Use of MEF2 binding molecules to inhibit HDAC-MEF2 interaction

Abstract

An isolated myocyte enhancer factor-2 (MEF2) binding molecule comprising an amino acid sequence of: X1 X2 X3 X4 A X6 A S X9 X10 V K X13 X14 L X16 E X18 X19 L X21 K X23 X24 X25 X26, in which X1 is S or G, or is removed; X2 is K or R; X3 is R or E; X4 is S or R; X6 is V or I; X9 is S or T; X10 is V or E; X13 is Q, M, or L; X14 is K or R; X16 is A or Q; X18 is V or F; X19 is I, V, or L; X21 is K, N, or S; X23 is Q, K, or S; X24 is Q or A, or is removed; X25 is A or T, or is removed; and X26 is A or K. In addition, use of the isolated MEF2 binding molecule to inhibit binding of histone deacetylase (HD AC) and MEF2 in a cell, and to inhibit HDAC- induced suppression of MEF2-dependent gene expression in a cell.

Description

Atty. Docket No. Georgetown.051. WOlTITLE USE OF MEF2 BINDING MOLECULES TO INHIBIT HDAC-MEF2 INTERACTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U. S. Provisional Application No. 63 / 720,123, filed on November 13, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said.xml copy, created on November 13, 2025, is named Georgetown_051_WOl-Sequence_Listing, and is 36,019 bytes in size.BACKGROUND OF THE INVENTION

[0003] Neurodegenerative diseases, which are characterized by the progressive injury of nerve cells, predominantly in the brain, affect millions of people globally. It is estimated that there are more than 55 million suffering from Alzheimer's and other dementias worldwide [1], with about 6.9 million having Alzheimer’s disease in the U. S. only [2], Further, there are more than 10 million worldwide living with Parkinson’s disease, including nearly one million in the U. S. [3], Significantly, most neurodegenerative diseases, including Alzheimer’s and Parkinson’s, currently have no cure. Therefore, there is an urgent need to develop new strategies to combat this public health issue.

[0004] Myocyte enhancer factor-2 (MEF2) proteins are involved in neuronal differentiation and survival in the central nervous system [4-8], MEF2 proteins are highly expressed in cerebellar granule neurons (CGNs) [9], and play a key role as a central regulator in various developmental programs, including control of neural crest development as well as neuronal differentiation and survival

[0010] , There are four types of MEF2 proteins in humans: MEF2A, MEF2B, MEF2C, and MEF2D

[0010] , Vertebrate MEF2 proteins, MEF2A, B, C and D, all have a highly conserved N-terminal region (residues 1-93) and a C-terminal region that isAtty. Docket No. Georgetown.051. WOlmore diverse

[0011] . Downregulation of certain genes by MEF2A has also been linked to characterize Alzheimer’s disease

[0012] ,

[0005] Histone deacetylases (HDACs) has been found to play critical role in the regulation of neuronal cell death via interactions with MEF2s

[0013] . Class II HDACs are a subclass of the HDAC family that are involved in regulating a variety of cellular processes, including cell differentiation, proliferation, and apoptosis

[0014] , Class Ila HDACs, which include HDAC4, HDAC5, HDAC7, and HDAC9 [15, 16], have highly conserved MEF2 binding domains

[0017] , This class of HDACs interact with MEF2s to repress MEF2-dependent gene expression

[0018] , HDAC-MEF2 transcription complexes have associations with neuronal survival as well as axon branching [13, 19, 20, 21], Protection of neurons from apoptotic death in neurodegenerative diseases occurs upon enhancement of MEF2 transcriptional activity

[0012] , In the event when MEF2 dissociates from HDAC, there is expression of prosurvival genes because MEF2 retains its activity

[0012] , Class Ila HDACs, with critical roles in regulation of neuronal apoptosis, are also abundantly expressed in the brain [17, 22-24], Therefore, development of new strategies to block binding of these HDACs to MEF2s will be an effective strategy to combat neurodegenerative diseases.SUMMARY OF INVENTION

[0006] The present invention is based in part on the development of new MEF2 binding molecules that can inhibit HDAC-MEF2 binding.

[0007] Thus, in one aspect, the present invention provides an isolated MEF2 binding molecule comprising an amino acid sequence of:Xi X2X3X4 A X6A S X9X10 V K X13 Xi4L Xi6E Xis X19 L X21 K X23 X24 X25 X26 (SEQ ID NO: 1), in which:Xi is S or G, or is removed;X2is K or R;X3 i s R or E;X4 is S or R;Xb is V or I;Atty. Docket No. Georgetown.051. WOlX9 is S or T;Xio is V or E;X13 is Q, M, or L;Xi4 is K or R;Xi6 is A or Q;Xis is V or F;X19 is I, V, or L;X21 is K, N, or S;X23 is Q, K, or S;X24 is Q or A, or is removed;X25 is A or T, or is removed; andX26 is A or K.

[0008] In some embodiments, the isolated MEF2 binding molecule comprises the amino acid sequence of SEQ ID NO: 1, in which X2 is K, X4 is S, Xs is V, X13 is Q or M, X14 is K, X19 is I or V, X21 is K or N, X23 is Q or K, X24 is Q or is removed, and X25 is A or is removed. In certain embodiments, the isolated MEF2 binding molecule comprises the amino acid sequence of SKRSAVASSVVKQKLAEVILKKQQAA (SEQ ID NO: 4). In other embodiments, the isolated MEF2 binding molecule comprises the amino acid sequence of KRSAVASSVVKQKLAEVILKKQQAALERTVHPN (SEQ ID NO: 5).

[0009] In some embodiments, the isolated MEF2 binding molecule comprises the amino acid sequence of SEQ ID NO: 1, in which Xi is S or G, X3 is E, X9 is T, Xio is E, Xi6 is Q, Xis is F, Xi9 is V or L, X21 is N or S, X23 is K or S, X24 is A or is removed, X25 is T or is removed, and X26 is K In certain embodiments, the isolated MEF2 binding molecule comprises the amino acid sequence of GKESAVASTEVKMKLQEFVLNKKK (SEQ ID NO: 6). In other embodiments, the isolated MEF2 binding molecule comprises the amino acid sequence of SKESAIASTEVKLRLQEFLLSKSK (SEQ ID NO: 7). In yet other embodiments, the isolated MEF2 binding molecule comprises the amino acid sequence of GRERAVASTEVKQKLQEFLLSKSATK (SEQ ID NO: 8).Atty. Docket No. Georgetown.051. WOl

[0010] Ill some embodiments, the isolated MEF2 binding molecule binds to MEF2A, MEF2B, MEF2C, or MEF2D. In certain embodiments, the isolated MEF2 binding molecule binds to MEF2A

[0011] In one aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the isolated MEF2 binding molecule of the present invention.

[0012] In another aspect, the present invention provides a vector comprising the isolated nucleic acid molecule of the present invention.

[0013] In yet another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention.

[0014] In one aspect, the present invention provides a composition comprising the isolated MEF2 binding molecule of the present invention and a carrier.

[0015] In a further aspect, the present invention provides a method of inhibiting binding of HDAC and MEF2 in a cell, the method comprising contacting the cell with the isolated MEF2 binding molecule of the present invention or with the composition of the present invention. In some embodiments, the I ID AC is selected from HDAC7, HDAC4, HDAC5, and HDAC9. In some embodiments, the MEF2 is selected from MEF2A, MEF2B, MEF2C, and MEF2D. In certain embodiments, the HD AC is HDAC7 and the MEF2 is MEF2A.

[0016] In yet a further aspect, the present invention provides a method of inhibiting HDAC-induced suppression of MEF2-dependent gene expression in a cell, the method comprising contacting the cell with the isolated MEF2 binding molecule of the present invention or the composition of the present invention. In some embodiments, the HD AC -induced suppression of MEF2-dependent gene expression is selected from HDAC7-induced suppression, HDAC4-induced suppression, HDAC5-induced suppression, and HDAC9-induced suppression. In some embodiments, the MEF2-dependent gene expression is selected from MEF2A-dependent gene expression, MEF2B-dependent gene expression, MEF2C-dependent gene expression, and MEF2D-dependent gene expression.Atty. Docket No. Georgetown.051. WOlBRIEF DESCRIPTION OF THE DRAWING FIGURES

[0017] FIG. 1 shows complex structures of the HDAC7-MEF2A complex predicted by AlphaFold2, as described in Example 1. Panel A shows HDAC7-MEF2A complex structures using full-length HDAC7 amino acid sequence. Panel B shows HDAC7-MEF2A complex structure predicted in Panel A (without zoom) with unstructured loops removed for molecular dynamics (MD) simulation. Panel C shows root mean square deviation (RMSD) measurements for 280 ns all atom MD simulation of the HDAC7-MEF2A complex in Panel B. Panel D shows HDAC7-MEF2A complex at 280 ns simulation. Panels E and F HDAC7-MEF2A complex structures using HDAC7 sequence with amino acids from position 1 to 200 (Panel E), and 72 to 172 (Panel F). Panel G shows HDAC7-MEF2A complex prepared for MD simulations. The MEF2A sequence with amino acids from positions 2 to 91 were used. Light orange and light blue colors represent two monomers of the MEF2A dimer.

[0018] FIG. 2 shows results relating to RMSD measurements of MD simulations for the HDAC7-MEF2A complex, as described in Example 1. Panel A shows RMSD measurements from 500 ns simulation trajectory of the HDAC7-MEF2A complex. Panel B shows HDAC7-MEF2A complex structure after 500 ns of simulation time.

[0019] FIG. 3 shows RMSD measurements for HDAC7 and MEF2A, both isolated and as a complex, as described in Example 1. Panel A shows RMSD measurements for HDAC7-MEF2A complex. Panel B shows RMSD measurements for individual HDAC7 in the HDAC7-MEF2A complex. Panel C shows RMSD measurements for individual MEF2A in the HDAC7-MEF2A complex in all four independent 500 ns runs of the HDAC7-MEF2A complex. Panel D shows RMSD measurements for isolated HDAC7 and MEF2A structures.

[0020] FIG. 4 shows representative results associated with the salt bridge responsible for establishing the linkage between HDAC7 and MEF2A, as described om Example 1. Panel A shows results relating to the salt bridge responsible for the HDAC7-MEF2A complex formation. Panel B shows location of LYS96 and ASP63 in HDAC7 and MEF2A, respectively, in which the amino acid residues are shown in licorice representations.

[0021] FIG. 5 shows data relating to salt bridges for the independent MD simulation runs for RUN2 (Panel A), RUN3 (Panel B), and RUN4 (Panel C), as described in Example 1.Atty. Docket No. Georgetown.051. WOl

[0022] FIG. 6 shows representative results associated with the hydrogen bond established by each atom pair from the analysis of the MD simulation run trajectory for RUN1, as described in Example 1. Panel A shows distance of hydrogen bonds formed between the residues in HDAC7 and MEF2A. The type of atoms that are predicted to establish the contacts are provided inside parentheses of each residue. Panel B shows representative interfacial complex structure showing locations of residues in both HDAC7 and MEF2A that form the hydrogen bonds. The crucial residues are shown in licorice representation and the black dotted lines represent the hydrogen bond

[0023] FIG. 7 shows distance of hydrogen bonds formed between the residues in HD AC7 and MEF2A from the analysis of the MD simulation ran trajectory for RUN2 (Panel A), RUN3 (Panel B), and RUN4 (Panel D), as described in Example 1.

[0024] FIG. 8 shows representative snapshots of hydrophobic residues in HDAC7 (purple texts) that establish hydrophobic interactions with MEF2A residues (orange texts for Chain A and blue texts for Chain B) (Panel A), and hydrophobic cavities (Cl, C2, and C3) formed by LEU residues in MEF2A (Panel B), as described in Example 1.

[0025] FIG. 9 shows multiple sequence alignment of class Ila HDACs (Panel A (SEQ ID NOS: 4 and 6-8)) and MEF2s (Panel B (SEQ ID NOS: 9-12)), as described in Example 1.

[0026] FIG. 10 shows the results associated with the effect of MEF2A-DNA binding on HDAC7-MEF2A binding, as described in Example 1. Panel A shows structure of the HDAC7-MEF2A-DNA complex at the end (500 ns) of MD simulation. Networks of hydrophobic interactions are shown in surface and CPK representations on the top of the “pyramid”. Panel B shows contact surface area between HDAC7 and MEF2A in the HDAC7-MEF2A (without DNA) and HDAC-MEF2A-DNA (with DNA) complexes. The red curve is the average contact area measured from rims RUN1-4, and the blue curve is the contact area between HDAC7 and MEF2A measured from the HD / XC7-MEF2A-DNA complex. All other traces in faint colors are contact areas measured for individual runs (RUN 1-4) of the HDAC7-MEF2A complex.

[0027] FIG. 11 shows results associated with the RMSD measurements of the HDAC7-MEF2A-DNA complex, as described in Example 1. Panel A shows RMSD measurements from the 500ns trajectory of FIDAC7-MEF2A-DNA complex. Panel B shows distances forAtty. Docket No. Georgetown.051. WOlsalt bridges and Panel C shows hydrogen bonds between HDAC7 and MEF2A in the HD AC 7-MEF 2 A -DNA com pl ex,

[0028] FIG. 12 shows MEF2A solvent-accessible surface area (SASA) measurements for MEF2A in the HDAC7-MEF2A complex (Panel A) and isolated MEF2A (Panel B), as described in Example 1.

[0029] FIG. 13 shows MEF2A radius of gyration (RG) measurements for MEF2A in the HDAC7-MEF2A complex (Panel A) and isolated MEF2A (Panel B), as described in Example 1

[0030] FIG. 14 shows representative SPR results associated with class Ila HDACs (HDAC9) binding to MEF2A, as described in Example 2. Panel A shows representative figure for HDACs-MEF2A bindings. Panel B shows the synthetic peptide (PT-HD7-Wt)-MEF2A binding. Insets in Panel B are PT-HD7-Wt bindings to glutathione-S-transferase (GST) and poly(ADP-ribose) polymerase 1 (PARP1). GST and PARP1 serve as negative control proteins. Colored lines are experimental data and dotted lines are theoretical fits.

