Psd-95 inhibitors and uses thereof

Abstract

This invention relates to compounds capable of binding to the PDZ domain of PSD-95 and their medical use as inhibitors of PSD-95-mediated protein-protein interactions.

Description

Technical Field

[0001] This invention relates to compounds capable of binding to the PDZ domain of PSD-95 and their medical use as inhibitors of PSD-95-mediated protein-protein interactions. Prior Technology

[0002] PSD-95 is a scaffold protein in neuronal synapses that interacts with N-methyl-d-aspartic acid (NMDA) receptors (Kornau et al., 1995) and neuronal nitric oxide synthase (nNOS) via its PSD-95 / Discs-large / ZO-1 (PDZ) domain. During cerebral ischemia, excessive glutamate release leads to overactivation of NMDA receptors and a detrimental increase in intracellular Ca2+ and NO levels, ultimately inducing excitotoxicity, resulting in neuronal death and brain damage (Aarts et al., 2002; Dawson et al., 1991; Huang et al., 1994; Sattler et al., 1999). PSD-95 is being investigated as a drug target for acute ischemic stroke (AIS) and related ischemic brain disorders.

[0003] A recent phase 3 clinical trial, ESCAPE-NA1 (Hill et al., 2020), examined the 20-meric peptide nerinetide (also known as Tat-NR2B9c or NA-1). This trial evaluated whether treatment with nerinetide, in addition to standard care (e.g., alteplase (tPA), tissue plasminogen activator), would improve clinical outcomes in patients with ischemic stroke undergoing endovascular thrombectomy (Hill et al., 2020). The study included a total of 1105 patients, with 659 receiving tPA and 446 not receiving treatment. While nerinetide was not effective in patients receiving alteplase, it improved functional outcomes, reduced mortality, and decreased infarct volume in the alteplase-untreated group (Hill et al., 2020). Recent studies have demonstrated that drug-drug interactions between nerinetide and tPA may explain the lack of effect in the tPA treatment group. Specifically, tPA generates cytoplasm, which is a serine protease that cleaves naine peptide (Mayor-Nunez et al., 2021). Therefore, compatibility of the drug with thrombolytic agents (which is the standard of care for AIS) is highly advantageous.

[0004] Nenetinide exhibits a relatively low affinity for the target PSD-95, prompting the design of dimeric compounds, such as the Tat-N-dimer and O-dimer, also known as UCCB01-144 (AB144) and UCCB01-125, respectively (Bach et al., 2009, 2012; WO 2010 / 004003, WO 2012 / 156308; Kucharz et al., 2017). In in vitro fluorescence polarization (FP) analysis, the Ki values ​​of PDZ1 and PDZ2 for nenetinide to PSD-95 ranged from 5 to 10 μM (Bach et al., 2012). In contrast, AB144 and UCCB01-125, due to their dimer structure and divalent nature, simultaneously bind to PDZ1 and PDZ2 of PSD-95, resulting in an affinity for PDZ1-2 (Ki values ​​of 4.6 nM and 9.5 nM, respectively) that is 500-1000 times greater than that for the monomer nainetide (Bach et al., 2012). In mice subjected to permanent middle cerebral artery occlusion (pMCAO), compared to the effect of saline, a single intravenous (iv) injection of AB144 (3 nmol / g) administered 30 min after ischemia reduced infarct volume by 40% and 37% at 6 h and 48 h post-ischemia survival, respectively (Bach et al., 2012). Furthermore, functional outcomes such as grip strength and rod rotation efficiency were improved and therefore associated with a reduction in infarct size. Under the same experimental conditions and dosage (3 nmol / g), nifedipine did not show significant neuroprotective properties (Bach et al., 2012). Similarly, UCCB01-125 without the Tat moiety did not reach the brain and did not show neuroprotective properties (Bach et al., 2012).

[0005] Therefore, PSD-95 is considered a particularly promising drug target for the treatment of acute conditions such as subarachnoid hemorrhage (SAH) and AIS. Importantly, PSD-95 is located intracellularly, so any drug targeting PSD-95 needs to effectively cross the cell membrane and bind to PSD-95. Summary of the Invention

[0006] The inventors have developed a series of novel compounds with high affinity for PSD-95. Compared to known PSD-95 inhibitors such as nifedipine, the compounds of the present invention exhibit improved cellular uptake and significantly higher cytoplasmic stability. Therefore, the compounds of the present invention can be used to treat excitotoxic diseases such as AIS and SAH, and are compatible with standard care (e.g., thrombolytic agents).

[0007] In one state sample, the present invention relates to a compound comprising: a. A first peptide (P1), comprising or consisting of the amino acid sequence X 2TX 3V (SEQ ID NO: 53), wherein X 2 is a group composed of E and S; X 3 can be freely chosen from groups composed of L, T, V, R, and D; b. A second peptide (P2) comprising or consisting of the amino acid sequence X 5TX 6V (SEQ ID NO: 55), wherein X 5 is a group composed of E and S; X 6 is a group composed of L, T, V, R, and D; and c. Cell-penetrating peptides (CPPs), selected from the following groups: i. Poly-L-arginine peptides (poly-Arg) consisting of 3 to 20 L-arginine residues; ii. yGrkkrrqrrr (SEQ ID NO: 9, D-TAT); iii. RQIKIWFQNRRMKWKK (SEQ ID NO: 14, L-Pen); iv. rqikiwfqnrrmkwkk (SEQ ID NO: 15, D-Pen); v. RKKRRRESRKKRRRES (SEQ ID NO: 17, L-DPV3); vi. LLIILRRRIRKQAHAHSK (SEQ ID NO: 18, L-pVEC); vii. KLALKLALKALKAALKLA (SEQ ID NO: 16, L-MAP); viii. PLIYLRLLRGQF (SEQ ID NO: 19, L-TP2); ix. |(Dap)KAPETALD| (SEQ ID NO: 21, MiniAp4); and x. |Ff(Nal2)RrRrQ|GABA-K (SEQ ID NO: 22, CPP12), CPP is attached to the linker via its C-terminus, and P1 and P2 are attached to the linker via their N-terminus, and the compound has the general structure of formula (I): Formula (I) Or a medically acceptable salt.

[0008] In another embodiment, the invention relates to compounds as defined herein, which are used as pharmaceuticals.

[0009] In another embodiment, the invention relates to compounds as defined herein, which are used to treat, prevent, alleviate, and / or delay the development of excitotoxic-related diseases (e.g., stroke, such as those selected from acute ischemic stroke and subarachnoid hemorrhage).

[0010] In another embodiment, the invention relates to compounds as defined herein, which are used to treat or prevent pain.

