WO2020252298A1 - Compositions and methods for targeting the nucleosome remodeling and deacetylase complex - Google Patents

Abstract

Provided are cell-permeable peptide mimetics, or synthetic peptides, of GATAD2A that are capable of binding MBD2 and disrupting the MBD2:GATAD2A interaction. Pharmaceutical compositions that include such synthetic peptides are suitable for treating conditions in subjects, including β-hemoglobinopathy and/or various cancers. Methods of treating subjects by administering such synthetic peptides and/or pharmaceutical compositions are also provided.

Description

DESCRIPTION

COMPOSITIONS AND METHODS FOR TARGETING THE NUCLEOSOME REMODELING AND DEACETYLASE COMPLEX

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent Application Serial No. 62/861 ,464, filed June 14, 2019, herein incorporated by reference in its entirety.

GRANT STATEMENT

This invention was made with government support under grant numbers GM1 18499-02 and DK1 15563-01 A1 awarded by National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

The subject matter disclosed herein relates generally to compositions and methods for targeting the Nucleosome Remodeling and Deacetylase (NuRD) complex. More specifically, disclosed herein are synthetic GATAD2A peptide and peptide mimetic compounds that disrupt the methylcytosine binding domain 2 (MBD2):NuRD complex.

BACKGROUND

The Nucleosome Remodeling and Deacetylase (NuRD) complex has been identified throughout the animal kingdom and plays a direct role in DNA methylation dependent gene silencing during normal development,⁷·⁸ reprogramming of pluripotent stem cells,^9-11 and aberrant silencing of tumor suppressor genes during carcinogenesis.³·¹² The methylcytosine binding domain 2 (MBD2) protein recruits and assembles the NuRD complex and thereby uniquely combines binding specificity for methylated DNA with histone deacetylation and chromatin remodeling.

Several diseases and conditions may be treatable by targeting the NuRD complex. For example, the NuRD complex may be targeted as a therapy for treating beta-hemoglobinopathies (sickle cell anemia and beta thalassemia) as well as multiple types of cancer (including breast, colon, glioblastoma, and hematopoietic). However, to date no known compositions effectively target the NuRD complex in vivo sufficient to treat such indications without significant side effects.

The present disclosure overcomes previous shortcomings in the art by providing compositions and methods for targeting the NuRD complex, and specifically the MBD2:NuRD complex, in living cells.

Accordingly, it is an object of the presently disclosed subject matter to provide compositions and methods for targeting the Nucleosome Remodeling and Deacetylase (NuRD) complex, including providing synthetic GATAD2A peptide and peptide mimetic compounds that disrupt the methylcytosine binding domain 2 (MBD2):NuRD complex. This and other objects are achieved in whole or in part by the presently disclosed subject matter. Further, an object of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Drawings and Examples.

SUMMARY

This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

In some embodiments, provided are synthetic peptide compounds, the synthetic peptide compounds comprising a synthetic peptide compound that disrupts GATAD2A/MBD2 coiled coil, wherein the synthetic peptide compound is configured to disrupt an interaction between methylcytosine binding domain 2 (MBD2) and GATAD2A. In some aspects, the synthetic peptide compound comprises a GATAD2A peptide mimetic compound configured to disrupt an interaction between methylcytosine binding domain 2 (MBD2) and Nucleosome Remodeling and Deacetylase (NuRD). In some aspects, the synthetic peptide compound is cell permeable. In some aspects, the synthetic peptide compound inhibits a MBD2:GATAD2A interaction intracellularly. In some aspects, the synthetic peptide compound comprises: a cell penetrating and/or a nuclear localization signal; a non-natural amino acid in place of or in addition to a methionine; an added cholic acid to a N-terminus of the peptide, or other adduct that promotes endosomal escape; and two or more residues introduced on an exposed surface of a helix of the peptide and capable of chemically reacting with a stapling agent, optionally two or more cysteine residues. In some aspects, the nuclear localization signal is from a SV40 large T-antigen. In some aspects, the nuclear localization signal promotes cell penetration, nuclear localization, and/or stronger binding affinity for the target as compared to a peptide without a nuclear localization signal. In some aspects, the non-natural amino acid comprises norleucine. In some aspects, the norleucine increases binding affinity, and/or reduces protease sensitivity.

In some aspects, the synthetic peptide further comprises one or more chemical staples between the two residues, optionally two cysteine residues. In some aspects, the one or more chemical staples improves binding, improves helicity, reduces proteolytic degradation and/or improves cell penetration as compared to the composition without the staples. In some aspects, the synthetic peptide further comprises a non-natural amino acid to increase stabilization of the peptide via the chemical staple.

In some embodiments, the synthetic peptide compound is selected from the group consisting of: YNIelKQLKEELRLEEAKLVLLKKLRQSQ-NH₂ (Peptide 17; SEQ ID NO: 1 ); KKKRKVYAIKQLKCELRCEEAKLVLLKKLRQSQ-NH2 (Peptide 18; SEQ ID NO: 2); YpalaOOGNIelKQLKCELRCEEAKLVLLKKLRQSQ-NH₂ (Peptide 19; SEQ ID NO: 3); KKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-NH₂ (Peptide 20; SEQ ID NO: 4); CA-

GKKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-NH₂ (Peptide 25; SEQ ID NO: 5); CA-KKKRKVYGNIelKQLKEELRLEEAKLVLLKKLRQSQ-NH₂ (Peptide 29; SEQ ID NO: 6); and a peptide having about 50% to about 99% homology to any of Peptides 17, 18, 19, 20, 25 and 29, wherein CA- comprises a cholic acid at the N- terminus, -NH₂ is a C-terminal amide, and C are linkage points for a chemical staple. In some aspects, the synthetic peptide compound is selected from the group consisting of: CA-GKKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ- NH₂ (Peptide 25; SEQ ID NO: 5); CA-

KKKRKVYGNIelKQLKEELRLEEAKLVLLKKLRQSQ-NH₂ (Peptide 29; SEQ ID NO: 6); and a peptide having about 80% to about 99% homology to Peptide 25 or Peptide 29,

wherein CA- comprises a cholic acid at the N-terminus, -Nhte is a C-terminal amide, and C are linkage points for a chemical staple.

In some aspects, the synthetic peptide comprises a binding affinity stronger than about 150 nM. In some aspects, the synthetic peptide compound is selected from:

; and

Ih some embodiments, provided herein are pharmaceutical compositions for use in treating a subject, the compositions comprising a synthetic peptide compound as disclosed herein. In some aspects, the composition further comprises a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier is pharmaceutically acceptable for use in humans. In some aspects, the pharmaceutical composition is adapted for treatment of a b-hemoglobinopathy and/or a cancer. In some aspects, the pharmaceutical composition is adapted for treatment of sickle cell anemia, b- thalassemia, breast cancer, colon cancer, brain cancer, acute leukemia, chronic leukemia and/or myelodysplastic syndromes. In some aspects, the pharmaceutical composition selectively induces apoptosis in one or more cancer cells by disrupting a NuRD complex in the one or more cancer cells.