[0031] FIG. 15 shows results associated with inhibition of HDACs-MEF2A binding, as described in Example 3. Panel A shows representative result showing 5 mM HDACs (HDAC9) binding to MEF2A in the absence (red) and presence of crosslinked PT-HD7-Wt (green). Panel B shows percentage (%) bindings of all 5 nM class Ila HD ACs to MEF2 A in the absence (100%, red) and presence of crosslinked PT-HD7-Wt (green). Data for GST (blue) and PARP1 (pink) are also shown as controls. Inset shows % binding of MEF2A to captured HDAC7 in the absence (100%, red) and presence of PT-HD7-Wt (green) in reverse orientation. Binding of PT-HD7-Wt (orange) and GST (blue) alone are shown as controls.

[0032] FIG. 16 shows results associated with binding MEF2A to the synthetic peptide (PT-HD7-Wt)at different concentrations, as described in Example 4. Panel A shows result showing 20 mM MEF2A binding to HDAC7 in the absence of presence of the synthetic peptide. Peptide concentration used werePT-HD7-Wt at concentrations of 37 nM, 333.3 nM, 1000 nM, and 3000 nM. Peptide only concentrations were injected for comparison. Panel B shows percentage (%) bindings of 20 mM MEF2A to HD / XC7 in the presence of the synthetic peptide at concentrations of 37 nM to 3000 nM.Atty. Docket No. Georgetown.051. WOl

[0033] FIG. 17 shows representative class Ila HDAC-MEF2 complex structures at the end of the 500 ns MD simulations, as described in Example 5. Panels A-O shows structures of HDAC4-MEF2A complex, HDAC4-MEF2B complex, HDAC4-MEF2C complex, HDAC4-MEF2D complex, HDAC5-MEF2A complex, HDAC5-MEF2B complex, FIDAC5-MEF2C complex, HDAC5-MEF2D complex, HDAC7-MEF2A complex, HDAC7-MEF2B complex, HDAC7-MEF2C complex, HDAC7-MEF2D complex, HDAC9-MEF2A complex, HDAC9-MEF2B complex, HDAC9-MEF2C complex, and HDAC9-MEF2D complex, respectively.

[0034] FIG. 18 shows representative human class Ila HDAC-MEF2 complexes at 500 nM all-atom MD simulations of protein data bank (PDB) structures, as described in Example 5. Panels A-E shows structures of HDAC4-MEF2A complex, HDAC4-MEF2D complex, HDAC5-MEF2D complex, HDAC7-MEF2D complex, and HDAC9-MEF2D complex, respectively.

[0035] FIG. 19 shows measurements and plots for class Ila HDAC-MEF2 complexes, as described in Example 5. Panels A-D show RMSD measurements for predicted class Ila HDAC4-MEF2 complexes, HDAC5-MEF2 complexes, HDAC-MEF2 complexes, and HDAC9-MEF2 complexes, respectively. Panels E-H show RMSD measurements for class Ila HDAC4-MEF2 complexes with crystal structures, HDAC5-MEF2 complexes with crystal structures, HDAC-MEF2 complexes with crystal structures, and HDAC9-MEF2 complexes with crystal structures, respectively. Panels I-L show RG measurements for class Ila HDAC4-MEF2 complexes with crystal structures, HDAC5-MEF2 complexes with crystal structures, HDAC-MEF2 complexes with crystal structures, and FIDAC9-MEF2 complexes with crystal structures, respectively. Panels M-P show potential energy vs. time plots for predicted class Ila HDAC4-MEF2 complexes, HDAC5-MEF2 complexes, HDAC-MEF2 complexes, and HDAC9-MEF2 complexes, respectively. Panels Q-T show potential energy vs. time plots for class Ila HDAC4-MEF2 complexes with crystal structures, HDAC5-MEF2 complexes with crystal structures, HDAC-MEF2 complexes with crystal structures, and HDAC9-MEF2 complexes with crystal structures, respectively. The same light-colored data correspond to the measurement from three different runs with the respective dark color as the average values for \each triplicate.

[0036] FIG. 20 shows RG measurements for class Ila HDAC4-MEF2 complexes (Panel A), FIDAC5-MEF2 complexes (Panel B), HDAC-MEF2 complexes (Panel C), and IIDAC9-Atty. Docket No. Georgetown.051. WOlMEF2 complexes (Panel D), as described in Example 5. The same light-colored data correspond to the measurement from three different runs with the respective dark color as the average values for each triplicate.

[0037] FIG. 21 shows cosine content calculations for the first three principal components (PCI, PC2, and PC3) for all class Ila HDAC4-MEF2 complexes (Panel A), HDAC5-MEF2 complexes (Panel B), HDAC-MEF2 complexes (Panel C), and HDAC9-MEF2 complexes (Panel D) that were simulated in study described in Example 5. PCI* plots correspond to the cosine contents calculated for the same systems with highly flexible terminal loops removed. The bars represent mean and the error bars represent s.d. values calculated from the cosine contents determined from three independent replica runs for each system.

[0038] FIG. 22 (SEQ ID NOS: 18-21) shows multiple sequence alignment of all MEF2s (Panel A) and locations of all amino acid residues in both class Ila HDACs and MEF2s that establish hydrophobic interactions in representative class Ila HDAC-MEF2 complexes (Panel B), as described in Example 5. HDACs and MEF2 proteins are zoomed-in near the binding interface to highlight the locations of the interacting residues.

[0039] FIG. 23 shows distance-time plots for hydrogen bonds between different amino acid residues in class Ila HDAC4-MEF2 complexes (Panel A), HDAC5-MEF2 complexes (Panel B), HDAC-MEF2 complexes (Panel C), and HD / XC9-MEF2 complexes (Panel D), as described in Example 5. The same light-colored data correspond to the measurement from three different runs with the respective dark color as the average values for each triplicate.

[0040] FIG. 24 shows distance-time plots for hydrogen bonds between different amino acid residues in HDAC4-MEF2A and HDAC4-MEF2D complexes with crystal structures (Panel A), and in HDAC9-MEF2D complex with crystal structures (Panel B), as described in Example 5. The same light-colored data correspond to the measurement from three different runs with the respective dark color as the average values for each triplicate.

[0041] FIG. 25 shows distance-time plots for salt bridges between different amino acid residues in predicted class Ila HDAC7-MEF2C complex (Panel A), and in HDAC9-MEF2B complex (Panel B), as described in Example 5. The same light-colored data correspond to the measurement from three different runs with the respective dark color as the average values for each triplicate.Atty. Docket No. Georgetown.051. WOl

[0042] FIG. 26 (SEQ ID NOS: 17-20) shows multiple sequence alignment of all MEF2s (Panel A), and locations of all amino acid residues in both class Ila HDACs and MEF2s that establish hydrogen bonds and a salt bridge in representative class Ila HDAC-MEF2 complexes (Panel B), as described in Example 5. HDACs and MEF2 proteins are zoomed near the binding interface to highlight the locations of the interacting residues.

[0043] FIG. 27 shows representative SPR sensorgranis for direct bindings of HDAC4 to MEF2A (Panel A), HDAC5 to MEF2A (Panel B), I IDAC7 to MEF2A (Panel C), and HDAC9 to MEF2A (Panel D), as described in Example 5. MEF2A was immobilized onto CMS chips. The continuous colored lines are experimental data and the black dotted lines are fit to the 1:1 kinetics binding model. Each concentration of all class Ila HDACs were injected in duplicate.DETAILED DESCRIPTION OF THE INVENTION

[0044] The practice of the present invention can employ, unless otherwise indicated, conventional techniques of biophysics, biochemistry, molecular biology, cell biology, and neurology, which are within the skill of the art.

[0045] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0046] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0047] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention.Documents incorporated by reference into this text are not admitted to be prior art.Atty. Docket No. Georgetown.051. WOlDefinitions

[0048] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0049] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0050] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone).Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0051] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of” are included.

[0052] Units, prefixes, and symbols are denoted in their Systeme International d’Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth.Likewise, a disclosed range is a disclosure of each individual value (z.e., intermediate) encompassed by the range, including integers and fractions. For example, a stated range of 5- 10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, and of 5.2, 7.5, 8.7, and so forth.

[0053] Unless otherwise indicated, the terms “at least” or “about” preceding a series of elements is to be understood to refer to every element in the series. The term “about” preceding a numerical value includes ± 10% of the recited value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentrati on range of about 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).Atty. Docket No. Georgetown.051. WOl

[0054] Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0055] The term “peptide” refers to a chain of two or more amino acids, and are typically less than 50 amino acids. The peptide can be linear or branched; it can comprise modified amino acids and non-amino acids can interrupt it. The term also encompass a peptide that has been modified naturally or by intervention; for example, disulfide bond formation, glycosyl tion, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation with a labeling component. Also included within the definition are, for example, a peptide containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. In certain embodiments, the peptide can occur as single chains or associated chains.

[0056] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g, a peptide) and its binding partner e.g., a target protein). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., peptide and target protein). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0057] The affinity or avidity of a peptide for a target protein can be determined experimentally using any suitable method known in the art, e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g, KINEXA® or BIACORE™ or OCTET® analysis). Direct binding assays as well as competitive binding assay formats can be readily employed (see, e.g., Berzofsky et al. (1984)

[0025] ; Kuby (1992)

[0026] ). The measured affinity of a particular peptide-target protein interaction can vary if measured under different conditions (e.g, salt concentration, pH, temperature). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD or Kd, Kon, Koff) are made with standardized solutions of peptide and target protein, and a standardized buffer, as known in the art.

[0058] The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in a given biological activity, including full blocking ofAtty. Docket No. Georgetown.051. WOlthe activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in biological activity. Accordingly, when the terms “inhibition” or “suppression” are applied to describe, e.g., an effect of an MEF2 binding molecule, the terms may refer to the ability of an MEF2 binding molecule statistically significantly decrease binding of other proteins to MEF2, such as binding of HD AC to MEF2, or may refer to the ability of an MEF2 binding molecule to statistically significantly decrease HD AC-induced suppression of MEF2-depdendent gene expression. Inhibition may be determined relative to an untreated control — for example, a control not treated with the MEF2 binding molecule. In some embodiments, the MEF2 binding molecule can inhibit binding of MEF2 to other proteins or can inhibit or prevent HD AC-induced suppression of MEF2-dependent gene expression by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or about 100%, as determined, for example, by co-immunopreciptation, surface plasmon resonance (SPR), affinity electrophoresis, or other assays known to those of skill in the art.

[0059] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0060] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g.. acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), a stabilizing agent e.g., human albumin), a preservative e.g., benzyl alcohol), an absorption promoter to enhance bioavailability and / or other conventional solubilizing or dispersing agents.

[0061] An “effective amount” of a binding molecule as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.Atty. Docket No. Georgetown.051. WOl

[0062] The MEF2 binding molecule can be naked or conjugated to other molecules such as toxins, labels, etc. The term “label” when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to a binding molecule, so as to generate a “labeled” binding molecule. The label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, as in the case of, for instance, an enzymatic label, can catalyze chemical alteration of a substrate compound or composition that is detectable.

[0063] Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully “treated” for a disease or disorder according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.

[0064] “Prevent” or “prevention” refer to prophylactic or preventative measures that prevent and / or slow the development or recurrence of a targeted pathologic condition or disorder. Thus, those in need of prevention include those prone to have or susceptible to the disorder, including those who have had the disorder and are susceptible to recurrence. In certain embodiments, a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms or pathology associated with the disease or disorder, or a later onset of symptoms or pathology associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.

[0065] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. InAtty. Docket No. Georgetown.051. WOlcertain embodiments, conservative substitutions in the amino acid sequences of the binding molecules of the invention do not abrogate the binding of the binding molecule to the protein(s), i.e., MEF2, to which the binding molecule binds. Methods of identifying conservative nucleotide and amino acid substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g., Brummell et al. (1993)

[0028] ; Kobayashi et al. (1999)

[0029] ; Burks et al. (1997)

[0030] ).

[0066] A “polynucleotide,” as used herein can include one or more “nucleic acids,” “nucleic acid molecules,” or “nucleic acid sequences,” and refers to a polymer of nucleotides of any length, and includes DNA and RNA The polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0067] The term “vector” means a construct, which is capable of delivering and, in some embodiments expressing, one or more gene(s) or sequence(s) of interest in a host cell.Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0068] An “isolated” peptide, binding molecule, polynucleotide, vector, or cell is in a form not found in nature. Isolated peptides, binding molecules, polynucleotides, vectors, or cells include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a peptide, binding molecule, polynucleotide, vector, or cell that is isolated is substantially pure. When used herein, the term “substantially pure” refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

[0069] The terms “identical” or percent “identity” in the context of two or more nucleic acids or peptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percentAtty. Docket No. Georgetown.051. WOlidentity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.

[0070] Other terms are defined elsewhere in this patent disclosure, or else are used in accordance with their usual meaning in the art.MEF2A Binding Molecules

[0071] The acronym “MEF2” refers to myocyte enhancer factor-2. MEF2 is a member of the MADS (MCM1, agamous, deficiens, SRF) family of transcription factors

[0011] . There are four types of MEF2 proteins in humans: MEF2A, MEF2B, MEF2C, and MEF2D

[0010] , The N-termini of MEF2 proteins contain the MADS-box and MEF2 domain, which cooperate to mediate dimerization, DNA binding, and cofactor interactions

[0030] and the C-terminal regions of MEF2 proteins function as a transcriptional activation domain

[0031] , It has been found that MEF2 always exists as a dimer

[0032] , and the two domains MADS and MEF2 are essential for the dimerization of this protein

[0033] , Vertebrate MEF2 proteins, MEF2A, B, C and D, all have a highly conserved N-terminal region (residues 1—93) and a C-terminal region that is more diverse.

[0072] The MEF2 proteins, as well as HD AC proteins that bind to MEF2, and the nucleotide sequences that encode them are well known in the art. For example, the human MEF2A amino acid sequence has GenBank / NCBI accession number Q02078, and the human HDAC7 amino acid sequence has GenBank / NCBI accession number Q8WUI4.

[0073] The present invention provides MEF2 binding molecules, e.g., synthetic peptides, that bind to MEF2 and inhibit or prevent binding of HD AC to MEF2.