[0011] In addition, this invention relates to a method for manufacturing compounds as defined herein. Simple Explanation of the Diagram

[0012] [picture] [1.] The affinity of O-PEG 4 dimers (compounds 1 to 7) for PSD-95 PDZ1-2, as determined by FP. Data are presented as mean + SEM, n = 3. [picture] [2.] Affinity of the dimer ligands fused to Ac-TAT and Ac-polyArg to PSD-95 PDZ1-2, as determined by FP. Data are presented as mean + SEM, n = 3. [picture] [3.] Affinity of N-PEG 4(IETDV) 2 fused to different CPP tags (SEQ ID 8 to 22) to PSD-95 PDZ1-2, as determined by FP. Data are presented as mean + SEM, n = 3. [picture] [4.] Affinity of NPEG 4-(KETLV) 2-dimer ligands fused to the polyArg CPP tag to PSD-95 PDZ1-2, as determined by FP. Data are presented as mean + SEM, n = 3. [picture] [5.] Half-lives of the dimer ligands fused to Ac-TAT and Ac-polyArg, as determined in in vitro cytoplasmic stability assays. Data are presented as mean + SEM, n = 3. [picture] [6.] Half-life of N-PEG 4(IETDV) 2 fused to different CPP tags (SEQ ID 8 to 22), as determined in in vitro cytoplasmic stability assays. Data are presented as mean + SEM, n = 3. [picture] [7.] Cellular uptake of the dimer ligands fused to Ac-TAT and Ac-polyArg, as measured by CAPA. Data are presented as mean + SEM, n = 3. [picture] [8.] Cellular uptake of N-PEG 4 (IETDV) 2 fused to different CPP tags (SEQ ID 8 to 22), as determined by CAPA. Data are presented as mean + SEM, n = 3. [picture] [9.] Cellular uptake of N-PEG 4 (IETDV) 2 and N-PEG 4 (KETLV) 2 fused to different polyArg CPP tags, as measured by CAPA. Data are presented as mean + SEM, n = 3. [picture] [10.] Half-lives of N-PEG 4(IETDV) 2 and N-PEG 4(KETLV) 2 fused to the polyArg CPP tag, as determined in human plasma stability analysis. Data are presented as mean ± SEM, n = 3. [picture] [11.] The general structure of the compound PDZ1-2 bound to PSD-95. R1 to R5 are amino acid side chains as described herein and R6 represents a CPP tag as described herein. Implementation

[0013] [] [definition] []

[0014] Acetylamine bond: As used in this article, an acetylamine bond is a chemical bond formed by the reaction between a carboxylic acid and an amine (and the accompanying elimination of water). When the reaction occurs between two amino acid residues, the resulting bond is called a peptide bond.

[0015] Includes: As used herein, the term "includes" should be understood in a manner that includes. Thus, by way of example, a composition comprising compound X may include compound X and, where appropriate, other compounds.

[0016] Dimer: As used herein, a dimer system refers to two identical or different chemical moieties associated by chemical or physical interactions. As examples, a dimer can be a homodimer, such as two identical chemical moieties linked by a linker. A dimer can also be a heterodimer, such as two different chemical moieties linked by a linker. An example of a dimer is the PSD-95 inhibitor of this invention, which is a compound comprising two peptides covalently linked by a linker, wherein these peptides are capable of simultaneously binding to or interacting with the PDZ1 and PDZ2 domains of PSD-95.

[0017] Dipeptide: As used in this article, a dipeptide refers to two natural or non-natural amino acids linked by peptide bonds.

[0018] Fatty acids: As used herein, fatty acids (abbreviated FA) generally refer to carboxylic acids having long aliphatic carbon chains, which may be saturated or unsaturated. Fatty acids may be selected from short-chain fatty acids (SCFA), medium-chain fatty acids (MCFA), long-chain fatty acids (LCFA), and very long-chain fatty acids (VLCFA). Short-chain fatty acids (SCFA) are fatty acids with an aliphatic tail of fewer than 6 carbons (i.e., butyric acid). Medium-chain fatty acids (MCFA) are fatty acids with an aliphatic tail of 6-12 carbons, which can form medium-chain triglycerides. Long-chain fatty acids (LCFA) are fatty acids with an aliphatic tail of 13 to 21 carbons. Very long-chain fatty acids (VLCFA) are fatty acids with an aliphatic tail of more than 22 carbons. The fatty acids of this invention may be any suitable fatty acid or fatty acid derivative known to those skilled in the art.

[0019] Non-protein amino acids: Non-protein amino acids, also known as non-coded, non-standard, or non-natural amino acids, are amino acids that are not encoded by the genetic code. A non-exhaustive list of non-protein amino acids includes γ-aminobutyric acid, 1-3-(2-naphthyl)alanine, 1-2,3-diaminopropionic acid, α-amino-n-butyric acid, n-valine, n-leucine, isoleucine, alloisoleucine, terleucine, α-amino-n-heptanoic acid, and pipecolic acid. (acids), α,β-diaminopropionic acid, α,γ-diaminobutyric acid, ornithine, allethamine, homocysteine, homoserine, β-alanine, β-amino-n-butyric acid, β-aminoisobutyric acid, α-aminoisobutyric acid, isovalerine, sarcosine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N-ethylalanine, N-methylβ-alanine, N-ethylβ-alanine, isoserine, and α-hydroxy-γ-aminobutyric acid.

[0020] Protein amino acids: Also known as natural amino acids, protein amino acids include alanine, cysteine, selenocysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartic acid, proline, pyrrolidine, glutamic acid, arginine, serine, threonine, valine, tryptophan, and tyrosine. Uppercase abbreviations indicate L-amino acids, while lowercase abbreviations indicate D-amino acids.

[0021] PDZ: As used in this article, 'PDZ' refers to postsynaptic density protein-95 (PSD-95), Drosophila homologous disc tumor inhibitor (DlgA), and closed band-1 protein (zo-1).

[0022] PSD-95: As used in this article, 'PSD-95' refers to postsynaptic density protein-95.

[0023] PSD-95 inhibitor: As used herein, "PSD-95 inhibitor" refers to a compound that binds to PDZ1, PDZ2, or both PDZ1 and PDZ2 of PSD-95 and inhibits protein-protein interactions in cells facilitated by these PDZ domains. An example of an interaction inhibited by a PSD-95 inhibitor is the formation of a ternary complex between nNOS, PSD-95, and the NMDA receptor. [Compound] []

[0024] In one embodiment, the present invention relates to a compound comprising a first peptide (P1) linked to a cell-penetrating peptide (CPP). In one embodiment, P1 is linked to the CPP via a linker. In one embodiment, the compound further comprises a second peptide (P2). In one embodiment, the CPP is linked to both P1 and P2 via a linker. Thus, in one embodiment, the CPP is linked to the linker via its C-terminus, and P1 and P2 are bound to the linker via their N-termini, and the compound has the following general structure of formula (I): . Formula (I)

[0025] Therefore, in one embodiment, the compound of the present invention comprises a first peptide (P1) and a second peptide (P2), wherein both P1 and P2 can simultaneously bind to the PDZ1 and PDZ2 domains of PSD-95, i.e., the compound is a dimer PSD-95 inhibitor.

[0026] In one embodiment, the present invention relates to compounds comprising a peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO: 53), wherein... a. X 2 is a group composed of E and S; and b. X 3 is a group composed of L, T, V, R, and D. Furthermore, P1 is linked to the cell-penetrating peptide (CPP).