In some embodiments, provided herein are methods of treating a subject, the methods comprising: providing a subject to be treated; and administering to the subject a synthetic peptide compound and/or pharmaceutical composition disclosed herein. In some aspects, the subject to be treated is a human subject. In some aspects, the subject to be treated is suffering from a b-hemoglobinopathy, optionally wherein the b-hemoglobinopathy is selected from sickle cell anemia and b-thalassemia. In some aspects, the subject to be treated is suffering from a cancer, optionally wherein the cancer is selected from breast cancer, colon cancer, brain cancer, acute leukemia, chronic leukemia and myelodysplastic syndromes. In some aspects, apoptosis is selectively induced in one or more cells in the subject by disrupting a NuRD complex in the one or more cells. In some aspects, the one or more cells comprise one or more cancer cells. In some aspects, apoptosis is not induced in non-cancerous cells.

These and other objects are achieved in whole or in part by the presently disclosed subject matter. Further, objects of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Drawings and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed subject matter can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (often schematically). In the figures, like reference numerals designate corresponding parts throughout the different views. A further understanding of the presently disclosed subject matter can be obtained by reference to an embodiment set forth in the illustrations of the accompanying drawings. Although the illustrated embodiment is merely exemplary of systems for carrying out the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of this presently disclosed subject matter, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and exemplify the presently disclosed subject matter.

For a more complete understanding of the presently disclosed subject matter, reference is now made to the following drawings in which:

Figure 1 is a schematic illustrating components and features of the disclosed synthetic peptides based on the GATAD2A coiled-coil.

Figure 2 is a schematic illustration of a NuRD Complex, comprising Retinoblastoma-binding protein 4/8 (RBBP4/8), Flistone deacetylase 1/2 (HDAC1/2), Metastasis-associated gene 1/2/3 (MTA 1/2/3), Methyl-binding domain 2/3 (MBD2/3), GATA zinc finger domain containing 2A/2B (GATAD2A/B), chromodomain helicase DNA-binding protein 3/4 (CFID3/4).

Figure 3 is a depiction of a nuclear magnetic resonance (NMR) structure of MBD2:GATAD2A, where MBD2 (light gray) and GATAD2A (dark gray) form an anti parallel coiled-coil.

Figure 4 is a schematic illustration of a design of a system as disclosed herein where the peptide mimic of GATAD2A disrupts the protein-protein interaction between MBD2 and GATAD2A, leading to re-expression of genes silenced by the NuRD complex.

Figure 5 is a schematic illustration of a peptide stapling, wherein the curved line represents a peptide sequence and SH represent cysteine residues.

Figure 6 is a bar graph depicting the results of a NanoBRET™ assay of peptide 20 (stapled) and peptide 25 (stapled). MBD2 is expressed as a fusion with nanoLuc and GATAD2A is expressed as a fusion with HaloTag enzyme. Disruption of the interaction between MBD2 and GATAD2A results in a loss of BRET signal. Cells were incubated with peptide dissolved in PBS at either 5 or 15 mM final concentration for 4 or 20 hr. Error bars represent standard deviation of 4 technical replicates.

Figure 7 is a bar graph depicting the results of a NanoBRET™ assay of 25_stapled, 29, and 30. Cells were incubated with peptide dissolved in PBS with a final concentration of 5, 10, or 15 mM for 4 or 20 hr. Error bars represent the standard deviation of 4 technical replicates.

Figure 8 is a schematic illustration of stapled peptide 25.

Figure 9 is a schematic illustration of peptide 29.

Figure 10 is a schematic illustration of a variation of peptide 29 shown in Figure 9, where peptide 29 is provided without the Nle at M144, and instead having Methionine in its place.

Figures 1 1 A-1 1 C include bar graphs of data showing that synthetic Peptide 29 inhibits growth in three cell lines (Fig. 1 1 A) and induces apoptosis (Figs. 1 1 B and 1 1 C) in leukemia cell lines. Peptide was added at varying concentrations to monocytic (U937), myeloid (MV41 1 ) and T-lymphoblastic (Jurkat) cell lines for 20 hours before measuring cell viability and apoptosis by both 7-AAD staining and PARP cleavage assays.

Figures 12A-12C include bar graphs of data showing that peptide 29 induces apoptosis in three different patient-derived primary acute leukemia cells.

Figures 13A-13D include bar graphs of data showing that peptide 29 does not induce apoptosis or inhibit growth of primary patient CD34+ bone marrow progenitor cells.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Definitions

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one skilled in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the present disclosure and the claims.

All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

Following long-standing patent law convention, the terms“a”,“an”, and“the” refer to“one or more” when used in this application, including the claims. Thus, for example, reference to "a cell" includes a plurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

As used herein, the term“about,” when referring to a value or to an amount of a composition, mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

The term“comprising”, which is synonymous with“including”“containing” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.

As used herein, the phrase“consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase“consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

As used herein, the phrase“consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

As used herein, the term“and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase“A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

As used herein, the term“substantially,” when referring to a value, an activity, or to an amount of a composition, mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±40%, in some embodiments ±30%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed apparatuses and devices.

A“compound,” as used herein, refers to any type of substance or agent that is commonly considered a drug, therapeutic, pharmaceutical, small molecule, or a candidate for use as the same, as well as combinations and mixtures of the above.

The use of the word“detect” and its grammatical variants is meant to refer to measurement of the species without quantification, whereas use of the word “determine” or“measure” with their grammatical variants are meant to refer to measurement of the species with quantification. The terms“detect” and“identify” are used interchangeably herein.

The term“inhibit,” as used herein, refers to the ability of a compound, agent, or method to reduce or impede a described function, level, activity, rate, etc., based on the context in which the term“inhibit” is used. Preferably, inhibition is by at least 10%, more preferably by at least 25%, even more preferably by at least 50%, and most preferably, the function is inhibited by at least 75%. The term“inhibit” is used interchangeably with“reduce” and“block.”

The term“material”, as used herein, refers to synthetic and natural materials such as matrix components. The term“materials and compounds” as used herein, refers to, inter alia, materials, compounds, cells, peptides, nucleic acids, drugs, matrix components, and imaging agents.

The term “modulate”, as used herein, refers to changing the level of an activity, function, or process. The term“modulate” encompasses both inhibiting and stimulating an activity, function, or process. The term “modulate” is used interchangeably with the term“regulate” herein.

The term “prevent,” as used herein, means to stop something from happening, or taking advance measures against something possible or probable from happening. In the context of medicine,“prevention” generally refers to action taken to decrease the chance of getting a disease or condition.

The term “regulate” refers to either stimulating or inhibiting a function or activity of interest. A“sample,” as used herein, refers to a biological sample from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, semen, tears, and urine. A sample can also be any other source of material obtained from a subject which contains cells, tissues, or fluid of interest.

