McI-I and hence assist in preventing inhibition of the pro-survival properties of McI-I by a pro-apoptotic molecule or the molecule may promote pro-apoptotic protein interaction and hence promote apoptosis.

Nucleic acid molecules encoding the fused proteins, screening protocols and molecules identified by the screening protocol including compositions comprising same all form aspects of the present invention.

Whilst the present disclosure is particularly exemplified in relation to McI-I, other pro- survival molecules such as BCI-X_L, Bcl-w and Al may also be employed.

A summary of sequence identifiers used throughout the subject specification is provided in Table 1. Abbreviations are defined in Table 2.

TABLE 1 Summary of sequence identifiers

TABLE 2

Abbreviations

BRIEF DESCRIPTION OF THE FIGURES

Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

Figure 1 is a representation of alignment of the BLR of mouse and human McI-I from residue 171 to 327 (hMcl-1 numbering). The mouse/human McI-I chimera is mouse McI- 1 from residue 171 to 208 (152 to 189 using niMcl-1 numbering) and then human McI-I from residue 209 to 327. The molecule is thus truncated at the N-terminus by 151 residues (mMcl-1 numbering) to retain the Bcl-2 like region of the molecule and at the C-terminus to remove the hydrophobic tail. This is cloned into the expression vector pGEX-6P3 (GE Biosciences) such that it can be expressed as a GST fusion protein which can be cleaved with PreScission Protease or HRV-3C protease. Residues highlighted in cyan represent those that differ between the chimera and hMcl-1, residues in orange are those that differ between the chimera and mMcl-1. It should be noted that the expressed protein contains GPLGS at N-terminus from protease site.

Figure 2 is a diagrammatical representation showing the structure of mMcl-1 detailing residues that are different in the mhMcl-1 chimera to hMcl-1. Residues different in the chimera to that of hsMcl-1 (i.e. mMcl-1 residues) are displayed in cyan. The BH3 domain is colored red, the BHl domain is colored green, the BH2 domain is colored yellow and the α3 - α4 loop is colored purple. The left image is a 180° rotation from that of the right.

Figure 3 is a tabulation representation showing binding characteristics of mMcl-1 ^BLR ( (mmoouussee)) aanndd mm//hhMMccll--11 ^BBLLRR ((cchh:imera) for a variety of BH3 only peptides as determined by isothermal titration calorimetry.

Figure 4 is a tabulation representation showing Binding differences for mMcl-1 ^BLR (mMcl-1) and m/h Mcl-1^BLR (m/hMcl-1) for mutant vs wildtype hBim peptides in a phage display assay. Figure 5 is a diagrammatical representation showing the crystal structure of the mhMcl- l^BLR:hBimBH3 complex. The mhMcl-1 chimera is shown displaying the surface of the molecule and the hBimBH3 as a cartoon in blue. For McI-I the BH3 domain is colored red, the BHl domain is colored green, the BH2 domain is colored yellow and the α3 - α4 loop is colored purple. The structure was obtained as follows: Seleno-methionine labelled mhMcl-l^BLR was complexed with hBim BH3 peptide and concentrated to 12 mg/ml. Crystals of the complex were grown in hanging drops at 22 °C with a reservoir solution consisting of 0.2 M ZnCl₂, 0.2 M imidazole pH 5.75 and 2 mM TCEP. Prior to flash freezing in liquid nitrogen crystals were equilibrated into cryoprotectant consisting of reservoir solution and increasing concentrations of trehalose to a final trehalose concentration of 30% (v/v). A MAD data set was collected to 1.56 A at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratories, USA. Data were integrated and scaled with HKL2000. Heavy atom sites were found using HKL2MAP. An initial model was built from the resulting map using Coot. Several rounds of building in Coot and refinement in Refmac5 were then used to produce the final model. Colored versions of this photograph are available from the patentees upon request.

Figures 6A through 6AC is a representation of the atomic structure co-ordinates of the nihMcl-l^BLR:LBIM BH3 complex shown in Figure 5.

,1

Figure 7 is a tabulated representation showing a library of compounds which are ranked by ability to inhibit interaction between McI-I and a peptide corresponding to the BH3 domain of Bim. DETAILED DESCRIPTION

Unless otherwise indicated, aspects of the subject invention are not limited to specific assay components, manufacturing methods, expression regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an agent" includes a single agent, as well as two or more agents; reference to "the invention" includes a single aspect or multiple aspects of the invention; and so forth.

A fusion (i.e. chimeric) molecule is provided which is useful for screening for agents which interact, associate or otherwise bind to the Bcl-2 like region (BLR) of a human anti- apoptotic (i.e. pro-survival) molecule of the Bcl-2 protein family such as but not limited to McI-I. Until generation of the fusion molecule described herein it was not possible to express human forms of, for example, McI-I. This inability has been overcome by the generation of a chimeric molecule comprising the BLR region for the human anti-apoptotic molecule fused at its N-terminal end to a portion of non-human homolog of the same or functionally equivalent molecule. Conveniently, although not necessary, the chimeric molecule further comprises a carrier molecule fused to the N-terminal region of the non- human homolog or the C-terminal region of the human anti-apoptotic molecule. One example of a carrier molecule is glutathione- S -transferase (GST).

