Polypeptides for inhibiting CDK5 phosphorylation activity

Description

PATENT APPLICATION-1-Inventors: J. Kent Werner, Jr., Albert Hunt, Jr., Ryan Spangler, Joseph Audie,Alexander Sergei Bayden, Yvonne AngellAttorney’s Docket No.: COTH-002-WO1Title: POLYPEPTIDES FOR INHIBITING CDK5 PHOSPHORYLATION ACTIVITYRELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 760,347, filed February 19, 2025. The entire teachings of the above application are incorporated herein by reference.FIELD OF THE DISCLOSURE

[0002] The disclosure relates to compositions, methods, processes, kits and devices for the design, preparation, manufacture, use and / or formulation of polypeptides for the treatment, prevention, diagnosis, and research of diseases, disorders, and / or conditions. The disclosure further embraces polypeptide compositions for delivery to a subject in need thereof.BACKGROUND

[0003] Cyclin-dependent kinase 5 (Cdk5) is a serine / threonine protein kinase primarily localized to neurons that is critical to nervous system development and function, with specific roles in supporting neuronal outgrowth, migration, synaptic health, and survival, as well as in the transport of intracellular cargoes and the patterning of cortical laminae (see, e.g., Dhavan and Tsai, Nat Rev Mol Cell Biol. 2001 Oct;2(10):749-59; and Cheung and Ip, Trends Cell Biol. 2012 Mar;22(3): 169-75; the contents of each of which are incorporated herein by reference in their entirety). Notably, the role of Cdk5 / P25 hyperactive complex in pathobiology is not limited to neurodegenerative disease, as growing evidence has implicated its activation in pain signaling in the peripheral nervous system, circadian rhythms, angiogenesis, and in oncology, e.g., pancreatic cancer, and beyond (see, e.g., Utreras et al., Drug Discov Today Ther Strateg. 2009 Sep;6(3): 105- 111 ; Liebl J. J Biol Chem. 2010 Nov 12;285(46):35932-43; Bosutti et al., PLoSAttorney Docket No.: COTH-002-WO1One. 2013 Sep 30;8(9):e75538; Eggers et al., Clin Cancer Res. 2011 Oct 1 ; 17(19):6140-50; and Kwak et al., J Biol Chem. 2013 Dec 27;288(52):36878-89; the contents of each of which are incorporated herein by reference in their entirety).

[0004] Despite tremendous therapeutic potential in the treatment of various nervous system and other diseases, disorders, and / or conditions (e.g., pain, cancer), no effective therapies exist for targeting Cdk5 protein kinase activity. While there have been drug discovery attempts to develop such therapies — as an example, ATP competitive small molecule Cdk5 inhibitors — these have largely failed, in part due to lack of sufficient selectivity leading to disruption of cellular functions, e.g., regulation of the cell cycle, attributed to other members of the Cdk family (see, e.g., Ahn et al., Chem Biol. 2005 Jul;12(7):811-23, the contents of which are incorporated herein by reference in their entirety). Overall, such efforts, typically involving Cdk5 inhibition, have been abandoned due to on- and off-target toxicity. Structural studies of the Cdk5 / P25 interface have also revealed that the complex buries a substantial portion of its surface area, a feature prohibitive to effective disruption using small molecule therapeutics (see, e.g., Tarricone et al., Mol Cell. 2001 Sep;8(3):657-69; Mapaelli et al., J Med Chem. 2005 Feb 10;48(3):671-9; Ahn et al., Chem Biol. 2005 Jul;12(7):811-23; the contents of each of which are incorporated herein by reference in their entirety). As Cdk5 / P25 is intracellular, it is also effectively intractable to protein or antibody therapy (Lee et al., Nature. 2000 May 18;405(6784):360-4, the contents of which are incorporated herein by reference in their entirety). Current Cdk5 inhibitors, small molecules, and antibody and protein treatments are suboptimal for therapeutic modulation of Cdk5-dependent activity and new therapeutic strategies are needed.

[0005] The present disclosure addresses this need by providing compositions and methods for novel polypeptides that have the potential to modulate Cdk5 / P25 hyperactivity and methods of using these polypeptides for the treatment, prevention, diagnosis and research of diseases, disorders and / or conditions. The present disclosure further embraces optimized polypeptide compositions for delivery to a subject in need thereof.SUMMARY OF THE DISCLOSURE

[0006] The disclosure provides Cdk5-inhibitory polypeptides. In some embodiments, the Cdk5-inhibitory polypeptides comprise an amino acid sequence having sequence identity to 10 to 24 consecutive amino acid residues of SEQ ID NO: 4. The Cdk5-inhibitory polypeptides may comprise an amino acid sequence having sequence identity to 10 to 24 consecutive amino acid- 2 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1residues of SEQ ID NO: 4 comprising one or more amino acid substitution compared to the amino acid sequence of SEQ ID NO: 4.

[0007] The Cdk5-inhibitory peptides may comprise one or more conservative amino acid substitution, or one or more unnatural amino acid substitution, or one or more amino acid modifications. In some embodiments, the Cdk5-inhibitory peptides comprise one or more amino acid substitution and / or one or more unnatural amino acid substitution and / or or one or more amino acid modifications. For example, the one or more amino acid substitution may enhance binding stability, stability of the Cdk5-inhibitory polypeptide in solution, alpha helicity of the Cdk5-inhibitory polypeptide, cell penetration of the Cdk5-inhibitory polypeptide, and / or solubility of the Cdk5-inhibitory polypeptide.

[0008] Examples of amino acid substitutions that can be implemented in the Cdk5-inhibitory polypeptides disclosed herein include:

[0009] a W5Y amino acid substitution at position 5 compared to SEQ ID NO: 4;

[0010] a D6(ABA), D6(AL4), D6(CTR), D6Q, D6E, D6L, D6M, D6(OME), D6(ORN), D6(SAR), D6T, D6W, or D6Y amino acid substitution at position 6 compared to SEQ ID NO: 4;

[0011] a C8(ABA), C8R, C8(CTR), C8Q, C8L, C8K, C8(ORN), C8W, or C8Y amino acid substitution at position 8 compared to SEQ ID NO: 4;

[0012] a V 1 l(HCS) amino acid substitution at position 11 compared to SEQ ID NO: 4;

[0013] an M15G, M15P, M15(PCO), or M15(HCS) amino acid substitution at position 15 compared to SEQ ID NO: 4;

[0014] an S17mS, SUH, or S17Mh amino acid substitution at position 17 compared to SEQ ID NO: 4;

[0015] an M19(HCS) amino acid substitution at position 19 compared to SEQ ID NO: 4; or

[0016] an A24M, A24V, A24L, A24(CYS-CYS), A24(HCY-HCY), A24R, A24K, A24(MDM), A24(NMI), (ORN), A24(2FM), A24(ESC), A24(NLE), A24(TFE), A24(YCP), or A24(26P) amino acid substitution at position 24 compared to SEQ ID NO: 4.

[0017] In some embodiments, the Cdk5-inhibitory polypeptides have an amino acid sequence having sequence identity to 10 to 24 consecutive amino acid residues of SEQ ID NO: 4 but with 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 4.

[0018] For instance, the Cdk5-inhibitory polypeptides may have an amino acid sequence having at least 79% identity to the amino acid sequence of SEQ ID NO: 4, at least 83% identity to the amino acid sequence of SEQ ID NO: 4, at least 87% identity to the amino acid sequence of- 3 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1SEQ ID NO: 4, at least 91% identity to the amino acid sequence of SEQ ID NO: 4, or at least at least 95% identity to the amino acid sequence of SEQ ID NO: 4.

[0019] Also provided herein are Cdk5-inhibitory peptides comprising an amino acid sequence having at least 79% sequence identity to the amino acid sequence of SEQ ID NO: 4, at least 83% sequence identity to the amino acid sequence of SEQ ID NO: 4, at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 4, at least 91% sequence identity to the amino acid sequence of SEQ ID NO: 4, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0020] The peptides can have one or more of the following amino acid substitutions compared to SEQ ID NO: 4:

[0021] a W5Y amino acid substitution at position 5 compared to SEQ ID NO: 4;

[0022] a D6(ABA), D6(AL4), D6(CTR), D6Q, D6E, D6L, D6M, D6(OME), D6(ORN), D6(SAR), D6T, D6W, or D6Y amino acid substitution at position 6 compared to SEQ ID NO: 4;

[0023] a C8(ABA), C8R, C8(CTR), C8Q, C8L, C8K, C8(ORN), C8W, or C8Y amino acid substitution at position 8 compared to SEQ ID NO: 4;

[0024] a V 1 l(HCS) amino acid substitution at position 11 compared to SEQ ID NO: 4;

[0025] an M15G, M15P, M15(PCO), or M15(HCS) amino acid substitution at position 15 compared to SEQ ID NO: 4;

[0026] an S17mS, SUH, or S17Mh amino acid substitution at position 17 compared to SEQ ID NO: 4;

[0027] an M19(HCS) amino acid substitution at position 19 compared to SEQ ID NO: 4; or

[0028] an A24M, A24V, A24L, A24(CYS-CYS), A24(HCY-HCY), A24R, A24K, A24(MDM), A24(NMI), (ORN), A24(2FM), A24(ESC), A24(NLE), A24(TFE), A24(YCP), or A24(26P) at position 24 compared to SEQ ID NO: 4.

[0029] In some embodiments, the Cdk5-inhibitory peptides provided herein have an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,- 4 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76.

[0030] Also provided are Cdk5-inhibitory peptides comprising various P5 truncations. For example, provided herein are Cdk5-inhibitory peptides having an amino acid sequence of SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. Various embodiments of the P5 truncation peptides comprise a C-terminal amide group modification. Various embodiments of the P5 truncation peptides comprise an N-terminal acetyl group modification. In some embodiments, the P5 truncation peptides comprise a C-terminal amide group modification and an N-terminal acetyl group modification.

[0031] Also provided herein are Cdk5-inhibitory polypeptides comprising an amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1. In some embodiments, the 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 comprise a Cdk5 helix domain. For example, the Cdk5 helix domain may comprise an amino acid sequence defined by amino acid S46 to amino acid L55 of SEQ ID NO: 1. In some embodiments, the Cdk5 helix domain comprises the amino acid sequence of SEQ ID NO: 81.

[0032] Some embodiments of the Cdk5-inhibitory polypeptides comprise an amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 but with one or more amino acid substitution compared to the amino acid sequence of SEQ ID NO: 81. The one or more amino acid substitution may comprise one or more conservative amino acid substitution, one or more unnatural amino acid substitution, or one or more amino acid modifications. In some embodiments, the disclosed Cdk5-inhibitory polypeptides comprise one or more conservative amino acid substitution and / or one or more unnatural amino acid substitution and / or or one or more amino acid modifications compared to SEQ ID NO: 81.

[0033] In some embodiments, the one or more amino acid substitution enhances binding stability, enhances stability of the Cdk5-inhibitory polypeptide in solution, alpha helicity of the Cdk5-inhibitory polypeptide, cell penetration of the Cdk5-inhibitory polypeptide, and / or enhances solubility of the Cdk5-inhibitory polypeptide.

[0034] Some embodiments of the Cdk5-inhibitory polypeptides comprise one or more amino acid substitution comprises as follows:

[0035] an amino acid substitution at position 1 of SEQ ID NO: 81 of: G1E, G1Y, GIA, GIF, G1Q, G1I, or GlS;- 5 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0036] an amino acid substitution at position 3 of SEQ ID NO: 81 of: P3E, P3L, P3I, P3Y, P3W, P3F, or P3Q;

[0037] an amino acid substitution at position 4 of SEQ ID NO: 81 of: S4A;

[0038] an amino acid substitution at position 5 of SEQ ID NO: 81 of: S5A;

[0039] an amino acid substitution at position 7 of SEQ ID NO: 81 of: L7A;

[0040] an amino acid substitution at position 8 of SEQ ID NO: 81 of: R8A;

[0041] an amino acid substitution at position 11 of SEQ ID NO: 81 of: Cl 1Y, Cl 1R, Cl IL, C11Q, orCHS;

[0042] an amino acid substitution at position 12 of SEQ ID NO: 81 of: L12A, L12F, L12W, L12S, L12Y, orL12R;

[0043] an amino acid substitution at position 13 of SEQ ID NO: 81 of: L13R;

[0044] an amino acid substitution at position 14 of SEQ ID NO: 81 of: K14A; or

[0045] an amino acid substitution at position 15 of SEQ ID NO: 81 of: E15W, E15A, E15Y, orE15Q.

[0046] The Cdk5-inhibitory polypeptides may comprise an amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 comprising the amino acid sequence of SEQ ID NO: 81 having 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 81.

[0047] Thus, the Cdk5-inhibitory polypeptides may comprise an amino acid sequence having at least 66% identity to the amino acid sequence of SEQ ID NO: 81, at least 73% identity to the amino acid sequence of SEQ ID NO: 81, at least 80% identity to the amino acid sequence of SEQ ID NO: 81, at least 87% identity to the amino acid sequence of SEQ ID NO: 81, or at least 93% identity to the amino acid sequence of SEQ ID NO: 81.

[0048] Also provided herein are Cdk5-inhibitory peptides comprising an amino acid sequence having at least 66% sequence identity to the amino acid sequence of SEQ ID NO: 81, at least 73% sequence identity to the amino acid sequence of SEQ ID NO: 81, at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 81, at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 81, or at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 81.

[0049] The Cdk5-inhibitory peptides may comprise a G1E, G1Y, GIA, GIF, G1Q, G1I, or G1S amino acid substitution at position 1 compared to SEQ ID NO: 81.

[0050] The Cdk5-inhibitory peptides may comprise a P3E, P3L, P3I, P3Y, P3W, P3F, or P3Q amino acid substitution at position 3 compared to SEQ ID NO: 81.- 6 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0051] The Cdk5-inhibitory peptides may comprise a S4A amino acid substitution at position 4 compared to SEQ ID NO: 81.

[0052] The Cdk5-inhibitory peptides may comprise a S5A amino acid substitution at position 5 compared to SEQ ID NO: 81.

[0053] The Cdk5-inhibitory peptides may comprise a L7A amino acid substitution at position 7 compared to SEQ ID NO: 81.

[0054] The Cdk5-inhibitory peptides may comprise a R8A amino acid substitution at position 8 compared to SEQ ID NO: 81.

[0055] The Cdk5-inhibitory peptides may comprise aCHY, C11R, C11L, C11Q, or CHS amino acid substitution at position 11 compared to SEQ ID NO: 81.

[0056] The Cdk5-inhibitory peptides may comprise a L12A, L12F, L12W, L12S, L12Y, or L12R amino acid substitution at position 12 compared to SEQ ID NO: 81.

[0057] The Cdk5-inhibitory peptides may comprise a L13R amino acid substitution at position 13 compared to SEQ ID NO: 81.

[0058] The Cdk5-inhibitory peptides may comprise a K14A amino acid substitution at position 14 compared to SEQ ID NO: 81.

[0059] The Cdk5-inhibitory peptides may comprise a E15W, E15A, E15Y, or E15Q amino acid substitution at position 15 compared to SEQ ID NO: 81.

[0060] Various embodiments of the Cdk5-inhibitory peptides provided herein have an amino acid sequence of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116.

[0061] The Cdk5-inhibitory polypeptide or peptides may inhibit Cdk5 / p25 activity and / or may not inhibit Cdk5 / p35 activity.BRIEF DESCRIPTION OF THE FIGURES

[0062] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.- 7 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0063] FIG. 1 shows HEK293 cells were transfected with optimal p25 / CDK5 and p35 / CDK5 (1:100 Donor: Acceptor ratio) and allowed to express overnight. Cells were labeled + / - 618 NanoBRET™ Ligand. 3 concentrations of control peptide (CT526) were electroporated into transfected cells. Cells were transferred to 96 well plate and NanoBRET™ substrate was added and measurements were collected on the GloMax® Discover instrument for up to 6 hours. Data was analyzed in Prism showing NanoBRET™ ratios (left panels) and normalized values (right panels).

[0064] FIG. 2 shows a plot of percent CDK5 / P25 complex formed versus concentration of Cdk5-inhibitory peptides (CT600 and CT526) or scrambled peptide as measured by ELISA.

[0065] FIG. 3 shows a plot of relative kinase activity versus concentration of CDK-5 inhibitory peptides (CT600 and CT526), scrambled negative control peptide, and tamoxifen positive control small molecule Cdk5 inhibitor.

[0066] FIG. 4 shows modulation of a cyclin-dependent kinase 5 (CDK5)-regulated phosphorylation network in a brain following administration of one or more exemplary peptides.

[0067] FIG. 5 shows dose-dependent disruption by peptides. Peptides containing unnatural amino acid substitution variants were tested in the STAT3 activity assay. These modifications yield peptides capable of disrupting the CDK5 / P25 complex.

[0068] FIGS. 6A-6B shows a comparison of PEG-modified peptides. FIG. 6A provides a comparison of CT526 and CT526PEG. FIG. 6B provides a comparison of CT600 and CT600PEG. Peptides containing pegylated variants were tested in the STAT3 activity assay. These modifications yield peptides capable of disrupting the CDK5 / P25 complex.

[0069] FIG. 7 shows brain slide immunohistochemistry was performed with an antiody against phospho-Tau 181 in the C9ORF72 AAV mouse model of Amyotrophic Lateral Sclerosis (ALS). A significant decrease in the percentage of phospho-Taul81-stained area was observed after treatment with two different doses of CT600. This result demonstrates that CT600 engages its target, the CDK5 / P25 complex, in vivo in mice, resulting in a significant reduction in tau hyperphosphorylation.DETAILED DESCRIPTIONI. INTRODUCTION

[0070] Cyclin-dependent kinase 5 (Cdk5) is a serine / threonine protein kinase primarily localized to neurons that is critical to nervous system development and function, with specific roles in supporting neuronal outgrowth, migration, synaptic health, and survival, as well as in the- 8 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1transport of intracellular cargoes and the patterning of cortical laminae (see, e.g., Dhavan and Tsai, Nat Rev Mol Cell Biol. 2001 Oct; 2(10):749-59; and Cheung and Ip, Trends Cell Biol. 2012 Mar; 22(3): 169-75; the contents of each of which are incorporated herein by reference in their entirety). Notably, the role of Cdk5 / P25 hyperactive complex in pathobiology is not limited to neurodegenerative disease, as growing evidence has implicated its activation in pain signaling in the peripheral nervous system, circadian rhythms, angiogenesis, and in oncology, e.g., pancreatic cancer, and beyond (see, e.g., Utreras et al., Drug Discov Today Ther Strateg. 2009 Sep;6(3): 105-111 ; Liebl J. J Biol Chem. 2010 Nov 12;285(46):35932-43; Bosutti et al., PLoS One.2013 Sep 30;8(9):e75538; Eggers et al., Clin Cancer Res. 2011 Oct 1; 17(19):6140-50; and Kwak et al., J Biol Chem. 2013 Dec 27; 288(52):36878-89; the contents of each of which are incorporated herein by reference in their entirety).

[0071] Despite vast therapeutic potential in the treatment of various nervous system and other diseases, disorders, and / or conditions (e.g., pain, cancer), no effective therapies exist for targeting Cdk5 protein kinase activity. While there have been drug discovery attempts to develop such therapies — as an example, ATP competitive small molecule Cdk5 inhibitors — these have largely failed, in part due to lack of sufficient selectivity leading to disruption of cellular functions, e.g., regulation of the cell cycle, attributed to other members of the Cdk family (see, e.g., Ahn et al., Chem Biol. 2005 Jul; 12(7):811-23, the contents of which are incorporated herein by reference in their entirety). Overall, such efforts, typically involving Cdk5 inhibition, have been abandoned due to on- and off-target toxicity. Structural studies of the Cdk5 / P25 interface have also revealed that the complex buries a substantial portion of its surface area, a feature prohibitive to effective disruption using small molecule therapeutics (see, e.g., Tarricone et al., Mol Cell. 2001 Sep;8(3):657-69; Mapaelli et al., J Med Chem. 2005 Feb 10;48(3):671-9; Ahn et al., Chem Biol.2005 Jul; 12(7):811-23; the contents of each of which are incorporated herein by reference in their entirety). As Cdk5 / P25 is intracellular, it is also effectively intractable to protein or antibody therapy (Lee et al., Nature. 2000 May 18;405(6784):360-4, the contents of which are incorporated herein by reference in their entirety). Current Cdk5 inhibitors, small molecules, and antibody and protein treatments are suboptimal for therapeutic modulation of Cdk5-dependent activity and new therapeutic strategies are needed.