[0027] In another embodiment, the invention relates to compounds comprising a first peptide (P1) linked to a second peptide (P2) via a linker, such as compounds having the general structure of formula (XXIV): Formula (XXIV) The linker, P1, and P2 are as defined herein. Preferably, P1 and P2 comprise or consist of KETLV (SEQ ID NO: 2), KETTV (SEQ ID NO: 3), KETVV (SEQ ID NO: 4), KETRV (SEQ ID NO: 5), ISTDV (SEQ ID NO: 6), or VETVV (SEQ ID NO: 7). Where appropriate, the linker further binds to a cell-penetrating peptide (CPP) or an albumin-binding moiety, thereby increasing the pathway for the peptide to cross the membrane or blood-brain barrier. The albumin-binding moiety can be any suitable chemical group for binding albumin. Preferably, the albumin-binding moiety is a fatty acid. In one embodiment, the linker further binds to the CPP, i.e., the compound has the general structure of formula (I).

[0028] In one state sample, the present invention relates to a compound comprising: a. A first peptide (P1), comprising or consisting of the amino acid sequence X 2TX 3V (SEQ ID NO: 53), wherein X 2 is a group composed of E and S; X 3 can be freely chosen from groups composed of L, T, V, R, and D; b. A second peptide (P2) comprising or consisting of the amino acid sequence X 5TX 6V (SEQ ID NO: 55), wherein X 5 is a group composed of E and S; X 6 is a group composed of L, T, V, R, and D; and c. Cell-penetrating peptides (CPPs), selected from the following groups: i. Poly-L-arginine peptides (poly-Arg) consisting of 3 to 20 L-arginine residues; ii. yGrkkrrqrrr (SEQ ID NO: 9, D-TAT); iii. RQIKIWFQNRRMKWKK (SEQ ID NO: 14, L-Pen); iv. rqikiwfqnrrmkwkk (SEQ ID NO: 15, D-Pen); v. RKKRRRESRKKRRRES (SEQ ID NO: 17, L-DPV3); vi. LLIILRRRIRKQAHAHSK (SEQ ID NO: 18, L-pVEC); vii. KLALKLALKALKAALKLA (SEQ ID NO: 16, L-MAP); viii. PLIYLRLLRGQF (SEQ ID NO: 19, L-TP2); ix. |(Dap)KAPETALD| (SEQ ID NO: 21, MiniAp4); and x. |Ff(Nal2)RrRrQ|GABA-K (SEQ ID NO: 22, CPP12), CPP is attached to the linker via its C-terminus, and P1 and P2 are attached to the linker via their N-terminus, and the compound has the general structure of formula (I): Formula (I) Or a medically acceptable salt. [, Binding peptide , ] [, P1 , ] [, and , ] [, P2 , ]

[0029] Peptides P1 and P2 have high affinity for the PDZ1 and PDZ2 domains of PSD-95. In one embodiment, the compound is a PSD-95 inhibitor, meaning that the compound can bind to one or more PDZ domains of PSD-95.

[0030] In one embodiment, the compound comprises a first peptide (P1) that contains or consists of the amino acid sequence X 2TX 3V (SEQ ID NO: 53), wherein a. X 2 is a group composed of E and S; and b. X 3 is a group composed of L, T, V, R and D.

[0031] In one embodiment, P1 comprises or consists of the sequence X1X2TX3V (SEQ ID NO: 54), wherein a. X 1 is a group composed of K, V, and I; b. X 2 is a group composed of E and S; and c. X 3 is a group composed of L, T, V, R and D.

[0032] In one embodiment, P1 comprises or is composed of IETTV (SEQ ID NO: 1). In one embodiment, P1 comprises or is composed of KETLV (SEQ ID NO: 2). In one embodiment, P1 comprises or is composed of KETTV (SEQ ID NO: 3). In one embodiment, P1 comprises or is composed of KETVV (SEQ ID NO: 4). In one embodiment, P1 comprises or is composed of KETRV (SEQ ID NO: 5). In one embodiment, P1 comprises or is composed of ISTDV (SEQ ID NO: 6). In one embodiment, P1 comprises or is composed of VETVV (SEQ ID NO: 7).

[0033] In one embodiment, P1 consists of 4 to 10 amino acid residues (e.g., 5 amino acid residues, e.g., 6 amino acid residues). Amino acid residues beyond those given in SEQ ID NO: 53 or SEQ ID NO: 54 may be protein amino acids or non-protein amino acids.

[0034] In one embodiment, the compound further comprises a second peptide (P2) comprising or consisting of the amino acid sequence X 5TX 6V (SEQ ID NO: 55), wherein a. X 5 selects a group composed of E and S; and b. X 6 is a group composed of L, T, V, R and D.

[0035] In one embodiment, P2 comprises or consists of the sequence X 4X 5TX 6V (SEQ ID NO: 56), wherein a. X 4 is a group composed of K, V, and I; b. X 5 selects a group composed of E and S; and c. X 6 is a group composed of L, T, V, R and D.

[0036] In one embodiment, P2 comprises or consists of IETTV (SEQ ID NO: 1). In one embodiment, P2 comprises or consists of KETLV (SEQ ID NO: 2). In one embodiment, P2 comprises or consists of KETTV (SEQ ID NO: 3). In one embodiment, P2 comprises or consists of KETVV (SEQ ID NO: 4). In one embodiment, P2 comprises or consists of KETRV (SEQ ID NO: 5). In one embodiment, P2 comprises or consists of ISTDV (SEQ ID NO: 6). In one embodiment, P2 comprises or consists of VETVV (SEQ ID NO: 7).

[0037] In one embodiment, P2 is composed of 4 to 10 amino acid residues (e.g., 5 amino acid residues, e.g., 6 amino acid residues). Amino acid residues beyond those given in SEQ ID NO: 55 or SEQ ID NO: 56 may be protein amino acids or non-protein amino acids.

[0038] In one embodiment, P1 and P2 are the same.

[0039] In one embodiment, if X2 is E, then X3 is not D. In one embodiment, if X5 is E, then X6 is not D. In one embodiment, P1 and / or P2 are not IEDTV (SEQ ID NO: 1).

[0040] In a preferred embodiment, peptides P1 and P2 are bound to a linker or CPP via their N-terminus. In one embodiment, the amino acid sequence X2TX3V (SEQ ID NO: 53) is the C-terminus of P1. In one embodiment, the amino acid sequence X5TX6V (SEQ ID NO: 55) is the C-terminus of P2. [, Cell-penetrating peptides , ] [, (CPP) , ]

[0041] The compounds of this invention comprise peptide P1, which is linked to a cell-penetrating peptide (CPP). The CPP is characterized by its ability to cross the blood-brain barrier (BBB) ​​and / or serous membrane of mammalian cells, thereby enabling intracellular delivery of cargo molecules (e.g., peptides, proteins, oligonucleotides) linked to it.