[0074] The terms “MEF2 binding molecule” or “binding molecule that binds to MEF2” refer to a binding molecule that is capable of binding MEF2 with sufficient affinity such that the binding molecule is useful for one of the applications described herein, including, but not limited to, in inhibiting binding of MEF2 to other proteins such as HD AC, or inhibiting or preventing HDAC-induced suppression of MEF2-dependent gene expression. Typically, a binding molecule that “specifically binds” to MEF2 binds to an unrelated, non-MEF2 protein to an extent of less than about 10% of the binding of the binding molecule to MEF2, asAtty. Docket No. Georgetown.051. WOlmeasured, e.g., by a radioimmunoassay (RIA), BIACORE™ (e.g., using recombinant MEF2 as the analyte and binding molecule as the ligand, or vice versa), KINEXA®, OCTET®, or other binding assays known in the art. In certain embodiments, binding molecule that binds to MEF2 has a dissociation constant (KD) of <1 pM, <100 nM, <10 nM, <1 nM, <0,1 nM, <10 pM, <1 pM, or <0.1 pM.

[0075] An MEF2 binding molecule of the invention includes a binding molecule comprising an amino acid sequence of:Xi X2X3X4AXe A S X9Xio V K Xi3Xi4 L Xi6 E Xis X19 L X21 K X23 X24X25 X26 (SEQ ID NO: 1), in which:Xi is S or G, or is removed;X2is K or R;X3is R or E;X4 is S or R;X6 is V or I;X9is S or T;X 10 is V or E;X13 is Q, M, or L;X14 is K or R;Xi6 is A or Q;Xis is V or F;X19 is I, V, or L;X21 is K, N, or S;X23is Q, K, or S;X24 is Q or A, or is removed;X25 is A or T, or is removed; andX26 is A or K.Atty. Docket No. Georgetown.051. WOl

[0076] In some embodiments, the MEF2 binding molecule comprises an amino acid sequence of SEQ ID NO: 1, in which X2 is K, X4 is S, Xe is V, X13 is Q or M, X14 is K, X19 is I or V, X2] is K or N, X23 is Q or K, X24 is Q or is removed, and X25 is A or is removed. Therefore, the MEP2 binding molecule may comprise the amino acid sequence of:Xi K X3S A V A S X9 X10 V K X13 K L Xi6E Xi8X19 L X21 K X23 X24 X25 X26 (SEQ ID NO: 2), in which:Xj is S or G, or is removed;X3 i s R or E;X9 is S or T;Xto is V or E;X13 is Q or M;Xi is A or Q;Xis is V or F;X19 is I or V;X21 is K or N;X23 is Q or K;X24 is Q or is removed;X25 is A or is removed; andX26 is A or K.

[0077] In some embodiments, the MEF2 binding molecule comprises the amino acid sequence of SKRSAVASSVVKQKLAEVILKKQQAA (SEQ ID NO: 4). In certain embodiments, the MEF2 binding molecule consists of the amino acid sequence of SKRSAVASSVVKQKLAEVILKKQQAA (SEQ ID NO: 4).

[0078] In some embodiments, the MEF2 binding molecule comprises the amino acid sequence of KRSAVASSVVKQKLAEVILKKQQAALERTVHPN (SEQ ID NO: 5). In certain embodiments, the MEF2 binding molecule consists of the amino acid sequence of KRSAVASSVVKQKLAEVILKKQQAALERTVHPN (SEQ ID NO: 5).Atty. Docket No. Georgetown.051. WOl

[0079] In some embodiments, the MEF2 binding molecule comprises the amino acid sequence of SEQ ID NO: 1, in which Xi is S or G, Xs is E, X9 is T, X10 is E, Xi6 is Q, Xi8 is F, X19 is V or L, X21 is N or S, X23 is K or S, X24 is A or is removed, X25 is T or is removed, and X26 is K. Therefore, the MEP2 binding molecule may comprise amino acid sequence of: Xi X2 E X4A X6A S T E V K X13 Xi4 L Q E F X19 L X21 K X23 X24X25 K (SEQ ID NO: 3), in whichXj is S or G;X2i s K or R;X4 is S or R;Xe is V or I;X13 is Q, M, or L;X14 is K or R;X19 is V or L;X21 is N or S;X23 is K or S;X24 is A or is removed;X25 is T or is removed.

[0080] In some embodiments, the MEF2 binding molecule comprises the amino acid sequence of GKESAVASTEVKMKLQEFVLNKKK (SEQ ID NO: 6). In certain embodiments, the MEF2 binding molecule consists of the amino acid sequence of GKESAVASTEVKMKLQEFVLNKKK (SEQ ID NO: 6).

[0081] In some embodiments, the MEF2 binding molecule comprises the amino acid sequence of SKESAIASTEX'KLRLQEFLLSKSK (SEQ ID NO: 7). In certain embodiments, the MEF2 binding molecule consists of the amino acid sequence of SKESAIASTEVKLRLQEFLLSKSK (SEQ ID NO: 7).

[0082] In some embodiments, the MEF2 binding molecule comprises the amino acid sequence of GRERAVASTEVKQKLQEFLLSKSATK (SEQ ID NO: 8). In certainAtty. Docket No. Georgetown.051. WOlembodiments, the MEF2 binding molecule consists of the amino acid sequence of GRERAVASTEVKQKLQEFLLSKSATK (SEQ ID NO: 8).

[0083] In other embodiments, the MEFT2 binding molecules comprises the amino acid sequence of KQHREQKLQQLKNKEKGKESAVASTEVKMKLQEFVLNKK (SEQ ID NO: 13); comprises the amino acid sequence of GEVKMKLQEFVLN (SEQ ID NO: 14); comprises the amino acid sequence of WGSGEVKLRLQEFLLS (SEQ ID NO: 15); comprises the amino acid sequence of GVVKQKI EVILKK (SEQ ID NO: 16); or comprises the amino acid sequence of GSGEVKQKLQEFLLSK (SEQ ID NO: 17). In certain embodim ents, the MEFT2 binding molecules consists of the amino acid sequence of KQHREQKLQQLKNKEKGKESAVASTEVKMKLQEFVLNKK (SEQ ID NO: 13); consists of the amino acid sequence of GEVKMKLQEFVLN (SEQ ID NO: 14); consists of the amino acid sequence of WGSGEVKLRLQEFLLS (SEQ ID NO: 15); consists of the amino acid sequence of GVVKQKLAEVILKK (SEQ ID NO: 16); or consists of the amino acid sequence of GSGEVKQKLQEFLLSK (SEQ ID NO: 17).

[0084] In addition to providing the specific MEF2 binding molecules provided above, the present invention also encompasses variants and equivalents of these MEF2 binding molecules. For example, such variants include variants of the specific sequences disclosed herein that comprise one or more substitutions, additions, deletions, or other mutations may be used. Such MEP2 binding molecules, i.e., having one or more substitutions, e.g., conservative substitutions, can be tested for binding to MEF2, and optionally tested for retained function, such as inhibition of binding of UD AC to MEF2, or inhibition or prevention of HD AC -induced suppression of MEF2-dependent gene expression.

[0085] In some embodiments, the MEP2 binding molecules may comprise additional chemical groups present at their amino and / or carboxy termini, to enhance the stability, bioavailability, and / or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides’ amino termini. Further, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides’ carboxy termini.

[0086] Moreover, the MEP2 binding molecules may be modified to alter their steric configuration. For example, the D-isomer of one or more of the amino acid residues of theAtty. Docket No. Georgetown.051. WOlpeptide may be used, rather than the usual L-isomer. Or, the MEP2 binding molecules may be modified chemically by reacting specific amino acids either before or after synthesis of the peptide. Examples for such modifications are well known in the art and are summarized, for example, in Chemical Reagents for Protein Modification (1995)

[0034] , which is incorporated herein by reference. Chemical modification of amino acids includes but is not limited to, modification by acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification of carboxyl groups and sulphydryl modification by performic acid oxidation of cysteine to cysteic acid, formation of mercurial derivatives, formation of mixed disulphides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide and carbamoyl ati on with cyanate at alkaline pH, although without limitation thereto. In this regard, the skilled person is referred to Chapter 15 of Current Protocols in Protein Science (1995)

[0035] for more extensive methodology relating to chemical modification of proteins.

[0087] The MEP2 binding molecules may comprise a salt form of peptides having the amino acid sequences described above. The non-natural salt form of the peptide mediates the solubility of the peptide, in particular in the context of pharmaceutical compositions comprising the peptides. A sufficient and at least substantial solubility of the peptide(s) is required in order to efficiently provide the peptides to the subject to be treated. Preferably, the salts are pharmaceutically acceptable salts of the peptides. These salts according to the invention include alkaline and earth alkaline salts such as salts of the Hofmeister series comprising as anions PO43-, SO42-, CH3COO, Cl-, Br, NO3, CIO4, and SCN-; and as cations NH4+, R+, K+, Na+, Cs+, Li+, Zn2+, Mg2+Ca2+, Mn2+, Cu2+, and Ba2+.

[0088] In some embodiments, the disclosure provides a composition, e.g., a pharmaceutical composition, comprising an MEF2 binding molecule of the invention, optionally further comprising one or more carriers, diluents, excipients, or other additives.Preparation of MEF2 Binding Molecules

[0089] The MEF2 binding molecules may be prepared by methods known in the art. For example, the MEF2 binding molecules may be synthesized by solid-phase peptide synthesis, such as the 9-fluorenylmethyloxy carbonyl- (Fmoc-)-polyamide mode or the tert-Atty. Docket No. Georgetown.051. WOlButyloxycarbonyl (Boc) mode of solid-phase peptide synthesis (see, e.g., Lukas et al. (1981)

[0036] ; Coin et al. (2007)

[0037] ; Jensen (2013)

[0038] ) and by references as cited therein. Or, the MEF2 binding molecules may be synthesized by liquid -phase peptide synthesis (see, e.g., Bayer & Mutter, 1972

[0039] ) or by a combination of solid phase and liquid phase methodologies as disclosed in Bruckdorfer et al. (2004)

[0040] , and the references as cited therein). Additional methods of preparing MEF2 binding molecules include through recombinant production, such as in E. coll.

[0041] ,

[0090] Purification may be performed by any one, or a combination of, techniques such as recrystallization, size exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography and (usually) reverse-phase high performance liquid chromatography using, e.g., acetonitrile / water gradient separation.

[0091] Analysis of peptides may be carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF mass spectrometric analysis.Polynucleotides Encoding MEF2 Binding Molecules, Preparation, and Expression Thereof

[0092] This disclosure provides certain polynucleotides comprising nucleic acid sequences that encode MEF2 binding molecules. The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and, if single stranded, can be the coding strand or non-coding (anti-sense) strand.

[0093] In certain embodiments, the polynucleotide can be isolated. In certain embodiments, the polynucleotide can be substantially pure. In certain embodiments, the polynucleotide can be cDNA or are derived from cDNA. In certain embodiments, the polynucleotide can be recombinantly produced. In certain embodiments, the polynucleotide can comprise the coding sequence for a mature polypeptide, fused in the same reading frame to a polynucleotide which aids, for example, in expression and optionally, secretion, of a polypeptide from a host ceil (e.g., a promoter or other regulatory sequence, a leader sequenceAtty. Docket No. Georgetown.051. WOlthat functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a pre-protein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotide can also encode an MEF2 binding pro-protein which is the mature protein plus additional 5’ amino acid residues.

[0094] The disclosure provides an isolated polynucleotide comprising a nucleic acid encoding an MEF2 binding molecule comprising an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% similarity to an amino acid sequence set forth herein, and / or comprising 1, 2, 3, 4, 5 or more amino acid substitutions, e.g., conservative substitutions, relative to an amino acid sequence set forth herein.

[0095] In certain embodiments the polynucleotide that comprises the coding sequence for the MEF2 binding molecule is fused in the same reading frame as a marker sequence that allows, for example, purification of the encoded polypeptide.

[0096] Polynucleotide variants are also provided. Polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coll).

[0097] The invention includes vectors comprising the polynucleotides described above. Suitable vectors are described elsewhere herein, and are known to those of ordinary skill in the art.

[0098] In certain embodiments, the disclosure provides a composition, e.g., a pharmaceutical composition, comprising a polynucleotide or vector as described above, optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0099] The disclosure further provides a host cell comprising a polynucleotide or vector of the invention, wherein the host cell can, in some instances, express a binding molecule that specifically binds to MEF2. Such a host cell can be utilized in a method of making an MEF2Atty. Docket No. Georgetown.051. WOlbinding molecule, where the method includes (a) culturing the host cell and (b) isolating the binding molecule from the host cell or from the culture medium, if the binding molecule is secreted by the host cell.

[0100] In some embodiments a nucleotide sequence encoding an MEF2 binding molecule can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a nucleotide oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5’ or 3’ overhangs for complementary assembly.

[0101] Once assembled (by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed, e.g., by nucleotide sequencing, restriction mapping, and / or expression of a biologically active polypeptide in a suitable host. In order to obtain high expression levels of a transfected gene in a host, the gene can be operatively linked to or associated with transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0102] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding MEF2 binding molecules. Recombinant expression vectors are replicable DNA constructs that have synthetic or cDNA-derived DNA fragments encoding an MEF2 binding molecule, operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translatedAtty. Docket No. Georgetown.051. WOlinto protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where a recombinant protein is expressed without a leader or transport sequence, the protein can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0103] The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host / vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as Ml 3, and filamentous single-stranded DNA phages.

[0104] Suitable host cells for expression of an MEF2 binding molecule include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of appropriate promoters.Prokaryotes include gram negative or gram-positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed.