The term“stimulate” as used herein, means to induce or increase an activity or function level such that it is higher relative to a control value. The stimulation can be via direct or indirect mechanisms. In one aspect, the activity or function is stimulated by at least 10% compared to a control value, more preferably by at least 25%, and even more preferably by at least 50%. The term“stimulator” as used herein, refers to any composition, compound or agent, the application of which results in the stimulation of a process or function of interest.

As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:

Amino Acid Codes and Functionally Equivalent Codons

Full Name 3-Letter 1 -Letter Functionally Equivalent Codons

Code Code

Aspartic Acid Asp D GAC; GAU

Glutamic Acid Glu E GAA; GAG

Lysine Lys K AAA; AAG

Arginine Arg R AGA; AGG; CGA; CGC; CGG; CGU

Histidine His H CAC; CAU

Tyrosine Tyr Y UAC; UAU

Cysteine Cys C UGC; UGU

Asparagine Asn N AAC; AAU

Glutamine Gin Q CAA; CAG

Serine Ser S ACG; AGU; UCA; UCC; UCG; UCU

Threonine Thr T ACA; ACC; ACG; ACU

Glycine Gly G GGA; GGC; GGG; GGU

Alanine Ala A GCA; GCC; GCG; GCU

Valine Val V GUA; GUC; GUG; GUU

Leucine Leu L UUA; UUG; CUA; CUC; CUG; CUU Isoleucine lie AUA; AUC; AUU

Methionine Met M AUG

Proline Pro P CCA; CCC; CCG; CCU

Phenylalanine Phe F UUC; UUU

Tryptophan Trp W UGG

The expression“amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. “Standard amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid residue”, “modified” or“unusual amino acids” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, nonstandard amino acids are represented by the full name thereof or by the abbreviation thereof as indicated in the following table:

Abbreviation Full Name

Aad 2-Aminoadipic acid

bAad 3-Aminoadipic acid

bAla or pAla beta-Alanine, beta-Aminoproprionic acid

Abu 2-Aminobutyric acid

4 Abu 4-Aminobutyric acid, piperidinic acid

Acp 6-Aminocaproic acid

Ahe 2-Aminoheptanoic acid

Aib 2-Aminoisobutyric acid

bAib 3-Aminoisobutyric acid

Apm 2-Aminopimelic acid

Dbu 2,4-Diaminobutyric acid

Des Desmosine

Dpm 2,2'-Diaminopimelic acid

Dpr 2,3-Diaminoproprionic acid

EtGly N-Ethylglycine

EtAsn N-Ethylasparagine

Hyl Hydroxylysine

aHyl allo-Hydroxylysine

3Hyp 3-Hydroxyproline 4Hyp 4-Hydroxyproline

Ide Isodesmosine

alle allo-lsoleucine

MeGly N-Methylglycine, sarcosine

Melle N-Methylisoleucine

MeLys 6-N-Methyllysine

MeVal N-Methylvaline

Nva Norvaline

Nle Norleucine

Orn Ornithine

As used herein, “synthetic amino acid” also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide’s circulating half-life without adversely affecting their activity. Additionally, a disulfide linkage may be present or absent in the peptides of the invention.

The term“amino acid” is used interchangeably with“amino acid residue,” and may refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Although the above structure does not show stereochemistry, as would be appreciated by one of ordinary skill in the art, such amino acids can occur in L- and D- forms. Moreover, the 20 standard amino acids are all in the L-form, and the amino acids used herein are all in the L-form (see, e.g. Figures 8 and 9) unless indicated otherwise. Amino acids may be classified into seven groups on the basis of the side chain R: (1 ) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, a secondary amino group in which the side chain is fused to the amino group.

The nomenclature used to describe the peptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified.

The term“basic” or“positively charged” amino acid, as used herein, refers to amino acids in which the R groups have a net positive charge at pH 7.0, and include, but are not limited to, the standard amino acids lysine, arginine, and histidine.

The term“staple” or“chemical staple”, as used herein, refers to any chemical structure, natural or synthetic, that can attach between two or more residues on a peptide. Such staples can prevent or decrease proteolytic degradation and/or improve cell penetration.

As used herein, an“analog” of a chemical compound is a compound that, by way of example, resembles another in structure but is not necessarily an isomer (e.g., 5-fluorouracil is an analog of thymine).

As used herein, the term“antisense oligonucleotide” or antisense nucleic acid means a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid which is present in a normal cell or in an affected cell. “Antisense” refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences. The antisense oligonucleotides of the invention include, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides.

The term “autologous”, as used herein, refers to something that occurs naturally and normally in a certain type of tissue or in a specific structure of the body. In transplantation, it refers to a graft in which the donor and recipient areas are in the same individual, or to blood that the donor has previously donated and then receives back, usually during surgery.

A“control” cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as a test cell, tissue, sample, or subject. The control may, for example, be examined at precisely or nearly the same time the test cell, tissue, sample, or subject is examined. The control may also, for example, be examined at a time distant from the time at which the test cell, tissue, sample, or subject is examined, and the results of the examination of the control may be recorded so that the recorded results may be compared with results obtained by examination of a test cell, tissue, sample, or subject. The control may also be obtained from another source or similar source other than the test group or a test subject, where the test sample is obtained from a subject suspected of having a disease or disorder for which the test is being performed.

A“test” cell, tissue, sample, or subject is one being examined or treated.

The term“delivery vehicle” refers to any kind of device or material, which can be used to deliver cells in vivo or can be added to a composition comprising cells administered to an animal. This includes, but is not limited to, implantable devices, aggregates of cells, matrix materials, gels, etc.

As used herein, a“derivative” of a compound refers to a chemical compound that may be produced from another compound of similar structure in one or more steps, as in replacement of H by an alkyl, acyl, or amino group.

As used herein, a“detectable marker” or a“reporter molecule” is an atom or a molecule that permits the specific detection of a compound comprising the marker in the presence of similar compounds without a marker. Detectable markers or reporter molecules include, e.g., radioactive isotopes, antigenic determinants, enzymes, nucleic acids available for hybridization, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, and molecules that provide for altered fluorescence-polarization or altered light-scattering.

A“disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a“disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

As used herein, an“effective amount” means an amount sufficient to produce a selected effect.

A “fragment” or “segment” is a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms“fragment” and“segment” are used interchangeably herein.

As used herein, a“functional” molecule is a molecule in a form in which it exhibits a property or activity by which it is characterized.

“Homologous” as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3'ATTGCC5' and 3'TATGGC share 50% homology.

As used herein,“homology” is used synonymously with“identity”.

The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403-410), and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site. BLAST nucleotide searches can be performed with the NBLAST program (designated “blastn” at the NCBI web site), using the following parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward = 1 ; expectation value 10.0; and word size = 1 1 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated“blastn” at the NCBI web site) or the NCBI“blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

As used herein“injecting or applying” includes administration of a compound of the invention by any number of routes and approaches including, but not limited to, topical, oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal approaches.