[0042] In one embodiment, the CPP is a poly-l-arginine peptide (poly-Arg). In one embodiment, the poly-Arg system consists of 3 to 20 L-arginine residues. In one embodiment, the poly-Arg system consists of 5 to 10 L-arginine residues (e.g., 7 to 10 L-arginine residues, such as 8 or 9 L-arginine residues). In one embodiment, the poly-Arg system consists of 3 to 10 L-arginine residues. In one embodiment, the CPP contains or consists of the amino acid sequence RRRRRRRRR (l-Arg 9, SEQ ID NO: 12). In one embodiment, the CPP contains or consists of the amino acid sequence RRRRRRR (l-Arg 8, SEQ ID NO: 57). In one embodiment, the CPP contains or consists of the amino acid sequence RRRRRRR (l-Arg 7, SEQ ID NO: 58). In one embodiment, the CPP comprises or consists of the amino acid sequence RRRRR (l-Arg 6, SEQ ID NO: 59). In one embodiment, the CPP comprises or consists of the amino acid sequence RRRRR (l-Arg 5, SEQ ID NO: 60). In one embodiment, the CPP comprises or consists of the amino acid sequence RRRR (l-Arg 4, SEQ ID NO: 61). In one embodiment, the CPP comprises or consists of the amino acid sequence RRR (l-Arg 3).

[0043] In one embodiment, the CPP is selected from the CPPs given in Table 1. [surface] [1.] Cell-penetrating peptide. [name] [sequence] [SEQ ID NO:] l-TAT YGRKKRRQRRR 8 d-TAT yGrkkrrqrrr 9 mTAT rRrGrKkRr 10 riTAT rrrqrrkkr 11 l-Arg 9 RRRRRRRRR 12 l-Arg 8 RRRRRRRR 57 l-Arg 7 RRRRRRR 58 l-Arg 6 RRRRRR 59 l-Arg 5 RRRRR 60 l-Arg 4 RRRR 61 l-Arg 3 RRR d-Arg 9 rrrrrrrrr 13 l-Pen RQIKIWFQNRRMKWKK 14 d-Pen rqikiwfqnrrmkwkk 15 l-MAP KLALKLalKalKAalKLA 16 l-DPV3 RKKRRRESRKKRRRES 17 l-pVEC LLIILRRRIRKQAHAHSK 18 l-TP2 PLIYLRLLRGQF 19 d-TP2 pliylrllrGqf 20 MiniAp4 |(Dap)KAPETALD| twenty one CPP12 |Ff(Nal2)RrRrQ|GABA-K twenty two

[0044] In one embodiment, CPP comprises a peptide selected from the group consisting of: poly-l-arginine peptides (poly-Arg), such as l-Arg 9; d-TAT; l-Pen; d-Pen; l-DPV3; l-pVEC; l-MAP; l-TP2; MiniAp4 and CPP12. In one embodiment, CPP is selected from the group consisting of: poly-l-arginine peptides (poly-Arg), such as l-Arg 9; d-TAT; l-Pen; d-Pen; l-DPV3; l-pVEC; l-MAP; l-TP2; MiniAp4 and CPP12. In one embodiment, CPP is d-TAT. In one embodiment, CPP is l-Pen. In one embodiment, CPP is d-Pen. In one embodiment, CPP is l-DPV3. In one embodiment, CPP is l-pVEC. In one embodiment, CPP is l-MAP. In one embodiment, CPP is l-TP2. In one embodiment, CPP is MiniAp4. In one embodiment, CPP is CPP12.

[0045] In one embodiment, the CPP contains no more than 20 amino acid residues, such as no more than 19, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, or no more than 7 amino acid residues.

[0046] In some embodiments, the CPP binds to a non-peptide portion. For example, the CPP may be methylated or acetylated. In some embodiments, when a peptide is defined herein as consisting of a specific amino acid residue sequence, the peptide does not bind to any other amino acid residue, but the peptide may bind to a non-peptide portion, provided that binding to the non-peptide portion does not result in another amino acid sequence. In some embodiments, the N-terminus of the CPP binds to a non-peptide portion. In some embodiments, the C-terminus of the CPP binds to a non-peptide portion. In some embodiments, the N-terminus of the CPP is acetylated, i.e., bound to the chemical structure CH3C(O)-. For example, the CPP may be a poly-Arg peptide consisting of 9 L-arginine residues, which is acetylated at the N-terminus. In some embodiments, the N-terminus of the CPP binds to a chloroalkyl tag (CA) having the following structure:

[0047] In one embodiment, the N-terminus of the CPP is methylated. In another embodiment, the N-terminus of the CPP is methylated. [, Connector , ] [, , ]

[0048] As used herein, the term "linker" refers to one or more atoms that form a link from one chemical entity to another. As an example, the "linker" mentioned herein can link peptides by forming a link with each N-terminus of peptides P1 and P2, which are bound to two PDZ domains. Multiple linkers are known in this art. Linkers can be, for example, chemical linkers or peptide linkers, or combinations thereof. In one embodiment, the linker contains an active functional group, such as an electrophilic or nucleophilic functional group, which can be used to link the linker to each peptide.

[0049] In one embodiment, the linker comprises one or more polyethylene glycol (PEG) units. PEG is a polymer of ethylene glycol having the chemical formula C₂nH₄n+₂Oₙ+₁ and the following repeating structures: Where n is an integer.

[0050] Therefore, the PEG unit has the following structure: .

[0051] For example, a polymer consisting of 4 PEG moieties or a polymer where PEG4 corresponds to 4 ethylene glycol moieties (n=4). []

[0052] The terms "PEG unit" and "PEG part" are used interchangeably in this document.

[0053] In one embodiment, at least one oxygen atom of a PEG unit is replaced by a nitrogen atom to obtain NPEG. As used herein, the term "ethylene glycol portion" refers to the structural unit constituting the PEG or NPEG linker.

[0054] In one embodiment, the N-termini of the two PDZ domain-binding peptides P1 and P2 are linked to each other via a linker comprising one or more PEG units, wherein at least one oxygen atom of the PEG unit is optionally replaced by a nitrogen atom. In one embodiment, P1 and / or P2 are individually linked to the PEG / NPEG unit via a spacer group, for example via a short alkane chain.

[0055] In one embodiment, the linker comprises one or more PEG units, wherein at least one oxygen atom of one PEG unit is replaced by a nitrogen atom to obtain NPEG.

[0056] In one embodiment, the linker comprises an NPEG unit and the CPP is directly or indirectly linked to the nitrogen atom in the NPEG linker backbone via chemical bonds. The nitrogen atom of the CPP linked to the NPEG linker can be mediated by: amide bonds, 1,3-dipolar cycloaddition (e.g., copper-catalyzed azide-alkyne cycloaddition), maleimine coupling, disulfide bonds, or amine reactive electrophilic groups selected from N-hydroxysuccinimine (NHS) esters, p-nitrophenyl esters, succinimine carbonate, p-nitrophenyl carbonate, succinimine carbamate, isocyanates, isothiocyanates, amide azides, sulfonyl chlorides, aldehydes, carbonates, imine esters, or acid anhydrides; and sulfo reactive groups selected from haloacetyl groups, alkyl halide derivatives, aziridine, acrylamide derivatives, and aromatics. In one embodiment, the linker comprises one or more PEG units, wherein at least one oxygen atom of one PEG unit is substituted with a nitrogen atom to obtain NPEG, and wherein the CPP is linked to the nitrogen atom of the linker via an amide bond. Alternatively, the linking of the CPP to the nitrogen atom of the linker comprising the NPEG unit may be mediated via a spacer group, wherein a suitable spacer group may be, for example, any amino acid; a short alkane chain; or a short PEG / NPEG chain.