[0105] Various mammalian or insect cell culture systems can be advantageously employed to express recombinant MEF2 binding molecules. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded,Atty. Docket No. Georgetown.051. WOlappropriately modified, and completely functional. Examples of suitable mammalian host cell lines include 293 cells (e.g., HEK-293, HEK-293T, AD293), the COS-7 lines of monkey kidney cells described by Gluzman (1981)

[0042] , and other cell lines including, for example, L cells, Cl 27, 3T3, Chinese ha ster ovary (CHO), HeLa, and BIIK cell lines. Mammalian expression vectors can comprise non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers (1988)

[0043] ,

[0106] MEF2 binding molecules produced by a transformed host can be purified according to any suitable method. Such standard methods include chromatography e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine (SEQ ID NO: 22), maltose binding domain, influenza coat sequence, and glutathione-S-transf erase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0107] For example, supernatants from systems that secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify an MEF2 binding molecule. Some or all of theAtty. Docket No. Georgetown.051. WOlforegoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

[0108] A recombinant MEF2 binding molecule produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.Use of MEF2 Binding Molecules

[0109] The present invention provides various methods of using the MEF2 binding molecules described herein. Such methods include, but are not limited to, use of the MEF2 binding molecules described herein for inhibition of binding of HD AC and MEF2, and inhibition or prevention of HD AC-induced suppression of MEF2-dependent gene expression. These methods may be conducted in a cell, and may be performed by contacting the cell with the MEF2 binding molecule, or a composition comprising the MEF2 binding molecule.Suitably, an effective amount of the MEF2 binding molecule is used to contact the cell.

[0110] Examples of a cell that may be used with the present invention include, but are not limited to, neurons, neural stem / progenitor cells (NSCs), and HeLa cells.

[0111] Inhibition of binding of HDAC and M EF2 may be evaluated using methods known in the art for assessing protein-protein interactions. Such methods may include, but are not limited to, co-immunoprecipitation, bimolecular fluorescence complementation, affinity electrophoresis, pull-down assays, label transfer, phage display, tandem affinity purification, chemical cross-linking in combination with matrix-assisted laser desorption / ionization (MALDI) mass spectrometry, proximity ligation assay, SPR, dual polarization interferometry, static light scattering, dynamic light scattering, flow-induced dispersion analysis, fluorescence polarization / anisotropy, fluorescence correlation spectroscopy, fluorescence resonance energy transfer, bio-layer interferometry, isothermal titrationAtty. Docket No. Georgetown.051. WOlcalorimetry, protein activity determination by nuclear magnetic resonance multi-nuclear relaxation measurements, microscale thermophoresis, and single color reflectometry.

[0112] Inhibition or prevention of HDAC-induced suppression of MEF2-dependent gene expression may be evaluated using methods known in the art for assessing gene expression. Such methods may include, but are not limited to, northern plotting, quantitative polymerase chain reaction (qPCR), microarrays, and RNA-seq.

[0113] In some embodiments, the MEF2 binding molecules provided herein are useful for the treatment of neurodegenerative diseases associated with suppression of MEF2 activity.Examples of such neurodegenerative diseases may include, but are not limited to, Alzheimer’s disease and Parkinson’s disease. For example, in one embodiment, the present invention provides a method of treatment, the method comprising administering to a subject in need thereof an MEF2 binding molecule, or a composition comprising an MEF2 binding molecule. In some such embodiments, the subject has Alzheimer’s disease or Parkinson’s disease.

[0114] Clinical response to administration of an MEF2 binding molecule can be assessed using standard screening techniques known in the art such as biomarkers and / or symptoms, and may depend on the neurodegenerative disease being treated. For example, Alzheimer’s disease may be evaluated based on one or more of presence / changes in amyloid-beta (AP)-related biomarkers (e.g., abnormal amyloid PET scan or low CSF Ap42 or AP42 / AP40 ratio), pathologic tau biomarkers, neurodegenerative / neuronal injury biomarkers, and cognitive symptoms (see Jack et al. (2018)

[0044] ).

[0115] Methods of preparing MEF2 binding molecules for administration to a subject, and methods of administering an MEF2 binding molecule to a subject, are well-known to those of ordinary skill in the art, or can be readily determined by those of ordinary skill in the art. For example, the route of administration of the MEF2 binding molecule can be, for example, oral, parenteral, by inhalation, or topical. The term “parenteral” as used herein includes, e.g, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, and vaginal administration. Oral dosage forms include, e.g, capsules, tablets, aqueous suspensions, and solutions. Nasal aerosol or inhalation dosage forms can be prepared, for example, as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorptionAtty. Docket No. Georgetown.051. WOlpromoters to enhance bioavailability, and / or other conventional solubilizing or dispersing agents.

[0116] The disclosed binding molecules can be formulated to facilitate administration and promote stability of the MEF2 binding molecules. Pharmaceutical compositions in accordance with the present invention can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, a “therapeutically effective amount” of an MEF2 binding molecule means an amount sufficient to achieve a benefit, e.g., to ameliorate symptoms of a disease or condition (e.g., a neurogenerative disease) or to inhibit proliferation of a cancer cell. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington 's Pharmaceutical Sciences (2000)

[0045] ).

[0117] The composition can be administered as a single dose, multiple doses, or over an established period of time in an infusion. Dosage regimens can also be adjusted to provide the optimum desired response e.g, a therapeutic or prophylactic response). The amount of an MEF2 / X binding molecule that can be combined with carrier materials to produce a dosage form will vary' depending upon many different factors, including means of administration, target site, physiological state of the patient (i.e., the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy), whether treatment is prophyl ctic or therapeutic, other medications administered, and whether the subject is a human or an animal. Usually, the subject is a human, but non-human mammals, including transgenic mammals, can also be treated. The amount of an MEF2 binding molecule to be administered is readily determined by one of ordinary skill in the art without undue experimentation, given this disclosure. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.Kits Comprising MEF2 Binding Molecules

[0118] This disclosure further provides kits that comprise an MEF2 binding molecule, which can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified MEF2 binding molecule in one or more containers. In some embodiments, the kit contains one or more of the components necessary' and / or sufficient toAtty. Docket No. Georgetown.051. WOlperform a detection assay, including controls, directions for performing assays, and any necessary software for analysis and presentation of results. In some embodiments, the kit further comprises instructions for use.

[0119] One skilled in the art will readily recognize that the disclosed MEF2 binding molecules can be readily incorporated into any of the established kit formats that are well known in the art.

[0120] Embodiments of the present disclosure can be further described and understood by reference to the following non-limiting “Examples,” which describe in the preparation of certain exemplary MEF2 binding molecules, some exemplary characterization of such molecules, and some exemplary methods for using such binding molecules. It will be apparent to those skilled in the art that many modifications to the specific description provided in the Examples can be practiced without undue experimentation and without departing from the scope of the present disclosure.EXAMPLESExample 1: Characterization of Molecular Interactions Between HDAC7 and MEF2A

[0121] A study was conducted to examine the interactions between HDAC7 and MEF2A using all-atom molecular dynamics (MD) simulations

[0046] . A model of the HDAC7-MEF2A complex was first generated, and then simulations were performed to analyze the interactions. Key amino acids were identified that are responsible for the formation of the HDAC7-MEF2A complex, including the amino acids that associate through hydrophobic interaction. Based on these HDAC7-MEF2A binding results, sequence alignment of class Ila HDACs and M EF2s were performed.MethodsSystem Preparation

[0122] The structure of the HDAC7-MEF2A complex was predicted with AlphaFold2 Colab

[0047] using the FASTA sequences of HDAC7 (UniProt (Consortium T. U, 2022) accession ID Q8WUI4) and MEF2A (UniProt (Consortium T. U., 2022) accession ID Q02078). TheAtty. Docket No. Georgetown.051. WOlAlphaFold2 prediction was obtained with three different sequence lengths for HDAC7, (i) a full-length sequence, (ii) only the first 200 amino acids, and (iii) amino acids from position 72 to 172, as well as the MEF2A amino acids from position 2 to 91. The HDAC7-MEF2A-DNA complex was modeled by structural overlapping of the DNA structure from the PDB ID 3KOV

[0048] with the HDAC7-MEF2A complex structure optimized with 500 ns all-atom MD simulations.MD Simulations

[0123] All-atom MD simulations of the HDAC7-MEF2A and HDAC7-MEF2A-DNA complexes were performed using NAMD software

[0049] and CHARMM36m force field

[0050] as used in previous studies [51 -54]. Briefly, the CHARMM-GUI web-server was used to prepare the simulation input files

[0055] , The systems were solvated in cubic boxes with TIP3 water model and 150 mM NaCl, The final solvated and ion neutralized HDAC7-MEF2A and HDAC7-MEF2A-DNA complexes contained 61663 and 51585 atoms, respectively. Each system was minimized for 10,000 steps with heavy atoms harmonically restrained and then equilibrated for 100 ps using the NVT ensemble. The production runs were then propagated with a time step of 2-fs.Data Analysis

[0124] The Visual Molecular Dynamics (VMD) software

[0056] was used to analyze simulation trajectories, visualize structures of proteins and DNA, and create figures. NAMD was used to calculate binding free energy using the MM / GBSA approach

[0057] as calculated in prior publications [50, 52], The following equation was used to compare binding free energy for the complex formation calculated using simplistic approach, with entropy term ignored

[0058] :SG=GHDAC7 - MEF2A ~ GHDAC7 ~ GMEF2Awhere GHDAC7-MEF2A or GHDAC7 or GMEF2A is the sum of energy contributions due to internal (bond, angle, dihedral, and improper), electrostatic, van der Waals, and solvation (polar and non-polar contributions) energies (G = E internal + E electrostatic + Evdw + E solvation)

[0059] , GraphPad Prism was used to plot graphs. A contact distance cutoff of 3.5 A and an angle cutoff of 30 degrees were used to analyze hydrogen bonding and the same atomic distanceAtty. Docket No. Georgetown.051. WOlcutoff was used to analyze salt bridges. Multiple sequence alignment was performed using COBALT

[0060] , Contact surface area was calculated as below

[0061] :SASAHDAC7+ SASAMEF2A-- SASAHDAC7-MEF2A2Contact Area = - 2where SASAHDAC7 corresponds to solvent-accessible surface area (SASA) of HDAC7 at any time, SASAMEF2A to SASA of MEF2A, and SASAHDAC7-MEF2A to SASA of the complex. VMD was used to measure the SASA values using the simulation trajectories. Carma was used to measure the radius of gyration (RG)

[0062] ,ResultsModeling of the HDAC7-MEF2A complex structure

[0125] FIG. 1 shows complex structures of the HDAC7-MEF2A complex predicted by AlphaFold2. The amino acid range used for MEF2A in all figures (FIG. 1, Panels A-D) was from 2 to 91, as found in the solved MEF2A dimer structure with amino acids from 2 to 91 (PDB ID 3K0V)

[0063] , This range is very' close to the reported amino acid region (1-86) that interacts with HDAC7

[0064] , This predicted model has an interface predicted template modelling (ipTM) score of 0.90. An ipTM score of approximately 0.75 or higher is generally related to model confidence

[0064] . It is not believed that the specific HDAC7 amino acids that interact with MEF2A, or a resolved HD / XC7 structure in the range that interacts with MEF2A, have been reported. A full-length HDAC7 sequence was first used to model the HDAC7-MEF2A complex structure.

[0126] FIG. 1, Panel A shows the HDAC7-MEF2A complex with a full-length HDAC7 sequence. Analysis of the predicted structure (zoomed view in FIG. 1, Panel A) shows that the amino acids in the HDAC7 helix (as enclosed by a rectangle in FIG. I, Panel A) strongly interact with the MEF2A dimer. The range of amino acids in this HDAC7 helix aligns well with the reported HDAC7 interaction range as discussed above. The other portions of HDAC7 were predicted as unstructured loops as displayed in full view and were not predicted to interact with MEF2A. Since loops do not appear to be important for the complex formation, these extended unstructured loops were removed to reduce system size and conducted an all atom MD simulation of the complex (FIG. 1, Panel B) for 280 ns. TheAtty. Docket No. Georgetown.051. WOlstructural integrity of the system was monitored via root mean square deviation (RMSD) measurements and simulation results were used to confirm the HDAC7-MEF2A binding interface as predicted in FIG. 1, Panel A. Besides initial reorganizations as revealed by RMSD measurements until —150 ns (FIG. 1, Panel C), structural integrity of the complex was maintained as revealed by fairly stable RMSD measurements beyond 150 ns. FIG. 1, Panel D shows the complex after 280 ns of the simulations of the HDAC7-MEF2A complex. The simulated complex (FIG. 1, Panel D) also shows that only the interacting helix and a few amino acid residues near to this helix are positioned to interact with MEF2A while other HDAC7 portions are moved away from MEF2A. The input HDAC7 amino acid range was narrowed from 1 to 200 and predicted the complex (FIG. 1, Panel E, ipTM=0.86). Based on the analysis of this complex (zoomed view in FIG. 2, Panel E), the strong interaction was again found to be around the same helix as observed in FIG. 1, Panel A. The other HDAC7 portion in the range from 1 to 200 was predicted as mostly unstructured and remained at a longer distance than 4.0 A from the MEF2A surface as displayed in full view. A residue¬ residue contact distance of 4.5 A or less can be considered as the direct interacting distance

[0065] , The input HDAC7 amino acid range from 72 to 172 was then selected and the HDAC7-MEF2A complex (ipTM=0.80) was again predicted, the resulting complex of which is shown in FIG. 1, Panel F. This prediction also showed the same HDAC7 helix and few amino acids in the loop (represented within a rectangle) connected to the helix interacts with MEF2A. All other amino acids in HDAC7 beyond the helix lie outside the range of 4.5 A. The HDAC7 portion beyond the helix and the loop region showing interaction with MEF2A were then removed to prepare the HDAC7-MEF2A complex structure to use as an input file for MD simulations. This truncation offers a much-reduced system size and reduces a great deal of computational cost. This final HDAC7-MEF2A complex structure (referred to as HDAC7-MEF2A complex hereafter) is shown in FIG. 1, Panel G.Simulation of the predicted HDAC7-MEF2A complex

[0127] All-atom MD simulations were performed for the HDAC7-MEF2A complex as shown in FIG. 1, Panel G. Four independent MD simulation runs (RUN1 -4) of the same system were completed. The structural integrity of the complex was monitored by measuring RMSD values. FIG. 2, Panel A shows RMSD measurements for a representative run (RUN1). FIG. 3 shows RMSD measurements from the remaining three runs (RUN2-4).Atty. Docket No. Georgetown.051. WOlRMSD measurements show that the complex structure was stable throughout the simulation, except during the initial times, which is expected since structural reorganizations take place to optimize the complex formation. RMSD measurements of the individual HDAC7 and MEF2A both in complex and isolated forms were also compared. The RMSD measurements show that the HDAC7-MEF2A complex is stable after —175 ns of simulations. The RMSD measurements of individual proteins were compared as well. FIG. 3, Panel B and FIG. 3, Panel C show RMSD measurements for individual HDAC7 and MEF2A, respectively, in the HDAC7-MEF2A complex. Slight fluctuations in RMSD measurements (FIG. 3, Panel B) were observed for HDAC7, which was expected due to flexible truncated HDAC7 structure, as compared to more stable RMSDs for MEF2A (FIG. 3, Panel C). In addition, RMSD values of both FIDAC7 and MEF2A in complex and isolated forms were compared. FIG. 3, Panel D shows RMSD measurements of HDAC7 and MEF2A in isolated form. The RMSD values for HDAC7 fluctuated less in the complex (FIG. 3, Panel B) as compared to the isolated form (FIG. 3, Panel D), likely due to the strong interaction of the HDAC7 helix with MEF2A that stabilized the complex. The RMSD values for MEF2A in isolated form (FIG. 3, Panel D) are very similar to the values in the complex form (FIG. 3, Panel C).