Hence, one aspect provides a chimeric molecule comprising a BLR from a human pro- survival protein of the Bcl-2 family of proteins and a portion of a non-human homolog thereof fused to the N-terminal region of the human protein wherein said chimeric molecule is produced in a cell in which a nucleic acid molecule encoding said chimeric molecule is expressed. More particularly, a fusion protein is provided comprising a C-terminal portion comprising a Bcl-2-like region (BLR) from a human pro-survival Bcl-2 protein and an N-terminal portion comprising a non-human homolog of the human pro-survival protein fused at its C-terminal end to the human pro-survival Bcl-2 protein said fusion protein further comprising a carrier protein fused to the N-terminal portion of the non-human homolog or the C-terminal portion of the human pro-survival Bcl-2 protein.

In one embodiment, the pro-survival protein is McI-I or a functional equivalent thereof and the non-human homolog is murine McI-I or a functional equivalent thereof.

Hence, a chimeric molecule is provided comprising a Bcl-2-like region (BLR) from human pro-survival protein McI-I of the Bcl-2 family of proteins and a portion of a non-human homolog thereof fused to the N-terminal region of McI-I wherein said chimeric molecule is capable of being produced in a cell in which a nucleic acid molecule encoding said chimeric molecule is expressed.

More particularly a chimeric molecule is provided comprising the BLR of human McI-I or its functional equivalent fused at its N-terminal end region to a portion of murine McI-I or its functional equivalent wherein the chimeric molecule is produced in a cell in which a nucleic acid molecule encoding said chimeric molecule is expressed.

The present invention is further directed to a fusion protein useful for screening for agents which modulate apoptosis of cells, said fusion protein comprising a C-terminal portion comprising a Bcl-2 homology region of human McI-I and an N-terminal portion comprising a carrier protein fused at its C-terminal end to a portion of murine McI-I .

In one embodiment, the portion of human McI-I lacks the N-terminal non-Bcl-2-like region and the C-terminal transmembrane anchor and is referred to herein as hMcl-l^BLR. The portion of its murine homolog comprises amino acids 152 to 189 (38 amino acid residues). The murine molecule portion is derived from a molecule, referred to herein as mMcl-l^BLR which lack the N-terminal non-Bcl-2-like region (amino acids 1 to 151) and the C-terminal transmembrane anchor (C-terminal 23 amino acids). Due to homology between the murine and human forms, there are only 9 amino acids which are of murine origin in the murine portion.

Hence, another aspect is directed to a chimeric molecule comprising the BLR of human McI-I comprising amino acid residues 209 through 327 inclusive fused at its N-terminal end to amino acid residues 152 through 189 inclusive of murine McI-I. This chimeric molecule is referred to herein as m/h McI- 1^BLR.

In a particular embodiment, the chimeric molecule further comprises a carrier protein fused to the N-terminal portion of the non-human portion or the C-terminal portion of the human protein. Conveniently, but not exclusively, the carrier portion is GST or a part or portion thereof. In addition, whilst GST or other carrier protein may be fused to the N- or C- terminal end portions of the chimeric molecule, one particular aspect includes the GST being fused to the N-terminal portion of the non-human homolog.

Hence, a fusion protein is provided useful for screening for modulators of apoptosis of cells, said fusion protein comprising an N-terminal region comprising GST fused to amino acids 152 to 189 of murine McI-I fused at its C-terminal end to amino acids of 209 to 327 of human McI-I.

Another aspect is directed to a chimeric protein of Formula I, represented in the order of N-terminal to C-terminal:

[X₁] n- [X₂] - [X₃] -[X₄J_1n (I)

wherein each OfX₁ and X₄ is a carrier protein or a part or portion thereof; n and m are 0 or 1 except n and m cannot both be 1 ; — is a peptide (amide) bond; X₂ is derived from an N-terminal truncated non-human pro-survival Bcl-2 family protein; and X₃ is derived from the human homolog of the non-human pro-survival protein.

In one embodiment, X₃ is or is derived from hMcl-l^BLR and X₂ is derived from mMcl-l^BLR.

Other examples of X₂/X₃ include BCI-X_L, Bcl-w and Al .

In another embodiment, the chimeric molecule comprises a GST or part thereof as X₁AX₄.

A further aspect provides a chimeric molecule comprising the amino acid sequence set forth in SEQ ID NO:4 and SEQ ID NO: 5 or an amino acid sequence having at least about 80% similarity to SEQ ID NO:4 or SEQ ID NO: 5 wherein the C-terminal region of said molecule comprises the BLR of human McI- 1.

Reference to "at least 80%" includes at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100%. The comparison may also be at the level of identity.

SEQ ID NO:4 is the amino acid sequence of the murine McI-I portion of the chimeric molecule and SEQ ID NO: 5 is the human McI-I portion of the chimeric molecule.

SEQ ID NOs:6 and 7 represent the amino acid sequences of mMcl-l^BLR and hMcl-l^BLR, respectively from residues 171 to 327.

Optionally, the chimeric molecule further comprises a GST portion having the amino acid sequence set forth in SEQ ID NO:3.

In an embodiment, therefore, the chimeric molecule comprises the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least 80% similarity thereto.

The chimeric molecule described herein may be generated by synthetic means such as the stepwise addition of one or more amino acids at a time. Alternatively, the molecule is made by recombinant means. Reference, therefore, to a "recombinant cell" includes a cell which comprises a nucleic acid molecule introduced to the cell or a precursor or relative of the cell wherein the nucleic acid molecule is expressible to generate the chimeric molecule. In one embodiment, the nucleic acid molecule is as set forth in SEQ ID NO:1 or is a nucleic acid molecule having at least 80% identity thereto or a nucleic acid molecule which hybridizes to SEQ ID NO:1 or a complementary form under low stringency conditions.