[0072] The present disclosure addresses this need by providing novel polypeptides, which may include any amino acid-based molecules (natural or non-natural), such as peptides, peptidomimetics, peptoids, and proteins, as further described herein, that have the potential to modulate Cdk5 / P25 hyperactivity and methods of using these polypeptides for the treatment,- 9 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1prevention, diagnosis and research of diseases, disorders and / or conditions. The present disclosure further embraces optimized polypeptide compositions for delivery to a subject in need thereof.Neurodegenerative Disease

[0073] From a behavioral standpoint, Cdk5 contributes to higher-order cognitive functions, including learning and memory, and hence, its proper function ultimately contributes to one’s ability to perform normal activities of everyday living (see, e.g., Fischer et al, Neuron. 2005 Dec 8;48(5): 825-38, Angelo et al., J Neurochem. 2006 Oct;99(2):353-70, Cheung and Ip, Trends Cell Biol. 2012 Mar;22(3): 169-75, the contents of each of which are incorporated herein by reference in their entirety). Given its multifunctional nature targeting more than a hundred proteins (including other protein kinases and phosphatases), Cdk5 activity is both tightly and topographically regulated under non-pathologic conditions, processes largely dependent on the binding of its neuron-specific, cyclin-regulated activators, P35 (“Cyclin-Dependent Kinase 5, Regulatory Subunit 1”; “CDK5R1”; Ensemble Gene ID: ENSG00000176749) and p39 (“Cyclin Dependent Kinase 5 Regulatory Subunit 2”; “CDK5R2”; Ensemble Gene ID:ENSG00000171450). Underscoring the importance of these co-activators in cdk5 modulation, P35 and p39 double-null mice, which exhibit a phenotype similar to Cdk5 knockout, display lamination defects and perinatal lethality, (Ko et al., J Neurosci. 2001 Sep 1 ;21(17):6758-71, the contents of which are incorporated herein by reference in their entirety).

[0074] The dysregulation and resultant hyperactivation of kinases such as Cdk5 has recently emerged as a key player in the etiology and progression neurodegenerative disorders, many of which lead to dementia. For instance, brain lesions such as extracellular amyloid beta (senile) plaques, and intracellular microtubule associated tau protein and neurofilament proteins phosphorylated at identified pathologic epitope(s), are histopathologic hallmarks of diverse neurodegenerative disorders, including Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD) (see, e.g. Cheung and Ip, Trends Cell Biol. 2012 Mar;22(3): 169-75, the contents of which are incorporated herein by reference in their entirety). Specifically, in AD, intracellular aggregates of hyperphosphorylated tau protein named neurofibrillary tangles (NFTs) track with clinical phenotypes in patients more strongly than do senile plaques (Bennet et al., Arch Neurol. 2004 Mar;61(3):378-84, Nelson et al., J Neuropathol Exp Neurol. 2009 Jan;68(l):l-14, the contents of each of which are incorporated herein by reference in their entirety). A number of additional phospho-tau species, e.g., soluble monomers- 10 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1and oligomers, have also been identified as key pathogenic entities in synaptic and neuronal destruction characteristic of AD. A growing heterogeneous group of diseases characterized by abnormally phosphorylated tau protein, the neurodegenerative tauopathies, includes diseases such as frontotemporal lobar degeneration, chronic traumatic encephalopathy, Progressive supranuclear palsy, corticobasal degeneration, and Pick disease (see, e.g., Iqbal et al., Nat Rev Neurol. 2016 Jan;12(l):15-27, the contents of which are incorporated herein by reference in their entirety). In each case, the abnormal phosphorylation of tau protein is a pivotal catalyst for disease progression.

[0075] Dysregulated Cdk5 activity may be linked to the development of phosphorylationdependent disease processes such as the formation of NFTs in AD. Mechanistically, a model of the role of Cdk5 in neurodegeneration suggests that first, cellular challenges such as oxidative stress, metabolic irregularities, amyloid beta toxicity, and inflammation trigger the influx of calcium into neurons, thereby activating calcium-activated protease calpain, which is in turn responsible for the cleavage of P35 into plO (n-terminus) and P25 (c-terminus; see, e.g., Patrick et al., J Biol Chem. 1998 Sep ll;273(37):24057-64, Patrick et al., Nature. 1999 Dec 9;402(6762):615-22, Lee et al., Nature. 2000 May 18;405(6784):360-4, Zheng et al., J Biol Chem. 2010 Oct 29;285(44):34202-12, the contents of each of which are incorporated herein by reference in their entirety). Next, P25 forms a more stable complex with Cdk5, which abnormally localizes to the cytoplasm and nucleus where it is hyperactivated. The cascade leading to formation of the Cdk5 / P25 complex is associated with a number of aberrant downstream signaling patterns: e.g., the plO domain sequesters P35 at the cytoplasmic face of the cell membrane and targets it for ubiquitination and degradation; the absence of plO extends the halflife of P25 (see, e.g., Patrick et al., J Biol Chem. 1998 Sep ll;273(37):24057-64, Patrick et al., Nature. 1999 Dec 9;402(6762):615-22, the contents of each of which are incorporated herein by reference in their entirety). Key among these patterns, however, is Cdk5 / P25-dependent tau hyperphosphorylation. Indeed, while it is estimated that tau possesses more than 70 phosphorylation sites, nearly a quarter of these may be phosphorylated, at least in part, by Cdk5. Of these, at least two — Ser202 and Ser205 — are unique to Cdk5 and are also responsible for priming subsequent phosphorylation at neighboring sites by glycogen synthase kinase 3-P (see, e.g., Shukla et al., Arch Med Res. 2012 Nov;43(8):655-62, Kimura et al., Front Mol Neurosci.2014 Jul 15;7:65,Tenreiro et al., Front Mol Neurosci. 2014 May 13;7:42, the contents of each of which are incorporated herein by reference in their entirety). Importantly, increased levels of P25 and Cdk5 activity have been identified in AD brain with approximately a 20-40 fold increase in- 11 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1the P25 / P35 ratio in four out of five AD patients (see, e.g., Patrick et al., Nature. 1999 Dec 9;402(6762):615-22, Tseng et al., FEBS Lett. 2002 Jul 17;523( l-3):58-62, the contents of each of which are incorporated herein by reference in their entirety), and Cdk5 genome-wide association study variants increase the risk of AD ( Arias- Vasquez et al., J Neurol. 2008 May;255(5):655-62, the contents of which are incorporated herein by reference in their entirety). In vitro work has demonstrated the conversion of P35 to P25, Cdk5 activation, tau phosphorylation and cell death in cortical neurons treated with amyloid beta, providing direct support for abnormal Cdk5 signaling in cell toxicity (see, e.g., Town et al., J Neurosci Res. 2002 Aug 1 ;69(3):362-72, Zheng et al., EMBO J. 2005 Jan 12;24(l):209-20, the contents of each of which are incorporated herein by reference in their entirety). Apart from tau phosphorylation, the introduction of P25 into primary neurons increases aberrant phosphorylation of APP and amyloid beta peptide secretions (Lui et al., FEBS Lett. 2003 Jul 17;547(l-3): 193-6, the contents of which are incorporated herein by reference in their entirety). Cell culture and AD mutant mouse model work involving the mutation of P35 cleavage site have demonstrated rescue of disease phenotypes, enhanced synaptic integrity and reduced levels of pathological tau and / or amyloid species (Seo et al., Cell.2014 Apr 10;157(2):486-498. 2014, Seo et al., J Neurosci. 2017 Oct 11;37(41):9917-99242017, the contents of each of which are incorporated herein by reference in their entirety).

[0076] Thus, in some embodiments, the compositions disclosed herein are useful in the treatment of neurodegenerative and other neurological disorders or conditions including ataxia(s), Alzheimer's Disease, frontotemporal dementia, dementia with Lewy bodies (DLB), Parkinson's disease, neuronal injury (stroke, traumatic brain injury (TBI) and epilepsy), progressive supranuclear palsy, amyotrophic lateral sclerosis (ALS), and / or limbic-predominant age-related TDP-43 encephalopathy (LATE).Proliferative Disorders

[0077] Polypeptides, including peptides, of the present disclosure target aberrant Cdk5-dependent activity associated with neurodegenerative disease, e.g., hyperphosphorylation of tau in AD, and other pathology by targeting protein-protein interactions such as that between P25 and Cdk5. Indeed, substantial progress has been made in disrupting this class of interactions toward the development of diverse therapeutics (See, e.g., Wetzler M. and Hamilton P. (2018). Peptides as Therapeutics in S. Koutsopoulos (Ed.), Peptide Applications in Biomedicine, Biotechnology and Bioengineering (pp. 215-230). Duxford, UK: Woodhead Publishing, the contents of which are incorporated herein by reference in their entirety). Generally speaking,- 12 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1small peptides have several advantages as a modality for disease treatment, key among which are safe catabolism and limited idiosyncratic toxicity. Indeed, the success rate of peptides is nearly double that of small molecules in clinical trials (DiMasi et al., Clin Pharmacol Ther. 2010 Mar;87(3):272-7, the contents of which are incorporated herein by reference in their entirety).

[0078] Pre-clinical studies from Pant and colleagues provide compelling evidence for a peptide-based approach to disruption of the Cdk5 / P25 complex toward reduced AD pathology, in particular, via specific targeting of the Cdk5 binding site, (see, e.g., Kesavapany et al., Biotechnol J. 2007 Aug;2(8):978-87, Shukla et al., FASEB J. 2013 Jan;27(l): 174-86, Sundaram et al., J Neurosci. 2013 Jan 2;33(l):334-43, Zheng et al., J Biol Chem. 2010 Oct 29;285(44):34202-12, Zheng et al., EMBO J. 2005 Jan 12;24(l):209-20, Zheng et al., PLoS One.2013 Sep 5;8(9):e63332, Zheng et al., Eur J Biochem. 2002 Sep;269(18):4427-34, and US Publication No. US8597660, the contents of each of which are incorporated herein by reference in their entirety). Initial in vitro work by the group demonstrated that a truncated form of P35 (aa 154-279), Cdk5 inhibitory peptide (CIP) inhibited Cdk5 activity. Follow-up studies revealed that a truncated 24-mer CIP peptide, P35 aa 245-277, when conjugated to an 11-aa transactivator of transcription (Tat) protein sequence, competed with P25 binding and inhibited cdk5 hyperactivation without disruption of physiological Cdk5 / P35. Subsequent in vivo validation work using a CIP-transgenic mouse demonstrated reduced selective inhibition of Cdk5 / P25 hyperactivation. In addition, in the 5xFAD mouse model of AD, a TAT-fusion applied to the truncated 24-mer (TFP5) inhibited Cdk5 / P25 hyperactivity, was associated with reduced AD pathology and inflammation, and rescued of cognitive and motor deficits. Further, PCT Pat. App. Pub. No. W02020102404 (incorporated herein by reference in its entirety) discloses use of the 24 amino acid peptide P5 for decreasing cancer cell viability, increasing apoptosis of cancer cells and treating cancer in subjects in need thereof. Together, these findings provide substantial basis for the need to further pursue the design and discovery of optimized peptides to modulate Cdk5 / P25 hyperactivity in neurodegenerative disease and other diseases, disorders, and / or conditions.

[0079] Thus, in some embodiments, the compositions disclosed herein are useful in the treatment of cancers including cancer of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart or adrenals. More particularly, cancers include solid tumor, sarcoma, carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,- 13 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi’s sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, a blood-bone tumor, acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, or multiple myeloma. Particular embodiments of cancer include glioblastoma and colorectal cancer.II. COMPOSITIONS OF THE DISCLOSUREPolypeptides of the Disclosure

[0080] The disclosure provides Cdk5-inhibiting polypeptides, which may include any amino acid-based molecules (natural or non-natural). As used herein, “polypeptide” embraces “peptides,” “peptidomimetics,” “peptoids,” and “proteins.” “Peptides” are traditionally considered to be polypeptides that range in size from about 4 to about 50 amino acids.Polypeptides larger than about 50 amino acids are generally termed “proteins.” The term “polypeptide” further embraces amino acid-based molecules comprising modifications including modified amino acid residues and / or unnatural amino acids, as further described herein.

[0081] In some embodiments, peptides and peptide variants or modifications and derivatives for selectively inhibiting Cdk5 / P25 activity are provided herein. In some instances, the Cdk5-inhibitory peptides are variants, modifications, and / or derivatives of the human Cdk5 protein sequence (SEQ ID NO: 1 or SEQ ID NO: 2). In some instances, the Cdk5-inhibitory peptides are variants, modifications, and / or derivatives of the human P35 protein sequence (SEQ ID NO: 3). In some instances, the Cdk5-inhibitory peptides are variants, modifications, and / or derivatives of the amino acid sequence of the P5 peptide (SEQ ID NO: 4), itself derived from the human P35- 14 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1protein. Amino acid sequences for SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 are provided in Table 1.

[0082] As used herein, “variants” refers to peptides having one or more amino acid difference between the variant peptide and a reference sequence. A variant peptide, for example, may include one or more amino acid substitution as compared to the reference sequence.

[0083] As used herein, “modifications” refers to peptides having one or more modified amino acid as compared to an unmodified reference sequence. A modified peptide, for example, may include one or more amino acid modification as compared to the reference sequence.Alternatively, or in addition, a modified peptide may include, for example, an added domain such as an added peptide label or other label (e.g., a detectable label), a peptide linker or other linker, or an added peptide or other moiety as compared to an unmodified reference sequence.

[0084] As used herein, “derivatives” refers to peptides having amino acid sequences derived from a parent polypeptide sequence such as a parent protein or a parent polypeptide. For instance, the peptide “P5” as described herein is a derivative of the peptide “P35” as described herein in that the amino acid sequence of P5 corresponds to the amino acid sequence of amino acids 254-277 of the parent P35 polypeptide sequence (i.e., P5 corresponds to K254-A277 of SEQ ID NO: 3).

[0085] The terms variant, modification, and derivative are not mutually exclusive. Some embodiments of the peptides provided herein are or comprise variants and / or modifications and / or derivatives of another (i.e., a reference) peptide. For instance, a peptide may include a single amino acid modification on a first residue compared to a reference sequence while a second amino acid residue is substituted with an alternative residue, such that the resulting peptide is both a modification and a variant of the reference peptide.Table 1. Human Cdk5 and P35 Amino Acid Sequences- 15 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0086] In some embodiments, Cdk5-inhibitory peptides of the present disclosure are fragments comprising 10 to 25 consecutive amino acid residues of SEQ ID NO: 1. For example, in the case of 10 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 10, aa 2 to aa 11, aa 3 to aa 12, etc., including up to aa 283 to aa 292 of SEQ ID NO: 1.

[0087] In the case of 11 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 11, aa 2 to aa 12, aa 3 to aa 13, etc., including up to aa 282 to aa 292 of SEQ ID NO: 1.

[0088] In the case of 12 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 12, aa 2 to aa 13, aa 3 to aa 14, etc., including up to aa 281 to aa 292 of SEQ ID NO: 1.

[0089] In the case of 13 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 13, aa 2 to aa 14, aa 3 to aa 15, etc., including up to aa 280 to aa 292 of SEQ ID NO: 1.

[0090] In the case of 14 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 14, aa 2 to aa 15, aa 3 to aa 16, etc., including up to aa 279 to aa 292 of SEQ ID NO: 1.

[0091] In the case of 15 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 15, aa 2 to aa 16, aa 3 to aa 17, etc., including up to aa 278 to aa 292 of SEQ ID NO: 1.

[0092] In the case of 16 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 16, aa 2 to aa 17, aa 3 to aa 18, etc., including up to aa 277 to aa 292 of SEQ ID NO: 1.- 16 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0093] In the case of 17 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 17, aa 2 to aa 18, aa 3 to aa 19, etc., including up to aa 276 to aa 292 of SEQ ID NO: 1.

[0094] In the case of 18 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 18, aa 2 to aa 19, aa 3 to aa 20, etc., including up to aa 275 to aa 292 of SEQ ID NO: 1.

[0095] In the case of 19 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 19, aa 2 to aa 20, aa 3 to aa 21, etc., including up to aa 274 to aa 292 of SEQ ID NO: 1.

[0096] In the case of 20 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 20, aa 2 to aa 21, aa 3 to aa 22, etc., including up to aa 273 to aa 292 of SEQ ID NO: 1.

[0097] In the case of 21 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 21, aa 2 to aa 22, aa 3 to aa 23, etc., including up to aa 272 to aa 292 of SEQ ID NO: 1.

[0098] In the case of 22 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 22, aa 2 to aa 23, aa 3 to aa 24, etc., including up to aa 271 to aa 292 of SEQ ID NO: 1.

[0099] In the case of 23 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 23, aa 2 to aa 24, aa 3 to aa 25, etc., including up to aa 270 to aa 292 of SEQ ID NO: 1.

[0100] In the case of 24 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 24, aa 2 to aa 25, aa 3 to aa 26, etc., including up to aa 269 to aa 292 of SEQ ID NO: 1.

[0101] In the case of 25 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 25, aa 2 to aa 26, aa 3 to aa 27, etc., including up to aa 268 to aa 292 of SEQ ID NO: 1.

[0102] In some embodiments, Cdk5-inhibitory peptides of the present disclosure are fragments comprising 10 to 25 consecutive amino acid residues of SEQ ID NO: 3. For example, in the case of 10 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 10, aa 2 to aa 11, aa 3 to aa 12, etc., including up to aa 298 to aa 307 of SEQ ID NO: 3.- 17 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0103] In the case of 11 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 11, aa 2 to aa 12, aa 3 to aa 13, etc., including up to aa 297 to aa 307 of SEQ ID NO: 3.

[0104] In the case of 12 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 12, aa 2 to aa 13, aa 3 to aa 14, etc., including up to aa 296 to aa 307 of SEQ ID NO: 3.

[0105] In the case of 13 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 13, aa 2 to aa 14, aa 3 to aa 15, etc., including up to aa 295 to aa 307 of SEQ ID NO: 3.

[0106] In the case of 14 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 14, aa 2 to aa 15, aa 3 to aa 16, etc., including up to aa 294 to aa 307 of SEQ ID NO: 3.

[0107] In the case of 15 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 15, aa 2 to aa 16, aa 3 to aa 17, etc., including up to aa 293 to aa 307 of SEQ ID NO: 3.

[0108] In the case of 16 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 16, aa 2 to aa 17, aa 3 to aa 18, etc., including up to aa 292 to aa 307 of SEQ ID NO: 3.

[0109] In the case of 17 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 17, aa 2 to aa 18, aa 3 to aa 19, etc., including up to aa 291 to aa 307 of SEQ ID NO: 3.

[0110] In the case of 18 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 18, aa 2 to aa 19, aa 3 to aa 20, etc., including up to aa 290 to aa 307 of SEQ ID NO: 3.

[0111] In the case of 19 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 19, aa 2 to aa 20, aa 3 to aa 21, etc., including up to aa 289 to aa 307 of SEQ ID NO: 3.

[0112] In the case of 20 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 20, aa 2 to aa 21, aa 3 to aa 22, etc., including up to aa 288 to aa 307 of SEQ ID NO: 3.

[0113] In the case of 21 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 21, aa 2 to aa 22, aa 3 to aa 23, etc., including up to aa 287 to aa 307 of SEQ ID NO: 3.- 18 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0114] In the case of 22 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 22, aa 2 to aa 23, aa 3 to aa 24, etc., including up to aa 286 to aa 307 of SEQ ID NO: 3.

[0115] In the case of 23 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 23, aa 2 to aa 24, aa 3 to aa 25, etc., including up to aa 285 to aa 307 of SEQ ID NO: 3.

[0116] In the case of 24 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 24, aa 2 to aa 25, aa 3 to aa 26, etc., including up to aa 284 to aa 307 of SEQ ID NO: 3.

[0117] In the case of 25 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 25, aa 2 to aa 26, aa 3 to aa 27, etc., including up to aa 283 to aa 307 of SEQ ID NO: 3.

[0118] In some embodiments, Cdk5-inhibitory peptides of the present disclosure are fragments comprising 10 to 24 consecutive amino acid residues of SEQ ID NO: 4. For example, in the case of 10 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 10, aa 2 to aa 11, aa 3 to aa 12, etc., including up to aa 15 to aa 24 of SEQ ID NO: 4.

[0119] In the case of 11 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 11, aa 2 to aa 12, aa 3 to aa 13, etc., including up to aa 14 to aa 24 of SEQ ID NO: 4.

[0120] In the case of 12 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 12, aa 2 to aa 13, aa 3 to aa 14, etc., including up to aa 13 to aa 24 of SEQ ID NO: 4.

[0121] In the case of 13 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 13, aa 2 to aa 14, aa 3 to aa 15, etc., including up to aa 12 to aa 24 of SEQ ID NO: 4.

[0122] In the case of 14 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 14, aa 2 to aa 15, aa 3 to aa 16, etc., including up to aa 11 to aa 24 of SEQ ID NO: 4.

[0123] In the case of 15 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 15, aa 2 to aa 16, aa 3 to aa 17, etc., including up to aa 10 to aa 24 of SEQ ID NO: 4.- 19 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0124] In the case of 16 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 16, aa 2 to aa 17, aa 3 to aa 18, etc., including up to aa 9 to aa 24 of SEQ ID NO: 4.