[0128] FIG. 2, Panel B shows the structure of the HDAC7-MEF2A complex after 500 ns of simulation time. The interfacial amino acids within 4.0 A contact distance are represented as sticks in each protein. The color of sticks represents the corresponding proteins and EF2A chains. There are several amino acids in each protein that establish interfacial contacts establishing different bonding interactions (discussed below) that are responsible for a strong association of the HDAC7 with the MEF2A dimer.Contribution of a salt bridge for the formation of the HDAC7-MEF2A complex

[0129] FIG. 4, Panel A shows the salt bridge responsible for establishing the linkage between HDAC7 and MEF2A. The salt bridge is consistent in all the four runs and the representative result in FIG. 4, Panel A is from the RUN!. Salt bridges from the other three runs (RUN2-4) are presented in FIG. 5. The average salt bridge distance for the last 50 ns (towards the end) of simulation trajectories in at least three runs is 3.5 A. These results hold for the simulation of the complex with full HDAC7 sequence with unstructured loops removed (FIG. 1, Panel D). All salt bridges that were not predicted in at least three of theAtty. Docket No. Georgetown.051. WOlfour independent runs were ignored. FIG. 4, Panel B shows the location of the amino acids in both HDAC7 and MEF2A.Hydrogen bonding between HDAC7 and MEF2A

[0130] The last 325 ns data of the simulation trajectories in each run to predict hydrogen bonds formed between HDAC7 and MEF2A, using the analysis protocol as described in the Methods section. When the RMSD measurements in all four runs were compared, it was found that RMSD values in at least three runs were stable beyond 175 ns after simulations were started, likely due to initial structural reorganizations. Therefore, it was opted to avoid the first 175 ns data in the analysis. In many cases, the same amino acid in one protein formed hydrogen bonds via different atoms with another protein. Only those individual bonds with occupancy greater than 25% in at least three of the four independent runs were considered. Table 1 shows a summary' of hydrogen bond occupancies calculated from each independent run. The occupancies shown in the Table 1 for each residue pair are the sum of all hydrogen bonding contributions due to different atoms of the same residue.

[0131] The distance vs. time graphs for each hydrogen bond established by each atom pair from the analysis of the RUN1 trajectory' are shown in FIG. 6, Panel A. The types of atoms are shown inside parentheses. Hydrogen bonds predicted from the analyses of other three different runs (RUN2-4) are plotted in FIG. 7.

[0132] Based on the analysis protocol, SER82 and LYS96 in HDAC7 are predicted to be crucial to form hydrogen bonds with ASP63 (Chain B) and ASP63 (Chain A), respectively, in MEF2A. Interestingly, SER82(HDAC7)-ASP63(MEF2A) hydrogen bond in RUN3 was predicted with a lower occupancy, which contributed to the reduction of the average occupancy (Table 1) for this hydrogen bond. The same residue pair LYS96(HDAC7)-ASP63(MEF2A) was predicted to establish both salt bridge (FIG.4, Panel A) and hydrogen bond (Table 1), this reiterated the importance of these two residues in the formation of the HDAC7-MEF2A complex. The same residue pairs form hydrogen bonds with full HDAC7 sequence with unstructured loops removed (FIG. 1 Panel D).Contribution due to hydrophobic interactions

[0133] The results show that there are a number of hydrophobic interactions between residues in HDAC7 and MEF2A across the interface. As shown in FIG. 8, Panel A, HDAC7Atty. Docket No. Georgetown.051. WOlinteracts with the residues in MEF2A that are located at both MEF2A chains. Surface representation in FIG. 8, Panel A shows a representative snapshot of a network of residues that establish hydrophobic interactions. The network of these hydrophobic interactions is extended across the binding interface along the HDAC7 helical length (perpendicular to the plane) and on both sides of the HDAC7 helix.

[0134] As shown in FIG. 8, Panel B, both LEU66 and LEU67 in MEF2A Chain A, LEU66 in MEF2A Chain A and LEU66 in MEF2A Chain B, and both L. EU66 and LEU67 in MEF2A Chain B form three hydrophobic cavities (labeled as Cl, C2, and C3, respectively). Each grove in these hydrophobic cavities are holding three different HDAC7 residues as shown in FIG. 8, Panel B. Table 2 lists all residues that are responsible for establishing the hydrophobic contacts. The hydrophobic residues listed in the Table 2 presented to establish contacts in the complex with full HDAC7 sequence with unstructured loops removed (FIG.1, Panel D). Based on the analysis of hydrophobic contacts, it is believed that the HDAC7-MEF2A binding interface is largely hydrophobic and this interaction mechanism determines a major specificity between these two proteins. A similar finding has been reported for the interaction mechanism between Cabinl and MEF2B

[0066] ,

[0135] Multiple sequence alignments for class Ila HDACs and MEF2s were also performed, as shown in FIG. 9. The residues that are conserved in at least three HDACs (see FIG. 9, Panel A) and MEF2s (see FIG. 9, Panel A) are highlighted in light blue color. The sequence alignment of HDACs show that most of the residues that the analysis predicted to establish HDAC7-MEF2A complex (FIG. 4, Panel A, Tables 2 and 3) are conserved in at least three HDACs. Moreover, all residues in MEF2A that were predicted to interact with HD / XC7 are conserved in all MEF2s. These results, therefore, are very helpful in investigating the interactions between other class Ila HDACs and MEF2s. Notably, all the amino acid residues in both HDAC7 and MEF2A that were predicted to establish HDAC7-MEF2A complex lie within the same exact range of amino acid ranges (2-72 in HDAC7 and 1-86 in MEF2A) as predicted experimentally

[0063] , It is believed that structural investigation is very limited with no ciystal structure of the functional HDAC7-MEF2A complex. Therefore, the results will be very helpful to the scientific community investigating this functionally important protein¬ protein complex formation.Atty. Docket No. Georgetown.051. WOlHDAC7-MEF2A interactions in presence ofDNA

[0136] Since MEF2 has regulatory roles

[0067] , the interaction of HDAC7 with MEF2A regulates the function of MEF2s

[0018] , and DNA bound MEF2C is also capable of binding to HDAC7

[0063] , the investigation was further extended to assess effect of MEF2A-DNA binding on HDAC7-MEF2A binding. FIG. 10, Panel A is the structure of the HDAC7- MEF2A-DNA complex at the end of 500 ns of MD simulations. The structure is in “pyramid” shape with the DNA as the base and HDAC7 on the top. The RMSD measurement of the complex as shown in FIG. 11, Panel A shows that the complex is fairly stable. As compared to FIG. 4, Panel A, there was one additional salt bridge formed between HDAC7 and MEF2A as depicted in FIG. 11, Panel B in the presence of DNA. Notably, this additional salt bridge between LYS76(HDAC7) and ASP63(MEF2A) was also present in two of the four independent runs without DNA. However, LYS76 lies on the front of a highly flexible HDAC7 loop (loop enclosed inside a rectangle in FIG. 1, Panel C). Therefore, it is believed that this additional salt bridge may not only be the determinant of the salt bridge strength between HDAC7 and MEF2A in the presence ofDNA.

[0137] The hydrogen bonds formed between HDAC7 and MEF2A in the HDAC7-MEF2A-DNA complex were also analyzed, and the results are presented in FIG. 11, Panel C and Table 3. All the residues that were predicted for the HDAC7-MEF2A complex (FIG. 6, Panel A, Table 1) are also present in the analysis of the HDAC7-MEF2A-DNA complex. There are a few more amino acid pairs predicted to form additional hydrogen bonds between HDAC7 and MEF2A in the HDAC7-MEF2A-DNA complex, as compared to what was predicted in the HDAC7-MEF2A only complex. The hydrogen bond between LYS76(HDAC7) and ASP63(MEF2A) as shown in Table 3 was also present in two of the four runs of HDAC7-MEF2A complex with occupancy of >38%, between ARG77(HDAC7) and ASP61(MEF2A) also present in two of the four runs with occupancy of >75%, between SER78(HDAC7) and ASP61 MEF2A) also present in two of the four runs with occupancy of >53%, and between SER78(HDAC7) and MET62(MEF2A) also present in two of the four runs with occupancy of >52%. It is to be noted that all these additional residues in HDAC7 lie on the front of a highly flexible loop (loop enclosed inside a rectangle in FIG. 1, Panel E) and the bonds are intermittent. Therefore, it is believed that these new bonds may not only be the determinants of the hydrogen bonding strength between HDAC7 and MEF2A in theAtty. Docket No. Georgetown.051. WOlpresence of DNA. Analysis of the hydrophobic network across the HDAC7-MEF2A interface in the HDAC7-MEF2A-DNA complex shows that the same residues (Table 2) contribute to the hydrophobic interactions. The only exception is that PRO75 in MEF2A was not found to be involved in the hydrophobic interaction for the HDAC7-MEF2A-DNA complex. The hydrophobic network is shown in FIG. 10, Panel A in surface and CPK representations on the top of the “Pyramid”.

[0138] FIG. 10, Panel B shows the measurement of the contact area for the two complexes without and with DNA. The traces in the faint colors correspond to the values for individual runs (RUN 1-4) of HDAC7-MEF2A simulations. The trend of the average contact area curve as well as the values from all four runs without DNA and with DNA are found to be similar. The average and standard deviation (S. D.) of all contact area values (values for each frame of entire 500 ns simulations) for red and blue traces were calculated, as shown in FIG. 10, Panel B (without and with DNA, respectively) and obtained 240.0 ± 11.9 A2 (Mean ± S. D.) for HDAC7-MEF2A binding in the HDAC7-MEF2A complex (red trace, without DNA) and 219.7 ± 17.3 A2 for HDAC7-MEF2A binding in the HDAC7-MEF2A-DNA complex (blue trace, with DNA). The similar values for the contact area for these two complexes (without and with DNA) show that structural changes of the HDAC7-MEF2A complex interface upon binding is not affected by the association of DNA to MEF2A. 250 ns all-atom simulations of isolated MEF2A structure were independently conducted, and solvent-accessible surface area (SAS A) of MEF2A in both complex (with HDAC7) and isolated forms were compared. The SASA measurements are shown in FIG. 12. The SAS A measurements in both systems are stable. At 250 ns, the SASA for MEF2A in the HDAC7-MEF2A complex was found to be 1150.8±16,8 A2 and that of isolated MEF2A as 1214.4 A2, As compared to these values in the complex structure, the higher value of SASA for the isolated form is due to confirmation flexibility of the MEF2A binding interface with more area exposed to solvent. The SASA value at the same time for MEF2A in HDAC7-MEF2A complex in the presence of DNA was obtained as 1151.8 A2, which is very similar to the value in the complex without DNA. The radius of gyration (RG) of the MEF2A structures both in complex and isolated forms were also examined. The RG values are stable (FIG. 13) throughout the simulations showing no significant structural changes. Significant differences in G values in both complex and isolated forms as well as in the complex with DNA were not observed. MM / GBSA binding free energy for HDAC7-MEF2A interactions were determined to be -54.0±8.6 kcal / mol in theAtty. Docket No. Georgetown.051. WOlabsence of DNA. A similar binding free energy of -57.5 kcal / mol between HDAC7 and MEF2A in the presence of DNA was also obtained,

[0139] Altogether, analysis of salt bridge, hydrogen bonds, contact area, SASA, RG, and MM / GBSA binding free energy suggest that the association of DNA to MEF2A has no significant effect in the interaction between these two functionally important proteins.Conclusion

[0140] The association of HDAC7 with MEF2A has an important cellular function of regulating MEF2s. MEF2A was selected and the molecular interactions between HDAC7 and MEF2A were characterized using all-atom MD simulations. First, the structure of the HDAC7-MEF2A complex using AlphaFold2 was predicted, and then MD simulations were utilized to investigate the interfacial interactions in the predicted complex. The salt-bridges, hydrogen-bonds, and hydrophobic interactions that stabilize the complex were identified. The results show a salt bridge between LYS96(HDAC7) and ASP63(MEF2A). Similarly, two residue pairs, SER82(HDAC7)-ASP63(MEF2A) and LYS96(HDAC7)-ASP63(MEF2A), were determined that participate in the formation of hydrogen bonds between the two proteins. An interfacial patch of hydrophobic interactions on both sides of HDAC7 and between the two helices of MEF2A dimer were found. This hydrophobic network spreads across the interfacial area along the entire length of the HDAC7 helix that is responsible for interacting with MEF2A. The results from the multiple sequence alignment show most of the HDAC7 residues that were predicted to be interacting with MEF2A are conserved in at least three class Ila HDACs and all residues in MEF2A are conserved in MEF2s. The results also revealed MEF2A-DNA binding does not significantly influence the recruitment of HDAC7