Hence, another aspect provides an isolated nucleic acid molecule encoding a chimeric molecule comprising a BLR from a human pro-apoptotic protein of the Bcl-2 family of proteins and a portion of a non-human homolog thereof fused to the N-terminal region of the human protein wherein said nucleic acid molecule is expressible in a cell to generate the chimeric molecule.

In a particular embodiment, the pro-survival protein is McI-I or a functional equivalent thereof and the non-human homolog is murine McI-I or a functional equivalent thereof.

In a particular embodiment, the nucleic acid molecule comprises a sequence of nucleotides encoding the BLR of human McI-I or its functional equivalent fused at its N-terminal end region to a portion of murine McI-I or its functional equivalent wherein the nucleic acid molecule is expressible in a recombinant cell to generate a chimeric molecule comprising portion of human McI-I and murine McI-I.

In another particular embodiment, the nucleic acid molecule encodes a chimeric molecule comprising the BLR of human McI-I comprising amino acid residues 209 through 327 inclusive fused at its N-terminal end to amino acid residues 152 through 189 inclusive of murine McM . This chimeric molecule is referred to herein as m/h McI- 1^BLR.

Hence, an isolated nucleic acid molecule is provided encoding a chimeric molecule comprising the BLR of human McI-I comprising amino acid residues 209 through 327 inclusive fused at its N-terminal end to amino acid residues 152 through 189 inclusive of murine McI-I, said nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO:1 or a nucleotide sequence having at least 80% identity to SEQ ID NO:1 after optimal alignment or a nucleic acid molecule capable of hybridizing to SEQ ID NO:1 or a complementary form thereof under low stringency conditions.

As indicated above, reference to "at least 80%" includes 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

The present invention extends to nucleic acid molecules, constructs comprising same and cells and viruses comprising the nucleic acid molecules or constructs. Such cells and viruses are referred to herein as a "recombinant cell" or a "recombinant virus".

The subject nucleic acid sequence or a part of a nucleic acid sequence may be introduced into a cell in a vector such that the nucleic acid sequence remains extrachromosomal. In such a situation, the nucleic acid sequence will be expressed by the cell from the extrachromosomal location. Vectors for introduction of nucleic acid sequence both for recombination and for extrachromosomal maintenance are known in the art and any suitable vector may be used. Methods for introducing nucleic acids into cells such as electroporation, calcium phosphate co-precipitation and viral transduction are known in the art.

In particular, a number of viruses have been used as nucleic acid vectors including papovaviruses (e.g. SV40, Madzak et al, J Gen Virol 75:1533-1536, 1992), adenovirus (Berkner, Curr Top Microbiol Immunol 158:39-66, 1992; Berkner et al, BioTechniques 6:616-629, 1988; Gorziglia and Kapikian, J Virol 55:4407-4412, 1992; Quantin et al, Proc Natl Acad Sci USA SP:2581-2584, 1992; Rosenfeld et al, Cell 65:143-155, 1992; Wilkinson et al, Nucleic Acids Res 20:233-2239, 1992; Stratford-Perricaudet et al, Hum Gene Ther 7:241-256, 1990; Schneider et al, Nat Genetics 75:180-183, 1998), vaccinia virus (Moss, Curr Top Microbiol Immunol 755:5-38, 1992; Moss, Proc Natl Acad Sci USA 93:11341-11348, 1996), adeno-associated virus (Muzyczka, Curr Top Microbiol Immunol 158:97-129, 1992; Ohi et al, Gene 89:279-282, 1990; Russell and Hirata, Nat Genetics 18:323-328, 1998), herpesviruses including HSV and EBV (Margolskee, Curr Top Microbiol Immunol 158:67-95, 1992; Johnson et al, J Virol 66:2952-2965, 1992; Fink et al, Hum Gene Ther 3:1-19, 1992; Breakefield and Geller, MoI Neurobiol 7:339-371, 1987; Freese et al, Biochem Pharmaco. 40:2189-2199, 1990; Fink et al, Ann Rev Neurosci 19:265-287, 1996), Antiviruses (Naldini et al, Science 272:263-267, 1996), Sindbis and Semliki Forest virus (Berglund et al, Biotechnology 11:916-920, 1993) and retroviruses of avian (Bandyopadhyay and Temin, MoI Cell Biol 4:749-754, 1984; Petropoulos et al, J Virol 5(5:3391-3397, 1992), murine (Miller, Curr Top Microbiol Immunol 158:1-24, 1992; Miller et al, MoI Cell Biol 5:431-437, 1985; Sorge et al, MoI Cell Biol 4:1730-1737, 1984; Mann and Baltimore, J Virol 54:401-407, 1985; Miller et al, J Virol 52:4337-4345, 1988) and human (Shimada et al, J Clin Invest 88: 1043- 1047, 1991; Helseth et al, J Virol 54:2416-2420, 1990; Page et al, J Virol 64:5270-5276, 1990; Buchschacher and Panganiban, J Virol 66:2731-2739, 1982) origin.

Non-viral nucleic acid transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor-mediated DNA transfer. Viral-mediated nucleic acid transfer can be combined with direct in vivo nucleic acid transfer using liposome delivery, allowing one to direct the viral vectors to particular cells. Alternatively, the retroviral vector producer cell line can be injected into particular tissue. Injection of producer cells would then provide a continuous source of vector particles.

In relation to nucleic acid molecules, the terms mutant, section, derivative, homolog, analog or mimetic have analogous meanings to the meanings ascribed to these forms in relation to proteinaceous molecules. In all cases, variant forms are tested for their ability to function as proposed herein using techniques which are set forth herein or which are selected from techniques which are currently well known in the art.