Unless otherwise specified, a“nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

As used herein,“parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

The term “pharmaceutical composition” shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

As used herein, the term “pharmaceutically acceptable carrier” means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject. In some embodiments, the subject is a human and thus, the pharmaceutically acceptable carrier is pharmaceutically acceptable for use in humans.

As used herein, the term“physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

A“subject” of diagnosis or treatment is an animal, including a human. It also includes pets and livestock.

As used herein, a“subject in need thereof” is a patient, animal, mammal, or human, who will benefit from the method of this invention.

The term“symptom,” as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a“sign” is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse and other observers.

A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs. A “therapeutically effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.

The term“topical application,” as used herein, refers to administration to a surface, such as the skin. This term is used interchangeably with “cutaneous application” in the case of skin. A“topical application” is a“direct application”.

By“transdermal” delivery is meant delivery by passage of a drug through the skin or mucosal tissue and into the bloodstream. Transdermal also refers to the skin as a portal for the administration of drugs or compounds by topical application of the drug or compound thereto. “Transdermal” is used interchangeably with “percutaneous.”

As used herein, the term“treating” may include prophylaxis of the specific injury, disease, disorder, or condition, or alleviation of the symptoms associated with a specific injury, disease, disorder, or condition and/or preventing or eliminating said symptoms. A“prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. “Treating” is used interchangeably with“treatment” herein. In the context of stroke, treating in accordance with the presently disclosed subject matter includes providing a decrease in the volume of infarcted brain.

Synthetic Peptide Compositions

Disclosed herein is a cell penetrating, synthetic peptide that disrupts the MBD2:NuRD complex in human cells. The presently disclosed subject matter is based at least in part on the observation that enforced expression of a small peptide from the GATAD2A protein (also known as p66a) could disrupt complex formation and block DNA methylation dependent gene silencing of fetal hemoglobin.¹ This effect has been a long-standing goal of the hemoglobin gene regulation field of science, since it represents a potent method for treating patients with b- hemoglobinopathies (sickle cell anemia and b-thalassemia). The presently disclosed subject matter validates the NuRD complex as a target for fetal hemoglobin regulation² and developed a modified peptide that can enter the cell and disrupt formation of the NuRD complex. The disclosed synthetic peptides can also be considered, and are sometimes interchangeably referred to, peptide mimetics. In some aspects, a peptide mimetic can be a molecule such as a peptide, a modified peptide or any other molecule that biologically mimics the activity of another compound or peptide, including for example, but not limited to, active ligands of hormones, cytokines, enzyme substrates, viruses or other bio-molecules.

The disclosed modified cell penetrant peptides were developed by testing a variety of adducts and modifications. In some embodiments, the disclosed synthetic peptides are based on the GATAD2A coiled-coil domain that incorporates one or more of the following features and as illustrated in Figure 1 :

1 . A cell penetrating and/or nuclear localizing adduct, such as the nuclear localization signal from the SV40 large T-antigen. This adduct can in some aspects promote cell penetration, nuclear localization, and increase binding affinity for the target.

2. A non-natural amino acid, for example norleucine (Nle), in place of or in addition to methionine. This modification can in some embodiments increase binding affinity, and/or in some embodiments reduce protease sensitivity.

3. An added cholic acid to the N-terminus or other adduct, e.g. a cyclic peptide, that promotes endosomal escape. This adduct can in some embodiments promote endosomal escape, which in some aspects can aid in for intracellular activity.

4. Optionally, in some instances, two or more cysteine residues (or other residues capable of chemically reacting with a stapling reagent) introduced on the exposed surface of the helix, and an added chemical staple between these cysteine residues. While not necessary for high-affinity binding, in some embodiments an optional staple can prevent proteolytic degradation and/or improve cell penetration.

While the primary goal of this research effort has been to treat b- hemoglobinopathies, extensive research has shown that the MBD2-NuRD complex is a potential therapeutic target for treating a wide variety of cancers. It has been shown that knockout of the MBD2 protein inhibits growth of triple-negative breast cancers,³ other laboratories have shown that knockout of MBD2 blocks the development of colon cancer in mouse models of Familial Adenomatous Polyposis.⁴ More recent work has shown that knockout of CHD4 (another component of MBD2-NuRD) sensitizes human leukemia to standard therapy and blocks colony formation in soft agar.⁵ A review summarizing the potential of targeting the MBD family of proteins, including additional disease targets, was recently published.⁶

Targeting the MBD2-NuRD Complex

The A/t/cleosome Remodeling and Deacetylase (NuRD) complex has been identified throughout the animal kingdom and plays a critical role in DNA methylation dependent gene silencing during normal development,^{7 8} reprogramming of pluripotent stem cells,^9-11 and aberrant silencing of tumor suppressor genes during carcinogenesis.^{3 12} The methylcytosine binding domain 2 (MBD2) protein recruits and assembles the NuRD complex (Figure 2) and thereby uniquely combines binding specificity for methylated DNA with histone deacetylation and chromatin remodeling. Disrupting MBD2 function restores expression of silenced genes while leading to only mild phenotypic changes.^{3 4 13} Applicants previously solved the structure of a key coiled-coil interaction between the MBD2 and GATAD2A proteins in NuRD and showed that enforced expression of the GATAD2A coiled-coil peptide (amino acid residues 138-178) blocks methylation dependent gene silencing by MBD2.¹ Applicants developed chemically modified cell permeable peptides that can inhibit the MBD2:GATAD2A interaction intracellularly. This chemically modified peptide has the potential to treat a variety of human diseases, including restoring expression of fetal hemoglobin to treat b-hemoglobinopathies (sickle cell anemia and b-thalassemia) as well as treating different cancer types including, but not limited to, breast, colon, and brain cancer and bone marrow neoplasms (acute and chronic leukemia and myelodysplastic syndromes).⁶

Although effective for treating myelodysplasia and acute myeloid leukemia, current anti-methylating agents block DNA methylation globally and are associated with significant off-target toxicity.^{14 15} In contrast, knockout of MBD2 in mouse models of cancer markedly decreases the development of tumors yet had only mild phenotypic effects.⁴ Hence targeting MBD2 should produce many of the benefits of DNA methylation inhibitors without the same level of toxicity. Inhibition of the MBD2-NuRD complex restores expression of methylated genes

MBD2-NuRD has been implicated in methylation dependent gene silencing in normal development.^{8 16} As direct evidence of this role, knockout of MBD2 in bUAO transgenic mice, a model of human globin regulation, leads to persistent expression of g-globin in the adult but otherwise causes minimal phenotypic changes.⁷’¹³ In fact, a recent study found that MBD2 knockout mice show only subtle changes in locomotor activity, weight, and nesting behavior.¹⁷ Persistent expression of g-globin is interesting as a possible treatment of sickle cell anemia. Individuals with the sickle cell mutation who continue to express higher levels of y- globin post-infancy are largely asymptomatic for sickle cell disease.¹⁸ _’ ¹⁹ The presence of g-globin prevents polymerization of sickle globin, making the reversal of g-globin gene silencing an intriguing target for sickle cell treatment.^18-20Thus, the results from MBD2 knockout experiments provide strong support for the hypothesis that inhibition of MBD2:GATAD2A can restore gene expression of methylated genes with significantly fewer off-target effects than current options.