[0057] In one embodiment, the linker comprises an NPEG unit and CPP is linked to the nitrogen atom of the linker via a amide bond. For example, in one embodiment, the compound has the general structure of formula (III): Equation (III) in CPP, P1, and P2 are as defined herein; p is an integer from 0 to 10; and q is an integer from 0 to 10.

[0058] In one embodiment, p = q. In one embodiment, p > q. In one embodiment, p < q. In one embodiment, the sum of p and q is an integer between 1 and 20. In one embodiment, p is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In one embodiment, p is an integer from 0 to 4. In one embodiment, q is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In one embodiment, q is an integer from 0 to 4. In one embodiment, the total number of ethylene glycol portions, p + q, is between 2 and 12, for example, 2, 4, 6, 8, 10, or 12. In one embodiment, the total number of ethylene glycol portions, p + q, is 4. In one embodiment, p is 2 and q is 2. [, Overall dimer structure , ] [, , ]

[0059] In one embodiment, the compound has the general structure of formula (XXV): Formula (XXV) in P1 and P2 are as defined herein; X a chooses freely , and A group that makes up; CPP is as defined in this article; p is an integer from 0 to 10; and q is an integer from 0 to 10.

[0060] In one embodiment, p and q are related to 2, and the compound has the general structure of formula (XXVI): . Formula (XXVI)

[0061] In one embodiment, the compound has the formula (XXVI), wherein Xa is -O- and P1 and P2 are KETLV (SEQ ID NO: 2), thereby forming compound 2, namely OPEG 4-(KETLV) 2.

[0062] In one embodiment, the compound has the formula (XXVI), wherein Xa is -O- and P1 and P2 are KETTV (SEQ ID NO: 3), thereby forming compound 3, namely OPEG 4-(KETTV) 2.

[0063] In one embodiment, the compound has the formula (XXVI), wherein Xa is -O- and P1 and P2 are KETVV (SEQ ID NO: 4), thereby forming compound 4, namely OPEG 4-(KETVV) 2.

[0064] In one embodiment, the compound has the formula (XXVI), wherein Xa is -O- and P1 and P2 are KETRV (SEQ ID NO: 5), thereby forming compound 5, namely OPEG 4-(KETRV) 2.

[0065] In one embodiment, the compound has the formula (XXVI), wherein Xa is -O- and P1 and P2 are ISTDV (SEQ ID NO: 6), thereby forming compound 6, namely OPEG 4-(ISTDV) 2.

[0066] In one embodiment, the compound has the formula (XXVI), wherein Xa is -O- and P1 and P2 are VETVV (SEQ ID NO: 7), thereby forming compound 7, namely OPEG 4-(VETVV) 2.

[0067] In one embodiment, the compound has the general structure of formula (V): Formula (V) in CPP is as defined in this article; X 1 is a group composed of K, V, and I; X 2 is a group composed of E and S; and X 3 can be freely chosen from groups composed of L, T, V, R, and D; X 4 is a group composed of K, V, and I; X 5 is a group composed of E and S; X 6 can be freely chosen from groups composed of L, T, V, R, and D; p is an integer from 0 to 10; and q is an integer from 0 to 10.

[0068] Compound (V) (where p and q are series 2) is further illustrated in Figure 11. R1 to R5 are amino acid side chains of peptides X1X2TX3V (SEQ ID NO: 54) and X4X5TX6V (SEQ ID NO: 56), and R6 represents a CPP tag as described herein. [, As , ] [, CPP , ] [, Gathering , ] [, -Arg , ]

[0069] In one embodiment, CPP is a poly-1-arginine peptide (poly-Arg). Therefore, in one embodiment, the compound has the following general structure of formula (II): Equation (II) Poly-Arg, linkers, P1 and P2 systems are as defined herein.

[0070] In one embodiment, CPP is a poly-L-arginine peptide (poly-Arg) composed of 3 to 20 L-arginine residues. In one embodiment, CPP is a poly-L-arginine peptide (poly-Arg) composed of 3 to 15 L-arginine residues. In one embodiment, CPP is a poly-L-arginine peptide (poly-Arg) composed of 3 to 12 L-arginine residues. In one embodiment, CPP is a poly-L-arginine peptide (poly-Arg) composed of 3 to 10 L-arginine residues. In one embodiment, the N-terminus of the poly-Arg is modified as appropriate, for example, acetylated as appropriate.

[0071] The compounds of the present invention containing poly-Arg CPP tags (e.g., l-Arg9 (SEQ ID NO: 12)) exhibit low CP50 values ​​(Example 4, Figures 7-9), indicating highly efficient cellular uptake, which demonstrates excellent BBB penetration and intracellular delivery. Surprisingly, compounds containing CPP l-Arg9 (SEQ ID NO: 12) exhibit in vitro cytoplasmic half-lives comparable to those containing d-amino acids or macrocyclic CPPs (Figures 5-6). Interestingly, the half-life of compounds containing l-Arg9 is significantly superior to that of compounds containing l-TAT (SEQ ID NO: 8) (Figure 5).

[0072] In one embodiment, CPP is a poly-Arg PEG linker comprising NPEG units, and CPP is linked to the nitrogen atom of the linker via amide bonds. Therefore, in one embodiment, the compound has the general structure of formula (IV): Formula (IV) in Poly-Arg, P1, and P2 systems are as defined herein; p is an integer from 0 to 10; and q is an integer from 0 to 10.

[0073] In one embodiment, p-system 2 and q-system 2. Therefore, in one embodiment, the compound has the general structure of formula (VII): Equation (VII) Poly-Arg, P1 and P2 are as defined in this paper.

[0074] In one embodiment, the compound has the general structure of formula (VI): Formula (VI) in X 1 is a group composed of K, V, and I; X 2 is a group composed of E and S; and X 3 can be freely chosen from groups composed of L, T, V, R, and D; X 4 is a group composed of K, V, and I; X 5 is a group composed of E and S; X 6 can be freely chosen from groups composed of L, T, V, R, and D; p is an integer from 0 to 10; and q is an integer from 0 to 10.

[0075] In one embodiment, p-system 2 and q-system 2. Therefore, in one embodiment, the compound has the general structure of formula (VIII): Formula (VIII) The poly-Arg, X1, X2, X3, X4, X5 and X6 series are as defined herein.

[0076] In one embodiment, CPP is L-Arg 9, p-based 2, and q-based 2. Therefore, in one embodiment, the compound has the general structure of formula (IX): Formula (IX) X1, X2, X3, X4, X5 and X6 are as defined in this document.