[0141] Overall, the study provides valuable insights into the molecular basis of the HDAC7-MEF2A complex formation, and the results as well as the approach can be useful for investigating the interactions between different class Ila HDACs and MEF2s.Atty. Docket No. Georgetown.051. WOlTable 1. Hydrogen bond occupancies obtained from the analysis from the last 325 ns trajectories of HDAC7-MEF2A simulations. The residues in italics are also predicted to establish salt bridges between HDAC7 and MEF2A.HDAC7 MEF2A Occupancy (%)Chain A Chain B RUN1 RUN2 RUN3 RUN4 Mean ± S. D. SER82 ASP63 72.8 73.7 10.5 72.7 57.4 ± 31.3LYS96 ASP63 58.5 50.3 65.3 72.3 61.6 ± 9.4Table 2. Amino acid residues in both HDAC7 and MEF2A that establish hydrophobic interactions between these two proteins.MEF2AHDAC7Chain A Chain BVAL80 PRO75 MET62ALA81 MET62, LEU66VAL85 LEU66, LEU67LEU89 LEU66 LEU66ALA90 LEU67ILE93 LEU66, LEU67Table 3. Hydrogen bond occupancies obtained from the analysis from the last 325 ns trajectories of HDAC7-MEF2A-DNA simulations. The residues in italics are the common residues obtained from the analysis of HDAC7-MEF2A simulation trajectories (Table 1),MEF2AHDAC7 Occupancy (%)Chain A Chain BLYS76 ASP83 66.5ARG77 ASP61 29.3SER78 ASP61 89.7SER78 MET62 84.7SER82 ASP63 51.5LYS96 ASP63 61.4Example 2: Validation of HDACs-MEF2A and Peptide Direct Bindings

[0142] To validate class Ila HDACs bindings to MEF2A, MEF2A was immobilized onto a CM5 chip surface as a ligand using standard amine coupling chemistry and injected the HDACs as analytes at various concentrations. In SPR technology, one of the two binding partners that is immobilized onto the sensor surface is termed as “ligand” and another partnerAtty. Docket No. Georgetown.051. WOlthat flows in solution over the ligand immobilized surface is termed as “analyte”. As expected, direct binding of HDAC7 and other three HDACs to immobilized MEF2A was observed, as represented by FIG. 14, Panel A. A KD value of 5.0 ± 0.5 nM (mean ± S. D. from three experiments) for HDAC4-MEF2A, 10.0 ± 2,4 nM for HDAC5-MEF2A, 3,5 ± 0.9 nM for HDAC7-MEF2A, and 19.1 ± 3.7 nM for HDAC9-MEF2A interactions were obtained. These results show that the MEF2A is a target biomolecule for the class Ila HDACs with nanomolar affinity.

[0143] In control experiments, injection of 10 nM HDACs to GST and PARP1 surfaces did not show binding (FIG. 14, Panel A). PARP1 was used as a negative control protein since this is also a nuclear protein and has a molecular weight of ~110 kDa

[0068] , which is similar to the molecular weight of MEF2 dimer (~55 kDa for MEF2 monomer)

[0069] , GST was also used as another control protein since the ligand ( MEF2A) was GST-tagged. The HDAC7-MEF2A binding was further confirmed by flipping the orientation of the ligand and analyte, and the ligand (HDAC7) was captured using anti-his antibody (very different surface chemistry); a similar KD value of 4.6 ± 0.9 nM was obtained. Based on the computational results, a parent peptide having an amino acid sequence of KRSAVASSVVKQKLAEVILKKQQAALERTVHPN (SEQ ID NO: 5) (named as “PT-HD7-Wt” hereafter) was designed and synthesized, and then injected onto MEF2A, GST, and PARP1 immobilized surfaces. FIG. 14, Panel B shows direct binding of the parent peptide to immobilized MEF2A with a KD value of 67.7±8.2 nM. Binding of PT-HD7-Wt to GST and PARP1 was not observed (FIG. 14, Panel B insets). A scrambled peptide for PT-HD7-Wt (named as PT-HD7-SCR hereafter) was also synthesized, and binding of this peptide to MEF2 A was tested. The same concentration range (as in FIG. 14, Panel B) of this peptide did not show binding to MEF2A, suggesting that the parent peptide shows a very specific binding to MEF2A with a nanomolar affinity.Example 3: SPR Blocking Assays

[0144] A representative figure from the results of blocking assays is shown in FIG. 15, Panel A. MEF2A was immobilized onto the chip surface using standard amine coupling chemistry. In the presence of PT-HD7-Wt crosslinked to MEF2A, binding amplitudes of 5 nM HDACs (green) were diminished as compared to 5 nM HDAC injections in the absence of PT-HD7-Atty. Docket No. Georgetown.051. WOlWt (red). As expected, there were no meaningful bindings observed for GST and PARP1. The blocking efficiency is presented in FIG, 15, Panel B for all HDACs considering the HDACs-MEF2A binding in the absence of PT-HD7-Wt as 100% binding.

[0145] These experiments showed that PT-HD7-Wt was able to impair the HDACs-MEF2A bindings. This observation was also confirmed by flipping ligand and analyte. HDAC7 was captured using anti-his capture chemistry and injected MEF2A in the absence (red, FIG. 15, Panel B inset) and presence (green, FIG. 15, Panel B inset) of PT-HD7-Wt In control injections, as shown in FIG. 15, Panel B inset, PT-HD7-Wt and GST only did not show meaningful or showed very' weak (likely due to bulk) binding to captured HDAC7. This experiment also confirmed that presence of PT-HD7-Wt specifically impaired the HDAC7-MEF2A binding.Example 4: Binding Assay

[0146] PT-HD7-Wt was tested at different concentrations ranging from 37 nM to 3000 nM for binding to MEF2A. The results are shown in FIG. 16. HDAC7 was captured onto a CM5 chip surface. 20 nM MEF2A was injected over the HDAC7 captured surface in the presence and absence of the peptide (PT-HD7-Wt). Injection of 20 nM MEF2A only binds to captured HDAC7. When 20 nM MEF2A was premixed with PT-HD7-Wt, the MEF2A-HDAC7 binding was increasingly inhibited as concentration of the peptide was increased. For comparison, only peptides at 1000 nM and 3000 nM were injected and that showed no binding to HDAC7. FIG. 16, Panel B shows the quantification of the MEF2A binding to captured HDAC7 in the absence and presence of PT-HD7-Wt.Example 5: Mapping Interactions Among Class Ila HDAC and MEF2

[0147] A study was conducted to examine the interactions between different class Ila HDACs (HDAC4, HDAC5, HDAC7, and HDAC9) and MEF2s (MEF2A, MEF2B, MEF2C, and MEF2D) using molecular dynamics (MD) [70, which is incorporated herein by reference], SPR-based direct binding experiments using four different purified class Ila HDACs and MEF2A were performed to validate the computational investigations.Atty. Docket No. Georgetown.051. WOlResultsPredicted class Ila HDAC-MEF2 complexes and their stability

[0148] 500 ns all-atom simulations of 15 different predicted class IIa HDAC-MEF2 complexes were performed. Complexes with human HDAC4-MEF2A, HDAC4-MEF2D, HDAC5-MEF2D, HDAC7-MEF2D, and HDAC9-MEF2D that have available crystal structures as controls were also chosen, which adds to 15 predicted complexes to make a total of 20 different simulation systems. Notably, three independent replica runs for each system (60 simulation trajectories in total) were conducted and the results were interpreted based on multitrajectory analyses.

[0149] FIG. 17 shows the representative complex structures at the end of the 500 ns simulations. The class Ila HDAC-MEF2 complex structures with crystal structures at the end of 500 ns simulations are shown in FIG. 18. PCA-based cluster analysis was performed, producing average structures from the simulation trajectories of all 15 different complexes that were studied. The representative average structures are provided (FIG. 18). In Example 1, it was predicted that only the truncated portion of HDAC7 (amino acids from position K76 to N108) was sufficient to form the HDAC7-MEF2A complex. The same range of the HDAC7 amino acids was used to prepare the simulation systems of complexes HDAC7 with MEF2B, MEF2C, and MEF2D, as was used to prepare the HDAC7-MEF2A complex that was used in all-atom simulations in Example 1.

[0150] A multiple sequence alignment results in Example 1 predicted that five out of six HDAC7 interacting residues were conserved in all class Ila HDACs, and the remaining one HDAC7 interacting residue is conserved in three class Ila HDACs. These alignment results provided guidance to select the range of amino acids that could potentially be used in the truncated HDAC4, HDAC5, and HDAC9 structures in complex with MEF2s. Moreover, the range of the HDAC7 portion that was predicted to interact with MEF2A in Example 1 was well within the range (from amino acid position 72 to 172) as outlined in a prior experimental prediction

[0063] , The published crystal structures, so far until this investigation, also presented only the truncated class Ila regions interacting with MEF2s. It is believed that the truncated class Ila HDACs regions were sufficient for their interactions with MEF2s. The range of the amino acids in these three class Ila HDACs were slightly extended from the conserved range of amino acids corresponding to HDAC7 residues interacting with MEF2A. Amino acidsAtty. Docket No. Georgetown.051. WOlwere used with positions from G161 to K184 for HDAC4, from S 173 to K196 for HDAC5, and from G133 to KI 58 for HDAC9 to create simulation inputs. The full-length class Ila HDAC-MEF2 complexes were first generated and it was confirmed that the same HDAC4 / 5 / 9 ranges as mentioned above were in close proximity to MEF2s. This truncation offered much less computational cost for each of the 45 all-atom simulations of the predicted complexes for 500 ns.

[0151] The stability of all class Ila HDAC-MEF2 complexes were monitored via measurements of the root-mean-square deviation (RMSD) and radius of gyration (RG). FIG.19, Panels A-D, show RMSD measurements, and FIG. 20, Panels A-D, represent RG measurements. Different shades represent different class Ila HDAC-MEF2 complexes.Since simulations for each class Ila HDAC-MEF2 complex were repeated three times, measurements from all three simulations are presented in lighter shades with the corresponding darker shades as the average values for each triplicate. The stability of RMSD and RG curves shows that the complexes were stable during the simulations. Single runs of a couple of complexes seemed to be fluctuating but stabilized toward the end of the simulations. FIG. 19 shows RMSD and RG measurements for the complexes with available crystal structures during this investigation.

[0152] While RMSD assesses better convergence and stable conformation and RG corresponds to compactness of simulated structures throughout the simulation [71-73], the evolution of potential energy profiles over the time course of the MD simulations were further analyzed, and the results are presented in FIG. 19. The potential energy vs time curve for each complex was stable, suggesting the stability of the simulated system

[0074] , The cosine content for the first three principal components (PCs; PC I, PC2, and PC3) for each class Ila HDAC-MEF2 complex was also calculated, and the results are presented in FIG. 21. In shorter simulations, due to insufficient convergence, initial PCs exhibit the shape of the cosine function [75-76], Therefore, cosine content that measures the closeness of the PC to a cosine shape can be used to assess the convergence of the MD simulation [76-78], The value of cosine content close to 1 for the first few PCs does not correspond to the convergence of a simulation system [76-78], As shown in FIG. 21, the average cosine content value for each class Ila HDAC-MEF2 complex was sufficiently lower than 1. For complexes with comparatively higher cosine content values for PCI, highly flexible terminal portions (awayAtty. Docket No. Georgetown.051. WOlfrom the binding interface) in HDACs and MEF2s were removed and the cosine content values were recalculated, which were found to be smaller than those calculated for the corresponding complete structures. Stable RMSD, RG, and potential energy profiles together with lower cosine content values for PCI, PC2, and PC3 as compared to 1 collectively suggest that the simulation systems are better stable and converged to produce a reliable prediction of interactions.Contribution of hydrophobic interactions for the formation ofHDAC-MEF2 complexes

[0153] The binding interface between class Ha HDACs and MEF2s in each class Ha HDAC-MEF2 complex was analyzed, and it was found that there were consistent hydrophobic interactions that are responsible for the formation of these complexes. Table 4 lists pairs of residues that are responsible for the hydrophobic interactions. These residues are the common residues that were found from analyses of all three all-atom MD simulation trajectories of each complex. It was found that L66 and L67 in all MEF2s consistently establish hydrophobic interactions with different class Ila HDACs. These residues were also in the list of residues that were predicted to establish hydrophobic interactions between HDAC7 and MEF2A in Example 1.

[0154] FIG. 22, Panel A shows that all residues in both chains A and B of all MEF2s that were predicted to establish hydrophobic interactions with class Ila HDACs are fully conserved in ail MEF2s. Table 5 lists the amino acid residues in both class Ila HDACs and MEF2s in the complexes with available crystal structures. Besides the A167(HDAC4)-M62(MEF2A) residue pair, V171-L67, L175-L66, and V179-L67 interacting pairs were found in both predicted and crystal structures of HDAC4-MEF2A complexes. Moreover, V171-L67 and L175-L66 interacting pairs were the same in both predicted and crystal structures of HDAC4-MEF2D; L187-L66, L191-L66 / L67, and V183-L66 / L67 in HDAC5-MEF2D; L89-L66 and V85-L66 / L67 in HDAC7-MEF2D; and L147-L66 and L151-L66 / L67 in HDAC9-MEF2D.

[0155] Notably, only amino acid residues from G169 to N181 in HDAC4, from W178 to S193 in HDAC5, from G83 to K96 in HDAC7, and from G139 to K154 in HDAC9 were available in the crystal structures for complexes of class Ila HDACs with MEF2D, in contrast to residues from G161 to K184 in HDAC4, residues from S173 to K196 in HDAC5, residues from K76 to N108 in HDAC7, and residues from G133 to K158 in HDAC9 that were used inAtty. Docket No. Georgetown.051. WOlthe corresponding predicted complexes. Table 6 shows the structural differences in class Ila HDACs between the available crystal structure and the predicted complex for each system. The amino acid residues in bold and underlined letters in predicted structures represent the ones that fall within the same range of amino acids in the available crystal structures. Since other amino acid residues are far away from the MEF2A interaction range, only the residues from position K145 in the HDAC4-MEF2A crystal structure were selected for subsequent simulations. It was also noticed that a few amino acids in other crystal structures were different from the sequences that were used to predict the corresponding complex. These structural differences might be a reason for a few discrepancies in the list of residue pairs that form hydrophobic interactions. Nevertheless, L66 and L67 in all MEF2s were found to be common in all simulations. This observation provides confidence in the prediction of complexes in this study. FIG. 22, Panel B represents the positions of all amino acid residues in both class Ila HDACs and MEF2s listed in Table 4 in the 3D structure of the class Ila HDAC-MEF2 complexes. Only one representative MEF2 (instead of all four MEF2s) was used to locate the position of amino acid residues, since the interacting residues were conserved.Contribution of hydrogen bonding and salt bridges

[0156] The analysis of hydrogen bonding between class Ila HDACs and MEF2s in each pair showed that hydrogen bonding moderately contributed to the formation of the class Ila HDAC-MEF2 complexes. The analysis predicted that D63 and T70 in all MEF2s consistently formed hydrogen bonds with residues in class Ila HDACs. Tables 7-10 shows the residues in both class Ila HDACs and MEF2s that formed hydrogen bonds with occupancies.