[0125] In the case of 17 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 17, aa 2 to aa 18, aa 3 to aa 19, etc., including up to aa 8 to aa 24 of SEQ ID NO: 4.

[0126] In the case of 18 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 18, aa 2 to aa 19, aa 3 to aa 20, etc., including up to aa 7 to aa 24 of SEQ ID NO: 4.

[0127] In the case of 19 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 19, aa 2 to aa 20, aa 3 to aa 21, etc., including up to aa 6 to aa 24 of SEQ ID NO: 4.

[0128] In the case of 20 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 20, aa 2 to aa 21, aa 3 to aa 22, etc., including up to aa 5 to aa 24 of SEQ ID NO: 4.

[0129] In the case of 21 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 21, aa 2 to aa 22, aa 3 to aa 23, etc., including up to aa 4 to aa 24 of SEQ ID NO: 4.

[0130] In the case of 22 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 22, aa 2 to aa 23, aa 3 to aa 24, etc., including up to aa 3 to aa 24 of SEQ ID NO: 4.

[0131] In the case of 23 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 23, aa 2 to aa 24, aa 3 to aa 25, etc., including up to aa 2 to aa 24 of SEQ ID NO: 4.

[0132] In the case of 24 amino acid (“aa”) fragments, Cdk5-inhibitory peptides of the disclosure may comprise aa 1 to aa 24, aa 2 to aa 25, aa 3 to aa 26, etc., including up to aa 1 to aa 24 of SEQ ID NO: 4.P35-Derived Cdk5-Inhibitory Peptides

[0133] Cdk5-inhibitory elements of fragments of the P35 peptide have been explored (see, e.g., Binukumar, B. K., et al. "Analysis of the inhibitory elements in the p5 peptide fragment of the CDK5 activator, P35, CDKR1 protein." Journal of Alzheimer's Disease 48.4 (2015): 1009-1017; incorporated herein by reference in its entirety). For example, U.S. Pat. App. Pub. No.- 20 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO120120115790 (incorporated herein by reference in its entirety) discloses a 24 amino acid peptide derived from P25 (a peptide of P35) with Cdk5-inhibitory activity, referred to as P5 with a protein transduction domain (PTD) and compositions and methods for reducing memory deficit in neurodegenerative disease (e.g., Alzheimer’s disease). Further, PCT Pat. App. Pub. No.W02020102404 discloses use of the 24 amino acid peptide P5 for decreasing cancer cell viability, increasing apoptosis of cancer cells and treating cancer in subjects in need thereof. Accordingly, P35-derived peptides are ripe targets for development of Cdk5 -inhibitory peptides. The present disclosure provides novel P35-derived peptides including modified and variant peptides based on the P5 amino acid sequence but further engineered to have improved Cdk5-inhibitory activity, referred to herein as “P5 variant peptides” or “P5 variants” for brevity (see, e.g., Table 2). The P5 variant peptides disclosed may have one or more amino acid substitution, insertion, or deletion compared to P5. In some embodiments, the P5 variant peptides further comprise one or more modification, detectable label, linker and / or spacer.

[0134] Thus, the Cdk5-inhibitory peptides described herein may include at least 15 consecutive amino acids from residues 154-279 of P35 (SEQ ID NO: 3), for example at least 18, at least 20, at least 24, at least 30, at least 40, at least 50, such as between 15-100, 20-90, 40-60, 15-30, 20-27, 23-25, including 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 2829, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 amino acids from residues 154-279 of P35 (SEQ ID NO: 3). The Cdk5-inhibitory peptides described herein may have the ability to reduce or inhibit Cdk5 activity (such as phosphorylation). In some embodiments, the Cck5-inhibitory peptides described herein have the ability to reduce the phosphorylation of one or more neuronal proteins that are regulated by and / or are functionally associated with Cdk5 signaling. For example, the Cdk5-inhibitory peptide may decrease the phosphorylation of one or more of microtubule- associated protein tau (MAPT), synaptic trafficking regulators (e.g., AGAP3), chromatin remodeling proteins (e.g., CHD4), phosphoinositide signaling proteins (e.g., MTMR7), cyclic AMP signaling components (e.g., ADCY8), synaptic adhesion proteins (e.g., NCAM2 and CADM1), and protein phosphatases involved in stress-kinase regulation (e.g., PPM1E). The Cdk5-inhibitor peptides described herein may have the ability to modulate neuronal signaling, synaptic function, cytoskeletal dynamics and / or intracellular signaling pathways.- 21 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0135] In some embodiments, the disclosed Cdk5-inhibitory peptide has at least 80%, such as 85%, 90%, 93%, 95%, 98%, 99% or greater sequence identity to the amino acid sequence set forth as SEQ ID NO: 4 and has the ability to reduce or inhibit Cdk5 activity (such as phosphorylation). In one example, the Cdk5-inhibitory peptide is P5 and comprises the amino acid sequence set forth as SEQ ID NO: 4 (residues spanning 154-279 of SEQ ID NO: 3). In another example, the Cdk5 -inhibitory peptide consists of the amino acid sequence set forth as SEQ ID NO: 4. Additional Cdk5-inhibitory peptides include Pl (spanning from E211-A277 of SEQ ID NO: 3), P2 (spanning M237-A277 of SEQ ID NO: 3), P3 (spanning E221-L267 of SEQ ID NO: 3), and P4 (spanning E221-L249 of SEQ ID NO: 3).

[0136] P5 variant peptides set forth in Table 2 were designed to comprise 1-4 (e.g., 1, 2, 3, or 4) amino acid substitutions and / or modifications compared to the reference P5 amino acid sequence. Additional P5 variant peptides set forth in Table 2 were designed as 10-16 (e.g., 10, 11, 12, 13, 14, 15, or 16) amino acid N-, C-, or N- and C-terminal truncations of the reference P5 amino acid sequence. P5 peptides were designed and their interaction and stability tested using a variety of programs and scoring functions: NLSdesign, NLSboltzmann, NLS sidechain, NLSnewton, NLSescore, NLSmscore and NLScscore.

[0137] Exemplary Cdk5-inhibitory peptides were designed based on the P5 peptide (see Table 2, P5 variant peptides and truncations) to improve inhibitory activity of the P5 peptide. Thus, the disclosure provides isolated P5 variant peptides, such as isolated peptides derived from the P5 peptide for use to treat neurodegenerative and / or proliferative disorders. In some examples, the isolated peptides of the disclosure include a PTD (e.g., YARAARRAARR, “PTD1”) and at least one spacer / linker moiety and / or one or more additional amino acids. Some embodiments of the isolated P5 variant peptides disclosed herein compete with hyperactive P25 regulatory molecule in binding to Cdk5 and thereby specifically inhibit Cdk5 / P25 activity (including phosphorylation activity).

[0138] In some embodiments, the Cdk5-inhibitory peptides disclosed herein (e.g., SEQ ID NO: 4) inhibit Cdk5 / p25 activity. In some embodiments, the Cdk5-inhibitory polypeptides disclosed herein (e.g., SEQ ID NO: 4) do not inhibit Cdk5 / p35 activity.

[0139] The one or more Cdk5-inhibitory peptides including P5 variant peptides can be attached to the PTD either directly or by a linker / spacer moiety by methods known to those of skill in the art. For example, recombinant DNA technology can be used to add the Cdk5-inhibitory peptide to a PTD to produce a disclosed peptide. Details of suitable recombinant DNA technology can be found, for example, in Sambrook et al. (ed.), Molecular Cloning: A- 22 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring, Harbor, N.Y., 1989. In one example, the PTD is added to the N-terminus of the Cdk5 -inhibitory peptide such as a P5 variant peptide. In another example, the PTD is added to the C-terminus of Cdk5-inhibitory peptide such as a P5 variant peptide. In specific embodiments, the PTD is covalently linked to the N-terminus or to the C-terminus of a Cdk5-inhibitory peptide.

[0140] The Cdk5-inhibitory peptides disclosed herein may comprise a P5 variant peptide having an amino acid sequence set forth in Table 2. In some embodiments, the P5 variant peptides comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth Table 2. In some embodiments, the P5 variant peptides have the ability to reduce or inhibit Cdk5 activity (such as phosphorylation). The disclosed P5 peptide variants may include a Cdk5-inhibitory domain linked to a PTD.

[0141] The Cdk5-inhibitory peptides disclosed herein may comprise a P5 truncation set forth in Table 2. For example, CT178, CT179, CT180, and CT181 represent P5 truncation peptides developed based on experimental and computational hot-spot analysis of P5.

[0142] In some embodiments, the disclosed isolated P5 variant peptides including P5 truncations include a label to assist with the detection of the peptide. For example, a peptide may include a sequence set forth in Table 2, a PTD, at least one spacer (linker) moiety and / or additional amino acid, and a label. Any label known to one of skill in the art can be employed that allows for peptide detection without interfering with the delivery or activity of the peptide. In one example, the peptide includes a fluorescent label. In one example, the fluorescent label is fluorescein isothiocyanate (FITC). In one example, the fluorescent label is fluorescein isothiocyanate Cy7. In another example, the disclosed isolated peptide has an amino acid sequence with at least 95% sequence identity to any one of the amino acid sequences set forth as SEQ ID NO: 10 through SEQ ID NO: 80. In some examples, the P5 variant peptides comprise an amino acid sequence set forth as any one of SEQ ID NO: 10 through SEQ ID NO: 80. In other examples, the P5 variant peptide consists of the amino acid sequence set forth as any one of SEQ ID NO: 10 through SEQ ID NO: 80. In yet further examples, the P5 variant peptide having the amino acid sequence set forth as any one of SEQ ID NO: 10 through SEQ ID NO: 80 is linked to a detectable label. For example, the isolated P5 variant peptide may be linked to FITC or Cy7 or other suitable label. The activity of the isolated polypeptide set forth as any one of SEQ ID NO: 10 through SEQ ID NO: 80 is not altered by the addition of a PTD, a linker molecule, and / or a- 23 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1detectable label. Thus, polypeptide sequences set forth as SEQ ID NO: 10 through SEQ ID NO: 80 linked to FITC have the ability to reduce or inhibit Cdk5 activity (such as phosphorylation).

[0143] Variant amino acid sequences may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth as one of SEQ ID NO: 10 through SEQ ID NO: 80 provided in Table 2. Abbreviations and other notations used in Table 2 are defined in the right-most column. Substituted amino acid residues as well as modified amino acid residues are indicated by parentheses in the amino acid sequences shown in Table 2.Table 2. Modified and Variant Peptides Based on P5- 24 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1- 25 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0144] Thus, some embodiments, of the Cdk5-inhibitory polypeptides provided herein comprise an amino acid sequence having sequence identity to 10 to 24 consecutive amino acid residues of SEQ ID NO: 4. The Cdk5-inhibitory polypeptides may comprise an amino acid sequence having sequence identity to 10 to 24 consecutive amino acid residues of SEQ ID NO: 4 comprising one or more amino acid substitution compared to the amino acid sequence of SEQ ID NO: 4.

[0145] The Cdk5-inhibitory peptides may comprise one or more conservative amino acid substitution, or one or more unnatural amino acid substitution, or one or more amino acid modifications. In some embodiments, the Cdk5-inhibitory peptides comprise one or more amino acid substitution and / or one or more unnatural amino acid substitution and / or or one or more amino acid modifications. For example, the one or more amino acid substitution may enhance binding stability, stability of the Cdk5-inhibitory polypeptide in solution, alpha helicity of the Cdk5-inhibitory polypeptide, cell penetration of the Cdk5-inhibitory polypeptide, and / or solubility of the Cdk5-inhibitory polypeptide.- 26 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0146] Examples of amino acid substitutions that can be implemented in the Cdk5-inhibitory polypeptides disclosed herein include:

[0147] a W5Y amino acid substitution at position 5 compared to SEQ ID NO: 4;

[0148] a D6(ABA), D6(AL4), D6(CTR), D6Q, D6E, D6L, D6M, D6(OME), D6(ORN), D6(SAR), D6T, D6W, or D6Y amino acid substitution at position 6 compared to SEQ ID NO: 4;

[0149] a C8(ABA), C8R, C8(CTR), C8Q, C8L, C8K, C8(ORN), C8W, or C8Y amino acid substitution at position 8 compared to SEQ ID NO: 4;

[0150] a V 1 l(HCS) amino acid substitution at position 11 compared to SEQ ID NO: 4;

[0151] an M15G, M15P, M15(PCO), or M15(HCS) amino acid substitution at position 15 compared to SEQ ID NO: 4;

[0152] an S17mS, SUH, or S17Mh amino acid substitution at position 17 compared to SEQ ID NO: 4;

[0153] an M19(HCS) amino acid substitution at position 19 compared to SEQ ID NO: 4; or

[0154] an A24M, A24V, A24L, A24(CYS-CYS), A24(HCY-HCY), A24R, A24K, A24(MDM), A24(NMI), (ORN), A24(2FM), A24(ESC), A24(NLE), A24(TFE), A24(YCP), or A24(26P) amino acid substitution at position 24 compared to SEQ ID NO: 4.

[0155] In some embodiments, the Cdk5-inhibitory polypeptides have an amino acid sequence having sequence identity to 10 to 24 consecutive amino acid residues of SEQ ID NO: 4 but with 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 4.

[0156] For instance, the Cdk5-inhibitory polypeptides may have an amino acid sequence having at least 79% identity to the amino acid sequence of SEQ ID NO: 4, at least 83% identity to the amino acid sequence of SEQ ID NO: 4, at least 87% identity to the amino acid sequence of SEQ ID NO: 4, at least 91% identity to the amino acid sequence of SEQ ID NO: 4, or at least at least 95% identity to the amino acid sequence of SEQ ID NO: 4.

[0157] Also provided herein are Cdk5-inhibitory peptides comprising an amino acid sequence having at least 79% sequence identity to the amino acid sequence of SEQ ID NO: 4, at least 83% sequence identity to the amino acid sequence of SEQ ID NO: 4, at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 4, at least 91% sequence identity to the amino acid sequence of SEQ ID NO: 4, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0158] The peptides can have one or more of the following amino acid substitutions compared to SEQ ID NO: 4:

[0159] a W5Y amino acid substitution at position 5 compared to SEQ ID NO: 4;- 27 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0160] a D6(ABA), D6(AL4), D6(CTR), D6Q, D6E, D6L, D6M, D6(OME), D6(ORN), D6(SAR), D6T, D6W, or D6Y amino acid substitution at position 6 compared to SEQ ID NO: 4;

[0161] a C8(ABA), C8R, C8(CTR), C8Q, C8L, C8K, C8(ORN), C8W, or C8Y amino acid substitution at position 8 compared to SEQ ID NO: 4;

[0162] a V 1 l(HCS) amino acid substitution at position 11 compared to SEQ ID NO: 4;

[0163] an M15G, M15P, M15(PCO), or M15(HCS) amino acid substitution at position 15 compared to SEQ ID NO: 4;

[0164] an S17mS, SUH, or S17Mh amino acid substitution at position 17 compared to SEQ ID NO: 4;

[0165] an M19(HCS) amino acid substitution at position 19 compared to SEQ ID NO: 4; or

[0166] an A24M, A24V, A24L, A24(CYS-CYS), A24(HCY-HCY), A24R, A24K, A24(MDM), A24(NMI), (ORN), A24(2FM), A24(ESC), A24(NLE), A24(TFE), A24(YCP), or A24(26P) at position 24 compared to SEQ ID NO: 4.

[0167] In some embodiments, the Cdk5-inhibitory peptides provided herein have an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76.

[0168] Also provided are Cdk5-inhibitory peptides comprising various P5 truncations. For example, provided herein are Cdk5-inhibitory peptides having an amino acid sequence of SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. Various embodiments of the P5 truncation peptides comprise a C-terminal amide group modification. Various embodiments of the P5 truncation peptides comprise an N-terminal acetyl group modification. In some embodiments, the P5 truncation peptides comprise a C-terminal amide group modification and an N-terminal acetyl group modification.- 28 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1Cdk5 Helix Peptides

[0169] Additional Cdk5-inhibitory peptides set forth in Table 3 were designed to comprise 15-amino acid variant peptides of the helix domain of human Cdk5 protein (i.e., amino acids 43-57 of SEQ ID NO: 1, or SEQ ID NO: 81) (referred to herein as “Cdk5 helix peptides”). The isolated Cdk5 helix peptides provided in Table 3 (i.e., SEQ ID NO: 81 through SEQ ID NO: 116) were designed to compete with or otherwise disrupt interaction between Cdk5 and P25 and thereby inhibit Cdk5 / P25 hyperactivity (including phosphorylation). Peptides having one (1) amino acid substitution compared to the reference Cdk5 helix amino acid sequence were designed. Additional Cdk5 helix peptides set forth in Table 3 were designed using an alanine scan technique to substitute various residues of SEQ ID NO: 81 with alanine (i.e., SEQ ID NO: 112 through SEQ ID NO: 116). Cdk5 helix peptides were designed and their interaction and stability tested using a variety of programs and scoring functions: NLSdesign, NLSboltzmann, NLSsidechain, NLSnewton, NLSescore, NLSmscore and NLScscore.

[0170] Thus, the disclosure provides isolated Cdk5 helix peptides, such as isolated peptides derived from the helix domain of human Cdk5 protein (i.e., amino acids 43-57 of SEQ ID NO: 1, or SEQ ID NO: 81) for use to treat neurodegenerative and / or proliferative disorders. In some examples, the isolated peptides of the disclosure include a PTD and at least one spacer / linker moiety and / or one or more additional amino acids. Some embodiments of the isolated Cdk5 helix peptides disclosed herein compete with hyperactive P25 regulatory molecule in binding to Cdk5 and thereby specifically inhibit Cdk5 / P25 activity (including phosphorylation activity).

[0171] The one or more Cdk5-inhibitory peptides including Cdk5 helix peptides can be attached to the PTD either directly or by a linker / spacer moiety by methods known to those of skill in the art. For example, recombinant DNA technology can be used to add the Cdk5-inhibitory peptide to a PTD to produce a disclosed peptide. Details of suitable recombinant DNA technology can be found, for example, in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring, Harbor, N.Y., 1989. In one example, the PTD is added to the N-terminus of the Cdk5 -inhibitory peptide such as a Cdk5 helix peptide. In another example, the PTD is added to the C-terminus of Cdk5-inhibitory peptide such as a Cdk5 helix peptide. In specific embodiments, the PTD is covalently linked to the N-terminus or to the C-terminus of a Cdk5-inhibitory peptide.

[0172] The Cdk5-inhibitory peptides disclosed herein may comprise a Cdk5 helix peptide having an amino acid sequence set forth in Table 3. In some embodiments, the Cdk5 helix- 29 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1peptides comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth Table 3. In some embodiments, the Cdk5 helix peptides have the ability to reduce or inhibit Cdk5 activity (such as phosphorylation). The disclosed P5 peptide variants may include a Cdk5-inhibitory domain linked to a PTD.

[0173] In some embodiments, the disclosed isolated Cdk5 helix peptides include a label to assist with the detection of the peptide. For example, a peptide may include sequence set forth in Table 3, a PTD, at least one spacer (linker) moiety and / or additional amino acid, and a label. Any label known to one of skill in the art can be employed that allows for peptide detection without interfering with the delivery or activity of the peptide. In one example, the peptide includes a fluorescent label. The fluorescent label is fluorescein isothiocyanate (FITC). In another example, the disclosed isolated peptide has an amino acid sequence with at least 95% sequence identity to any one of the amino acid sequences set forth as SEQ ID NO: 81 through SEQ ID NO: 116. In some examples, the Cdk5 helix peptides comprise an amino acid sequence set forth as any one of SEQ ID NO: 81 through SEQ ID NO: 116. In other examples, the Cdk5 helix peptide consists of the amino acid sequence set forth as any one of SEQ ID NO: 81 through SEQ ID NO: 116. In yet further examples, the Cdk5 helix peptide having the amino acid sequence set forth as any one of SEQ ID NO: 81 through SEQ ID NO: 116 is linked to a detectable label. The isolated Cdk5 helix peptide is linked to FITC. The activity of the isolated polypeptide set forth as any one of SEQ ID NO: 81 through SEQ ID NO: 116 is not altered by the addition of a PTD, a linker molecule, and / or a detectable label. Thus, polypeptide sequences set forth as SEQ ID NO: 81 through SEQ ID NO: 116 linked to FITC have the ability to reduce or inhibit Cdk5 activity (such as phosphorylation).

[0174] Variant amino acid sequences may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth as one of SEQ ID NO: 81 through SEQ ID NO: 116.