[0077] In one embodiment, the compound is selected from the group consisting of formulas (X) to (XVI): Formula (X) Formula (XI) Formula (XII) Formula (XIII) Formula (XIV) Formula (XV) Formula (XVI)

[0078] In one embodiment, any of peptides P1, P2, and / or CPP is further modified. For example, in one embodiment, the N-terminus of CPP is acetylated as appropriate. Thus, in one embodiment, the compound is selected from the group consisting of formulas (XVII) to (XXIII): Formula (XVII) Formula (XVIII) Formula (XIX) Formula (XX) Formula (XXI) Formula (XXII) Formula (XXIII) [, Specific compounds , ] [, , ]

[0079] In one embodiment, the compound has the general structure of CPP-NPEG 4-(P1)(P2), i.e., the compound has the formula (XXVIII): Formula (XXVIII) Furthermore, CPP, P1, and P2 are as defined in Table 2. In one embodiment, the compounds are selected from the group consisting of compounds 9 to 63. [surface] [2.] The compounds illustrated in this invention. [Compound] [Mode] [P, 1, ] [、P , 2, ] [CPP] [] 8 (AB144) IETDV (SEQ ID NO: 1) l -TAT YGRKKRRQRRR- (SEQ ID NO: 8) 9 IETDV (SEQ ID NO: 1) l-TAT Ac-YGRKKRRQRRR- (SEQ ID NO: 23) 10 IETDV (SEQ ID NO: 1) l-TAT CA-YGRKKRRQRRR- (SEQ ID NO: 24) 11 KETLV (SEQ ID NO: 2) l-TAT Ac-YGRKKRRQRRR- (SEQ ID NO: 23) 12 KETLV (SEQ ID NO: 2) l-TAT CA-YGRKKRRQRRR- (SEQ ID NO: 24) 13 KETTV (SEQ ID NO: 3) l-TAT Ac-YGRKKRRQRRR- (SEQ ID NO: 23) 14 KETTV (SEQ ID NO: 3) l-TAT CA-YGRKKRRQRRR- (SEQ ID NO: 24) 15 KETVV (SEQ ID NO: 4) l-TAT Ac-YGRKKRRQRRR- (SEQ ID NO: 23) 16 KETVV (SEQ ID NO: 4) l-TAT CA-YGRKKRRQRRR- (SEQ ID NO: 24) 17 KETRV (SEQ ID NO: 5) l-TAT Ac-YGRKKRRQRRR- (SEQ ID NO: 23) 18 KETRV (SEQ ID NO: 5) l-TAT CA-YGRKKRRQRRR- (SEQ ID NO: 24) 19 ISTDV (SEQ ID NO: 6) l-TAT Ac-YGRKKRRQRRR- (SEQ ID NO: 23) 20 ISTDV (SEQ ID NO: 6) l-TAT CA-YGRKKRRQRRR- (SEQ ID NO: 24) 21 VETVV (SEQ ID NO: 7) l-TAT Ac-YGRKKRRQRRR- (SEQ ID NO: 23) 22 VETVV (SEQ ID NO: 7) l-TAT CA-YGRKKRRQRRR- (SEQ ID NO: 24) 23 (XVII) KETLV (SEQ ID NO: 2) l-Arg 9 Ac-RRRRRRRRR- (SEQ ID NO: 25) 24 KETLV (SEQ ID NO: 2) l-Arg 9 CA-RRRRRRRRR- (SEQ ID NO: 26) 25 (XVIII) KETTV (SEQ ID NO: 3) l-Arg 9 Ac-RRRRRRRRR- (SEQ ID NO: 25) 26 KETTV (SEQ ID NO: 3) l-Arg 9 CA-RRRRRRRRR- (SEQ ID NO: 26) 27 (XIX) KETVV (SEQ ID NO: 4) l-Arg 9 Ac-RRRRRRRRR- (SEQ ID NO: 25) 28 KETVV (SEQ ID NO: 4) l-Arg 9 CA-RRRRRRRRR- (SEQ ID NO: 26) 29 (XX) KETRV (SEQ ID NO: 5) l-Arg 9 Ac-RRRRRRRRR- (SEQ ID NO: 25) 30 KETRV (SEQ ID NO: 5) l-Arg 9 CA-RRRRRRRRR- (SEQ ID NO: 26) 31 (XXI) ISTDV (SEQ ID NO: 6) l-Arg 9 Ac-RRRRRRRRR- (SEQ ID NO: 25) 32 ISTDV (SEQ ID NO: 6) l-Arg 9 CA-RRRRRRRRR- (SEQ ID NO: 26) 33 (XXII) VETVV (SEQ ID NO: 7) l -Arg 9 Ac-RRRRRRRRR- (SEQ ID NO: 23) 34 VETVV (SEQ ID NO: 7) l -Arg 9 CA-RRRRRRRRR- (SEQ ID NO: 26) 35 (XXIII) IETDV (SEQ ID NO: 1) l -Arg 9 Ac-RRRRRRRRR- (SEQ ID NO: 25) 36 IETDV (SEQ ID NO: 1) l -Arg 9 CA-RRRRRRRRR- (SEQ ID NO: 26) 38 IETDV (SEQ ID NO: 1) d-TAT Ac-ygrkkrrqrrr- (SEQ ID NO: 27) 39 IETDV (SEQ ID NO: 1) d-TAT CA-ygrkkrrqrrr- (SEQ ID NO: 28) 40 IETDV (SEQ ID NO: 1) mTAT Ac-rRrGrKkRr- (SEQ ID BO: 29) 41 IETDV (SEQ ID NO: 1) mTAT CA-rRrGrKkRr- (SEQ ID NO: 30) 42 IETDV (SEQ ID NO: 1) riTAT Ac-rrrqrrkkr- (SEQ ID NO: 31) 43 IETDV (SEQ ID NO: 1) riTAT CA-rrrqrrkkr- (SEQ ID NO: 32) 44 IETDV (SEQ ID NO: 1) d-Arg Ac-rrrrrrrrrr- (SEQ ID NO: 33) 45 IETDV (SEQ ID NO: 1) d-Arg CA-rrrrrrrrrr- (SEQ ID NO: 34) 46 IETDV (SEQ ID NO: 1) l-Pen Ac-RQIKIWFQNRRMKWKK- (SEQ ID NO: 35) 47 IETDV (SEQ ID NO: 1) l-Pen CA-RQIKIWFQNRRMKWKK- (SEQ ID NO: 36) 48 IETDV (SEQ ID NO: 1) d-Pen Ac-rqikiwfqnrrmkwkk- (SEQ ID NO: 37) 49 IETDV (SEQ ID NO: 1) d-Pen CA-rqikiwfqnrrmkwkk- (SEQ ID NO: 38) 50 IETDV (SEQ ID NO: 1) l-DPV3 Ac-RKKRRRESRKKRRRES- (SEQ ID NO: 39) 51 IETDV (SEQ ID NO: 1) l-DPV3 CA-RKKRRRESRKKRRRES- (SEQ ID NO: 40) 52 IETDV (SEQ ID NO: 1) l-pVEC Ac-LLIILRRRIRKQAHAHSK- (SEQ ID NO: 41) 53 IETDV (SEQ ID NO: 1) l-pVEC CA-LLIILRRRIRKQAHAHSK- (SEQ ID NO: 42) 54 IETDV (SEQ ID NO: 1) l-MAP Ac-KLALKLALKALKAALKLA- (SEQ ID NO: 43) 55 IETDV (SEQ ID NO: 1) l-MAP CA-KLALKLALKALKAALKLA- (SEQ ID NO: 44) 56 IETDV (SEQ ID NO: 1) l-TP2 Ac-PLIYLRLLRGQF- (SEQ ID NO: 45) 57 IETDV (SEQ ID NO: 1) l-TP2 CA-PLIYLRLLRGQF- (SEQ ID NO: 46) 58 IETDV (SEQ ID NO: 1) d-TP2 Ac-pliylrllrGqf- (SEQ ID NO: 47) 59 IETDV (SEQ ID NO: 1) d-TP2 CA-pliylrllrGqf- (SEQ ID NO: 48) 60 IETDV (SEQ ID NO: 1) MiniAp4 Ac-|(Dap)CAPITAL|- (SEQ ID NO: 49) 61 IETDV (SEQ ID NO: 1) MiniAp4 CA-|(Dap)CAPITAL|- (SEQ ID NO: 50) 62 IETDV (SEQ ID NO: 1) CPP12 |Ff(Nal2)RrRrQ|GABA-K- (SEQ ID NO: 51) 63 IETDV (SEQ ID NO: 1) CPP12 |Ff(Nal2)RrRrQ|GABA-K(CA)- (SEQ ID NO: 52) 64 IETDV (SEQ ID NO: 1) l-Arg 7 CA-RRRRRRR- (SEQ ID NO: 68) 65 IETDV (SEQ ID NO: 1) l-Arg 5 CA-RRRRR- (SEQ ID NO: 70) 66 IETDV (SEQ ID NO: 1) l-Arg 3 CA-RRR- 67 KETLV (SEQ ID NO: 2) l-Arg 7 Ac-RRRRRRR- (SEQ ID NO: 63) 68 KETLV (SEQ ID NO: 2) l-Arg 7 CA-RRRRRRR- (SEQ ID NO: 68) 69 KETLV (SEQ ID NO: 2) l-Arg 6 Ac-RRRRRR- (SEQ ID NO: 64) 70 KETLV (SEQ ID NO: 2) l-Arg 6 CA-RRRRRR- (SEQ ID NO: 69) 71 KETLV (SEQ ID NO: 2) l-Arg 5 Ac-RRRRR- (SEQ ID NO: 65) 72 KETLV (SEQ ID NO: 2) l-Arg 5 CA-RRRRR- (SEQ ID NO: 70) [[ID=?1]] 73 KETLV (SEQ ID NO: 2) l-Arg 4 Ac-RRRR- (SEQ ID NO: 66) 74 KETLV (SEQ ID NO: 2) l-Arg 4 CA-RRRR- (SEQ ID NO: 71) 75 KETLV (SEQ ID NO: 2) l-Arg 3 CA-RRR-