[0157] FIG. 23 shows the hydrogen bonding distances between atoms in different residues in both class Ila HDACs and MEF2s. Measurements from three independent simulations (same light colors) and their average values (corresponding dark colors) are presented. It was found that some hydrogen bonds were intermittent, as shown in FIG. 23, Panel D. As shown in Tables 7-10, the analysis did not predict hydrogen bond formation between all class Ila HDACs and MEF2s, unlike all class Ila HDACs showing hydrophobic interactions with different MEF2s. Table 11 shows a list of amino acid residues that established hydrogen bonds between residues in class Ila HDACs and MEF2s with crystal structures, and FIG. 24Atty. Docket No. Georgetown.051. WOlshows distance-time plots for corresponding hydrogen bonds. While residues establishing hydrogen bonds in HDAC4-MEF2A and HDAC4-MEF2D in crystal structures were also included in the results for corresponding predicted complexes, there were inconsistencies in the results for HDAC7-MEF2D and HDAC9-MEF2D structures in both crystals and predicted complexes that formed hydrogen bonds. These inconsistencies were likely due to structural differences. Notably, the range of amino acids in class Ila HDACs were longer in the predicted complexes as compared to what was found in the ciystal structures of class Ila HDACs with MEF2D.

[0158] The residues in bold text (Tables 9 and 10) that form hydrogen bonds between K96 in HDAC7 and D63 in MEF2D, as well as KI 54 in HDAC9 and D63 in MEF2B, also formed salt bridges for the predicted complexes, as shown in FIG. 25. This observation reinforces the importance of these residues in the corresponding complex formations. The analysis did not predict any salt bridges for the crystal structures, likely due to structural differences. The predicted salt bridges were quite few as compared to hydrogen bonds. Even though more hydrogen bonding than salt bridges was predicted, the hydrogen bonds were not as many as predicted hydrophobic interactions, suggesting that hydrogen bonding and salt bridges are not the main interaction mechanisms of the complex formations between class Ila HDACs and MEF2s. FIG. 26 shows the location of amino acid residues that form hydrogen bonds in the 3D structures of class Ila HDAC-MEF2 complexes. FIG. 26, Panel A shows that the amino acid residues in MEF2s that establish hydrogen bonding with different class Ila HDACs were conserved in all MEF2s. Therefore, only a representative MEF2 structure was used in complex with different HDACs.Binding affinities of class Ila HDAC-MEF2 interactions

[0159] MM / GBSA binding free energies were calculated for all 15 predicted complexes and five ciystal structures that were studied to quantify binding affinities between the class Ila HDACs and MEF2s. Even though the MM / GBSA binding free energy values are generally overestimated, the binding free energy values still offered a good comparison of binding affinities among different complexes

[0051] , The MM / GBSA binding free energies in Table 12 show that the class Ila HDAC-MEF2 complexes binded with a comparable affinity. The same was true for the HDAC7-MEF2A complex reported in Example 1. Table 13 lists the MM / GBSA binding free energy values for complexes with crystal structures.Atty. Docket No. Georgetown.051. WOl

[0160] Very similar affinity values for both predicted and the complex with the available crystal structure for the HDAC4-MEF2A complex were observed. However, the binding affinities for other complexes with crystal structures were weaker than the values for the corresponding structures that were predicted. Similar affinity (MM / GBSA binding free energy) values was obtained when extra amino acids were ignored from the simulation trajectories in the predicted complexes to match the amino acid residues available in the crystal structures. This implied that the weaker affinities for these crystal structures were due to the structural differences (fewer available class Ila HDACs amino acid residues).Interfacial contact analysis was also performed, and the results are presented in Table 14. As shown in Table 14, the number of interfacial contacts for the crystal structures involving MEF2D was much lower compared to the corresponding predicted structures. The higher number of interfacial contacts corresponded to a stronger affinity

[0079] , The results from the interfacial contact analysis thus supported weaker affinities for the ciystal structures with MEF2D as compared to the corresponding predicted structures. A similar number of contacts for both the crystal and predicted complexes of HDAC4-MEF2A were found. In the simulation of HDAC4-MEF2A ciystal structure, HDAC4 amino acid residues that reside along a long helical chain and are far away from the MEF2A interacting site were not used. This truncation offered a shorter simulation time. However, a slightly longer range (G145 to K183) in the ciystal was used as compared to the corresponding predicted structure (G161 to KI 84). The affinity of the complex formation was found to be -48.6 ± 2.3 kcal / mol (Table 13), which was very similar to what was calculated for the corresponding predicted complex. Moreover, analyses of binding interactions (Table 5 and Table 11) also did not predict any residues in HDAC4 outside the range used in the predicted complex. This justified the truncation of the HDAC4-MEF2A crystal structure for simulations.Surface Plasmon Resonance (SPR) Validations

[0161] The SPR-based technique was very useful to validate direct binding between biomolecules investigated using another method [52-54, 80, 81], Since all amino acid residues in MEF2s that were involved in hydrophobic interactions (FIG. 22) and hydrogen bonding, as well as salt bridges (FIG. 26), were conserved in all MEF2s, MEF2A was selected as a representative MEF2 and performed direct binding experiments with ail four class Ila HDACs. SPR experiments were repeated three times for each complex formation.Atty. Docket No. Georgetown.051. WOl

[0162] FIG. 27 shows representative SPR sensorgranis for the direct binding of class Ila HDACs to MEF2A immobilized onto CM5 chip surfaces. Each concentration of all of the class Ila HDACs was injected in duplicate. The continuous lines shown in FIG. 27 correspond to experimental data. We fitted the experimental data to the 1: 1 kinetic binding model. The dotted lines in FIG. 27 represent the fits. The association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD, affinity) values derived via SPR data fitting are provided in FIG. 27. The ka, kd, and KD values are listed as mean ± standard deviation from three independent experiments. The KD values for each class Ila HDAC-MEF2A complex were fairly comparable to nanomolar affinity. Comparable MM / GBSA binding free energy values listed in Table 12 agreed with the fairly comparable nanomolar affinities as obtained using SPR. A direct comparison of the MM / GBSA affinity value with the corresponding SPR affinity was not recommended. The discrepancy arose due to technical differences in the two methods. In MD simulations, the complex moved freely in an aqueous cubic box. In SPR experiments, one of the binding partners (MEF2A in this case) was restricted to the chip surface, and hence, the complex formed as a result of binding. Nevertheless, the similarity of binding affinities in SPR experiments validated the similarity in MM / GBSA values as observed in MM / GBSA calculations. Slightly higher SPR affinities (lower KD values) for HDAC4-MEF2A and HDAC7-MEF2A direct bindings might have corresponded to a larger number of hydrogen bonds formed for these complexes, as shown in Tables 7 and 9 and in Example 1. There were two pairs of amino acid residues predicted for the HDAC7-MEF2A complex that establish hydrogen bonding in Example 1. Moreover, the lack of hydrogen bonds HDAC9-MEF2A in Table 10 as compared to other class Ila HDACs might correspond to slightly weaker affinity (higher experimental KD value) for the HDAC9-MEF2A complex formation. Altogether, these SPR experiments not only validated the class Ila HDAC-MEF2A binding experimentally but also provided confidence in the computational results.Materials and MethodProteins, Sensorchips, and Reagents

[0163] HDAC4 (catalog no. MBS953633), HDAC5 (catalog no. MBS388238), and HDAC9 (catalog no. MBS2030545) were purchased from MyBioSource (San Diego, CA). HDAC7 (catalog no. LS-G22737) and MEF2A (catalog no. LS-G29304) were purchased from LSBioAtty. Docket No. Georgetown.051. WOl(Newark, CA). Series S CM5 sensor chips (catalog no. 29149603), amine coupling kit (catalog no. BR100050), and HBS-P+ buffer (catalog no. BRI 00671) were purchased from Cytiva (Marlborough, MA).System Preparation

[0164] UniProt

[0082] FASTA sequences (accession ID P56524 for HDAC4, accession ID Q9UQL6 for HDAC5, accession ID Q8WUI4 for HDAC7, accession ID Q9UKV0 for HDAC9, accession ID Q02078 for MEF2A, accession ID Q02080 for MEF2B, accession ID Q06413 for MEF2C, and accession ID QI 4814 for MEF2D) corresponding to all class Ila HDACs and MEF2s were used to predict 15 different complexes of class Ila HDACs (HDAC4, HDACS, HDAC7, and HDAC9) with all four MEF2s (4 HDAC4-MEF2 complexes, 4 HDAC5-MEF2 complexes, 3 HDAC7-MEF2 complexes, and 4 HDAC9-MEF2 complexes) using AlphaFold Colab.

[0047] , The remaining HDAC7-MEF2A complex was investigated in Example 1. The entire sequence of class Ila HDACs and MEF2s amino acids from positions 2 to 91 was supplied as input sequences during the generation of the full-length complex structures. Based on these results and multiple sequence alignments in Example 1, the truncated region of all class Ila HDACs in complex with all MEF2s was chosen for all-atom simulations. Also used were PDB ID 7XUZ

[0083] (HDAC4-MEF2A), PDB ID 8PDE

[0084] (HDAC4-MEF2D), PDB ID 8Q9P

[0084] (HDAC5-MEF2D), PDB ID 8Q9Q

[0084] (HDAC7-MEF2D), and PDB ID 8Q9R

[0084] (HDAC9-MEF2D) for all-atom MD simulations of published crystal structures containing human HDACs and human MEF2s. MD Simulations

[0165] All-atom MD simulations of each individual complex were carried out utilizing the NAMD software

[0085] (version 2.14 or 3.0) and CHARMM36m

[0050] force field as used in Example 1 and other prior investigations [51-54], The simulation input files were prepared using the CHARMM-GUI Web server

[0055] , Cubic boxes filled with TIP3 water model were used for solvation with the addition of 150 mM NaCl for neutralization. The size of the solvated cubic boxes for all 20 different systems varied from 73 × 73 × 73 Å3with a total number of atoms of 36,118 to 92 × 92 × 92 Å3with a total number of atoms of 72,803. The solvated and ion-neutralized systems were minimized for 10,000 steps with heavy atoms restrained harmoniously, followed by 100 ps equilibration under the NVT ensemble with 1 fs time step at 300 K. The long-range interactions were calculated using the Particle MeshAtty. Docket No. Georgetown.051. WOlEwald (PME) method

[0086] , The nonbonded interactions were cut off at 12 A. Production runs for analysis were executed using a time step of 2 fs for 500 ns at 300 K, 1 atm pressure, and Langevin dynamics with a damping constant of 1 ps1under the NPT conditions. Three independent replica runs were conducted for each complex. Root-mean -square deviation (RMSD), radius of gyration (RG), potential energy measurements, and cosine content calculations were performed to assess the stability and convergence of MD simulation trajectories.Surface Plasmon Resonance (SPR)

[0166] All SPR measurements were conducted using a Biacore T200 instrument (Marlborough, MA) with CM5 chips at 25 °C. MEF2A proteins were diluted in 10 mM sodium acetate buffer (pH 5.0) and immobilized onto the CM5 chips using standard amine coupling chemistry'. A neighboring flow cell (FC) was activated and deactivated using the same surface chemistry as the FC used to immobilize MEF2A, but no proteins were immobilized onto the reference FC. HBS-P (10 mM Hepes pH 7.4, 150 mM NaCl, 0.05% surfactant P20), which was 10* diluted from HBS-P+, was used as the immobilization running buffer (buffer that runs in the background during immobilization). Different concentrations of class Ila HDACs (40-1.25 nM, 2-fold dilutions) were prepared in kinetics buffer (buffer that was used to dilute the class Ila HDACs as well as runs in background during binding) over the reference and MEF2A immobilized surfaces. Each concentration of all class Ila HDACs was injected in duplicate to monitor technical reproducibility. HBS-P was used as the kinetics running buffer for binding of HDAC7 and HDAC9 to MEF2A. HBS-P supplemented with 1% (v / v) glycerol was used as the kinetics buffer for binding of HDAC4 and HDAC5 to MEF2A to avoid glycerol mismatch that was included in the storage buffer of HDAC4 and HDAC5 A flow rate of 30 pL / min was maintained during injections of all class Ila HDACs. The contact and dissociation times used for class Ila HDAC-MEF2A bindings were 90 and 300 s, respectively. A 20 s pulse of a regeneration solution containing 1: 500 H3PO4 (HsPO4:ddH2O, v / v) was injected for surface regeneration. All SPR sensorgrams obtained for analysis were both blank (buffer only response) and reference (response corresponding to the reference FC) subtracted. All SPR experiments were repeated in three independent experiments.Atty. Docket No. Georgetown.051. WOlData Analysis

[0167] The Visual Molecular Dynamics (VMD) software

[0056] was used to analyze all MD simulation trajectories, visualize complex structures, root-mean-square deviation (RMSD) measurements, interfacial contact analysis, and generate 3D figures of complexes.Measurements of radius of gyration (RG) and PCA-based cluster analysis were carried out using Camia

[0062] , The NAMD Energy plugin in VMD was used to calculate the potential energy. The MD Analysis tool was used to calculate cosine contents [76, 87], NAMD was used to calculate binding free energies using the MM / GBSA method

[0057] and to compare binding affinities using a simplistic approach, as was adopted for the HDAC7-MEF2A complex in Example 1 A 2.8 A distance cutoff was used in VMD to analyze hydrophobic interactions, 3.5 A distance and 30° angle cutoffs for hydrogen bonding, and a 3.5 A distance cutoff for salt bridges and interfacial contacts. COBALT