[0175] In some embodiments, Cdk5 helix peptides disclosed in Table 3 were designed to compete with P25 for binding to Cdk5. In some embodiments, Cdk5 helix peptides disclosed in Table 3 were designed to stabilize the helix peptide in solution. In some embodiments, Cdk5 helix peptides disclosed in Table 3 were designed to introduce a kink in the tertiary structure and / or in the quaternary structure of the helix peptide in solution alone or in complex with Cdk5 protein. For instance, in some embodiments of the Cdk5 helix peptides disclosed in Table 3, interface-pointing residues between P25 and Cdk5 were substituted to enhance binding. In other- 30 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1instances, interface-avoiding residues present at the P25 / Cdk5 interface were substituted to enhance helix stability and / or solubility (i.e., to enhance alpha helicity of the Cdk5-inhibitory polypeptide). In some embodiments, Cdk5 helix peptides disclosed in Table 3 were designed to enhance cell penetration of the Cdk5-inhibitory polypeptide.

[0176] Principles guiding design of Cdk5 helix peptides set forth in Table 3 include, e.g., model-based Cdk5 / P25 complex formation having 1,289 contacts, 7 hydrogen bonds, buried solvent-accessible surface area (SASA) (=2686.07 A2), and AG equivalent to approximately -20.1 kcal / mol.

[0177] In silico modeling was used to position the Cdk5 helix reference sequence (SEQ ID NO: 81) close to P25 helix, which contributes approximately 50% of binding affinity to P25 (contacts = 563 (44%); Hbonds = 4 (57%); buried SASA = 1,184.47 A2(44%); AG equivalent to approximately -10.1 kcal / mol (50%)).

[0178] Overall, five (5) P25-pointing residues were identified as candidates for mutation / substitution to enhance binding stability of the Cdk5 helix peptides provided in Table 3. These residues include (with reference to their position in Cdk5 protein, SEQ ID NO: 1): G43, P45, C53, L54, and E57. Ten (10) diverse amino acids with high helix propensities were tested at all 5 sites. Amino acids were positioned and modeled using rotamer library and energy minimization, statistical potential free energy scoring (SP), empirical free energy scoring (E), force field based free energy scoring (F), and consensus scoring using SP + E + F. Substitutions implemented to enhance binding stability of Cdk5 helix peptide to P25 include the following:

[0179] G43E, G43Y, G43A, G43F, G43Q, G43I, G43S

[0180] P45E, P45L, P45I, P45Y, P45W, P45F, P45Q

[0181] C53Y, C53R, C53L, C53Q, C53S

[0182] L54A, L54F, L54W, L54S, L54Y, L54R

[0183] E57W, E57A, E57Y, E57Q

[0184] Additional substitutions were identified that may further stabilize Cdk5 helix:

[0185] G43E (N-terminus stabilization)

[0186] P45E, P45L, P45I, P45Y, P45W, P45F, P45Q (to remove Pro residue)

[0187] C53R (i, i+3 contact with E51)

[0188] L54R (i, i+4 contact with E51)

[0189] E57W, E57A, E57Y, E57Q (to remove Glu at C-terminus)

[0190] Unbound Cdk5 helix was predicted to be stable in solution. Bound Cdk5 helix was predicted to be stabilized by five (5) “hotspot residues”: S46, S47 (1 hbond), L49, R50 (2- 31 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1hbonds), and K56 (2 hbonds). Alanine scan was performed to substitute these hotspot residues with alanine (SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, and SEQ ID NO: 116 provided in Table 3).

[0191] Substituted amino acid residues are indicated by parentheses in the amino acid sequences shown in Table 3.Table 3. Cdk5 Helix Peptides- 32 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0192] Thus, provided herein are Cdk5-inhibitory polypeptides comprising an amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1. In some embodiments, the 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 comprise a Cdk5 helix domain. For example, the Cdk5 helix domain may comprise an amino acid sequence defined by amino acid S46 to amino acid L55 of SEQ ID NO: 1. In some embodiments, the Cdk5 helix domain comprises the amino acid sequence of SEQ ID NO: 81.

[0193] Some embodiments of the Cdk5-inhibitory polypeptides comprise an amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 but with one or more amino acid substitution compared to the amino acid sequence of SEQ ID NO: 81. The one or more amino acid substitution may comprise one or more conservative amino acid substitution, one or more unnatural amino acid substitution, or one or more amino acid modifications. In some embodiments, the disclosed Cdk5-inhibitory polypeptides comprise one or more conservative amino acid substitution and / or one or more unnatural amino acid substitution and / or or one or more amino acid modifications compared to SEQ ID NO: 81.

[0194] In some embodiments, the one or more amino acid substitution enhances binding stability, enhances stability of the Cdk5-inhibitory polypeptide in solution, and / or enhances solubility of the Cdk5-inhibitory polypeptide.

[0195] Some embodiments of the Cdk5-inhibitory polypeptides comprise one or more amino acid substitution comprises as follows:

[0196] an amino acid substitution at position 1 of SEQ ID NO: 81 of: G1E, G1Y, GIA, GIF, G1Q, G1I, or GlS;

[0197] an amino acid substitution at position 3 of SEQ ID NO: 81 of: P3E, P3L, P3I, P3Y, P3W, P3F, or P3Q;

[0198] an amino acid substitution at position 4 of SEQ ID NO: 81 of: S4A;

[0199] an amino acid substitution at position 5 of SEQ ID NO: 81 of: S5A;

[0200] an amino acid substitution at position 7 of SEQ ID NO: 81 of: L7A;

[0201] an amino acid substitution at position 8 of SEQ ID NO: 81 of: R8A;

[0202] an amino acid substitution at position 11 of SEQ ID NO: 81 of: Cl 1Y, Cl 1R, Cl IL, C11Q, orCHS;- 33 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0203] an amino acid substitution at position 12 of SEQ ID NO: 81 of: L12A, L12F, L12W, L12S, L12Y, orL12R;

[0204] an amino acid substitution at position 13 of SEQ ID NO: 81 of: L13R;

[0205] an amino acid substitution at position 14 of SEQ ID NO: 81 of: K14A; or

[0206] an amino acid substitution at position 15 of SEQ ID NO: 81 of: E15W, E15A, E15Y, orE15Q.

[0207] The Cdk5-inhibitory polypeptides may comprise an amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 comprising the amino acid sequence of SEQ ID NO: 81 having 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 81.

[0208] Thus, the Cdk5-inhibitory polypeptides may comprise an amino acid sequence having at least 66% identity to the amino acid sequence of SEQ ID NO: 81, at least 73% identity to the amino acid sequence of SEQ ID NO: 81, at least 80% identity to the amino acid sequence of SEQ ID NO: 81, at least 87% identity to the amino acid sequence of SEQ ID NO: 81, or at least 93% identity to the amino acid sequence of SEQ ID NO: 81.

[0209] Also provided herein are Cdk5-inhibitory peptides comprising an amino acid sequence having at least 66% sequence identity to the amino acid sequence of SEQ ID NO: 81, at least 73% sequence identity to the amino acid sequence of SEQ ID NO: 81, at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 81, at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 81, or at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 81.

[0210] The Cdk5-inhibitory peptides may comprise a G1E, G1Y, GIA, GIF, G1Q, G1I, or G1S amino acid substitution at position 1 compared to SEQ ID NO: 81.

[0211] The Cdk5-inhibitory peptides may comprise a P3E, P3L, P3I, P3Y, P3W, P3F, or P3Q amino acid substitution at position 3 compared to SEQ ID NO: 81.

[0212] The Cdk5-inhibitory peptides may comprise a S4A amino acid substitution at position 4 compared to SEQ ID NO: 81.

[0213] The Cdk5-inhibitory peptides may comprise a S5A amino acid substitution at position 5 compared to SEQ ID NO: 81.

[0214] The Cdk5-inhibitory peptides may comprise a L7A amino acid substitution at position 7 compared to SEQ ID NO: 81.

[0215] The Cdk5-inhibitory peptides may comprise a R8A amino acid substitution at position 8 compared to SEQ ID NO: 81.- 34 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0216] The Cdk5-inhibitory peptides may comprise a Cl 1Y, Cl 1R, Cl IL, Cl IQ, or Cl IS amino acid substitution at position 11 compared to SEQ ID NO: 81.

[0217] The Cdk5-inhibitory peptides may comprise a L12A, L12F, L12W, L12S, L12Y, or L12R amino acid substitution at position 12 compared to SEQ ID NO: 81.

[0218] The Cdk5-inhibitory peptides may comprise a L13R amino acid substitution at position 13 compared to SEQ ID NO: 81.

[0219] The Cdk5-inhibitory peptides may comprise a K14A amino acid substitution at position 14 compared to SEQ ID NO: 81.

[0220] The Cdk5-inhibitory peptides may comprise a E15W, E15A, E15Y, or E15Q amino acid substitution at position 15 compared to SEQ ID NO: 81.

[0221] Various embodiments of the Cdk5-inhibitory peptides provided herein have an amino acid sequence of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.Amino Add Substitutions

[0222] Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.Examples of conservative substitutions are shown below.

[0223] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Some embodiments of the Cdk5-inhibitory peptides disclosed herein comprise one or more conservative amino acid substitutions as shown in Table 4.Table 4. Conservative Amino Acid Substitutions- 35 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0224] Non-conservative substitutions, on the other hand, may give rise to significant changes in protein properties. Non-conservative substitutions include, for example: (a) a hydrophilic residue, for example, seryl or threonyl, substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, substituted for (or by) one not having a side chain, for example, glycine. The effect of any non-conservative amino acid substitution on a polypeptide’s structure and / or function generally must be determined, i.e., empirically and / or by in silico assessment.Unnatural Amino Adds

[0225] As used herein, the term “amino acid” includes the residues of the natural amino acids as well as unnatural amino acids. The twenty natural proteinogenic amino acids are identified and referred to herein by either the one-letter or three-letter designations as follows: aspartic acid (Asp:D), isoleucine (Ile:I), threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q) methionine (Met:M), asparagine (Asn:N). Naturally occurring amino acids exist in their levorotary (L) stereoisomeric forms. Amino acids referred to herein are L-- 36 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1stereoisomers except where otherwise indicated. The term “amino acid” also includes amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as a (C1-C6) alkyl, phenyl or benzyl ester or amide; or as an alpha-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, Greene, T. W.; Wutz, P. G. M., Protecting Groups In Organic Synthesis; second edition, 1991, New York, John Wiley & sons, Inc., and documents cited therein, the contents of each of which are herein incorporated by reference in their entirety). Cdk5-inhibitory peptides of the present disclosure may also include modified amino acids.

[0226] “Unnatural” amino acids have side chains or other features not present in the 20 naturally-occurring amino acids listed above and include, but are not limited to: N-methyl amino acids (e.g., N-methyl serine or N-methyl histidine, N-methyl-L-isoleucine), N-alkyl amino acids, alpha, alpha substituted amino acids, beta-amino acids, alpha-hydroxy amino acids, D-amino acids, and other unnatural amino acids known in the art (See, e.g., Josephson et al., (2005) J. Am. Chem. Soc. 127: 11727-11735; Forster, A.C. et al. (2003) Proc. Natl. Acad. Sci. USA 100: 6353-6357; Subtelny et al., (2008) J. Am. Chem. Soc. 130: 6131-6136; Hartman, M.C.T. et al. (2007) PLoS ONE 2:e972; and Hartman et al., (2006) Proc. Natl. Acad. Sci. USA 103:4356-4361). Further unnatural amino acids useful for the optimization of Cdk5-inhibitory peptides of the present disclosure include, but are not limited to 2- (methylamino)-4- (methylsulf anyl)butanoic acid (“MDM”), (S)-l-(3-methylisoxazol-4-yl)ethanone (“PCO”), L-Glutamic acid 5-methyl ester (“OME”), (R)-2-methyl-2-aminooctanoic acid (“AL4”), (S)-2-amino-6-oxopimelic acid (“26P”), 1,2,3,4-tetrahydroisoquinoline-l-carboxylic acid, l-amino-2,3-hydro-lH-indene-l-carboxylic acid, homolysine, homoarginine, homoserine, 2-aminoadipic acid, 3-aminoadipic acid, betaalanine, aminopropionic acid, 2-aminobutyric acid (“ABA”), 4-aminobutyric acid, 5-aminopentanoic acid, 5 -aminohexanoic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, desmosine, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxy lysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylpentylglycine, naphthylalanine, ornithine (“ORN”), pentylglycine, thioproline, norvaline, tert-butylglycine (also known as tert-leucine), phenylglycine, azatryptophan, 5-azatryptophan, 7-azatryptophan, 4-fluorophenylalanine, penicillamine, sarcosine (“SAR”), homocysteine, 1-aminocyclopropanecarboxylic acid, 1 -aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, 4-aminotetrahydro-2 / 7-- 37 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1pyran-4-carboxylic acid, (S)-2-amino-3-(l / / -tetrazol-5-yl)propanoic acid, cyclopentylglycine, cyclohexylglycine, cyclopropylglycine, rpco-methyl-arginine, 4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine, 5-fluoro tryptophan, 5-chloro tryptophan, citrulline (i.e., L(+)-citrulline, “CTR”), 4-chloro-homophenylalanine, homophenylalanine, 4-aminomethyl-phenylalanine, 3-aminomethyl-phenylalanine, octylglycine, norleucine, tranexamic acid, 2-amino pentanoic acid, 2-amino hexanoic acid, 2-amino heptanoic acid, 2-amino octanoic acid, 2-amino nonanoic acid, 2-amino decanoic acid, 2-amino undecanoic acid, 2-amino dodecanoic acid, aminovaleric acid, and 2-(2-aminoethoxy)acetic acid, pipecolic acid, 2-carboxy azetidine, hexafluoroleucine, 3-Fluorovaline, 2-amino-4,4-difluoro-3-methylbutanoic acid, 3-fluoro- isoleucine, 4-fluoroisoleucine, 5-fluoroisoleucine, 4-methyl-phenylglycine, 4-ethyl-phenylglycine, 4-isopropyl-phenylglycine, (S)-2-amino-5-azidopentanoic acid (also referred to herein as “X02”), (S)-2-aminohept-6-enoic acid (also referred to herein as “X30”), (S)-2-aminopent-4-ynoic acid (also referred to herein as “X31”), (S)-2-aminopent-4-enoic acid (also referred to herein as “XI 2”), (5)-2-amino-5-(3-methylguanidino) pentanoic acid, (S)-2-amino-3-(4-(aminomethyl)phenyl)propanoic acid, (5)-2-amino-3-(3-(aminomethyl)phenyl)propanoic acid, ( )-2-amino-4-(2-aminobenzo[ ]oxazol-5-yl)butanoic acid, (S)-leucinol, (S)-valinol, (S)-tert-leucinol, (7?)-3-methylbutan-2-amine, (5)-2-methyl- 1 -phenyl propan- 1 -amine, and (S)-N,2-dimethyl-l-(pyridin-2-yl)propan-l-amine, (5)-2-amino-3-(oxazol-2-yl)propanoic acid, (5)-2-amino-3-(oxazol-5-yl)propanoic acid, (S)-2-amino-3-(l,3,4-oxadiazol-2-yl)propanoic acid, (5)-2-amino-3-(l,2,4-oxadiazol-3-yl)propanoic acid, (5)-2-amino-3-(5-fluoro-l / / -indazol-3-yl)propanoic acid, and (5)-2-amino-3-(l / / -indazol-3-yl)propanoic acid, (S)-2-amino-3-(oxazol-2-yl)butanoic acid, (S)-2-amino-3-(oxazol-5-yl) butanoic acid, (5)-2-amino-3-(l,3,4-oxadiazol-2-yl) butanoic acid, (S)-2-amino-3-(l,2,4-oxadiazol-3-yl) butanoic acid, (5)-2-amino-3-(5-fluoro-l / / -indazol-3-yl) butanoic acid, and ( )-2-amino-3-(l / / -indazol-3-yl) butanoic acid, 2-(2’MeOphenyl)-2-amino acetic acid, tetrahydro 3-isoquinolinecarboxylic acid and stereoisomers thereof (including, but not limited, to D and L isomers).

[0227] Various embodiments of the Cdk5-inhibitory peptides disclosed herein (e.g., in Table 2 and Table 3) comprise one or more amino acid substitution, wherein an amino acid is substituted with a synthetic or non-naturally occurring amino acid residue (“unnatural amino acids”), such as those listed above. For instance, unnatural amino acid substitutions may be introduced into P5 to create covalent bonds between P5 and Cdk5 Cys 53, including, e.g., 4-cyano-phenylalanine (4CF), nitrilo-l-methionine (4CY), or 2-cyano-phenylalanine (CNP2).- 38 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0228] Unnatural amino acids for use in the polypeptides of the disclosure may include L-non-polar amino acids, acrylamides, nitrile-containing amino acids, longer versions of cysteine (L and D), and proline derivatives or analogs (for instance, useful for stabilizing by stapling around a kink within the Cdk5 helix region).

[0229] Additional unnatural amino acids that are useful in the optimization of Cdk5-inhibitory peptides of the present disclosure include but are not limited to fluorinated amino acids wherein one or more carbon bound hydrogen atoms are replaced by fluorine. The number of fluorine atoms included can range from 1 up to and including all of the hydrogen atoms. Examples of such amino acids include but are not limited to 2,2,2-trifluoroethanol (TFE), 3-fluoroproline, 3,3-difluoroproline, 4-fluoroproline, 4,4-difluoroproline, 3,4-difluroproline, 3, 3,4,4-tetrafluoroproline, 4-fluoro tryptophan, 5-fluro tryptophan, 6-fluoro tryptophan, 7-fluorotryptophan, and stereoisomers thereof.

[0230] Further unnatural amino acids that are useful in the optimization of Cdk5-inhibitory peptides of the present disclosure include but are not limited to those that are disubstituted at the a-carbon. These include amino acids in which the two substituents on the a-carbon are the same, for example a-amino isobutyric acid, and 2-amino-2-ethyl butanoic acid, as well as those where the substituents are different, for example a-methylphenylglycine and a-methylproline. Further, the substituents on the a-carbon may be taken together to form a ring, for example 1-aminocyclopentanecarboxylic acid, 1- aminocyclobutanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, 3-aminotetrahydrofuran-3-carboxylic acid, 3-aminotetrahydropyran-3-carboxylic acid, 4-aminotetrahydropyran-4-carboxylic acid, 3-aminopyrrolidine-3-carboxylic acid, 3-aminopiperidine-3-carboxylic acid, 4-aminopiperidinnne-4-carboxylix acid, and stereoisomers thereof.

[0231] Additional unnatural amino acids that are useful in the optimization of Cdk5-inhibitory peptides of the present disclosure include but are not limited to analogs of tryptophan in which the indole ring system is replaced by another 9 or 10 membered bicyclic ring system comprising 0, 1, 2, 3 or 4 heteroatoms independently selected from N, O, or S. Each ring system may be saturated, partially unsaturated, or fully unsaturated. The ring system may be substituted by 0, 1, 2, 3, or 4 substituents at any substitutable atom. Each substituent may be independently selected from H, F, Cl, Br, CN, COOR, CONRR’, oxo, OR, NRR’. Each R and R’ may be independently selected from H, C1-C20 alkyl, or C1-C20 alkyl-O-Cl-20 alkyl.

[0232] Unnatural amino acids may be purchased from Sigma-Aldrich (St. Louis, MO), Bachem (Torrance, CA) or other suppliers. Unnatural amino acids may further include any of- 39 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1those listed in Table 2 of US patent publication US 2011 / 0172126, the contents of which are incorporated herein by reference in their entirety. In some embodiments, Cdk5-inhibitory peptides described herein comprise an N-terminal capping acetyl group (ACE).

[0233] In some embodiments, analogs of tryptophan (also referred to herein as “tryptophan analogs”) may be useful in the optimization of Cdk5-inhibitory peptides of the present disclosure. Tryptophan analogs may include, but are not limited to 5 -fluoro tryptophan [(5-F)W], 5-methyl-O-tryptophan [(5-MeO)W], 1 -methyltryptophan [(1-Me-W) or (l-Me)W], D-tryptophan (D-Trp), azatryptophan (including, but not limited to 4-azatryptophan, 7-azatryptophan and 5-azatryptophan,) 5-chloro tryptophan, 4-fluorotryptophan, 6-fluorotryptophan, 7-fluoro tryptophan, and stereoisomers thereof. Except where indicated to the contrary, the term “azatryptophan” and its abbreviation, “azaTrp,” as used herein, refer to 7-azatryptophan.

[0234] Essentially any amino acid that, when attached to an appropriate tRNA, can be assembled into a polymer by natural or mutant ribosomes can be used (see Sando, S. et al., (2007) J. Am. Chem. Soc. 129:6180-6186; Dedkova, L. et al. (2003) J. Am. Chem. Soc. 125: 6616-6617; Josephson, K., Hartman, M.C.T., and Szostak, J.W. (2005) J. Am. Chem. Soc.127:11727-11735; Forster, A.C. et al. (2003) Proc. Natl. Acad. Sci. USA 100:6353-6357;Subtelny, A.O., Hartman, M.C.T., and Szostak, J.W. (2008) J. Am. Chem. Soc. 130:6131-6136; and Hartman, M.C.T. et al. (2007) PLoS ONE 2:e972).