When in nucleic acid form, a derivative comprises a sequence of nucleotides having at least 80% identity to a parent molecule, such as a nucleic acid sequence encoding a binding partner of the present invention, or a section thereof. A "section" of a nucleic acid molecule is defined as having a minimal size of at least about 5 nucleotides or preferably about 10 nucleotides or more preferably at least about 15 nucleotides. This definition includes all sizes in the range of 5-15 nucleotides including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides as well as greater than 15 nucleotides including 50, 100, 300, 500, 1000 or 2000 nucleotides or nucleic acid molecules having any number of nucleotides within these values. Having at least about 80% identity means, having optimal alignment, a nucleic acid molecule comprises at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with a reference sequence.

The terms "similarity" or "identity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and amino acid sequence comparisons are made at the level of identity rather than similarity.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al (Nucl Acids Res 25:3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994- 1998, Chapter 15).

The terms "sequence similarity" and "sequence identity" as used herein refer to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, GIy, VaI, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present disclosure, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity. The nucleic acid molecules disclosed and contemplated herein are also capable of hybridizing to other genetic molecules. Reference herein to "hybridizes" refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. For example, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below. In alternative aspects, nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low).

Reference herein to a "low stringency" includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as "medium stringency", which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or "high stringency", which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T_m = 69.3 + 0.41 (G+C)% (Marmur and Doty, JMo/ Biol 5:109-118, 1962). However, the T_n, of a duplex nucleic acid molecule decreases by I⁰C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur J Biochem 4(5:83-88, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows:low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42⁰C; a moderate 99

stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.

The terms "nucleic acid", "nucleotide" and "polynucleotide" include RNA (mRNA, tRNA, rRNA, siRNA), DNA (genomic DNA, cDNA), synthetic forms and mixed polymers, both sense and/or antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g. phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g. polypeptides), intercalators (e.g. acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g. α-anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

The chimeric molecule contemplated herein is useful for screening for agents which interact with the BLR of the human pro-survival protein such as but not limited to human McI-I. A variety of assays may be employed, for example, the chimeric protein is immobilized to beads and contacted with potential ligands. The ligands may be from a natural source such as extracts of plant material, coral, soil or microorganisms. Alternatively, chemical synthetic libraries, protein libraries or fragments or derivatives of pro-apoptotic molecules may be sources of potential ligands. Binding of the ligands is determined by a variety of methods including the use of labels or magnetic resonance or other spectrometric devices. Ligands are then tested for antagonistic or agonistic properties with respect to the human pro-survival molecule. A method of identifying agents useful for modulating apoptosis of cells in a subject is also contemplated herein. These methods of identification comprise screening naturally produced libraries, chemical produced libraries, as well as combinatorial libraries, phage display libraries and in vitro translation-based libraries comprising an expressed form of m/hMcl-1. The capability of the subject agents, whether they be proteinaceous or non- proteinaceous, to modulate the pro-survival activity of McI-I may be assessed via a number of screening methods which would be well known to a person skilled in the art. One method of screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing m/hMcl-l^BLR. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between a target and the agent being tested, or examine the degree to which the formation of a complex between a target and a known ligand is aided or interfered with by the agent being tested. Another method uses sensitive spectrometric assays to detect the pressure of binding to the human portion of the chimeric molecule.

One form of assay involves competitive binding assays. In such competitive binding assays, the target is typically labeled. Free target is separated from any putative complex and the amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested to target molecule. One may also measure the amount of bound, rather than free, target. It is also possible to label the agent rather than the target.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a target and is described in detail in Geysen (International Patent Publication No. WO 84/03564). Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with a target and washed. Bound target molecule is then detected by methods well known in the art. This method may be adapted for screening for non-peptide, chemical entities. This aspect, therefore, extends to combinatorial approaches to screening for agents capable of modulating the level of expression of the target proteins or genes encoding same. Two-hybrid screening is also useful in identifying other members of a biochemical or genetic pathway associated with a target. Two-hybrid screening conveniently uses Saccharomyces cerevisiae and Saccharomyces pombe. Target interactions and screens for agonists and antagonists can be carried out using the yeast two-hybrid system, which takes advantage of transcriptional factors that are composed of two physically separable, functional domains. The most commonly used is the yeast GAL4 transcriptional activator consisting of a DNA binding domain and a transcriptional activation domain. Two different cloning vectors are used to generate separate fusions of the GAL4 domains to genes encoding potential binding proteins. The fusion proteins are co-expressed, targeted to the nucleus and if interactions occur, activation of a reporter gene (e.g. lacZ) produces a detectable phenotype. In the present case, for example, S. cerevisiae is co-transformed with a library or vector expressing a cDNA GAL4 activation domain fusion, and a vector expressing a target gene fused to GAL4. If lacZ is used as the reporter gene, co-expression of the fusion proteins will produce a blue color. Small molecules or other candidate compounds which interact with a target will result in loss of color of the cells. Reference may be made to the yeast two-hybrid systems as disclosed by Munder et al (Appl Microbiol Biotechnol 52:311-320, 1999) and Young et al, Nat Biotechnol 16:946-950, 1998). Molecules thus identified by this system are then re-tested in animal cells.

Agents are therefore provided which modulate the level of pro-survival activity of human McI-I identified by an assay comprising screening for agents which bind to the BLR of human McI-I in a fusion construct comprising an N-terminal region comprising GST fused to a fusion protein useful for screening for modulators of apoptosis of cells, said fusion protein comprising an N-terminal region comprising GST fused to amino acids 152 to 189 of murine McI-I fused at its C-terminal end to amino acids of 209 to 327 of human McI-I. The fusion construct may alternatively comprise the GST fused to the C-terminal end portion of human McI- 1.