Hypermethylation of tumor suppressor genes represents a pro-oncogenic change found in a wide range of malignancies

Importantly, the NuRD complex binds to the largest number of aberrantly hypermethylated CpG islands in cancer as compared to other MBD proteins^22-24 and silences expression of tumor suppressor genes.^{3 12} Genetic knockdown or knockout of MBD2 in a variety of cancer models, ranging from breast cancer to glioblastoma, markedly reduces the growth of neoplastic cells with only limited effects on normal cells.³ _’ ^25-27 As such, NuRD represents an exciting therapeutic target to slow tumor growth and prevent metastasis with a low level of systemic toxicity.²⁸

MBD2: GA TAD2A

An interaction key for NuRD silencing activity is between the coiled-coil domain of MBD2 and the N-terminal conserved region 1 (CR1 ) of GATAD2A.²⁹ It has been shown that disruption of this interaction through overexpression of GATAD2A CR1 rescues expression of fetal globin in cultured cells, though not to the same degree as MBD2 knockdown.¹ Through co-immunoprecipitation experiments, it was discovered that enforced expression of FLAG tagged GATAD2A CR1 captured all NuRD components except the CHD3 protein and endogenous GATAD2A, leading to the hypothesis that GATAD2A acts as a protein link between CHD3 and the rest of the NuRD complex, and losing the CHD3 interaction with the rest of the complex is what causes loss of silencing.¹ The defined structure of both domains involved in the MBD2:GATAD2A interaction make this a more accessible target for therapy.

MBD2 and GATAD2A form an anti-parallel coiled-coil (Figure 3). Coiled-coil interactions are formed when two or more a-helices bind and essentially form a coil of coils.³⁰’³¹ Further probing of this interaction through truncations of GATAD2A shows the central 25 amino acids of GATAD2A CR1 are sufficient to bind MBD2, while only the N-terminal portion or C-terminal portion of GATAD2A CR1 does not bind.¹

Development of a synthetic inhibitor of MBD2.GA TAD2A

As disclosed herein, Applicants have developed a cell-permeable peptide mimetic of GATAD2A that is capable of binding MBD2 and disrupting the MBD2:GATAD2A interaction and thereby function as a novel molecular therapeutic for treating b-hemoglobinopathies and cancer, depicted in Figure 4.

The central 26 amino acids capable of disrupting the MBD2:GATAD2A interaction, residues 144-169, acted as a scaffold for the development of this peptide. With the structural information available, it was decided to replace residues at the i and i+4 positions along the solvent-exposed face with a covalent link, or staple. Without being bound by any particular theory or mechanism of action, this should prevent the staple from interfering with the binding interface and provide better stabilization of the peptide in the appropriate secondary structure. Peptides are often quickly degraded in a proteolytic environment such as the cytoplasm of the cell, and stapling also helps to protect peptides from this proteolytic degradation. The stapling reagent utilized in these studies is a cysteine-reactive reagent and provides a thioether linked staple as shown in Figure 5. Table 1 : Table of Peptide Sequences.

Sequences are modifications of Residues 144-169 of GATAD2A. Letters represent the one-letter amino acid codes, with the exceptions noted. Peptides have either a free N-terminus or the N-terminus is capped with FAM, indicated in the text by #_FAM. “CA-“: cholic acid at the N-terminus.“-NH2” indicates C-terminal amide. Additions to the parent sequence are underlined. Mutations are in bold. Cysteine residues (C) are the linkage points for peptide staples.

Synthetic Peptide Compounds

In some embodiments, disclosed herein are synthetic peptide compounds.

These synthetic peptide compounds can in some aspects comprise synthetic peptide compound comprising a synthetic peptide compound that disrupts GATAD2A/MBD2 coiled coil, wherein the synthetic peptide compound is configured to disrupt an interaction between methylcytosine binding domain 2 (MBD2) and GATAD2A. The synthetic peptide compound can comprise a GATAD2A peptide mimetic compound configured to disrupt an interaction between methylcytosine binding domain 2 (MBD2) and Nucleosome Remodeling and Deacetylase (NuRD). Moreover, these synthetic peptide compounds can in some aspects comprise a GATAD2A peptide mimetic compound, wherein the GATAD2A peptide mimetic compound is configured to disrupt an interaction between methylcytosine binding domain 2 (MBD2) and Nucleosome Remodeling and Deacetylase (NuRD). In some embodiments, the GATAD2A peptide mimetic compound is cell permeable. In some aspects, the GATAD2A peptide mimetic compound inhibits a MBD2:GATAD2A interaction intracellularly. In some embodiments, the GATAD2A peptide mimetic compound comprises a GATAD2A coiled-coil domain.

In some embodiments, such synthetic peptide compounds can comprise a cell penetrating and/or a nuclear localization signal, a non-natural amino acid in place of or in addition to a methionine, an added cholic acid to a N-terminus of the peptide, or other adduct, e.g. a cyclic peptide, that promotes endosomal escape, and two or more residues introduced on an exposed surface of a helix of the peptide and capable of chemically reacting with a stapling agent, optionally two or more cysteine residues. In some aspects, the nuclear localization signal is from a SV40 large T-antigen. In some embodiments, the nuclear localization signal promotes cell penetration, nuclear localization, and/or increases binding affinity for the target. In some embodiments, the non-natural amino acid comprises norleucine. In some embodiments, the norleucine increases binding affinity, and/or reduces protease sensitivity. In some embodiments, the peptide further comprises one or more chemical staples between the two cysteine residues. In some embodiments, the one or more chemical staples improves binding, improves helicity, reduces proteolytic degradation and/or improves cell penetration. In some embodiments, the peptide further comprises a non-natural amino acid to increase stabilization of the peptide via the chemical staple.

In some embodiments, a synthetic peptide compound as disclosed herein can comprise a peptide as follows: YNIelKQLKEELRLEEAKLVLLKKLRQSQ-Nh (Peptide 17; SEQ ID NO. 1 ); KKKRKVYAIKQLKCELRCEEAKLVLLKKLRQSQ-NH2 (Peptide 18; SEQ ID NO. 2);

YpalaOrnOrnGNIelKQLKCELRCEEAKLVLLKKLRQSQ-NH2 (Peptide 19; SEQ ID NO. 3); KKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-NH₂ (Peptide 20; SEQ ID NO. 4); CA-GKKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-Nh (Peptide 25; SEQ ID NO. 5); and CA- KKKRKVYGNIelKQLKEELRLEEAKLVLLKKLRQSQ-NH₂ (Peptide 29; SEQ ID NO. 6), wherein CA- comprises a cholic acid at the N-terminus, -NH2 is a C-terminal amide, and C are linkage points for a chemical staple. In some aspects, such a peptide is preferably selected from CA-

GKKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-NH₂ (Peptide 25; SEQ ID NO. 5); CA-KKKRKVYGNIelKQLKEELRLEEAKLVLLKKLRQSQ-Nh (Peptide 29; SEQ ID NO. 6); and a peptide having about 50% to about 99% homology, or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, to any of Peptides 17, 18, 19, 20, 25 and 29, wherein CA- comprises a cholic acid at the N-terminus, -NH2 is a C-terminal amide, and C are linkage points for a chemical staple. In some aspects, such a peptide compound comprises a binding affinity stronger than about 150 nM.