[0235] When unnatural amino acids are desired, it may be advantageous to use a purified translation system that lacks endogenous aminoacylated tRNAs (Shimizu, Y. et al. (2001) Nat. Biotech. 19:751-755; Josephson, K., Hartman, M.C.T., and Szostak, J.W. (2005) J. Am. Chem. Soc. 127: 11727-11735; Forster, A.C. et al. (2003) Proc. Natl. Acad. Sci. USA 100: 6353-6357). If unnatural amino acids are used with an in vitro translation system based on a lysate or extract, it may be desirable to deplete the extract of endogenous tRNAs, as previously described (see Jackson, R.J., Napthine, S., and Brierley, I. (2001) RNA 7:765-773). A system based on purified E. coli translation factors is commercially available (PUREXPRESS™; New England Biolabs, Ipswich, MA). These systems are useful for translation with unnatural amino acids to produce peptidomimetics.

[0236] When using natural amino acids with an in vitro translation system based on a lysate or extract, translation is dependent on the enzymatic charging of amino acids onto tRNAs by tRNA synthetases, all of which are components of the extracts. Alternatively, in vitro translation systems that use purified translation factors and ribosomes, or tRNA-depleted extracts, require that aminoacylated tRNAs be provided. In these instances, purified or in vitro synthesized tRNAs- 40 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1can be charged with amino acids using chemical (see Frankel, A., Millward, S.W., and Roberts, R.W. (2003) Chem. Biol. 10:1043-1050) or enzymatic procedures (Josephson, K., Hartman, M.C.T., and Szostak, J.W. (2005) J. Am. Chem. Soc. 127: 11727-11735; Murakami, H. et al. (2006) Nat. Methods 3:357-359).Amino Add Modifications

[0237] Some embodiments of the Cdk5-inhibitory peptides disclosed herein comprise one or more amino acid modifications. The present disclosure contemplates variants and derivatives of polypeptides presented herein. These include substitutional, insertional, deletional, and covalent variants and derivatives.

[0238] In some embodiments, homocysteine may be useful in stabilizing the kink in P5.

[0239] A large hydrophobic pocket on Cdk5 may be filled from P5 position 24 using apolar unnatural residues such as 2-allyl-glycine (2AG), s-(difhioromethyl)-homocysteine (2FM), adamanthane (ADAM), 2-aminoheptanoic acid (AHP), 3-cyclohexyl-alanine (ALC), 5-bromo-l-isoleucine (BIU), 3-chloro-l-alanine (C2N), cyclohexylglycine (CHG), diethylalanine (DILE), ethionine (ESC), 3-fluoro-valine (FVAL), homoleucine (HLEU), homophenylalanine (HPE), norleucine (NLE), norvaline (NVA), (2s)-2-amino-4,4-difluorobutanoic acid (OBF), styrylalanine (STYA), 5,5,5-trifluoro-leucine (TFLE), or 3-cyclopentyl-alanine (CPA3).

[0240] Cdk5 residues Argl20 / Glu57 may be reached from P5 Position 24 to improve stability using unnatural amino acid homoarginine (HRG), cysteine-s-acetamide (YCM), 2-aminoadipic acid (UNI), or (2s)-2,8-diaminooctanoic acid (HHK).

[0241] Cdk5 residues Argl20 / Glu57 may be reached from P5 Position 24 to improve stability using a (homo)cysteine-(homo)cysteine bridge (HCS-HCS) and / or a cysteine-cysteine bridge (CYS-CYS).N-Terminal Modifications

[0242] Modified amino acid residues useful for the optimization of Cdk5-inhibitory peptides of the present disclosure include, but are not limited to those which are chemically blocked (reversibly or irreversibly); chemically modified on their N-terminal amino group or their side chain groups; chemically modified in the amide backbone, as for example, N-methyl amino acids, D (unnatural amino acids) and L (natural amino acids) stereoisomers; or residues wherein the side chain functional groups are chemically modified to another functional group. In some embodiments, modified amino acids include without limitation, methionine sulfoxide;- 41 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1methionine sulfone; aspartic acid-(beta-methyl ester), a modified amino acid of aspartic acid; N-ethylglycine, a modified amino acid of glycine; alanine carboxamide; and / or a modified amino acid of alanine. In another example, the N-terminus is acetylated.C-Terminal Modifications

[0243] Modified amino acid residues useful for the optimization of Cdk5-inhibitory peptides of the present disclosure include, but are not limited to C-terminal amide group (NH2) modifications, N-alkyl amides, aldehydes, esters, p-Nitroanilide (pNA), and 7-Amino-4-Methylcoumarin (AMC).Other Modifications

[0244] The present disclosure contemplates further modifications of the Cdk5-inhibitory peptides that do not affect the ability of the peptides to selectively inhibit Cdk5 / P25 activity. Such modifications include amino acid substitutions, insertions or deletions, and modifications, for example, to reduce antigenicity of the peptides, to enhance the stability of the peptides and / or to improve the pharmacokinetics of the peptides. Further modifications may result in a polypeptide that differs by only a small number of amino acids. Such modifications include insertions (for example, of 1-3 or more residues), or substitutions that do not interfere with the ability of the peptides to inhibit Cdk5 / P25 activity.

[0245] Modified amino acid residues useful for the optimization of Cdk5-inhibitory peptides of the present disclosure include, but are not limited to those which are chemically blocked (reversibly or irreversibly); chemically modified on their N-terminal amino group or their side chain groups; chemically modified in the amide backbone, as for example, N-methyl amino acids, D (unnatural amino acids) and L (natural amino acids) stereoisomers; or residues wherein the side chain functional groups are chemically modified to another functional group. In some embodiments, modified amino acids include without limitation, methionine sulfoxide; methionine sulfone; aspartic acid-(beta-methyl ester), a modified amino acid of aspartic acid; N-ethylglycine, a modified amino acid of glycine; alanine carboxamide; and / or a modified amino acid of alanine. Unnatural amino acids may be purchased from Sigma- Aldrich (St. Louis, MO), Bachem (Torrance, CA) or other suppliers. Unnatural amino acids may further include any of those listed in Table 2 of US patent publication US 2011 / 0172126, the contents of which are incorporated herein by reference in their entirety.

[0246] Another modification is based on the cross-linking of amino acids to produce cyclic structures. Cyclic regions in a polypeptide contain a rigid domain, which reduces conformational- 42 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1flexibility and degrees of rotational freedom, leading to very high affinity binding to target proteins. A number of methods for cyclizing a polypeptide are available to those skilled in the art and are incorporated herein by reference. Typically, the chemical reactivity of specific amino acid side chains and / or the carboxyl or amino termini of the polypeptide are exploited to crosslink two sites of the polypeptide to produce a cyclic molecule. In one method, the thiol group of a cysteine residue is cross-linked with another cysteine residue to form a disulfide bond. In some embodiments, thiol groups of cysteine residues react with bromomethyl groups of poly(bromomethyl)benzene molecules to form stable linkages (see Timmerman, P. et al., (2005) ChemBioChem 6:821-824, the contents of which are herein incorporated by reference in their entirety). Poly(bromomethyl)benzene molecules of the present invention may include, but are not limited to l,2-bis(bromomethyl)benzene, l,3-bis(bromomethyl)benzene and 1,4-bis(bromomethyl)benzene. Bis-, tris- and tetrakis(bromomethyl)benzene molecules, for example, can be used to generate bridging moieties to produce polypeptides with one, two or three loops, respectively. Bromomethyl groups of a poly(bromomethyl)benzene molecule may be arranged on the benzene ring on adjacent ring carbons (ortho- or o-), with a ring carbon separating the two groups (meta- or m-) or on opposite ring carbons (para- or p-). In some embodiments, m-bis(bromomethyl)benzene (also referred to herein as m-dibromoxylene) is utilized in the formation of cyclic polypeptides. In some embodiments, o-bis(bromomethyl)benzene (also referred to herein as o-dibromoxylene) or p-bis(bromomethyl)benzene (also referred to herein as p-dibromoxylene) are utilized in the formation of cyclic polypeptides. In some embodiments, thiol groups of cysteine residues react with other reagents comprising one or more bromo functional groups to form stable linkages. Such reagents may include, but are not limited to poly(bromomethyl)pyridines (including, but not limited to 2,6-bis(bromomethyl)pyridine), poly(bromomethyl)alkylbenzenes (including, but not limited to l,2-bis(bromomethyl)-4-alkylbenzene) and / or (E)-l,4-dibromobut-2-ene.

[0247] In another exemplary method, a side chain amino group and a terminal amino group are cross-linked with disuccinimidyl glutarate (see Millward, S.W. et al., J. Am. Chem. Soc. 127:14142-14143, 2005). In other approaches, cyclization is accomplished by forming a thioether bond between two sites on the polypeptide (see Timmerman, P. et al., (2005) ChemBioChem 6:821-824; incorporated by reference herein in its entirety). An enzymatic method relies on the reaction between (1) a cysteine and (2) a dehydroalanine or dehydrobutyrine group, catalyzed by a lantibiotic synthetase, to create the thioether bond (see Levengood, M.R. and Van der Donk, W.A., Bioorg. and Med. Chem. Lett. 18:3025-3028, 2008). The dehydro- 43 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1functional group can also be generated chemically by the oxidation of selenium containing amino acid side chains incorporated during translation (see Seebeck, F.P. and Szostak, J.W. J. Am. Chem. Soc. 2006).

[0248] Various modifications to reduce immunogenicity and / or improve the half-life of therapeutic proteins are known in the art. For example, the peptides can undergo glycosylation, isomerization, or deglycosylation according to standard methods known in the art. Similarly, the peptides can be modified by non-naturally occurring covalent modification for example by addition of polyethylene glycol moieties (pegylation) or lipidation, e.g., myristoylation. In one example, the peptides are conjugated to polyethylene glycol to improve their pharmacokinetic profiles. Conjugation can be carried out by techniques known to those skilled in the art (see, for example, Deckert et al., Int. J. Cancer 87: 382-390, 2000; Knight et al., Platelets 15: 409-418, 2004; Leong et al., Cytokine 16: 106-119, 2001; and Yang et al., Protein Eng. 16: 761-770, 2003). In some embodiments, antigenic epitopes can be identified and altered by mutagenesis. Methods of identifying antigenic epitopes are known in the art (see for example, Sette et al., Biologicals 29:271-276, 2001), as are methods of mutating such antigenic epitopes. Further, modifications may be incorporated to decrease the toxicity of the disclosed peptides. The general toxicity of the peptides according to the present disclosure can be tested according to methods known in the art.

[0249] Optionally, the peptides of the present disclosure may further include a series of consecutive amino acids encoding a domain (a protein tag; for example, a myc- or his-tag) that facilitates the isolation and purification of the peptide. An “isolated” peptide means a peptide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. In some embodiments, a peptide of this disclosure is purified, i.e., essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities. For instance, domains that are useful in the isolation of a peptide include a histidine domain (which can be isolated using nickel-chelating resins), an S-peptide domain (which can be isolated using an S-protein, see Kim et al. Protein Sci. 2:348-356, 1993), and a chitin binding domain (which can bind to chitin beads, see Chong et al. Gene 192:271-281, 1997; and Watanabe et al. J. Bacteriol. 176:4465-4472, 1994). In some embodiments, the domain is present at the carboxy terminal end of the peptide. In some embodiments, the domain can be cleaved from the remainder of the peptide by the use of a protease or self-cleaving sequence.- 44 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1Detectable Labels

[0250] The disclosed peptides can include one or more labels, for example to permit detection of the peptides. In various applications, a disclosed Cdk5-inhibitory peptide includes a label (e.g., a detectable label) conjugated to the peptide, e.g., covalently bound to the peptide. A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the peptide or composition, e.g., in a tissue or sample. Thus, a labeled peptide or composition provides an indicator of the presence or concentration of such in a sample. The disclosure is not limited to the use of particular detectable labels, although examples are provided.

[0251] A detectable label associated with a Cdk5-inhibitory peptide disclosed herein can be detected either directly or indirectly. A detectable label can be detected by any known or yet to be a discovered mechanism including absorption, emission and / or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

[0252] Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Invitrogen, e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies, Invitrogen Detection Technologies, Molecular Probes, Eugene, Oreg.). Examples of fluorophores that can be attached (for example, chemically conjugated) to a Cdk5-inhibitory peptide or composition are provided in U.S. Pat. No. 5,866,366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-- 45 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitro tyro sine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron®. Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives.

[0253] Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997 ; J. Biol.Chem. 274:3315-22, 1999), as well as GFP, Lissamine™, diethylaminocoumarin, fluorescein chloro triazinyl, naphthofluorescein, 4,7 -dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Invitrogen Detection Technologies, Molecular Probes (Eugene, Oreg.) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6,130,101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No.5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

[0254] In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOT™- 46 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1(obtained, for example, from QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.; see also, U.S. Pat. Nos. 6,815,064, 6,682,596 and 6,649,138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and / or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can be coupled to a variety of biological molecules (including dNTPs and / or nucleic acids) or substrates by techniques described in, for example, Bruchez et al. (1998) Science 281:2013-6, Chan et al. (1998) Science 281:2016-8, and U.S. Pat. No. 6,274,323.

[0255] Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927,069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622;6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018;5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003 / 0165951 as well as PCT Publication No. 99 / 26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Invitrogen (Carlsbad, Calif.).

[0256] Additional detectable labels include, for example, radioisotopes (such as 3H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd 3+, and liposomes.

[0257] Detectable labels that can be used with the disclosed Cdk5-inhibitory peptides and pharmaceutical compositions also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, P-galactosidase, P-glucuronidase or p-lactamase. Where the detectable label includes an enzyme, a chromogen, Anorogenic compound, or luminogenic compound can be used in combination with the enzyme to generate a detectable signal (numerous of such compounds are commercially available, for example, from Invitrogen Corporation, Eugene Oreg.). Particular examples of chromogenic compounds include diaminobenzidine (DAB), 4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl- 47 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP / NBT, fast red, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-P-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-P-galactopyranoside (X-Gal), methylumbelliferyl-P-D-galactopyranoside (MU-Gal), p-nitrophenyl-a-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-P-D-glucuronide (X-Gluc), 3-amino-9-ethyl carbazol (AEC), fuchsin, iodonitro tetrazolium (INT), tetrazolium blue and tetrazolium violet.Spacers / Linkers

[0258] According to the present disclosure, a Cdk5-inhibitory peptide or variant thereof may be attached, such as covalently attached to another Cdk5-inhibitory peptide through an appropriate linker or spacer. For example, an optional spacer / linker moiety and / or additional amino acid(s) may be added to any region of the isolated peptide, including, but not limited to, the N- or C-terminus of the peptide or TAT provided that such moieties do not interfere with delivery and / or function.

[0259] Depending on such factors as the molecules to be linked, and the conditions in which the peptide is being administered (such as if the peptide is being used in a method of detection), the linker can vary in length and composition for optimizing such properties as flexibility and stability. The linker may be a peptide heterologous to the Cdk5-inhibitory peptide or PTD (for example, TAT) sequence. In some examples, a linker is a peptide such as poly-lysine, polyglutamine, poly-glycine, poly-proline or any combination thereof. In some examples, the peptide linker can be designed to be either hydrophilic or hydrophobic in order to enhance the desired inhibitory activity of the Cdk5-inhibitory peptide sequence, thereby inhibiting Cdk5 hyperphosphorylation. The peptide linker and the Cdk5-inhibitory peptides herein can be encoded as a single fusion polypeptide.

[0260] In some examples, the linker acts as a molecular bridge to link a peptide to a detectable label. The linker or spacer can serve, for example, simply as a convenient way to link the two entities, as a means to spatially separate the two entities, to provide an additional functionality to the peptide, or a combination thereof. For example, it may be desirable to spatially separate the peptide and the detectable label to prevent the detectable label from interfering with the activity of the peptide and / or vice versa. The linker can also be used to provide a stability sequence, a molecular tag, or various combinations thereof. In one example, the linker is one or more glycines, such as 2-10 or 4-6, including 2, 3, 4, 5, 6, 7, 8, 9 or 10 glycine residues.- 48 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0261] The selected linker can be bifunctional or polyfunctional, e.g., contain at least a first reactive functionality at, or proximal to, a first end of the linker that is capable of bonding to, or being modified to bond to, peptides or variants thereof provided herein and a second reactive functionality at, or proximal to, the opposite end of the linker that is capable of bonding to, or being modified to bond to, e.g., a PTD, such as a TAT PTD, or other functional domain. The two or more reactive functionalities can be the same (i.e., the linker is homobifunctional) or they can be different (i.e., the linker is heterobifunctional). A variety of bifunctional or polyfunctional cross-linking agents are known in the art that are suitable for use as linkers (for example, those commercially available from Pierce Chemical Co., Rockford, Ill.). Alternatively, these reagents can be used to add the linker to a Cdk5-inhibitory peptide and / or PTD domain, such as a TAT PTD.

[0262] The length and composition of the linker / spacer can be varied considerably provided that it can fulfill its purpose as a molecular bridge. The length and composition of the linker are generally selected taking into consideration the intended function of the linker, and optionally other factors such as ease of synthesis, stability, resistance to certain chemical and / or temperature parameters, and biocompatibility. For example, the linker or spacer should not significantly interfere with the delivery of the Cdk5-inhibitory peptide, such as the delivery of the Cdk5-inhibitory peptide to the brain, or with the activity of the peptide relating to regulating one or more signs or symptoms of a neurodegenerative disease.

[0263] Linkers suitable for use according to the present disclosure may be branched, unbranched, saturated, or unsaturated hydrocarbon chains, including peptides as noted above. In some embodiments of the present disclosure, the linker is a branched or unbranched, saturated or unsaturated, hydrocarbon chain having from 1 to 100 carbon atoms, wherein one or more of the carbon atoms is optionally replaced by — O — or — NR — (wherein R is H, or Cl to C6 alkyl), and wherein the chain is optionally substituted on carbon with one or more substituents selected from the group of (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, amide, azido, cyano, nitro, halo, hydroxy, oxo (=0), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0264] Examples of suitable linkers include, but are not limited to, peptides having a chain length of 1 to 100 atoms, and linkers derived from groups such as ethanolamine, ethylene glycol, polyethylene with a chain length of 6 to 100 carbon atoms, polyethylene glycol with 3 to 30 repeating units, phenoxyethanol, propanolamide, butylene glycol, butyleneglycolamide, propyl phenyl, and ethyl, propyl, hexyl, steryl, cetyl, and palmitoyl alkyl chains.- 49 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0265] The linker may be a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 50 carbon atoms, wherein one or more of the carbon atoms is optionally replaced by — O — or — NR — (wherein R is as defined above), and wherein the chain is optionally substituted on carbon with one or more substituents selected from the group of (Cl-C6) alkoxy, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, amide, hydroxy, oxo (=0), carboxy, aryl and aryloxy.

[0266] In another example, the linker is an unbranched, saturated hydrocarbon chain having from 1 to 50 carbon atoms, wherein one or more of the carbon atoms is optionally replaced by O — or — NR — (wherein R is as defined above), and wherein the chain is optionally substituted on carbon with one or more substituents selected from the group of (C1-C6) alkoxy, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, amide, hydroxy, oxo (=0), carboxy, aryl and aryloxy.

[0267] In a specific example, the linker is a peptide having a chain length of 1 to 50 atoms. In another embodiment, the linker is a peptide having a chain length of 1 to 40 atoms. As known in the art, the attachment of a linker or spacer to a peptide need not be a particular mode of attachment or reaction. Various reactions providing a product of suitable stability and biological compatibility are acceptable.Protein Transduction Domains

[0268] In some embodiments, the Cdk5-inhibitory peptides of the disclosure comprise a protein transduction domain (PTD). PTDs constitute a family of polypeptides that facilitate protein transduction across membranes in a receptor-independent manner (Wadia and Dowdy, Curr. Protein Pept. Sci. 4(2):97-104, 2003). This phenomenon was originally described for the human immunodeficiency virus (HlV)-encoded trans activator of transcription (TAT) protein, which was shown to cross membranes and initiate transcription. It was then discovered that the portion of the TAT protein that was required for the transduction of the protein was only an 11 amino acid polypeptide: YGRKKRRQRRR (SEQ ID NO: 5). When fused with other proteins, the TAT peptide has been demonstrated to deliver these proteins, varying in size from 15 to 120 kDa, into cells in tissue culture (Frankel and Pabo, Cell 55(6): 1189-93, 1988; Green and Loewenstein, J. Gen. Microbiol. 134(3):849-55, 1988; Vives et al., J. Biol. Chem.272(25): 16010-7, 1997; Yoon et al., J. Microbiol. 42(4):328-35, 2004; Cai et al., Eur. J. Pharm. Sci. 27(4):311-9, 2006). As disclosed herein, the TAT peptide facilitates a Cdk5-inhibitory peptide to cross the BBB and localize in the brain.- 50 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0269] Other TAT polypeptide sequences include, but are not limited to SEQ ID NO: 6 (YARAARRAARR, “PTD1” herein), SEQ ID NO: 7 (YGRKRRQRRR, “PTD2” herein), SEQ ID NO: 8 (RKKRRQRRR, “PTD3” herein), or SEQ ID NO: 9 (KKKKKKKKK, “PTD4” herein). Thus, any one of the Cdk5-inhibitory peptides provided herein, including any of those in Table 2 or Table 3 herein, may further comprise a TAT polypeptide sequence such as that of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Typically, the TAT polypeptide sequence will be at the C-terminus of a Cdk5-inhibitory peptide disclosed herein, but a TAT polypeptide sequence may be at the N- or at the C-terminus of a Cdk5-inhibitory peptide disclosed herein.