The agent may, in one aspect, be a proteinaceous or non-proteinaceous molecule and may be based on the structure of McI-I or a molecule which interacts with McI-I such as Bak, Bim, Bad, Bid, Noxa or Puma may be identified following natural produce screening or the screening of a chemical library.

The terms "agent", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" may be used interchangeably herein to refer to a substance that induces a desired pharmacological and/or physiological effect, i.e. agonizing or antagonizing McI-I pro-survival activity. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "agent", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The agents of the present invention may be any proteinaceous molecules such as peptides, polypeptides and proteins or non-proteinaceous molecules such as nucleic acid molecules and small to large natural or synthetically derived organic and inorganic molecules.

In relation to proteinaceous molecules, including peptides, polypeptide and proteins, without distinction, the terms include mutants, parts, derivatives, homologs, analogs or mimetics of one or more of McI-I itself or of the pro-apoptotic molecules Bak, Bim, Bad, Bid, Noxa and/or Puma. By "modulating" is meant an agent which upregulates or down- regulates protein activity. In one embodiment, the agent promotes dissociation of McI-I and/or BCI-XL from Bak. In another embodiment, the agent interacts with the BH3 binding groove of McI-I to which the BH3 domain of pro-apoptotic molecules interact.

Accordingly, mutant forms of McI-I as well as other Bcl-2 proteins which interact with McI-I are contemplated by the present invention and may be naturally occurring or artificially generated variants of the agent, such as but not limited to mutant forms of McI- 1 or Bak, Bim, Bid or Puma, comprising one or more amino acid substitutions, deletions or additions. Mutants may be produced by mutagenesis or other chemical methods or generated recombinantly or synthetically. Alanine scanning is a useful technique for identifying important amino acids (Wells, Methods Enzymol 202:2699-2705, 1991). In this technique, an amino acid residue is replaced by alanine and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the polypeptide. Mutants are tested for their ability to regulate angiogenesis and for other qualities such as longevity, binding affinity, dissociation rate and ability to cross biological membranes.

The present invention extends to parts of Bak, Bim, Bad, Bid, Noxa or Puma which are capable of binding to the BLR of human McI-I. Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in

Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell

Scientific Publications. Alternatively, peptides can be produced by digestion of an amino acid sequence of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-

C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Any such part, section or fragment, irrespective of its means of generation, is also to be understood as being encompassed by the term "derivative" as used herein.

Thus derivatives, or the singular derivative, encompass parts, mutants, homologs, fragments, analogs as well as hybrid or fusion molecules and glycosylaton variants. Derivatives also include molecules having a percent amino acid sequence identity over a window of comparison after optimal alignment. Preferably, the percentage similarity between a particular sequence and a reference sequence is at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater. Preferably, the percentage similarity between species, functional or structural homologs of the instant agents is at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater. Percentage similarities or identities between 60% and 100% are also contemplated such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

Analogs of the agents contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs. This term also does not exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as those given in Table 1) or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell and/or cross the blood- brain barrier.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using A- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy- 5 -phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids, contemplated herein is shown in Table 3. TABLE 3

Codes for non-conventional amino acids

Non-conventional Code Non-conventional Code amino acid amino acid

α-ammobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-rαethylleucine Dmleu α-napthylalanine Anap Non-conventional Code Non-conventional Code amino acid amino acid

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dniorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtφ N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnnαala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(I -hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NMs

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-ammobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-metliylornithine Diimorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(I -methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyρhenyl)gIycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet Non-conventional Code Non-conventional Code amino acid amino acid

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine MnIe

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhin N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n = 1 to n = 6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodϋmide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and N _α-methylamino acids, introduction of double bonds between C_α and Cp atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

Mimetics are another useful group of agents to test for an ability to modulate McI-I pro- survival activity such as mimetics of one or more of Bak, Bim, Bad, Bid, Noxa or Puma. The term is intended to refer to a substance which has some chemical similarity to the molecule it mimics but which antagonizes or agonizes its interaction with a target (i.e. McI-I). A peptide mimetic may be a peptide-containing molecule that mimics elements of protein secondary structure (Johnson et al, Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et al, Eds., Chapman and Hall, New York, 1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions such as those of antibody and antigen, enzyme and substrate or scaffolding proteins. A peptide mimetic, therefore, is designed to permit molecular interactions similar to the natural molecule.

The designing of mimetics to a pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g. peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. First, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. As described hereinbefore, Alanine scans of peptides are commonly used to refine such peptide motifs. These parts or residues constituting the active region of the compound are known as its "pharmacophore".

Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.

In a variant of this approach, the three-dimensional structure of a receptor and ligand are modelled. This can be especially useful where the receptor and/or ligand change conformation on binding, allowing the model to take account of this in the design of the mimetic. Modeling can be used to generate agents which interact with the linear sequence or a three-dimensional configuration.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, for example, enhance or interfere with the function of a polypeptide in vivo (see, e.g. Hodgson, BioTechnology P: 19-21,

1991). In one approach, one first determines the three-dimensional structure of a protein of interest by x-ray crystallography, by computer modeling or most typically, by a combination of approaches. Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of homologous proteins.

In yet another embodiment, a method is provided of promoting apoptosis, said method comprising administering to a subject an agent which is capable of inhibiting the pro- survival activity of human McI-I.