“Synthetic peptides or polypeptides” means a peptide that was made using chemical reagents rather than by an organism. In some aspects, a synthetic peptide or polypeptide can also mean a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods and solution-phase synthesis methods are known to those of skill in the art. Alternatively or in addition, synthetic peptides or polypeptides can be prepared using molecular biology techniques, such as with expression vectors comprising a nucleotide sequence encoding an amino acid sequence and suitable transformed microorganisms. The presently disclosed subject matter thus encompasses the use of recombinant peptides. Examples of solid phase peptide synthesis methods include the BOC method that utilized tert-butyloxcarbonyl as the a-amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protect the a-amino of the amino acid residues, both methods of which are well- known by those of skill in the art.

To ensure that the proteins or peptides obtained from either chemical or biological synthetic techniques is the desired peptide, analysis of the peptide composition should be conducted. Such amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or additionally, the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequencers which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine definitely the sequence of the peptide.

Prior to its use, the peptide can be purified to remove contaminants. In this regard, it will be appreciated that the peptide will be purified to meet the standards set out by the appropriate regulatory agencies. Any one of a number of a conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high-pressure liquid chromatography (HPLC) using an alkylated silica column such as C4 -,C8- or C18- silica. A gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion-exchange chromatography can be also used to separate peptides based on their charge.

Substantially pure peptide obtained as described herein may be purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al.1990.

Peptide Modification and Preparation

Peptide preparation is described in the Examples. It will be appreciated, of course, that the proteins or peptides of the presently disclosed subject matter may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C1 -C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH2), and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide’s C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without effect on peptide activity.

Acid addition salts of the presently disclosed subject matter are also contemplated as functional equivalents. Thus, a peptide in accordance with the presently disclosed subject matter treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the peptide is suitable for use in the presently disclosed subject matter.

The presently disclosed subject matter also provides for modifications of the disclosed peptides. Modified peptides can differ from the disclosed peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. To that end, one or more conservative amino acid changes typically have no effect on peptide function.

Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Also included are peptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or non-standard synthetic amino acids. The peptides of the presently disclosed subject matter are not limited to products of any of the specific exemplary processes listed herein.

It will be appreciated, of course, that the peptides, derivatives, or fragments thereof may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the presently disclosed subject matter are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.

As discussed, modifications or optimizations of peptides of the presently disclosed subject matter are within the scope of the application. Specifically, a peptide sequence identified can be modified to optimize its potency, pharmacokinetic behavior, stability and/or other biological, physical and chemical properties.

Amino Acid Substitutions In certain embodiments, the disclosed methods and compositions may involve preparing peptides with one or more substituted amino acid residues.

In various embodiments, the structural, physical and/or therapeutic characteristics of peptide sequences may be optimized by replacing one or more amino acid residues.

Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the presently disclosed subject matter are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.

The skilled artisan will be aware that, in general, amino acid substitutions in a peptide typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions). The properties of the various amino acids and effect of am ino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.

For example, one can make the following isosteric and/or conservative amino acid changes in the parent polypeptide sequence with the expectation that the resulting polypeptides would have a similar or improved profile of the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: including alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid, S-cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from C1 - 10 carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions.

Substitution of aromatic-substituted hydrophobic amino acids: including phenylalanine, tryptophan, tyrosine, biphenylalanine, 1 -naphthylalanine, 2- naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy-substituted forms of the previous listed aromatic amino acids, illustrative examples of which are: 2- 3- or 4-aminophenylalanine, 2-, 3- or 4- chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4- methoxyphenylalanine, 5-amino- 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2’- 3’-, or 4’-amino-, 2’-, 3’-, or 4’-chloro-, 2,3, or 4-biphenylalanine, 2’, -3’,- or 4’-methyl- 2, 3 or 4-biphenylalanine, and 2- or 3-pyridylalanine.

Substitution of amino acids containing basic functions: including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or aryl-substituted (from C1 -C10 branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms (such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position for example. Compounds that serve as illustrative examples include: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)- alanine, N,N-gamma, gamma’-diethyl-homoarginine. Included also are compounds such as alpha methyl arginine, alpha methyl 2,3-diaminopropionic acid, alpha methyl histidine, alpha methyl ornithine where alkyl group occupies the pro-R position of the alpha carbon. Also included are the amides formed from alkyl, aromatic, heteroaromatic (where the heteroaromatic group has one or more nitrogens, oxygens, or sulfur atoms singly or in combination) carboxylic acids or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives) and lysine, ornithine, or 2,3- diaminopropionic acid.

Substitution of acidic amino acids: including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine.

Substitution of hydroxyl containing amino acids: including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine. It is also understood that the amino acids within each of the categories listed above can be substituted for another of the same group.

For example, the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157: 105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1 .9); alanine (+1 .8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1 .3); proline (-1 .6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making conservative substitutions, the use of amino acids whose hydropathic indices are within +1-2 is preferred, within +/- 1 are more preferred, and within +/- 0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Patent No. 4,554, 101 ). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-0.1 ); alanine (-0.5); histidine (-0.5); cysteine (- 1 .0); methionine (-1 .3); valine (-1 .5); leucine (-1 .8); isoleucine (-1 .8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine. The effect of various amino acid residues on protein secondary structure is also a consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha-helical, beta-sheet or reverse turn secondary structure has been determined and is known in the art (see e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251 -276; 1979, Biophys. J., 26:367- 384).

Based on such considerations and extensive empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. For example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gin, asn, lys; Asn (N) his, asp, lys, arg, gin; Asp (D) asn, glu; Cys (C) ala, ser; Gin (Q) glu, asn; Glu (E) gin, asp; Gly (G) ala; His (H) asn, gin, lys, arg; lie (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gin, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed. For interior residues, conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; lie and Val; Val and Leu; Leu and lie; Leu and Met; Phe and Tyr; Tyr and Trp. (See e.g., PROWL Rockefeller University website). For solvent exposed residues, conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and lie; lie and Val; Phe and Tyr. Various matrices have been constructed to assist in selection of amino acid substitutions, such as the PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in an encoded peptide sequence are well known and a matter of routine experimentation for the skilled artisan, for example by the technique of site-directed mutagenesis or by synthesis and assembly of oligonucleotides encoding an amino acid substitution and splicing into an expression vector construct.