[0270] Examples of Cdk5-inhibitory peptides comprising a TAT polypeptide sequence include: a Cdk5-inhibitory peptide having amino acid sequence as set forth in SEQ ID NO: 10 further comprising at the C-terminal end a TAT polypeptide sequence having amino acid set forth in SEQ ID NO: 6 ; a Cdk5-inhibitory peptide having amino acid sequence as set forth in SEQ ID NO 81 further comprising at the C-terminal end a TAT polypeptide sequence having amino acid set forth in SEQ ID NO: 6; a Cdk5-inhibitory peptide having amino acid sequence as set forth in SEQ ID NO: 79 further comprising at the C-terminal end a TAT polypeptide sequence having amino acid set forth in SEQ ID NO: 6; a Cdk5-inhibitory peptide having amino acid sequence as set forth in SEQ ID NO: 80 further comprising at the C-terminal end a TAT polypeptide sequence having amino acid set forth in SEQ ID NO: 6; a Cdk5-inhibitory peptide having amino acid sequence as set forth in SEQ ID NO: 77 further comprising at the C-terminal end a TAT polypeptide sequence having amino acid set forth in SEQ ID NO: 6; and a Cdk5-inhibitory peptide having amino acid sequence as set forth in SEQ ID NO: 78 further comprising at the C-terminal end a TAT polypeptide sequence having amino acid set forth in SEQ ID NO: 6.

[0271] The Cdk5-inhibitory peptides provided herein need not comprise a PTD polypeptide sequence having 100% identity to those set forth as SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The current disclosure contemplates use of a modified or variant PTD polypeptide sequence in which one or more amino acids differ from one or more of the PTD peptide sequences provided herein. However, the modified or variant PTD polypeptide sequence contemplated for use retains the capacity to facilitate protein transduction across membranes. Methods of preparing PTD fusion proteins and expression vectors comprising PTD fusion proteins are known in the art (see, for example, U.S. Pat. Nos. 7,094,407 and 7,060,673, herein incorporated by reference in their entirety). Thus, as described herein, PTD polypeptides are useful to facilitate delivery of Cdk5-inhibitory peptides across the BBB. Provided herein are PTD- 51 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1polypeptides fused to Cdk5-inhibitory peptides to allow the fusion proteins to cross the BBB and localize within the brain.

[0272] Other PTDs are known in the art and can be used in the compositions and methods described herein. Examples of such PTDs include, but are not limited to, peptides from the VP22 protein of herpes simplex virus (HSV) type 1 (Elliott et al., Cell 88:223-233, 1997); the UL-56 protein of HSV-2 (U.S. Pre-Grant Publication No. 2006 / 0099677); the Vpr protein of HIV- 1 (U.S. Pre-Grant Publication No. 2005 / 0287648); the third helix of the Drosophila Antennapedia homeobox gene (Derossi et al., J. Biol. Chem. 269:10444-10450, 1994; Schwarze et al., Trends Pharmacol. Sci. 21:45-48, 2000); the transportan protein (Pooga, FASEB J. 12:67-77, 1998; Hawiger, Curr. Opin. Chem. Biol. 3:89-94, 1999). A number of artificial peptides also are known to function as PTDs, such as poly-arginine, poly-lysine and others (see, for example, U.S. PreGrant Publication Nos. 2006 / 0106197; 2006 / 0024331; 2005 / 0287648; and 2003 / 0125242;Zhibao et al., Mol. Ther. 2:339-347, 2000; and Laus et al., Nature Biotechnol. 18:1269-1272, 2000). Each of the above-listed publications, and PTD sequences disclosed therein, is herein incorporated by references in its entirety.Nucleic Acids

[0273] The present disclosure also concerns nucleic acid constructs including polynucleotide sequences that encode the isolated polypeptides, including peptides, disclosed herein, such as isolated nucleic acid molecules and vectors including such nucleic acid molecules. These polynucleotides include DNA, cDNA and RNA sequences, which encode the polypeptide of interest. Thus, this disclosure encompasses polynucleotides encoding the amino acid sequences comprising any of the Cdk5 -inhibitory peptides described above, for example peptides comprising a Cdk5-inhibitory peptide such as any of those provided in Table 2 or Table 3.

[0274] The nucleic acid constructs can include polynucleotides that encode heterologous polypeptides in addition to those set forth above, for example peptides that include peptide linkers or other moieties to aid in the purification, detection (such as heterologous fluorescent protein sequences, such as green fluorescent protein and the like), and / or attachment of the peptides to a solid surface (such as GST, biotin, avidin or streptavidin).

[0275] The coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence such that, while the nucleotide sequence is altered, it nevertheless encodes a peptide having an amino acid sequence the same as the disclosed peptide sequences, for example for optimization of expression in a host cell, such as a bacterial host cell,- 52 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1such as E. coli. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from encoding sequences disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences.

[0276] To produce such nucleic acid constructs, polynucleotide sequences encoding peptides are inserted into a suitable expression vector, such as a plasmid expression vector. Procedures for producing polynucleotide sequences encoding the peptides disclosed herein and for manipulating them in vitro are well known to those of skill in the art, and can be found (see for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y., 1994).

[0277] A wide variety of cloning and in vitro amplification methodologies are well known to persons skilled in the art. A nucleic acid encoding a polypeptide can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self- sustained sequence replication system (3SR) and the QP replicase amplification system (QB). Methods for the manipulation and insertion of the nucleic acids of this disclosure into vectors are well known in the art (see for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y., 1994). PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).

[0278] A polynucleotide sequence encoding the disclosed peptides can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. A promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences (which can be) near the start site of transcription, such as in the case of a polymerase II type promoter (a TATA element). A promoter also can include distal enhancer or repressor elements, which can be located as much as several thousand base- 53 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1pairs from the start site of transcription. Both constitutive and inducible promoters are included (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987).

[0279] The polynucleotides including recombinant DNA can be incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or can exist as a separate molecule (for example a cDNA) independent of other sequences. Typically, the nucleic acid constructs encoding the peptides of this disclosure are plasmids. However, other vectors (for example, viral vectors, phage, cosmids, etc.) can be utilized to replicate the nucleic acids. In the context of this disclosure, the nucleic acid constructs typically are expression vectors (for example, prokaryotic, eukaryotic, or mammalian expression vectors) that contain a promoter sequence, which facilitates the efficient transcription of the inserted genetic material by the host cell. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences (encoding, for example, a selectable marker) that allow phenotypic selection of the transformed cells.

[0280] DNA sequences encoding the polypeptides including Cdk5-inhibitory peptides of this disclosure can be expressed in vitro by DNA transfer into a suitable host cell. Thus, also disclosed are host cells that include vectors encoding the peptides of this disclosure. The cell may be prokaryotic or eukaryotic. The term host cell also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

[0281] Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells, which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC12 method using procedures well known in the art. Alternatively, MgC12 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

[0282] When the host is a eukaryote, methods of transfection of DNA such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors can be used.Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding the polypeptide of interest, and a second foreign DNA molecule encoding a selectable phenotype (selectable marker). Another method is to use a eukaryotic viral vector, such as simian virus 40- 54 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1(SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed„ 1982).III. PHARMACEUTICAL COMPOSITIONS

[0283] Provided herein are pharmaceutical compositions including any of the disclosed compounds, such as the disclosed polypeptides including Cdk5-inhibitory peptides or nucleic acids in combination with (i.e., formulated with) a pharmaceutically acceptable carrier or vehicle. In some embodiments, the pharmaceutical composition includes a Cdk5-inhibitory peptide having an amino acid sequence set forth as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76. In some embodiments, the pharmaceutical composition includes a Cdk5-inhibitory peptide having an amino acid sequence set forth as SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116. In some embodiments, the pharmaceutical compositions include a Cdk5-inhibitory peptide set forth herein covalently linked to a detectable label such as FITC, Cy7, or other detectable label.- 55 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0284] The pharmaceutical composition may also include one or more agents or drugs known to be therapeutically active in the treatment of a disorder or disease. These agents may include steroids, anti-inflammatory compounds, immunosuppressive compounds, and / or antioxidant compounds.

[0285] In some embodiments, the pharmaceutical composition is administered intraperitoneally. In other embodiments, the pharmaceutical composition is administered orally. Additional routes of administration may include transdermal, venous, intranasal, and inhalation routes. Inhalational preparations can include aerosols, particulates, and the like. In general, the goal for particle size for inhalation is about one (1) micron or less for alveolar absorption. For administration by inhalation, the pharmaceutical compositions are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for instance, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0286] Methods for preparing pharmaceutical compositions are known to those skilled in the art (see Remington’s Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa., 1995). Preparations for parenteral (including intraperitoneal) administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. In some embodiments, the composition includes a carrier which facilitates the peptide to cross the blood-brain barrier. In some embodiments, the composition includes a carrier which facilitates the peptide to penetrate a tumor microenvironment.

[0287] Pharmaceutical compositions for oral use can be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. Such compositions can be prepared according to standard- 56 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1methods known to the art for the manufacture of pharmaceutical compositions and may contain one or more agents selected from the group of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically acceptable and palatable preparations. Tablets contain the active ingredient in admixture with suitable non-toxic pharmaceutically acceptable excipients including, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as com starch, or alginic acid; binding agents, such as starch, gelatin or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Pharmaceutical compositions for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

[0288] Pharmaceutical compositions can include pharmaceutically acceptable salts of the disclosed peptides. Pharmaceutically acceptable salts of the presently disclosed compounds include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine (“ORN”), choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts are also inclusive of the free acid, base, and zwitterionic forms. Description of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002).

[0289] The peptides described herein can be administered to a subject for therapeutic treatment of a neurodegenerative disorder, such as a neurodegenerative disease. Thus, a therapeutically effective amount of a composition comprising one or more of the disclosed peptides is administered to a subject already suffering from a neurodegenerative disorder,- 57 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1including a neurodegenerative disease (such as ALS, AD, PD or a combination thereof), in an amount sufficient to improve a sign or a symptom of the disorder.

[0290] The peptides described herein can be administered to a subject for therapeutic treatment of a proliferative disorder, such as cancer. Thus, a therapeutically effective amount of a composition comprising one or more of the disclosed peptides is administered to a subject suffering from a proliferative disorder in an amount sufficient to improve a sign or a symptom of the disorder.

[0291] Generally a suitable dose is about 1 milligram per kilogram (mg / kg) to about 100 mg / kg, such as a dose of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg, administered parenterally, for example intraperitoneally (ip), or orally. Other higher or lower doses may be used, such as from about 0.001 mg / kg to about 1 g / kg, such as about 0.1 mg / kg to about 500 mg / kg, including about 0.5 mg / kg to about 200 mg / kg. For example, a suitable dose is about 1 mg / kg to about 100 mg / kg, such as a dose of about 1 mg / kg, about 10 mg / kg, about 20 mg / kg, about 50 mg / kg, or about 100 mg / kg administered ip. Unit dosage forms are also contemplated, for example 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg unit dosages. Unit dosages may be formulated for parenteral, e.g., ip, oral, or other administration.

[0292] Single or multiple administrations of the composition comprising one or more of the disclosed peptides can be carried out with dose levels and pattern being selected by the treating physician. Generally, multiple doses are administered. In some embodiments, the composition is administered parenterally, for example intraperitoneally, once per day. However, the composition can be administered twice per day, three times per day, four times per day, six times per day, every other day, twice a week, weekly, or monthly. In some examples, the composition is administered parenterally, for example intraperitoneally, for two or more consecutive days, for example for 3, 4, 5, 6, 7, 8, 9, 10, or more consecutive days. In other examples, the composition is administered parenterally, for example intraperitoneally, once per day for two or more consecutive days. In a specific example, the composition is administered parenterally, for example intraperitoneally, once per day for at least three consecutive days. Treatment can- 58 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1continue for at least a month, for example for two or three months, sometimes for six months or a year, and may even continue indefinitely, i.e., chronically. Repeat courses of treatment are also contemplated.

[0293] In sone embodiments, the pharmaceutical composition is administered without sequential or concurrent administration of a second agent for the treatment of a neurodegenerative disorder. In one specific, non-limiting example, one or more of the disclosed peptides is administered without sequential or concurrent administration of other agents, such as without sequential or concurrent administration of an additional agent also known to target the neurodegenerative disorder. In other specific non-limiting examples, a therapeutically effective amount of a disclosed pharmaceutical composition is administered sequentially or concurrently with an additional agent, including an additional neurodegenerative disorder therapy (such as, but not limited to, monoclonal antibodies, an anti-inflammatory agent, such as glatiramer acetate, an antioxidant, such as lipoic acid). For example, the disclosed compounds are administered in combination with (sequentially or concurrently) protease and / or proteasome inhibitors, antioxidants, anti-inflammatory drugs or combinations thereof.IV. DEFINITIONS

[0294] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those skilled in the art in practicing subject matter of the present disclosure. The singular forms “a,” “an,” and “the” refer to one or more than one, unless the context clearly dictates otherwise. For example, the term “comprising an amino acid” includes single or plural amino acid molecules or residues and is considered equivalent to the phrase “comprising at least amino acid molecule.” The term “or” refers to a single element of stated alternative elements or a combination of two or more elements unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements.

[0295] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one skilled in the art to which this disclosure relates. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. For example, conventional methods well known in the art to which a disclosed invention pertains are described in various general and more specific references,- 59 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

[0296] Additional terms commonly used in molecular genetics can be found in Benjamin Lewin, Genes V published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0297] All sequences associated with GenBank, NCBI, UniProt, or Ensembl Accession Nos. or equivalent genomic are protein databases mentioned herein are incorporated by reference in their entirety to the extent permissible by applicable rules and / or law.

[0298] The term “administer” or “administration” and the like mean: To provide or give a subject an agent, such as a disclosed peptide or pharmaceutical composition, by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. A particular type of administration is intraperitoneal (ip).

[0299] The term “Alzheimer's disease” (abbreviated “AD”) means: A progressive brain disorder that occurs gradually and results in memory loss, behavioral and personality changes, and a decline in mental abilities. These losses are related to the death of brain cells and the breakdown of the connections between them. The course of this disease varies from person to person, as does the rate of decline. On average, AD patients live for 8 to 10 years after they are diagnosed, though the disease can last up to 20 years. AD advances by stages, from early, mild forgetfulness to a severe loss of mental function. At first, AD destroys neurons in parts of the brain that control memory, especially in the hippocampus and related structures. As nerve cells in the hippocampus stop functioning properly, short-term memory fails. AD also attacks the cerebral cortex, particularly the areas responsible for language and reasoning.- 60 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0300] The term “amyotrophic lateral sclerosis” (“ALS”) means: A progressive, usually fatal, neurodegenerative disease caused by the degeneration of motor neurons. As a motor neuron disease, the disorder causes muscle weakness and atrophy throughout the body as both the upper and lower motor neurons degenerate, ceasing to send messages to muscles. Unable to function, the muscles gradually weaken, develop fasciculations (twitches) because of denervation, and eventually atrophy because of that denervation. The subject may ultimately lose the ability to initiate and control all voluntary movement except for the eyes. ALS is also known as Lou Gehrig's disease.

[0301] The term “blood-brain barrier” (“BBB”) means: The barrier formed by epithelial cells in the capillaries that supply the brain and central nervous system (“CNS”). This barrier selectively allows entry of substances such as water, oxygen, carbon dioxide, and nonionic solutes such as glucose, alcohol, and general anesthetics, while blocking entry of other substances. Some small molecules, such as amino acids, are taken across the barrier by specific transport mechanisms.

[0302] The term “cyclin dependent kinase 5” (“Cdk5”) means: A member of the cyclin dependent kinase (Cdk) family of serine / threonine kinases, most of which are key regulators of the cell cycle. Unlike mitotic Cdks, Cdk5 plays a primary role in brain development, neuronal migration, neurite outgrowth and axon patterning. Cdk5 activity is regulated through associating with its neuron-specific activators, P35 and P39. Cdk5 has also been implicated as a player in learning and memory. Normally, Cdk5 activity is tightly regulated but under conditions of neuronal stress it is deregulated leading to hyperactivity, neuronal pathology and cell death. Hyperactivity of Cdk5 may be involved in neurodegenerative disorders. As used herein, a cyclin dependent kinase 5 inhibitory domain, is a domain that reduces or inhibits one or more biological functions of Cdk5, such as kinase activity (phosphorylation). Cdk5 refers to the amino acid sequence of the protein product of Ensemble Gene ID: ENSG00000164885 (i.e., NP_004926.1, UniProtKB identifier: Q00535). “Cdk5-inhibitory polypeptide” or “Cdk5-inhibitory peptide” as used herein refers to a polypeptide or peptide, as the case may be, which reduces or inhibits one or more biological functions of Cdk5, such as kinase activity (phosphorylation), including inhibiting or reducing interaction of Cdk5 and P25 to inhibit or reduce Cdk5 / P25 based hyperphosphorylation activity. The terms “reducing” or “inhibiting” as used herein in reference to a biological function are intended to have broad meaning and may involve, e.g., interfering with a physical interaction between two molecular species, blocking a functional group of a molecular species, blocking a functional target of a molecular species, blocking the formation of- 61 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1a functional molecular species, or otherwise causing the degradation or dysfunction of a molecular species. For example, a number of proteins are regulated by, or are functionally associated with, Cdk5. Modulation of Cdk5, e.g., by reducing the phosphorylation of Cdk5, may result in the reduction of phosphorylation of those proteins. For example, microtubule-associated protein tau (MAPT), synaptic trafficking regulators (e.g., AGAP3), chromatin remodeling proteins (e.g., CHD4), phosphoinositide signaling proteins (e.g., MTMR7), cyclic AMP signaling components (e.g., ADCY8), synaptic adhesion proteins (e.g., NCAM2 and CADM1), and protein phosphatases involved in stress-kinase regulation (e.g., PPM1E) may exhibit decreased phosphorylation as a result of decreasing the phosphorylation of Cdk5, e.g., by administering one or more Cdk5-inhibitory polypeptide or Cdk5-inhibitory peptide.

[0303] The term “label” or “detectable label” means: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule, for example by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a nucleic acid molecule or polypeptide, thereby permitting detection of the nucleic acid molecule or polypeptide. Examples of labels include, but are not limited to, radioactive isotopes, nitroimidazoles, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. ( Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). In some embodiments, the label is a fluorophore (“fluorescent label”).Fluorophores are chemical compounds, which when excited by exposure to a particular wavelength of light, emits light (i.e., fluoresces), for example at a different wavelength.Fluorophores can be described in terms of their emission profile, or “color.” Green fluorophores, for example Cy3, FITC, and Oregon Green, are characterized by their emission at wavelengths generally in the range of 515-5401. Red fluorophores, for example Texas Red, Cy5 and tetramethylrhodamine, are characterized by their emission at wavelengths generally in the range of 590-6901. In some examples, a disclosed peptide is labeled.

[0304] The term “linker” means: A relatively short series of amino acids (for example, between 2 and 150 amino acids) that separates elements or domains of a fusion protein.Examples of specific linkers can be found, for instance, in Hennecke et al. ( Protein Eng. 11:405-410, 1998); and U.S. Pat. Nos. 5,767,260 and 5,856,456. Linkers may be repetitive or non-repetitive. One classical repetitive linker used in the production of single chain Fvs (SCFvs) is- 62 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1the (Gly 4Ser) 3 (or (GGGGS) 3 or (G 4S) 3) linker. Non-repetitive linkers also have been produced and methods for the random generation of such linkers are known (Hennecke et al., Protein Eng. 11:405-410, 1998). In addition, linkers may be chosen to have more or less secondary character (e.g., helical character, U.S. Pat. No. 5,637,481) depending on the conformation desired in the final fusion protein.