Hence, the agents and methods described and contemplated herein facilitate the development of methods and pharmaceutical compositions for preventing and/or treating a range of cancers or necrotic conditions. Examples of cancers include, without being limited to, ABLl protooncogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukaemia, acute myeloid leukaemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukaemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukaemia, chronic myeloid leukaemia, colorectal cancers, cutaneous T-cell lymphoma, dermatofibrosarcoma- protuberans, desmoplastic-small-round-cell-tumor, ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anaemia, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell tumors, gestational- trophoblastic-disease, glioma, gynaecological cancers, haematological malignancies, hairy cell leukaemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhan's-cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukaemia, li-fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer- (nsclc), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral-neuroectodermal-tumors, pituitary cancer, polycythemia vera, prostate cancer, rare-cancers-and-associated-disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell-carcmoma-(skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-φladder), transitional-cell-cancer- (renal-pelvis-/-ureter), trophoblastic cancer, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom 's- macroglobulinemia or Wilms' tumor.

Reference herein to "treatment" may mean a reduction in the severity of an existing disease or condition. The term "treatment" is also taken to encompass "prophylactic treatment" to prevent the onset of a disease or condition. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylactic treatment" does not necessarily mean that the subject will not eventually contract a disease or condition.

A "subject" as used herein refers to humans and non-human primates (e.g. gorilla, macaque, marmoset), livestock animals (e.g. sheep, cow, horse, donkey, pig), companion animals (e.g. dog, cat), laboratory test animals (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animals (e.g. fox, deer), reptiles or amphibians (e.g. cane toad), fish

(e.g. zebrafish) and any other organisms (e.g. C. elegans) who can benefit from the agents of the present invention. There is no limitation on the type of animal that could benefit from the presently described agents. The most preferred subject of the present invention is a human. A subject regardless of whether it is a human or non-human organism may be referred to as a patient, individual, animal, host or recipient.

The agents of the present invention can be combined with one or more pharmaceutically acceptable carriers and/or diluents to form a pharmacological composition.

Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rates of the pharmaceutical compositions of the invention. Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the peptides or polypeptides, or excipients or other stabilizers and/or buffers. Detergents can also used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. Pharmaceutically acceptable carriers and formulations for peptides and polypeptide are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., Remington's Pharmaceutical Sciences, 18^th Edition, Mack Publishing Company, Easton, PA, 1990 ("Remington's").

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g., phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the modulatory agent of the invention and on its particular physio-chemical characteristics.

Administration of the agent, in the form of a pharmaceutical composition, may be performed by any convenient means known to one skilled in the art. Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, orally, rectally, patch and implant.

For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Due to their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier, see, e.g, International Patent Publication Number WO 96/11698.

Agents of the present invention, when administered orally, may be protected from digestion. This can be accomplished either by complexing the agent with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the agent in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are well known in the art, see, e.g. Fix, Pharm Res 75:1760-1764, 1996; Samanen et al, J Pharm Pharmacol 45:119-135, 1996; U.S. Patent Number 5,391,377, describing lipid compositions for oral delivery of therapeutic agents.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. 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 polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. 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 superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the agents in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the 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 the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

For parenteral administration, the agent may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the agents are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used for delivering the agent. Such penetrants are generally known in the art e.g. for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories e.g. Sayani and Chien, Crit Rev Ther Drug

Carrier Syst 73:85-184, 1996. For topical, transdermal administration, the agents are formulated into ointments, creams, salves, powders and gels. Transdermal delivery systems can also include patches.

For inhalation, the agents of the invention can be delivered using any system known in the art, including dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like, see, e.g., Patton, Nat Biotech 7(5:141-143, 1998; product and inhalation delivery systems for polypeptide macromolecules by, e.g., Dura Pharmaceuticals (San Diego, CA), Aradigm (Hayward, CA)₅ Aerogen (Santa Clara, CA), Inhale Therapeutic Systems (San Carlos, CA), and the like. For example, the pharmaceutical formulation can be administered in the fonn of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another aspect, the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include, for example, air jet nebulizers.

The agents of the invention can also be administered in sustained delivery or sustained release mechanisms, which can deliver the formulation internally. For example, biodegradable microspheres or capsules or other biodegradable polymer configurations capable of sustained delivery of an agent can be included in the formulations of the invention (e.g. Putney and Burke, Nat Biotech 7(5:153-157, 1998).

In preparing pharmaceuticalsm, a variety of formulation modifications can be used and manipulated to alter pharmacokinetics and biodistribution. A number of methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art. Examples of such methods include protection of the compositions of the invention in vesicles composed of substances such as proteins, lipids (for example, liposomes), carbohydrates, or synthetic polymers. For a general discussion of pharmacokinetics, see, e.g., Remington's.

In one aspect, the pharmaceutical formulations comprising agents of the present invention are incorporated in lipid monolayers or bilayers such as liposomes, see, e.g., U.S. Patent Numbers 6,110,490; 6,096,716; 5,283,185 and 5,279,833. The invention also provides formulations in which water-soluble modulatory agents of the invention have been attached to the surface of the monolayer or bilayer. For example, peptides can be attached to hydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing liposomes (e.g. Zalipsky et al, Bioconjug Chem (5:705-708, 1995). Liposomes or any form of lipid membrane, such as planar lipid membranes or the cell membrane of an intact cell e.g. a red blood cell, can be used. Liposomal formulations can be by any means, including administration intravenously, transdermally (Vutla et al, J Pharm Sci 85:5-8, 1996), transmucosally, or orally. The invention also provides pharmaceutical preparations in which the agents of the invention are incorporated within micelles and/or liposomes (Suntres and Shek, J Pharm Pharmacol 46:23-28, 1994; Woodle et al, Pharm Res 9:260- 265, 1992). Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art see, e.g., Remington's; Akimaru et al, Cytokines MoI Ther 7:197-210, 1995; Alving et al, Immunol Rev 145:5-31, 1995; Szoka and Papahadjopoulos, Ann Rev Biophys Bioeng 9:467-508, 1980; U.S. Patent Numbers 4, 235,871, 4,501,728 and 4,837,028.