[0080] In one embodiment, the compound has the formula (XXVII): 第77行原文缺失左括号,推测为“(XXVII)”,翻译为“式(XXVII)” Formula (XXVII) 注:原文第31行标签“ ”疑似有误,已按原样保留并翻译。若有其他特殊要求或修正,请随时告知。Furthermore, P1 and P2 are as defined in Table 3. In one embodiment, the compound is selected from the group consisting of compounds 2 to 7. [surface] [3.] The compounds illustrated in this invention. [Compound] [Mode] [P, 1, ] [、] [P, 2, ] [name] 1 (UCCB01-125) IETDV (SEQ ID NO: 1) OPEG 4-(IETDV) 2 2 (XXIX) KETLV (SEQ ID NO: 2) OPEG 4-(KETLV) 2 3 (XXX) KETTV (SEQ ID NO: 3) OPEG 4-(KETTV) 2 4 (XXXI) KETVV (SEQ ID NO: 4) OPEG 4-(KETVV) 2 5 (XXXII) KETRV (SEQ ID NO: 5) OPEG 4-(KETRV) 2 6 (XXXIII) ISTDV (SEQ ID NO: 6) OPEG 4-(ISTDV) 2 7 (XXXIV) VETVV (SEQ ID NO: 7) OPEG 4-(VETVV) 2

[0081] In one embodiment, the compound is selected from the group consisting of formulas (XXIX) to (XXXIV): Formula (XXIX) Formula (XXX) Formula (XXXI) Formula (XXXII) Formula (XXXIII) Formula (XXXIV)

[0082] In one embodiment, P1 and P2 are composed of the amino acid sequence IETDV (SEQ ID NO: 1) and CPP is selected from the group consisting of: D-Pen (rqikiwfqnrrmkwkk, SEQ ID NO: 15); L-pVEC (LLIILRRRIRKQAHAHSK, SEQ ID NO: 18); L-TP2 (PLIYLRLLRGQF, SEQ ID NO: 19); MiniAp4 (|(Dap)KAPETALD|, SEQ ID NO: 21); and CPP12 (|Ff(Nal2)RrRrQ|GABA-K, SEQ ID NO: 22). [, Salt and prodrug , ] [, , ]

[0083] As defined herein, compounds may be in the form of pharmaceutically acceptable salts or prodrugs of the compound. In one embodiment of the invention, compounds as defined herein may be formulated as pharmaceutically acceptable addition salts or hydrates of the compound, such as (but not limited to) K+, Na+, and non-salts, such as H+. [, Pharmaceutical Composition , ] [, , ]

[0084] In one embodiment, this invention relates to pharmaceutical compositions comprising compounds as defined herein. The formulation of the compounds of this invention into pharmaceutical compositions is well known in the art and is further described in Gennaro (ed.), 2000, Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams & Wilkins (2000); and Ansel et al., 1999, Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippincott Williams & Wilkins Publishers. [, Membrane permeability , ] [, , ]

[0085] Because PSD-95 is located intracellularly, efficient transmembrane penetration is essential for any drug targeting PSD-95. To evaluate the cellular permeability and cytoplasmic delivery of compounds as defined herein, cellular chloroalkane penetration assay (CAPA) (Peraro et al., 2018) can be used. This assay utilizes a modified haloalkane dehalogenase designed to covalently bind chloroalkane (CA) molecules. Cytoplasmic delivery is reported using HeLa cell lines expressing fusion proteins containing HaloTag, green fluorescent protein (GFP), and mitochondrial targeting peptides. The general format of CAPA is a pulse-tracking analysis (Deprey and Kritzer, 2020). Cells expressing the HaloTag enzyme are co-cultured with CA-tagged peptides. When these CA peptides penetrate the cell membrane and reach the cytoplasm, they bind to and react with HaloTag (pulse step). After a washing step, cells are co-cultured with a quantitative amount of CA-tagged dye that has penetrated the cell membrane and reacted with the remaining unreacted HaloTag sites (tracking step). Flow cytometry was used to measure the fluorescence intensity of cells, and the measured fluorescence was inversely proportional to the amount of CA peptide reaching the penetrated cells, thus allowing for the evaluation of cytoplasmic delivery. The obtained data are typically expressed as CP 50 values, which represent the concentration at which 50% cell penetration was observed. The CP 50 values ​​of compounds can be measured as described in Example 4. Example 4 shows that cellular uptake of the compounds varies greatly depending on the CPP used, with measured CP 50 values ​​ranging from 86.1 µM (compound 22) to 0.68 µM (compound 30) (Figure 7). Furthermore, the cellular uptake efficiency of NPEG 4 (IETDV) 2, bound to a series of CPP tags (SEQ ID NO 8 to 22), produces an unpredictable range of CP 50 values ​​(Figure 8).