[0305] The term “modulate” or “modulating” and the like means: To adjust, alter, regulate an activity, a degree or rate of such. Modulating can be increasing or a decreasing an activity. A pharmaceutical composition, including one or more of the disclosed Cdk5-inhibitory peptides, may be administered to modulate (reduce or inhibit) one or more signs or symptoms of a neurodegenerative disorder or disease.

[0306] The term “multiple sclerosis” (“MS”) means: A slowly progressive CNS disease characterized by disseminated patches of demyelination in the brain and spinal cord, resulting in multiple and varied neurological symptoms and signs, usually with remissions and exacerbation. An increased family incidence suggests genetic susceptibility, and women are somewhat more often affected than men. The symptoms of MS include weakness, lack of coordination, paresthesias, speech disturbances, and visual disturbances, most commonly double vision. More specific signs and symptoms depend on the location of the lesions and the severity and destructiveness of the inflammatory and sclerotic processes. Relapsing-remitting multiple sclerosis is a clinical course of MS that is characterized by clearly defined, acute attacks with full or partial recovery and no disease progression between attacks. Secondary-progressive multiple sclerosis is a clinical course of MS that initially is relapsing-remitting, and then becomes progressive at a variable rate, possibly with an occasional relapse and minor remission. Primary progressive multiple sclerosis presents initially in the progressive form. A clinically isolated syndrome is the first neurologic episode, which is caused by inflammation / demyelination at one or more sites in the CNS.

[0307] The term “neurodegenerative disorder” or “neurodegenerative disease” means: Any type of disorder or disease that is characterized by the progressive deterioration of the nervous system. In some cases, a neurodegenerative disease is associated with axon damage. Axon damage includes axon degeneration and a reduction in axon density, for example in the white matter of the caudal spinal cord. White matter tissue damage includes axons undergoing Wallerian-like degeneration, reduced nerve fiber density, and demyelination. White matter tissue damage can be determined by histological examination of white matter, for example from the ventrolateral or dorsal thoracic spinal cord. White matter tissue damage may also be determined- 63 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1by MRI. Evidence of axonal damage can be inferred from presence of abnormal MRI signals, such as permanentiy decreased T 1 signals (“black holes”), decreased n-acetyl aspartate (NAA) and whole brain atrophy.

[0308] Axon damage also includes decreased neurofilament phosphorylation (NF-P) (see e.g., Trapp et al., N. Engl. J. Med. 338:278-285, 1998). Neurofilaments in myelinated axons are normally heavily phosphorylated. NF-P can be determined by immunohistochemical staining. A reduction in NF-P reflects demyelination and axon damage. Decreasing axon damage in a subject includes a reduction in white matter tissue damage as compared with an untreated subject, such as a reduction in the decrease in NF-P as compared with an untreated subject. Decreasing axon damage also encompasses preventing axon damage and repair of axon damage. Repair of axon damage in a subject includes a reduction in white matter tissue damage or a reduction in the decrease in NF-P as compared with an earlier time point, for example prior to beginning treatment with other compounds used to treat an axonal disorder, including a neurodegenerative disease.

[0309] A neurodegenerative disorder may be a disorder associated with Cdk5 hyperphosphorylation. In some examples, a neurodegenerative disorder, such as AD, PD, AES, or a combination thereof is associated with an increase in neurofilament phosphorylation (such as hyperphosphorylation of neurofilament protein M or H(NF-M / H), for example at least a 2-fold increase in neurofilament M or H phosphorylation as compared to phosphorylation levels in a control, or non-diseased sample). In some examples, AD is associated with a 5- to 8-fold increase in neurofilament M phosphorylation or a 2- to 8-fold increase in neurofilament H phosphorylation. In some examples, a neurodegenerative disorder is ALS, AD, corticobasal degeneration, Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV- associated dementia, Kennedy's disease, Krabbe's disease, Eewy Body dementia, MS, PD, tropical ataxic neuropathy, ALS / PD, lathyrism, primary lateral sclerosis, spinal muscular atrophy or any combination thereof. Neurodegenerative diseases include AD, ALS, and PD.

[0310] The term “nucleic acid” means: A polymer composed of nucleotide units (nucleosides) (ribonucleosides, deoxyribonucleosides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleosides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without- 64 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

[0311] Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single- stranded nucleotide sequence is the 5 '-end; the left-hand direction of a doublestranded nucleotide sequence is referred to as the 5 '-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5 '-end of the RNA transcript are referred to as “upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as “downstream sequences.”

[0312] “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0313] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0314] “Recombinant nucleic acid” refers to a nucleic acid having nucleotide sequences that are not naturally joined together. This includes nucleic acid vectors comprising an amplified or- 65 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1assembled nucleic acid which can be used to transform a suitable host cell. A host cell that comprises the recombinant nucleic acid is referred to as a “recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, for example a “recombinant polypeptide.” A recombinant nucleic acid may serve a non-coding function (for example a promoter, origin of replication, ribosome-binding site, etc.) as well.

[0315] The term “nucleotide” means: The fundamental unit of nucleic acid molecules. A nucleotide includes a nitrogen-containing base attached to a pentose monosaccharide with one, two, or three phosphate groups attached by ester linkages to the saccharide moiety.

[0316] The major nucleotides of DNA are deoxyadenosine 5 '-triphosphate (dATP or A), deoxy guano sine 5 '-triphosphate (dGTP or G), deoxycytidine 5 '-triphosphate (dCTP or C) and deoxythymidine 5 '-triphosphate (dTTP or T). The major nucleotides of RNA are adenosine 5'-triphosphate (ATP or A), guanosine 5 '-triphosphate (GTP or G), cytidine 5 '-triphosphate (CTP or C) and uridine 5 '-triphosphate (UTP or U).

[0317] Nucleotides include those nucleotides containing modified bases, modified sugar moieties and modified phosphate backbones, for example as described in U.S. Pat. No. 5,866,336 to Nazarenko et al. (herein incorporated by reference).

[0318] Examples of modified base moieties which can be used to modify nucleotides at any position on its structure include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N~6-sopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5 '-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5-oxyacetic acid methylester, uracil-5 -oxy acetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

[0319] Examples of modified sugar moieties which may be used to modify nucleotides at any position on its structure include, but are not limited to: arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof.- 66 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0320] The term “parenteral” means: Administered outside of the intestine, for example, not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application. In one example, parenteral refers to intraperitoneal administration.

[0321] The term “Parkinson's disease” (“PD”) means: An idiopathic, slowly progressive, degenerative CNS disorder characterized by slow and decreased movement, muscular rigidity, resting tremor, and postural instability. The loss of substantia nigra neurons, which project to the caudate nucleus and putamen, results in the depletion of the neurotransmitter dopamine in these areas.

[0322] The term “peptide” means: Any compound composed of amino acids or amino acid analogs chemically bound together. Peptide as used herein includes oligomers of amino acids, amino acid analogs, or small and large peptides and any chain of amino acids, regardless of length or post-translational modification (such as glycosylation or phosphorylation). In one example, a peptide is two or more amino acids joined by a peptide bond. Typically, a peptide consists of fewer than fifty amino acids; for example, consisting of approximately 7 to approximately 40 amino acids, consisting of approximately 7 to approximately 30 amino acids, consisting of approximately 7 to approximately 20 amino acids. In one example, a peptide consists of 24 amino acids and is referred to as P5 (SEQ ID NO: 4). “Peptide” applies to amino acid polymers, to naturally occurring amino acid polymers, and non-naturally occurring amino acid polymers as well as those in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. Peptides include peptoids and other peptidomimetics.

[0323] A “polypeptide” is a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide. The term polypeptide encompasses peptides.- 67 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0324] The term “soluble” in reference to a peptide or a polypeptide refers to a form of such peptide or polypeptide (or portion thereof) that is not inserted into a cell membrane.

[0325] The term “pharmaceutical composition” means: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. A pharmaceutical composition can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional compound induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject). In a particular example, a pharmaceutical agent is an agent that significantly reduces one or more symptoms associated with a neurodegenerative disease.

[0326] The terms “pharmaceutically acceptable carriers” or “pharmaceutically acceptable vehicles” include conventional ones familiar to those skilled in the art of polypeptide formulation. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more peptides provided herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In some embodiments, the carrier is one that allows the therapeutic compound to cross the blood-brain barrier. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0327] Protease: An enzyme that catalyzes the hydrolysis of peptide bonds, for example peptide bonds in a protein. Examples of proteolytic enzymes include endoproteases, such as trypsin, chymotrypsin, endoprotease ArgC, endoprotease aspN, endoprotease gluC, thermolysin, and endoprotease lysC. The specific bonds cleaved by an endoprotease or a chemical protein cleavage agents may be more specifically referred to as “endoprotease cleavage sites” and “chemical protein cleavage agent sites,” respectively. Proteins typically contain one or more intrinsic protein cleavage agent sites recognized by one or more protein cleavage agents by virtue- 68 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1of the amino acid sequence of the protein. A protease inhibitor is an agent that inhibits the activity of a protease.

[0328] The term “protein transduction domain” (“PTD”) means: A polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a cell membrane, including the blood-brain barrier (BBB). In one example, the protein transduction domain is an HIV trans-activator of transcription (TAT) protein transduction domain which facilitates the transport of one of the disclosed Cdk5 -inhibitory peptides across the BBB. PTDs can be naturally occurring or synthetically produced.

[0329] The term “purified” as used herein does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide (such as a Cdk5-inhibitory polypeptide), protein or other active compound is one that is isolated in whole or in part from naturally associated proteins and other contaminants, in which the polypeptide or other active compound is purified to a measurable degree relative to its un-purified state, for example, relative to its purity within a cell extract or chemical synthesis checker.

[0330] In certain embodiments, the term “substantially purified” refers to a polypeptide, protein, peptide, or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components. Such purified preparations can include materials in covalent association with the polypeptide, such as glycoside residues or materials admixed or conjugated with the polypeptide, which may be desired to yield a modified derivative or analog of the polypeptide or to produce a combinatorial therapeutic formulation, conjugate, fusion protein or the like. The term purified thus includes such desired products as peptide and protein analogs or mimetics or other biologically active compounds wherein additional compounds or moieties are bound to the polypeptide in order to allow for the attachment of other compounds and / or provide for formulations useful in therapeutic treatment or diagnostic procedures.

[0331] Generally, substantially purified polypeptides, proteins, peptides, or other active compounds represent more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the respective compound with additional ingredients in a complete pharmaceutical formulation for therapeutic administration. Additional ingredients can include a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other like co-ingredients. More typically, the polypeptide or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species- 69 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation can be essentially homogeneous, wherein other macromolecular species are less than 1%.

[0332] The term “recombinant” means: A nucleic acid or polypeptide that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical or biochemical synthesis. One of ordinary skill in the art will appreciate that many different recombinant polynucleotides and recombinant polypeptides may be created by molecular engineering.

[0333] The term “sequence identity” means: The similarity between two nucleic acid sequences, or two amino acid sequences, expressed in terms of the similarity between the sequences. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch J. Mol. Biol. 48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins & Sharp Gene 73: 237-244, 1988; Higgins & Sharp CABIOS 5: 151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al. Meth. Mol. Bio. 24, 307-31, 1994.Altschul et al. ( J. Mol. Biol. 215:403-410, 1990), presents a detailed consideration of sequence alignment methods and homology calculations.

[0334] The term “subject” means: Any living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.

[0335] The term “therapeutically effective” as in an amount or concentration means: An amount of a composition that alone, or together with an additional therapeutic agent(s), is sufficient to achieve a desired effect or response in a subject, or in a cell, being treated with the agent. The effective amount of the agent will be dependent on several factors, including, but not limited to the subject or cells being treated, and the manner of administration of the therapeutic composition. In one example, a therapeutically effective amount or concentration is one that is sufficient to prevent advancement, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease, such as a neurodegenerative disease. In one example, a desired effect or response is to reduce or inhibit one or more symptoms associated with hyperphosphorylation by Cdk5 / P25. The one or more symptoms do- 70 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1not have to be completely eliminated for the composition to be therapeutically effective. For example, a composition can decrease the symptom by a desired amount, for example by at least 20%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100%, as compared to the symptom in the absence of the composition. In some examples, a desired response is to reduce or inhibit Cdk5 phosphorylation by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100%, as compared to the Cdk5 phosphorylation in the absence of the disclosed peptides.

[0336] A therapeutically effective amount of a disclosed pharmaceutical composition can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. For example, a therapeutically effective amount of such agent can vary from about 100 pg to about 100 mg per kg body weight if administered intravenously. In some embodiments, a therapeutically effective amount is between 10 to 100 mg / kg body weight administered ip, including 20 to 80 mg / kg body weight, 30 to 70 mg / kg body weight, or 40 to 60 mg / kg, such as about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, 70 mg / kg, about 80 mg / kg or about 90 mg / kg.

[0337] The term “transformed” and the like refers to a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection (e.g., with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0338] The term “vector” means: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

[0339] The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, objects and advantages of the- 71 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1disclosure will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. 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 disclosure belongs. In the case of conflict, the present description will control.

[0340] The present disclosure is further illustrated by the following non-limiting examples.EXAMPLESExample 1. Peptide Screening

[0341] A cellular tau assay is performed to assess tau phosphorylation in the presence of Cdk5-inhibitory peptides of the disclosure. Biophysical assays such as microscale thermophoresis to assess biophysical binding properties of Cdk5-inhibitory peptides are performed. Candidate Cdk5-inhibitory peptides having superior efficacy and binding properties are selected for further assessment.Example 2. Metabolite Studies

[0342] Metabolite detection studies are performed to find active species of Cdk5-inhibitory peptides.Example 3. Pharmacokinetics

[0343] Cryofluorescence tomography (CFT) imaging and pharmacokinetic studies are performed to assess Cdk5-inhibitory peptide function.Example 4. PS19 (P301L tau transgenic mouse) Study

[0344] A study is performed to validate the preliminary efficacy of Cdk5-inhibitory peptides in the PS 19 tauopathy mouse model.Example 5. NanoBRET™ Assay Testing Peptide Inhibition of CDK5:p25 and CDK5:p35 First AspectDelivery of positive control peptide via electroporation

[0345] 1. Bulk transfect HEK293 cells with optimal orientations and ratios of CDK5:p35 and CDK5:p25 pairs and add NanoBRET™ ligand.- 72 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0346] 2. Harvest cells and electroporate with 3 concentrations of peptide inhibitor or vehicle control

[0347] 3. Plate cells in 96 well and measure BRET kinetically up to 6 hours.Testing of CDK5 / p25 and CDK5 / p35 NanoBREI™ pair with peptide via electroporation

[0348] 1. Optimized pairs: NL-p25 / CDK5-HT and NL-p35 / CDK5-HT. Donor Acceptor ratio at 1:100.

[0349] 2. Hek293 cells were plated into 6 well format at a density of 4x10A5 cells / ml, 2ml each. 2 wells each.

[0350] 3. Use standard FuGENE®HD protocol: Mix 200pl Opti-MEM Reduced Serum Medium: use 4pg of HT vector and 4pl of diluted NLuc vectors @ 1:100. Add 12ul FuGENE®HD and incubate ~10 mins.

[0351] 4. Transfer ~100ul / well of the DNA / transfection reagent to each well. Incubate overnight @ 37C 5% CO2.

[0352] 5. The next day cells were labeled + / - NanoBRET™ 618 ligand and incubated overnight.

[0353] 6. CT526 peptide was prepared as lOmM solution in water. It was further diluted to ImM in water.

[0354] 7. For electroporation: 500uM final: add 5ul lOmM stock to cells. IOOUM final: add lOul of ImM stock, lOuM final: add lul of ImM stock to cells. For no treatment add up to lOul water to cells.

[0355] 8. Remove medium from cells and wash with 1 ml DPBS

[0356] 9. Add 0.3 ml 0.25% trypsin-EDTA and incubate at room temperature until cells lift, remove trypsin.

[0357] 10. Add 1 ml phenol red free Medium / 4% FBS, disperse cells in well. POOL like cells as needed. Count cells.

[0358] 11. Spin cells and resuspend to ~2 x 106 cells / lOOul Minis Ingenio reagent. Transfer to cuvette and add the peptide to respective cuvettes.

[0359] 12. Follow HEK293 program on the Biorad Gene Pulser.

[0360] 13. Add 1ml of OPTI 4% FBS media to cuvette and let cells rest several mins.

[0361] 14. Collect cells and dilute to 2E5 / ml and dispense lOOul / well. 4 reps

[0362] 15. Make a 5x Nano-Gio® substrate mixture (50uM) (1:100 dilution) by adding 60ul of Nano-Gio® substrate to 6ml of Opti-MEM (no serum).- 73 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0363] 16. Add 25ul to each well and read plate on GloMax® Discover instrument using Kinetic NanoBRET™ 618 protocol for up to 6 hours.

[0364] 17. Results are reported as NanoBRET™ Ratios and normalized to control (no treatment).Conclusions (see FIG. 1)

[0365] 1. Testing of control peptide CT526 delivered by electroporation into HEK293 cells expressing the optimal NanoBRET™ vectors for p25 / CDK5 and p35 / CDK5 has been completed.

[0366] 2. A time-dependent decrease in CDK5 / p25 NanoBRET™ signal was observed at the lOOpM and 500pM peptide concentrations.

[0367] 3. CDK5 / p35 NanoBRET™ pair showed only slight decrease in signal with 500pM concentration of peptide, which was present and time zero and did not decrease further over time.

[0368] 4. The kinetic time course was measured out to 6 hours however the raw donor acceptor values past 3 hours are too low to accurately calculate NanoBRET™ values.Example 6: Human CDK5 Receptor Binding Assay

[0369] Purpose: To provide evidence that Cdk5-inhibitory polypeptides inhibit CDK5 / P25 binding.

[0370] Method: An ELISA was prepared. Briefly, a high-binding plate was coated with human CDK5 (Sino Biological, Inc. Cat # 10897 -H09B) diluted in IX PBS to 0.5 pg / mL.

[0371] The plate was incubated overnight at room temperature (RT).

[0372] After incubation, the plate was washed four times with IX PBS + 0.05% Tween-20 (wash buffer).

[0373] 3% bovine serum albumin (BSA) was applied to the entire plate. The plate was incubated for > 1 hour at RT.

[0374] After incubation, the plate was washed four times with wash buffer.

[0375] Next, a serial dilution of each Cdk5-inhibitory polypeptide was prepared in ultrapure water.

[0376] In parallel, P25 protein (Abeam Cat # abl25653) was diluted to 0.5 pg / mL in IX PBS.

[0377] The dissolved peptide and P25 solution were combined in a dilution plate and were incubated for 30 minutes at RT.- 74 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0378] The peptide / P25 solution was transferred to the high-binding plate and incubated for 1.5 h at RT with 500 rpm shaking, allowing for the interaction between CDK5 and P25 to occur in the presence or absence of the peptides.

[0379] After incubation, a biotinylated anti-p35 primary antibody (Invitrogen Cat# MAS-14834) was diluted to 0.5 pg / mL and added to the plate. Notably, the anti-P35 antibody used recognizes the P25 truncation product as well.

[0380] A further 1 h incubation was performed at RT with 500 rpm shaking before washing four times.

[0381] A Horseradish Peroxidase-streptavidin conjugate was added and incubated for 30 minutes at RT with 500 rpm shaking.

[0382] After incubation, the plate was washed four times before development with tetramethylbenzidine (TMB). The plate was developed at RT for 30 minutes with 500 rpm shaking before 10% sulfuric acid was added to stop color development. The plate was read at 450 -650 nm.Conclusions (FIG. 2)

[0383] Cdk5-inhibitory polypeptides CT600 (SEQ ID NO: 117) and CT526 (SEQ ID NO: 10) disrupted CDK5 / P25 binding by -30%, while a scrambled control peptide had little to no effect.

[0384] Importantly, a dose-dependent inhibition of CDK5 / P25 binding was observed. The disruption of the CDK5 / P25 complex is indicated by a decrease in the optical density of the ELISA, with a higher value indicating a higher concentration of the formed CDK5 / P25 complex.Example 7: STAT3 Activity Assay

[0385] Purpose: To demonstrate the efficacy of CD K5 -inhibitory peptides at inhibiting CDK5 kinase activity in live cells.

[0386] Method: Human HEK Blue IL-6 cells (HEK 293 IL-6 reporter) at the third passage were seeded at IxlO4cells / well in 100 pl DMEM media containing 10% heat-inactivated FBS and 2X Gentamicin and incubated overnight at 37°C in 5% CO2.

[0387] In a separate dilution plate, all peptides and Tamoxifen (positive control) were added at 4X the desired final concentration and serially diluted in assay media (60 pl / well).

[0388] Fifty pl of each diluted peptide or Tamoxifen was added to separate wells of the HEK BlueIL-6 plate and the plate was incubated for one hour at 37°C.- 75 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0389] Fifty pl of Human IL-6 was added to the cell plate at 4X the final concentration. Final assay volume was 200 ul / well. The plate was incubated for 24 hours at 37 °C.