In some embodiments, a synthetic peptide compound as disclosed herein can comprise a peptide as follows:

Stapled Peptide 25 (see also Fig. 8)

Peptide 29 (see also Fig. 9)

In some embodiments, Peptide 29 can also be provided without the Nle at

M144, and instead having Methionine in its place, as shown in Figure 10 and below.

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions for use in treating a subject, the compositions comprising a synthetic peptide compound as disclosed herein. In some embodiments, such a pharmaceutical composition can further comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is pharmaceutically acceptable for use in humans. In some embodiments, the pharmaceutical composition is adapted for treatment of a b-hemoglobinopathy and/or a cancer. In some embodiments, the pharmaceutical composition is adapted for treatment of sickle cell anemia, b- thalassemia, breast cancer, colon cancer, brain cancer, acute leukemia, chronic leukemia and/or myelodysplastic syndromes.

Treatment Methods

Also provided herein are methods of treating a subject. Such methods can in some aspects comprise providing a subject to be treated, and administering to the subject a synthetic peptide compound and/or pharmaceutical composition of any of the above claims. In some embodiments, the subject to be treated is a human subject. In some embodiments, the subject to be treated is suffering from a b-hemoglobinopathy, optionally wherein the b-hemoglobinopathy is selected from sickle cell anemia and b-thalassemia. In some embodiments, the subject to be treated is suffering from a cancer, optionally wherein the cancer is selected from breast cancer, colon cancer, brain cancer, acute leukemia, chronic leukemia and myelodysplastic syndromes.

EXAMPLES

The following examples are included to further illustrate various embodiments of the presently disclosed subject matter. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the presently disclosed subject matter. EXAMPLE 1

Evaluating binding affinity of the synthetic peptide mimetics Peptide 17 was synthesized to act as the starting point for what would be considered the base dissociation constant (Kd ) of the disclosed synthetic peptides. It is the native sequence except for a single mutation, M144 to norleucine, a structural isostere of methionine, an amino acid that is prone to oxidation. Peptide 17 had a Kd of 90 ± 3 nM. Peptide 19 was synthesized with two cysteine mutations (E150C, L154C) for the stapling reaction and two ornithine residues to aid solubility of the peptide. After stapling, the cysteine mutations led to a peptide with a similar affinity of 120 ± 7 nM compared to 90 ± 3 nM for peptide 17 (Table 2).

To create a cell-permeable version of the stapled peptide, a nuclear localization signal (NLS) was added to peptide 17, giving peptide 20. This led to a small improvement in binding affinity. Mutating M144 to alanine, the canonical amino acid with the highest helical propensity, while keeping the NLS and same cysteine mutations created peptide 18. This single mutation of the norleucine (Nle) to alanine led to two-fold weaker binding; therefore, comparing peptides 18 and 20 shows that the M144 alanine mutation is detrimental to binding compared to the M144 norleucine mutation (Table 2). Table 2: Binding Affinities Measured by Isothermal Titration Calorimetry (ITC).

otherwise indicated. CA-: cholic acid is attached to the N-terminus. Data represent the average of 2 replicates, and the error is the standard deviation of the replicates. (-) there are no cysteines for stapling. (^*) only one replicate. NB: no binding detected at concentrations used.

EXAMPLE 2

Evaluating Cell Penetration

To test for functional cell penetration, an intracellular NanoBRET assay of stable interaction between MBD2 and GATAD2A coiled-coil domains was developed.³³ Disruption of this interaction in the cell is expected to give a reduced BRET (bioluminescence resonance energy transfer) signal. Peptide 20, a stapled peptide containing the M144Nle mutation and the NLS for nuclear localization and/or cell penetration, showed minimal activity in the BRET assay (Figure 6). Fluorescence microscopy studies suggested that this is because peptide 20 is largely trapped in endosomes. Addition of cholic acid to the N-terminus of the peptide,³⁴ generating peptide 25, allowed for endosomal escape of the peptide, resulting in a reduction in the NanoBRET signal, which indicates disruption of the GATAD2A:MBD2 interaction.

The addition of the NLS and cholic acid to peptide 17, which contains no stapling sites, resulted in peptide 29. Comparing the binding affinity of peptide 17 and peptide 29 shows the NLS and cholic acid caused the binding of peptide 29 to become two-fold stronger (Table 2). Using the intracellular NanoBRET assay, both peptides 25 (stapled with NLS and cholic acid) and 29 (NLS and cholic acid) showed activity (Figure 7). Peptide 30 acted as a negative control with glycine mutations at the binding interface, which prevented binding to MBD2 as verified by ITC, Table 2. The presence of the NLS and cholic acid on peptide 30 and keeping the net charge the same as 29 allowed this to act as a proper negative control.

Peptide 25 and peptide 29 both show a dose dependent response and are still active after 20 hours, while the negative control 30 shows no signal change. Without being bound by any particular theory or mechanism of action, the slightly greater activity of peptide 29 compared to 25 is most likely explained by the tighter binding affinity.

Peptide 25 (Figure 8) and peptide 29 (Figure 9) are both capable of disrupting the MBD2:GATAD2A interaction intracellulary. The addition of the NLS and cholic acid created cell-permeable peptides, while also increasing binding affinity. The mutation of M144 to norleucine instead of alanine also increased binding affinity. In some embodiments, Peptide 29 can also be provided without the Nle at M144, and instead having Methionine in its place, as shown in Figure 10.

EXAMPLE 3

Evaluating Biological Efficacy

Previous work has shown that knockdown of the CHD4 protein from the NuRD complex sensitizes leukemic cells to DNA damaging chemotherapy (daunorubicin)⁵. Because GATAD2A/B acts as structural link between the CHD4 and MBD2 proteins, tests were conducted to determine whether Peptide 29 can act to“chemically” knock down CHD4, and thus inhibit the growth of leukemia cells in tissue culture. As can be seen in Figs 1 1 A-1 1 C, as compared to the negative control (Peptide 30), Peptide 29 induces apoptosis in three different human leukemia cell lines (monocytic - U937, myeloid - MV41 1 , and T-lymphoblastic leukemia - Jurkat). Importantly, Peptide 29 also induces apoptosis in primary patient-derived acute myeloid and T-lymphoblastic leukemia (Figs. 12A-12C). Yet, the peptide does not inhibit cell growth or induce apoptosis in CD34+ patient-derived bone marrow progenitor cells (Fig. 13). Together, these studies indicate that the peptide effectively disrupts NuRD and selectively induces apoptosis in a variety of leukemia cells but not in normal bone marrow progenitors, i.e. non-cancerous cells.

REFERENCES

All references listed herein including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (e.g., GENBANK® database entries and all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

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(34) Beaudoin, S.; Rondeau, A.; Martel, O.; Bonin, M.-A.; van Lier, J. E.; Leyton, J. V. Mol. Pharm. 2016, 13 (6), 1915-1926. It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is:

1 . A synthetic peptide compound, the synthetic peptide compound comprising a synthetic peptide compound that disrupts GATAD2A/MBD2 coiled coil, wherein the synthetic peptide compound is configured to disrupt an interaction between methylcytosine binding domain 2 (MBD2) and GATAD2A.