The pharmaceutical compositions of the invention can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisorial in nature and are adjusted depending on the particular therapeutic context, patient tolerance, etc. The amount of agent adequate to accomplish this is defined as the "effective amount". The dosage schedule and effective amounts for this use, i.e., the "dosing regimen" will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., Remington's; Egleton and Davis, Peptides 75:1431-1439, 1997; Langer, Science 249:1527-1533, 1990.

In accordance with these methods, the agents and/or pharmaceutical compositions contemplated herein may be co-administered with one or more other agents. Reference herein to "co-administered" means simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. Reference herein to "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of agents and/or pharmaceutical compositions. Co-administration of the agents and/or pharmaceutical compositions may occur in any order.

Aspects of the present invention are further described by the following non-limiting examples.

EXAMPLE 1

Generation of chimeric m/lι Mcl-1^BLR )

A chimera of mMcl-l^BLR and hMcl-l^BLR was made using the restriction enzyme Sphl. This site is conserved in the DNA sequence of mMcl-1 and hMcl-1 (nucleotide 636 in niMcl-1 and nucleotide 693 in hMcl-1). The protein encoded by this chimeric construct

(hereafter referred to as "m/hMcl-lBLR") is human sequence with the exception of nine amino acids towards the N-terminus of the protein as detailed in Figure I₅ see also SEQ ID

NOs:6 (mMcl-l^BLR) and 7 (hMcl-1 ^BLR), respectively. Importantly, this leaves the region of the protein within and near the BH3 binding site of the protein as human sequence (Figure

2). M/hMcl-l^BLR can be expressed and purified with similar efficacy to the mMcl-l^BLR. It is anticipated that further combinations of mMcl-1 residues and hMcl-1 residues within this region may be made to make the chimera even more human like whilst maintaining the ability to be expressed and purified.

EXAMPLE 2 Binding characteristics

Isothermal titration calorimetry was used to compare the binding characteristics of m/hMcl-l^BLR and mMcl-1 ^BLR to peptides corresponding to the BH3 regions of various BH3 only proteins of the Bcl-2 family (Figure 3). This demonstrates that m/hMcl-l^BLR is able to bind to BH3 peptides with similar but not identical efficacy to mMcl-1 ^BLR and would therefore be useful as a screening target for compounds which could inhibit this interaction. EXAMPLE 3 Binding to Bim

Phage display binding competition assays were performed to compare the binding of m/hMcl-l^BLR and mMcl-l^BLR to mutations of the Bim BH3 only peptide (Figure 4). These demonstrate subtle differences in the binding characteristics of these proteins for small differences in BH3 binding. These differences may be important when screening for compounds that inhibit the interaction of hMcl-1 with BH3 only proteins.

EXAMPLE 4

3 dimensional structure of mhMcl-l^BLR complexed with IιBimBH3 peptide

The crystal structure of the mhMcl-l^BLR:hBimBH3 domain complex was solved with MAD phasing using seleno-methionine incorporated protein to 1.56 A (Figure 5). Seleno- methionine labelled mhMcl-l^BLR was complexed with hBim BH3 peptide and concentrated to 12 mg/ml. Crystals of the complex were grown in hanging drops at 22 ⁰C with a reservoir solution consisting of 0.2 M ZnCl₂, 0.2 M imidazole pH 5.75 and 2 mM TCEP. Prior to flash freezing in liquid nitrogen crystals were equilibrated into cryoprotectant consisting of reservoir solution and increasing concentrations of trehalose to a final trehalose concentration of 30% (v/v). A MAD data set was collected to 1.56 A at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratories, USA. Data were integrated and scaled with HKL2000. Heavy atom sites were found using HKL2MAP. An initial model was built from the resulting map using Coot. Several rounds of building in Coot and refinement in Refmac5 were then used to produce the final model. The atomic structure co-ordinates are shown in Figures 6A through 6AC. EXAMPLE 5 Compound identification

Both m/hMcl-l^BLR and mMcl-l^BLR) were used to identify compounds that inhibit interaction between McI-I and the Bim BH3 peptide. Compounds that show the highest activity in the m/hMcl-l^BLR assay are not the same as those that show the highest activity in the mMcl-l^BLR assay. As m/hMcl-l^BLR is more representative of the hMcl-1 than mMcl-l^BLR, and as it contains a BH3 binding groove which is composed of human residues only, it is considered to be a superior tool for drug discovery programs than the mMcl-l^BLR protein.

A library of compounds were screened for the ability to inhibit interaction between McI-I and a peptide corresponding to the BH3 domain of Bim. The top 10 hits when mmMcl- 1^BLR is used as the target protein are listed in Figure 7. The third column displays the ranking of the same compound when the library is screened with mMcl-l^BLR as the target protein. Compounds listed as >131 were not within the top 131 ranked compounds in the m/hMcl-l^BLR assay.