[0060] was used in multiple sequence alignments. Biacore T200 evaluation software version 3.2.1 (Marlborough, MA) was used to fit SPR sensorgrams using a 1:1 kinetics binding model. GraphPad Prism (Boston, MA) was used to plot the graphs.Table 4. Hydrophobic residues in HDAC-MEF2 complexes.HDACsMEF2sHDAC4 HDAC5 HDAC6 HDAC7 Name Chain AA AA AA AA AA M62 A 167L66, L67 V171 LI 51 AL66 L175 L147 MEF2AL67 L191L66 L175 LI 87 L147 BL67 V179 VI 43 L66 L175 LI 87 L147 A L67 VI 79, LI 80MEF2B L66, L67 LI 91 VI 43 L66 LI 75 LI 87 L89 LI 47 BL67 V171 VI 83 LI 51 L66 LI 75 LI 87, LI 91 VI 43, LI 47 MEF2C A L67 VI 79L66, L67 V85Atty. Docket No. Georgetown.051. WOlL66 LI 75 LI 87 L89, 193 LI 47, LI 51 L67 V17IBL66, L67 VI 83P75 Al 37 L66 LI 75 LI 87 L89 LI 47 A L67 VI 71 LI 92L66, L67 L191 V85MEF2DL66 LI 75 L187 L89 LI 47 B L67 193L66, L67 VI 83 LI 51Table 5. Hydrophobic residues in class Ila HDAC-MEF2 complexes with crystal structures.HDACsMEF2sHDAC4 HDACS HDAC6 HDAC7 Name Chain AA AA AA AA AA L66 LI 75AL67 VI 79MEF2AL66 LI 75BL67 VI 71L66 VI 79 L187 L89 L147 A L67 LI 80L66, L67 L191 VI 43 MEF2DL66 LI 75 LI 87 L89 LI 47 B L67 V171 L152L66, L67 VI 83 V85 L151Atty. Docket No. Georgetown.051. WOlTable 6. Comparison of amino acid residues in available crystal structures and that were used to predict different class Ila HDAC-MEF2 complexes. The amino acid residues in bold and underlined letters for predicted structures represent the ones that fall within the same range of amino acids in the available crystal structures. The amino acid residues before position K145 from the available crystal structure were not considered for the simulation of HDAC4-MEF2A crystal. The texts inside parentheses for complex structures correspond to PDB ID and chain (PDB ID, chain) used in simulations. The letters in italics represent differences in class Ila HDAC amino acids in the sequences of crystal and predicted structures.Complex Structures Class Ha HDAC Sequences145- Crystal KQHREQKLQQLKNKEKGKESAVASTEVKMKLQEFVL HDAC4- (7XUZ, A) NKK-183MEF2A (SEQ IDNO: 13)161 -GKESAVASTEVKMKLQEFVLNKKK- 184 Predicted(SEQ ID NO: 6)Crystal 169-GEVKMKLQEF VLN- 181HDAC4- (8PDE, C) (SEQ ID NO: 14)MEF2D 161 -GKESAVASTEVKMKLQEFVLNKKK- 184Predicted(SEQ ID NO: 6)Crystal 178-JTGSGEV’KLRLQEFLLS- 193 HDAC5- (8Q9P, X) (SEQ ID NO: 15)MEF2D 173-SKFSAMSTEVKLRLOEFLLSKSK- 196Predicted(SEQ ID NO: 7)Crystal 83 -G WK QK L AE VILKK-96HDAC7- (8Q9Q, X) (SEQ ID NO: 16)MEF2D 75 - SKR S A V A SNV VKOKLAE VILKK00 / \ A - 100Predicted(SEQ ID NO: 4)Crystal 139-GSGE VKQKLQEFLLSK- 154 HDAC9- (8Q9R, C) (SEQ ID NO: 17)MEF2D 133 -GRERA VA S7EVKOKLOEFLLSKA1 K- 158Predicted(SEQ ID NO: 8)Atty. Docket No. Georgetown.051. WOlTable 7. Amino acid residues responsible for the formation of hydrogen bonding in class Ila HDAC4-MEF2 complex with hydrogen bonding occupancies (occupancy values are listed as mean ± standard deviation (s.d.) from three independent runs of the complex. The residues in the bold text are also predicted to establish salt bridges).MEF2s HDAC4Name Chain AA AA % OccupancyD63 SI 68 66.0 ± 10.4A MEF2A T70B T70 Q176 52.0 ± 3.0D63MEF2B AT70 Q176 32.3 ± 7.5D63MEF2C AT70 Q176 30.2 ± 5.9A D63MEF2D D63BT70Q176 27.7 ± 6.3Table 8. Amino acid residues responsible for the formation of hydrogen bonding in class I la HDAC5-MEF2 complex with hydrogen bonding occupancies (occupancy values are listed as mean ± standard deviation (s.d.) from three independent runs of the complex. The residues in the bold text are also predicted to establish salt bridges).MEF2s HDAC5Name Chain AA AA % OccupancyD63A MEF2A T70 Q188 26.0 ± 8.0B T70D63 SI 80 69.1 ± 7.3MEF2B AT70D63MEF2C AT70A D63MEF2D D63BT70Atty. Docket No. Georgetown.051. WOlTable 9. Amino acid residues responsible for the formation of hydrogen bonding in class Ila HDAC7-MEF2 complex with hydrogen bonding occupancies (occupancy values are listed as mean ± standard deviation (s.d.) from three independent runs of the complex. The residues in the bold text are also predicted to establish salt bridges).MEF2s HDAC7Name Chain AA AA % OccupancyD63A MEF2A T70B T70D63MEF2B AT70D63 S82 59.6 ± 4.3MEF2C AT70A D63 S82 90.7 ± 15.3MEF2D D63 K96 56.8 ± 4.4BT70Table 10. Amino acid residues responsible for the formation of hydrogen bonding in class Ila HDAC9-MEF2 complex with hydrogen bonding occupancies (occupancy values are listed as mean ± standard deviation (s.d.) from three independent runs of the complex. The residues in the bold text are also predicted to establish salt bridges).MEF2s HDAC9Name Chain AA AA % OccupancyD63A MEF2A T70B T70D63 K154 57.1 ± 13.5MEF2B AT70D63MEF2C AT70A D63MEF2D D63BT70Atty. Docket No. Georgetown.051. WOlTable 11. Amino acid residues responsible for the formation of hydrogen bonding in class Ila HDAC4-MEF2 complex and HDAC9-MEF2 complex, and hydrogen bonding occupancies for complexes with crystal structures. The occupancy values are listed as mean ± standard deviation (s.d.) from the three independent runs of each complex.MEF2s (Cryst als) HDAC4 (Crystal) HDAC9 (Crystal) Name Chain AA AA % Occupancy AA % Occupancy MEF2A B D63 S168 39.7 + 10.5A T70 Q176 20.4 + 4.7MEF2DT70 QI 48B 27.2 ± 0.9Table 12. Binding free energies calculated using the MM / GBSA approach listed as mean ± standard deviation from the three independent rims of each complex.MEF2s HDACsHDAC4 AG HDAC5 AG HDAC7 AG HDAC9 AG Name(kcal / mol) (kcal / mol) (kcal / mol) (kcal / mol) MEF2A -51.6 ± 3.5 -48.9 + 3.2 -49.8 ± 4.4 MEF2B -54.7 ± 7.1 -52.3 ± 4.5 -51.7 ± 8.3 -49.8 ± 4.6 MEF2C -54.1 ± 7.2 -48.8 ± 7.3 -45.6 ± 2.1 -48.9 ± 2.5MEF2D -49.3 ± 6.9 -49.1 ± 6.0 -53.3 ± 8.5 -49.8 ± 5.7Table 13. MM / GBSA binding free energies listed as mean ± standard deviation (s.d.) from the three independent runs of each complex with crystal structures. The MM / GBSA values inside parentheses are calculated for modelled complex by removing extra residues from the complex to match amino acid residues as presented in the corresponding crystal structure.MEF2s HDACsHDAC4 AG HDAC5 AG HDAC7 AG HDAC9 AG Name(kcal / mol) (kcal / mol) (kcal / mol) (kcal / mol) MEF2A -48.6 ± 2.3-28.9 ± 0.9 -35.1 ± 0.6 -26.5 ± 0.7 -34.5 + 0.8 MEF2D(-37.8 ± 3.3)(-27.6 + 1.1) (-25.5 ± 0.5) (-35.0 + 2.0)Atty. Docket No. Georgetown.051. WOlTable 14. Comparison of number of interfacial contacts between the predicted complexes and corresponding complexes with crystal structures. The number of contact values are listed as mean ± standard deviation (s.d.) from the three independent runs of each complex.MEF2s HDACsName HDAC4 HDAC5 HDAC7 HDAC9 MEF2A Predicted 71,0 + 6.1Crystal 62.0 + 6.1Predicted 69,3 + 2.9 67.3 + 5.0 77.3 + 7.0 75.7 + 4.9MEF2D Crystal 35.7 + 1.5 40.7 + 3.2 38.3 + 4.2 36.7 + 0.6Atty. Docket No. Georgetown.051. 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Claims

Atty. Docket No. Georgetown.

051. WOlCLAIMS1. An isolated myocyte enhancer factor-2 (MEF2) binding molecule comprising an amino acid sequence of:Xi X2 X3X4A X6A S X9X10 V K X13 X14 L Xi6E Xis X19 L X21 K X23 X24X25 X26 (SEQ ID NO: 1),wherein:Xi is S or G, or is removed;X2 is K or R;X3 is R or E;X4 is S or R;X6is V or I;X9is S or T;X10 is V or E;X13 is Q, M, or L;X14 is K or R;Xi6 is A or Q;Xis is V or F;X19 is I, V, or L;X21 is K, N, or S;X23 is Q, K, or S;X24 is Q or A, or is removed;X25 is A or T, or is removed; andX26 is A or K.

2. The isolated MEF2 binding molecule according to claim 1, comprising the amino acid sequence of:Xi K X3S A V A S X9X10 V K X13 K L Xi6E Xis X19 L X21 K X23 X24X25 X26 (SEQ ID NO: 2),whereinXi is S or G, or is removed;X3 is R or E;Atty. Docket No. Georgetown.

051. WOlX9is S or T;Xio is V or E;X13 is Q or M;Xi6 is A or Q;Xis is V or F;X19 is I or V;X21 is K or N;X23 is Q or K;X24 is Q or is removed;X25 is A or is removed; andX26 is A or K.

3. The isolated MEF2 binding molecule according to claim 1 or 2, comprising the amino acid sequence of SKRSAVASSVVKQKLAEVILKKQQAA (SEQ ID NO: 4).

4. The isolated MEF2 binding molecule according to claim 1 or 2, comprising the amino acid sequence ofKRSAVASSVVKQKLAEVILKKQQAALERTVHPN (SEQ ID NO: 5).

5. The isolated MEF2 binding molecule according to claim 1, comprising the amino acid sequence of:Xi X2 E X4A X6A S T E V K X13 X14 L Q E F X19 L X21 K X23 X24X25 K (SEQ ID NO: 3), whereinXi is S or G;X2 is K or R;X4 is S or R;X6is V or I;X13 is Q, M, or L;X14 is K or R;X19 is V or L;X21 is N or S;X23 is K or S;Atty. Docket No. Georgetown.

051. WOlX24 is A or is removed;X25 is T or is removed.

6. The isolated MEF2 binding molecule according to claim 1 or 5, comprising the amino acid sequence of GKES AVASTEVKMKLQEFVLNKKK (SEQ ID NO: 6).

7. The isolated MEF2 binding molecule according to claim 1 or 5, comprising the amino acid sequence of SKESAIASTEVKLRLQEFLLSKSK ( SEQ ID NO: 7).

8. The isolated MEF2 binding molecule according to claim 1 or 5, comprising the amino acid sequence of GRERAVASTEVKQKLQEFLLSKS ATK (SEQ ID NO: 8).

9. The isolated MEF2 binding molecule according to any one of claims 1-8, wherein the isolated peptide binds to MEF2A, MEF2B, MEF2C, or MEF2D.

10. An isolated MEF2 binding molecule consisting of an amino acid sequence of SKRSAVASSVVKQKLAEVILKKQQAA (SEQ ID NO: 4).

11. An isolated MEF2 binding molecule consisting of an amino acid sequence of KRSAVASSVVKQKLAEVILKKQQAALERTVHPN (SEQ ID NO: 5).

12. The isolated MEF2 binding molecule according to claim 10 or 11, wherein the isolated peptide binds to MEF2A.

13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the isolated MEF2 binding molecule according to any one of claims 1-12.

14. A vector comprising the isolated nucleic acid molecule according to claim 13.

15. A host cell comprising the isolated nucleic acid molecule according to claim 13 or a vector according to claim 14.Atty. Docket No. Georgetown.

051. WOl16. A composition comprising the isolated MEF2 binding molecule of any one of claims 1-12 and a carrier.

17. A method of inhibiting binding of histone deacetylase (HD AC) and myocyte enhancer factor-2 (MEF2) in a cell, the method comprising contacting the cell with the isolated MEF2 binding molecule according to any one of claims 1-12.

18. The method according to claim 17, wherein the HDAC is selected from HDAC7, HDAC4, HDAC5, and HDAC9.

19. The method according to claim 17, wherein the MEF2 is selected from MEF2A, MEF2B, MEF2C, and MEF2D.

20. The method according to any one of claims 17-19, wherein the HDAC is HDAC7 and the MEF2 is MEF2A.

21. A method of inhibiting histone deacetylase (HDAC)-induced suppression of myocyte enhancer factor-2 (MEF2)-dependent gene expression in a cell, the method comprising contacting the cell with the isolated MEF2 binding molecule according to any one of claims 1-12.

22. The method according to claim 21, wherein the HDAC -induced suppression of MEF2-dependent gene expression is selected from HDAC7-induced suppression, HDAC4-induced suppression, HDAC5-induced suppression, and HDAC9-induced suppression.

23. The method according to claim 21 or 22, wherein the MEF2-dependent gene expression is selected from MEF2A-dependent gene expression, MEF2B-dependent gene expression, MEF2C-dependent gene expression, and MEF2D-dependent gene expression.

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