[0390] Fifty pl was removed from each well and transferred to a new plate.The level of alkaline phosphatase in each well was determined using standard procedures. The quantification was performed by reading the plate at 405 nm.Conclusions (FIG. 3)

[0391] CDK5 phosphorylates STAT3 generating phosphoSTAT3. In HEK Blue IL-6 cells, the phosphorylation of STAT3 produces secreted embryonic alkaline phosphatase (SEAP). The production of SEAP is measured by ELISA, allowing for a quantitative readout of CDK5 kinase activity.

[0392] A decrease in phosphorylation of CDK5 target phosphoSTAT3 was observed with CT600 and CT526. A scrambled peptide minimally inhibited the phosphorylation of STAT3 while the therapeutic peptides reduced phosphorylation of STAT3 by 25-30%.

[0393] A known small molecule inhibitor of CDK5 / P25, tamoxifen, decreased phosphorylation of STAT3 by 15-20%.

[0394] The inventive CDK5-inhibitory peptides clearly disrupt the phosphorylating activity of CDK5 / P25 to a greater extent than current known small molecule inhibitors of CDK5 / P25.Example 8: Modulation of a CDK5-regulated phosphorylation network in neural tissue

[0395] FIG. 4 illustrates modulation of a cyclin-dependent kinase 5 (CDK5)-regulated phosphorylation network in neural tissue following administration of the disclosed agents. CDK5 is a proline-directed serine / threonine kinase involved in neuronal signaling, synaptic organization, cytoskeletal regulation, and intracellular signaling pathways.

[0396] As shown schematically, treatment results in coordinated decreases in phosphorylation across multiple neuronal proteins that are known to be regulated by, or functionally associated with, CDK5 signaling. Representative proteins exhibiting decreased phosphorylation include microtubule-associated protein tau (MAPT), synaptic trafficking regulators (e.g., AGAP3), chromatin remodeling proteins (e.g., CHD4), phosphoinositide signaling proteins (e.g., MTMR7), cyclic AMP signaling components (e.g., ADCY8), synaptic adhesion proteins (e.g., NCAM2 and CADM1), and protein phosphatases involved in stress-kinase regulation (e.g., PPM IE). These proteins are enriched for proline-directed serine / threonine motifs characteristic of CDK5 substrates.- 76 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0397] Importantly, as further illustrated in FIG. 4, the observed decreases in phosphorylation occur independently of corresponding changes in total protein abundance. Total protein levels for representative CDK5-associated substrates remain substantially unchanged following treatment, indicating that the observed effects reflect modulation of phosphorylation state rather than altered protein expression, degradation, or cellular toxicity.

[0398] The coordinated nature of the phosphorylation changes across multiple CDK5-associated substrates is consistent with modulation of a CDK5-regulated signaling network rather than inhibition of a single downstream target. Such network-level modulation may result in downstream functional effects on neuronal signaling, synaptic function, cytoskeletal dynamics, and intracellular signaling pathways.

[0399] In certain embodiments, the phosphorylation changes depicted in FIG. 4 may be quantified using one or more pharmacodynamic readouts, including composite phosphorylation signatures derived from multiple CDK5-associated substrates, individual substrate phosphorylation levels, or combinations thereof. These readouts may be used to assess target engagement, dose response, treatment efficacy, or modulation of disease-relevant signaling pathways.Equivalents and Scope

[0400] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0401] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.- 77 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO1

[0402] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed.

[0403] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0404] In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0405] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

[0406] While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the disclosure.- 78 - 4932-9691-4063, v. 1

Claims

Attorney Docket No.: COTH-002-WO1CLAIMSWhat is claimed is:

1. A Cdk5-inhibitory polypeptide comprising an amino acid sequence having sequence identity to 10 to 24 consecutive amino acid residues of SEQ ID NO: 4.

2. The Cdk5-inhibitory polypeptide of claim 1, wherein the amino acid sequence having sequence identity to 10 to 24 consecutive amino acid residues of SEQ ID NO: 4 comprises an amino acid sequence having one or more amino acid substitution compared to the amino acid sequence of SEQ ID NO: 4.

3. The Cdk5-inhibitory polypeptide of claim 1 or 2, wherein the one or more amino acid substitution comprises one or more conservative amino acid substitution.

4. The Cdk5-inhibitory polypeptide of claim 2 or 3, wherein the one or more amino acid substitution comprises one or more unnatural amino acid substitution.

5. The Cdk5-inhibitory polypeptide of any one of claims 2-4, wherein the Cdk5-inhibitory polypeptide comprises one or more amino acid modifications.

6. The Cdk5-inhibitory polypeptide of any one of claims 2-5, wherein the one or more amino acid substitution enhances binding stability.

7. The Cdk5-inhibitory polypeptide of any one of claims 2-6, wherein the one or more amino acid substitution enhances stability of the Cdk5-inhibitory polypeptide in solution.

8. The Cdk5-inhibitory polypeptide of any one of claims 2-7, wherein the one or more amino acid substitution enhances alpha helicity of the Cdk5-inhibitory polypeptide.

9. The Cdk5-inhibitory polypeptide of any one of claims 2-8, wherein the one or more amino acid substitution enhances cell penetration of the Cdk5-inhibitory polypeptide.- 79 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO110. The Cdk5-inhibitory polypeptide of any one of claims 2-9, wherein the one or more amino acid substitution enhances solubility of the Cdk5-inhibitory polypeptide.

11. The Cdk5-inhibitory polypeptide of any one of claims 2-10, wherein the one or more amino acid substitution comprises a W5Y amino acid substitution at position 5 compared to SEQ ID NO: 4.

12. The Cdk5-inhibitory polypeptide of any one of claims 2-10, wherein the one or more amino acid substitution comprises a D6(ABA), D6(AL4), D6(CTR), D6Q, D6E, D6L, D6M, D6(OME), D6(ORN), D6(SAR), D6T, D6W, or D6Y amino acid substitution at position 6 compared to SEQ ID NO: 4.

13. The Cdk5-inhibitory polypeptide of any one of claims 2-10, wherein the one or more amino acid substitution comprises a C8(ABA), C8R, C8(CTR), C8Q, C8L, C8K, C8(ORN), C8W, or C8Y amino acid substitution at position 8 compared to SEQ ID NO: 4.

14. The Cdk5-inhibitory polypeptide of any one of claims 2-10, wherein the one or more amino acid substitution comprises a Vll(HCS) amino acid substitution at position 11 compared to SEQ ID NO: 4.

15. The Cdk5-inhibitory polypeptide of any one of claims 2-10, wherein the one or more amino acid substitution comprises an M15G, M15P, M15(PCO), or M15(HCS) amino acid substitution at position 15 compared to SEQ ID NO: 4.

16. The Cdk5-inhibitory polypeptide of any one of claims 2-10, wherein the one or more amino acid substitution comprises an S17mS, SUH, or S17Mh amino acid substitution at position 17 compared to SEQ ID NO: 4.

17. The Cdk5-inhibitory polypeptide of any one of claims 2-10, wherein the one or more amino acid substitution comprises an M19(HCS) amino acid substitution at position 19 compared to SEQ ID NO: 4.- 80 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO118. The Cdk5-inhibitory polypeptide of any one of claims 2-10, wherein the one or more amino acid substitution comprises an A24M, A24V, A24L, A24(CYS-CYS), A24(HCY-HCY), A24R, A24K, A24(MDM), A24(NMI), (ORN), A24(2FM), A24(ESC), A24(NLE), A24(TFE), A24(YCP), or A24(26P) amino acid substitution at position 24 compared to SEQ ID NO: 4.

19. The Cdk5-inhibitory polypeptide of any one of claims 2-18, wherein the amino acid sequence having sequence identity to 10 to 24 consecutive amino acid residues of SEQ ID NO: 4 comprises 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 4.

20. The Cdk5-inhibitory polypeptide of any one of claims 2-19, comprising an amino acid sequence having at least 79% identity to the amino acid sequence of SEQ ID NO: 4.

21. The Cdk5-inhibitory polypeptide of any one of claims 2-20, comprising an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 4.

22. The Cdk5-inhibitory polypeptide of any one of claims 2-21, comprising an amino acid sequence having at least 87% identity to the amino acid sequence of SEQ ID NO: 4.

23. The Cdk5-inhibitory polypeptide of any one of claims 2-22, comprising an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO: 4.

24. The Cdk5-inhibitory polypeptide of any one of claims 2-23, comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 4.

25. A Cdk5-inhibitory peptide comprising an amino acid sequence having at least 79% sequence identity to the amino acid sequence of SEQ ID NO: 4.

26. The Cdk5-inhibitory peptide of claim 25 having an amino acid sequence of at least 83% sequence identity to the amino acid sequence of SEQ ID NO: 4.

27. The Cdk5-inhibitory peptide of claim 25 having an amino acid sequence of at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 4.- 81 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO128. The Cdk5-inhibitory peptide of claim 25 having an amino acid sequence of at least 91% sequence identity to the amino acid sequence of SEQ ID NO: 4.

29. The Cdk5-inhibitory peptide of claim 25 having an amino acid sequence of at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4.

30. The Cdk5-inhibitory peptide of claim 25, comprising a W5Y amino acid substitution at position 5 compared to SEQ ID NO: 4.

31. The Cdk5-inhibitory peptide of claim 25, comprising a D6(ABA), D6(AL4), D6(CTR), D6Q, D6E, D6L, D6M, D6(OME), D6(ORN), D6(SAR), D6T, D6W, or D6Y amino acid substitution at position 6 compared to SEQ ID NO: 4.

32. The Cdk5-inhibitory peptide of claim 25, comprising a C8(ABA), C8R, C8(CTR), C8Q, C8L, C8K, C8(ORN), C8W, or C8Y amino acid substitution at position 8 compared to SEQ ID NO: 4.

33. The Cdk5-inhibitory peptide of claim 23, comprising a Vll(HCS) amino acid substitution at position 11 compared to SEQ ID NO: 4.

34. The Cdk5-inhibitory peptide of claim 25, comprising an M15G, M15P, M15(PCO), or M15(HCS) amino acid substitution at position 15 compared to SEQ ID NO: 4.

35. The Cdk5-inhibitory peptide of claim 25, comprising an S17mS, SUH, or S17Mh amino acid substitution at position 17 compared to SEQ ID NO: 4.

36. The Cdk5-inhibitory peptide of claim 25, comprising an M19(HCS) amino acid substitution at position 19 compared to SEQ ID NO: 4.

37. The Cdk5-inhibitory peptide of claim 25, comprising an A24M, A24V, A24L, A24(CYS-CYS), A24(HCY-HCY), A24R, A24K, A24(MDM), A24(NMI), (ORN), A24(2FM), A24(ESC), A24(NLE), A24(TFE), A24(YCP), or A24(26P) at position 24 compared to SEQ ID NO: 4.- 82 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO138. A Cdk5-inhibitory peptide having an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76.

39. A Cdk5-inhibitory peptide having an amino acid sequence of SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80.

40. The Cdk5-inhibitory peptide of claim 39, comprising a C-terminal amide group modification.

41. The Cdk5-inhibitory peptide of claim 39 or 40, comprising an N-terminal acetyl group modification.

42. The Cdk5-inhibitory peptide of any one of claims 39-41, comprising a C-terminal amide group modification and an N-terminal acetyl group modification.

43. A Cdk5-inhibitory polypeptide comprising an amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1.

44. The Cdk5-inhibitory polypeptide of claim 43, wherein the 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 comprise a Cdk5 helix domain.- 83 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO145. The Cdk5-inhibitory polypeptide of claim 43 or 44, wherein the Cdk5 helix domain comprises an amino acid sequence defined by amino acid S46 to amino acid L55 of SEQ ID NO: 1.

46. The Cdk5-inhibitory polypeptide of any one of claims 43-45, wherein the Cdk5 helix domain comprises the amino acid sequence of SEQ ID NO: 81.

47. The Cdk5-inhibitory polypeptide of any one of claims 43-46, wherein the amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 comprises an amino acid sequence having one or more amino acid substitution compared to the amino acid sequence of SEQ ID NO: 81.

48. The Cdk5-inhibitory polypeptide of claim 47, wherein the one or more amino acid substitution comprises one or more conservative amino acid substitution.

49. The Cdk5-inhibitory polypeptide of claim 47 or 48, wherein the one or more amino acid substitution comprises one or more unnatural amino acid substitution.

50. The Cdk5-inhibitory polypeptide of any one of claims 47-49, wherein the Cdk5-inhibitory polypeptide comprises one or more amino acid modifications.

51. The Cdk5-inhibitory polypeptide of any one of claims 47-50, wherein the one or more amino acid substitution enhances binding stability.

52. The Cdk5-inhibitory polypeptide of any one of claims 47-51, wherein the one or more amino acid substitution enhances stability of the Cdk5-inhibitory polypeptide in solution.

53. The Cdk5-inhibitory polypeptide of any one of claims 47-52, wherein the one or more amino acid substitution enhances alpha helicity of the Cdk5-inhibitory polypeptide.

54. The Cdk5-inhibitory polypeptide of any one of claims 47-53, wherein the one or more amino acid substitution enhances cell penetration of the Cdk5-inhibitory polypeptide.- 84 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO155. The Cdk5-inhibitory polypeptide of any one of claims 47-54, wherein the one or more amino acid substitution enhances solubility of the Cdk5-inhibitory polypeptide.

56. The Cdk5-inhibitory polypeptide of any one of claims 47-55, wherein the one or more amino acid substitution comprises an amino acid substitution at position 1 of SEQ ID NO: 81 of: G1E, G1Y, GIA, GIF, G1Q, G1I, or G1S.

57. The Cdk5-inhibitory polypeptide of any one of claims 47-56, wherein the one or more amino acid substitution comprises an amino acid substitution at position 3 of SEQ ID NO: 81 of: P3E, P3L, P3I, P3Y, P3W, P3F, or P3Q.

58. The Cdk5-inhibitory polypeptide of any one of claims 47-57, wherein the one or more amino acid substitution comprises an amino acid substitution at position 4 of SEQ ID NO: 81 of: S4A.

59. The Cdk5-inhibitory polypeptide of any one of claims 47-58, wherein the one or more amino acid substitution comprises an amino acid substitution at position 5 of SEQ ID NO: 81 of: S5A.

60. The Cdk5-inhibitory polypeptide of any one of claims 47-59, wherein the one or more amino acid substitution comprises an amino acid substitution at position 7 of SEQ ID NO: 81 of: L7A.

61. The Cdk5-inhibitory polypeptide of any one of claims 47-60, wherein the one or more amino acid substitution comprises an amino acid substitution at position 8 of SEQ ID NO: 81 of: R8A.

62. The Cdk5-inhibitory polypeptide of any one of claims 47-61, wherein the one or more amino acid substitution comprises an amino acid substitution at position 11 of SEQ ID NO: 81 of: CHY, C11R, C11L, C11Q, or CHS.

63. The Cdk5-inhibitory polypeptide of any one of claims 47-62, wherein the one or more amino acid substitution comprises an amino acid substitution at position 12 of SEQ ID NO: 81 of: L12A, L12F, L12W, L12S, L12Y, or L12R.- 85 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO164. The Cdk5-inhibitory polypeptide of any one of claims 47-63, wherein the one or more amino acid substitution comprises an amino acid substitution at position 13 of SEQ ID NO: 81 of: L13R.

65. The Cdk5-inhibitory polypeptide of any one of claims 47-64, wherein the one or more amino acid substitution comprises an amino acid substitution at position 14 of SEQ ID NO: 81 of: K14A.

66. The Cdk5-inhibitory polypeptide of any one of claims 47-65, wherein the one or more amino acid substitution comprises an amino acid substitution at position 15 of SEQ ID NO: 81 of: E15W, E15A, E15Y, or E15Q.

67. The Cdk5-inhibitory polypeptide of any one of claims 47-66, wherein the amino acid sequence having sequence identity to 10 to 25 consecutive amino acid residues of SEQ ID NO: 1 comprises the amino acid sequence of SEQ ID NO: 81 having 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NO: 81.

68. The Cdk5-inhibitory polypeptide of any one of claims 47-67, comprising an amino acid sequence having at least 66% identity to the amino acid sequence of SEQ ID NO: 81.

69. The Cdk5-inhibitory polypeptide of any one of claims 47-68, comprising an amino acid sequence having at least 73% identity to the amino acid sequence of SEQ ID NO: 81.

70. The Cdk5-inhibitory polypeptide of any one of claims 47-69, comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 81.

71. The Cdk5-inhibitory polypeptide of any one of claims 47-70, comprising an amino acid sequence having at least 87% identity to the amino acid sequence of SEQ ID NO: 81.

72. The Cdk5-inhibitory polypeptide of any one of claims 47-71, comprising an amino acid sequence having at least 93% identity to the amino acid sequence of SEQ ID NO: 81.- 86 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO173. A Cdk5-inhibitory peptide comprising an amino acid sequence having at least 66% sequence identity to the amino acid sequence of SEQ ID NO: 81.

74. The Cdk5-inhibitory peptide of claim 73 having an amino acid sequence of at least 73% sequence identity to the amino acid sequence of SEQ ID NO: 81.

75. The Cdk5-inhibitory peptide of claim 73 having an amino acid sequence of at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 81.

76. The Cdk5-inhibitory peptide of claim 73 having an amino acid sequence of at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 81.

77. The Cdk5-inhibitory peptide of claim 73 having an amino acid sequence of at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 81.

78. The Cdk5-inhibitory peptide of claim 73, comprising a G1E, G1Y, GIA, GIF, G1Q, G1I, or G1S amino acid substitution at position 1 compared to SEQ ID NO: 81.

79. The Cdk5-inhibitory peptide of claim 73, comprising a P3E, P3L, P3I, P3Y, P3W, P3F, or P3Q amino acid substitution at position 3 compared to SEQ ID NO: 81.

80. The Cdk5-inhibitory peptide of claim 73, comprising a S4A amino acid substitution at position 4 compared to SEQ ID NO: 81.

81. The Cdk5-inhibitory peptide of claim 73, comprising a S5A amino acid substitution at position 5 compared to SEQ ID NO: 81.

82. The Cdk5-inhibitory peptide of claim 73, comprising a L7A amino acid substitution at position 7 compared to SEQ ID NO: 81.

83. The Cdk5-inhibitory peptide of claim 73, comprising a R8A amino acid substitution at position 8 compared to SEQ ID NO: 81.- 87 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO184. The Cdk5-inhibitory peptide of claim 73, comprising aCHY, C11R, C11L, C11Q, or CHS amino acid substitution at position 11 compared to SEQ ID NO: 81.

85. The Cdk5-inhibitory peptide of claim 73, comprising a L12A, L12F, L12W, L12S, L12Y, or L12R amino acid substitution at position 12 compared to SEQ ID NO: 81.

86. The Cdk5-inhibitory peptide of claim 73, comprising a L13R amino acid substitution at position 13 compared to SEQ ID NO: 81.

87. The Cdk5-inhibitory peptide of claim 73, comprising a K14A amino acid substitution at position 14 compared to SEQ ID NO: 81.

88. The Cdk5-inhibitory peptide of claim 73, comprising a E15W, E15A, E15Y, or E15Q amino acid substitution at position 15 compared to SEQ ID NO: 81.

89. A Cdk5-inhibitory peptide having an amino acid sequence of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116.

90. The Cdk5-inhibitory polypeptide of any one of claims 1-72, wherein the polypeptide inhibits Cdk5 / p25 activity.

91. The Cdk5-inhibitory polypeptide of any one of claims 1-72, wherein the polypeptide inhibits phosphorylation of one or more proteins selected from the group consisting of microtubule-associated protein tau (MAPT), synaptic trafficking regulators, chromatin remodeling proteins, phosphoinositide signaling proteins, cyclic AMP signaling components, synaptic adhesion proteins, and protein phosphatases involved in stress-kinase regulation.- 88 - 4932-9691-4063, v. 1Attorney Docket No.: COTH-002-WO192. The Cdk5-inhibitory polypeptide of any one of claims 1-72, wherein the polypeptide does not inhibit Cdk5 / p35 activity.

93. The Cdk5-inhibitory peptide of any one of claims 73-89, wherein the peptide inhibits Cdk5 / p25 activity.

94. The Cdk5-inhibitory peptide of any one of claims 73-89, wherein the polypeptide inhibits phosphorylation of one or more proteins selected from the group consisting of microtubule-associated protein tau (MAPT), synaptic trafficking regulators, chromatin remodeling proteins, phosphoinositide signaling proteins, cyclic AMP signaling components, synaptic adhesion proteins, and protein phosphatases involved in stress-kinase regulation.

95. The Cdk5-inhibitory peptide of any one of claims 73-89, wherein the peptide does not inhibit Cdk5 / p35 activity.- 89 - 4932-9691-4063, v. 1

Images