Those skilled in the art will appreciate that aspects of the invention described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that aspects of the invention include all such variations and modifications. Aspects of the invention also include all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features. BIBLIOGRAPHY

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Claims

CLAIMS:

1. A chimeric molecule comprising a Bcl-2-like region (BLR) from human pro- survival protein McI-I of the Bcl-2 family of proteins and a portion of a non-human homolog thereof fused to the N-terminal region of McI-I wherein said chimeric molecule is capable of being produced in a cell in which a nucleic acid molecule encoding said chimeric molecule is expressed.

2. The chimeric molecule of Claim 1 comprising amino acids 209 through 327 of human McI-I.

3. The chimeric molecule of Claim 1 or 2 wherein the non-human homolog of McI-I is murine McI-I.

4. The chimeric molecule of Claim 3 comprising amino acid residues 152 through 189 of murine McI-I fused to the N-terminal region of human McI-I.

5. The chimeric molecule of any one of Claims 1 to 4 further comprising a carrier protein fused to the N-terminal region of the non-human homolog of McI-I or the C- terminal region of human McI-I .

6. The chimeric molecule of Claim 5 wherein the carrier protein is GST or a fragment or derivative thereof.

7. The chimeric molecule of Claim 5 or 6 immobilized to a solid support via the carrier molecule.

8. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding the chimeric molecule of any one of Claim 1 to 7.

9. The isolated nucleic acid molecule of Claim 8 comprising the nucleotide sequence set forth in SEQ ID NO:1 or a nucleotide sequence having at least 80% identity thereto or is capable of hybridizing to SEQ ID NO:1 or a complementary form under low stringency conditions.

10. The nucleic acid molecule of Claim 8 or 9 when part of a genetic construct.

11. A genetically modified cell comprising the nucleic acid molecule of Claim 8 or 9 or 10.

12. A chimeric protein of Formula I₅ represented in the order of N-terminal to C- terminal:

[Xi] _n- [X₂] - [X₃] - [X₄Jm (T)

wherein each OfX₁ and X₄ is a carrier protein or a part or portion thereof; n and m are 0 or 1 except n and m cannot both be 1 ;

- is a peptide bond;

X₂ is derived from an N-terminal truncated non-human pro-survival Bcl-2 family protein; and

X₃ is derived from the human homolog of the non-human pro-survival protein.

13. The chimeric protein of Claim 12 wherein X₃ is or is derived from hMcl-l^BLR and

X₂ is derived from mMcl-1 BLR

14. The chimeric protein of Claim 12 or 13 wherein Xi and X₄ is GST or a portion thereof.

15. A chimeric molecule comprising the amino acid sequence set forth in SEQ ID NO:4 and SEQ ID NO:5 or an amino acid sequence having at least about 80% similarity to SEQ ID NO:4 or SEQ ID NO:5 wherein SEQ ID NO:4 is the amino acid sequence of a murine McI-I portion and SEQ ID NO: 5 is the amino acid sequence of a human McI-I portion wherein the C-terminal region of said chimeric molecule comprises the BLR of human McI-I.

16. The chimeric molecule of Claim 15 further comprising SEQ ID NOs. -6 and 7 which represent the amino acid sequences of mMcl-l^BLR and hMcl-l^BLR, respectively from residues 171 to 327.

17. The chimeric molecule of Claim 15 or 16 wherein the chimeric molecule further comprises a GST portion fused to the N-terminal end having the amino acid sequence set forth in SEQ ID NO:3.

18. The chimeric molecule of Claim 15 or 16 or 17 wherein the chimeric molecule comprises the amino acid sequence set forth in SEQ ID NO. -2 or an amino acid sequence having at least 80% similarity thereto.

19. An assay to detect a ligand of the BLR of a human anti-apoptotic molecule said assay comprising contacting a potential source of ligands to be tested with a chimeric molecule comprising a BLR from a human pro-survival protein of the Bcl-2 family of proteins and a portion of a non-human homolog thereof fused to the N-terminal region of the human protein wherein said chimeric molecule is produced in a cell in which a nucleic acid molecule encoding said chimeric molecule is expressed and screening for binding of a ligand to said chimeric molecule.

20. The assay of Claim 19 wherein the human pro-survival molecule is McI-I .

21. The assay of Claim 20 wherein the portion of McI-I comprises amino acids 209 through 327.

22. The assay of Claim 20 wherein the non-human homolog of McI-I is murine McI-I .

23. The assay of Claim 22 wherein the chimeric molecule comprises amino acid residues 152 through 189 of murine McI-I fused to the N-terminal region of human McI-I.

24. The assay of any one of Claims 19 to 23 wherein the chimeric molecule further comprises a carrier protein fused to the N-terminal region of the non-human homolog of the pro-survival protein.

25. The assay of Claim 24 wherein the carrier protein is GST or a fragment or derivative thereof.

26. The assay of Claim 24 or 25 wherein the chimeric molecule is immobilized to a solid support via the carrier molecule.

27. A ligand identified by the assay of any one of Claims 24 to 26.

28. The ligand of Claim 27 wherein the ligand is an antagonist of the pro-survival protein.

29. The ligand of Claim 27 wherein the ligand is an agonist of the pro-survival protein.

30. The ligand of Claim 27 or 28 or 29 wherein the pro-survival protein is human McI- 1.

31. Use of the three-dimensional co-ordinates of a mhMcl-l:hBimBH3 complex set forth in Figures 6A through 6AC for computer assisted in-silico screening or design of agents which interact with human Mcl-2.

32. An isolated agent identified using the three-dimensional co-ordinates of Claim 31.

33. A composition comprising the agent of Claim 32 and one or more pharmaceutically acceptable carriers and/or diluents.