2. The synthetic peptide compound of claim 1 , wherein the synthetic peptide compound comprises a GATAD2A peptide mimetic compound configured to disrupt an interaction between methylcytosine binding domain 2 (MBD2) and Nucleosome Remodeling and Deacetylase (NuRD).

3. The synthetic peptide compound of any of the above claims, wherein the synthetic peptide compound is cell permeable.

4. The synthetic peptide compound of any of the above claims, wherein the synthetic peptide compound inhibits a MBD2:GATAD2A interaction intracellularly.

5. The synthetic peptide compound of any of the above claims, wherein the synthetic peptide compound comprises:

a cell penetrating and/or a nuclear localization signal;

a non-natural amino acid in place of or in addition to a methionine;

an added cholic acid to a N-terminus of the peptide, or other adduct that promotes endosomal escape; and

two or more residues introduced on an exposed surface of a helix of the peptide and capable of chemically reacting with a stapling agent, optionally two or more cysteine residues.

6. The synthetic peptide compound of claim 5, wherein the nuclear localization signal is from a SV40 large T-antigen.

7. The synthetic peptide compound of claim 6, wherein the nuclear localization signal promotes cell penetration, nuclear localization, and/or stronger binding affinity for the target as compared to a peptide without a nuclear localization signal.

8. The synthetic peptide compound of claim 5, wherein the non-natural amino acid comprises norleucine.

9. The synthetic peptide compound of claim 8, wherein the norleucine increases binding affinity, and/or reduces protease sensitivity.

10. The synthetic peptide compound of claim 5, further comprising one or more chemical staples between the two residues, optionally two cysteine residues.

1 1 . The synthetic peptide compound of claim 10, wherein the one or more chemical staples improves binding, improves helicity, reduces proteolytic degradation and/or improves cell penetration as compared to the composition without the staples.

12. The synthetic peptide compound of claim 1 1 , further comprising a non-natural amino acid to increase stabilization of the peptide via the chemical staple.

13. The synthetic peptide compound of any of the above claims, wherein the synthetic peptide compound is selected from the group consisting of:

YNIelKQLKEELRLEEAKLVLLKKLRQSQ-NH₂ (Peptide 17; SEQ ID NO: 1 ); KKKRKVYAIKQLKCELRCEEAKLVLLKKLRQSQ-NH2 (Peptide 18; SEQ ID NO: 2);

YpalaOOGNIelKQLKCELRCEEAKLVLLKKLRQSQ-NH₂ (Peptide 19; SEQ ID NO: 3);

KKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-NH₂ (Peptide 20; SEQ ID NO: 4); CA-GKKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-Nhte (Peptide 25; SEQ ID NO: 5);

CA-KKKRKVYGNIelKQLKEELRLEEAKLVLLKKLRQSQ-NH₂ (Peptide 29; SEQ ID NO: 6); and

a peptide having about 50% to about 99% homology to any of Peptides 17,

18, 19, 20, 25 and 29,

wherein CA- comprises a cholic acid at the N-terminus, -NH2 is a C-terminal amide, and C are linkage points for a chemical staple.

14. The synthetic peptide compound of any of the above claims, wherein the synthetic peptide compound is selected from the group consisting of:

CA-GKKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-Nh (Peptide 25; SEQ ID NO: 5);

CA-KKKRKVYGNIelKQLKEELRLEEAKLVLLKKLRQSQ-NH₂ (Peptide 29; SEQ ID NO: 6); and

a peptide having about 80% to about 99% homology to Peptide 25 or Peptide 29,

wherein CA- comprises a cholic acid at the N-terminus, -NH₂ is a C-terminal amide, and C are linkage points for a chemical staple.

15. The synthetic peptide compound of claim 14, wherein the synthetic peptide compound comprises Peptide 25 or Peptide 29.

16. The synthetic peptide compound of any of the above claims, wherein the synthetic peptide comprises a binding affinity stronger than about 150 nM.

17. The synthetic peptide compound of any of the above claims, wherein the synthetic peptide compound is selected from:

; and

18. A pharmaceutical composition for use in treating a subject, the composition comprising a synthetic peptide compound of any of the above claims.

19. The pharmaceutical composition of claim 17, wherein the composition further comprises a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of any of claims 18-19, wherein the pharmaceutically acceptable carrier is pharmaceutically acceptable for use in humans.

21. The pharmaceutical composition of any of claims 18-20, wherein the pharmaceutical composition is adapted for treatment of a b-hemoglobinopathy and/or a cancer.

22. The pharmaceutical composition of claim 21 , wherein the pharmaceutical composition is adapted for treatment of sickle cell anemia, b- thalassemia, breast cancer, colon cancer, brain cancer, acute leukemia, chronic leukemia and/or myelodysplastic syndromes.

23. The pharmaceutical composition of any of claims 18-22, wherein the pharmaceutical composition selectively induces apoptosis in one or more cancer cells by disrupting a NuRD complex in the one or more cancer cells.

24. A method of treating a subject, the method comprising:

providing a subject to be treated; and

administering to the subject a synthetic peptide compound and/or pharmaceutical composition of any of the above claims.

25. The method of claim 24, wherein the subject to be treated is a human subject.

26. The method of any of claims 24-25, wherein the subject to be treated is suffering from a b-hemoglobinopathy, optionally wherein the b- hemoglobinopathy is selected from sickle cell anemia and b-thalassemia.

27. The method of any of claims 24-25, wherein the subject to be treated is suffering from a cancer, optionally wherein the cancer is selected from breast cancer, colon cancer, brain cancer, acute leukemia, chronic leukemia and myelodysplastic syndromes.

28. The method of any of claims 24-27, wherein the synthetic peptide compound is selected from:

CA-GKKKRKVYGNIelKQLKCELRCEEAKLVLLKKLRQSQ-Nhte (Peptide 25; SEQ ID NO: 5);

CA-KKKRKVYGNIelKQLKEELRLEEAKLVLLKKLRQSQ-NH₂ (Peptide 29; SEQ ID NO: 6); and

a peptide having about 80% to about 99% homology to Peptide 25 or Peptide 29,

wherein CA- comprises a cholic acid at the N-terminus, -Nhte is a C-terminal amide, and C are linkage points for a chemical staple.

29. The method of any of claims 24-28, wherein the synthetic peptide compound comprises Peptide 25 or Peptide 29.

30. The method of any of claims 24-28, wherein the synthetic peptide compound is selected from:

>

; and

31. The method of any of claims 24-30, wherein apoptosis is selectively induced in one or more cells in the subject by disrupting a NuRD complex in the one or more cells.

32. The method of claim 31 , wherein the one or more cells comprise one or more cancer cells.

33. The method of claim 32, wherein apoptosis is not induced in non- cancerous cells.