[0086] In one embodiment, the CP 50 value of the compound is not greater than 250 µM, for example not greater than 200 µM, for example not greater than 150 µM, for example not greater than 100 µM, for example not greater than 80 µM, for example not greater than 70 µM, for example not greater than 60 µM, for example not greater than 50 µM, for example not greater than 40 µM, for example not greater than 30 µM, for example not greater than 20 µM, for example not greater than 15 µM, for example not greater than 10 µM, for example not greater than 5 µM. Preferably, the CP 50 value of the compound is not greater than 60 µM. [, Cytoplasmic stability , ] [, , ]

[0087] Ischemic stroke (also known as cerebral ischemia or cerebral ischemia) is usually caused by a blockage in the arteries that supply blood to the brain. The blockage reduces blood flow and oxygen to the brain, leading to brain cell damage or death. Blockages in blood vessels can be removed using a series of mechanical devices or by delivering "clot-dissolving agents" intravenously or intra-arterially. These clot-dissolving agents contain tissue plasminogen activator (tPA), which generates plasminogen from plasminogen. Examples of recombinant tPA include alteplase, reteplase, and tenecteplase, and other thrombolytic agents that break down clots include streptokinase, urokinase, and desmotaplase.

[0088] In one embodiment, the compound of the present invention is administered to an individual receiving tPA or recombinant tPA, which is standard care for AIS. Therefore, compatibility of the compound with tPA administration (including the generation of cytoplasm, which is a serine protease) is necessary.

[0089] In Example 3, the in vitro cytoplasmic stability of the compounds of the present invention was determined. In one embodiment, the half-life of the compounds in the presence of pf cytoplasmic acid in the cytoplasmic stability analysis described in Example 3 was at least 10 min, for example at least 30 min, for example at least 1 h, for example at least 2 h, for example at least 3 h, for example at least 4 h, for example at least 5 h, for example at least 6 h, for example at least 7 h, for example at least 8 h, for example at least 9 h, for example at least 10 h, for example at least 15 h, for example at least 20 h, for example at least 30 h. [, right , ] [, PSD-95 , ] [, Of , ] [, PDZ1-2 , ] [, Affinity , ] [, , ]

[0090] Preferably, the compounds of the present invention have an affinity for PDZ1-2 of PSD-95 within the nemerol concentration range, thereby making them highly potent inhibitors. Affinity for PDZ1-2 of PSD-95 is a key factor in reducing the critical drug concentration required to achieve a therapeutic effect, which is particularly important when the drug must cross the blood-brain barrier (BBB) ​​to reach its target, as the BBB often limits the accumulation of drug concentration at the target site.

[0091] In some embodiments, the compounds of the present invention are dimer PSD-95 inhibitors that simultaneously bind to PDZ1 and PDZ2, which explains their high affinity for these domains.

[0092] As described in Example 2, the obtained compound of the present invention has an affinity for PDZ1-2 of PSD-95 in the low nemer concentration range and is similar to l-TAT-NPEG 4(IETDV) 2.

[0093] In one embodiment, the Ki value of compound for PDZ1-2 of PSD-95 is not greater than 100 nM, for example not greater than 80 nM, for example not greater than 70 nM, for example not greater than 60 nM, for example not greater than 50 nM, for example not greater than 40 nM, for example not greater than 30 nM, for example not greater than 20 nM, for example not greater than 10 nM. [Medical Use] []

[0094] In one state, the compounds of the present invention, as defined herein, are used as pharmaceuticals.

[0095] The PDZ1 and PDZ2 domains of PSD-95 interact with several proteins, including simultaneously binding to NMDA-type ionotropic glutamate receptors and the nitric oxide (NO)-producing enzyme nNOS. NMDA receptors are a major mediator of systemic excitotoxicity, namely glutamate-mediated neurotoxicity, which is involved in neurodegenerative diseases and acute brain injury. PSD-95 binds to NMDA receptors (mainly GluN2A and GluN2B subunits) and nNOS via PDZ1 and PDZ2, respectively. Activation of NMDA receptors leads to calcium ion influx, which activates nNOS, thereby generating NO. Therefore, PSD-95 mediates a specific link between NMDA receptor activation and NO production, which, if prolonged, can be harmful to cells and is a key promoter of glutamate-mediated neurotoxicity. It is known that targeting PSD-95 to inhibit the ternary complex of nNOS / PSD-95 / NMDA receptor interaction can prevent ischemic brain injury in mice by impairing the functional link between calcium ion entry and NO production, while maintaining the physiological functions of the NMDA receptor (e.g., ion flow and pro-survival signaling pathways). Specific inhibition of excitotoxicity can be achieved by using PSD-95 inhibitors to disrupt the intracellular nNOS / PSD-95 / NMDA receptor complex.

[0096] The compounds of this invention are PSD-95 inhibitors and therefore can inhibit excitotoxicity. Consequently, the compounds of this invention can be used to treat a variety of diseases, specifically neurological diseases, and particularly those mediated in part by excitotoxicity. These diseases and disorders include stroke, epilepsy, hypoxia, traumatic CNS injuries unrelated to stroke (e.g., traumatic brain injury and spinal cord injury), other cerebral ischemia, Alzheimer's disease, and Parkinson's disease.

[0097] In one embodiment, the invention relates to compounds as defined herein for the prevention, treatment, mitigation, and / or delay of excitotoxic-related diseases and their development. In one embodiment, the excitotoxic-related disease is stroke. In one embodiment, the excitotoxic-related disease is ischemic stroke. In one embodiment, the excitotoxic-related disease is cerebral ischemia. In one embodiment, the excitotoxic-related disease is acute ischemic stroke. In one embodiment, the excitotoxic-related disease is subarachnoid hemorrhage.

[0098] In one embodiment, the present invention relates to the use of a compound as defined herein for the manufacture of a medicament for the prevention, treatment, relief of excitotoxicity-related diseases and / or delay of their development.

[0099] In one embodiment, the present invention relates to a method for preventing, treating, alleviating, and / or delaying the development of excitotoxic diseases, the method comprising administering a therapeutically effective amount of a compound as defined herein.

[0100] In one embodiment, the present invention relates to compounds for reducing and / or counteracting the damaging effects of excitotoxicity. In one embodiment, the compound is used to reduce the damaging effects of stroke. In one embodiment, the compound is used to treat the damaging effects of acute ischemic stroke. In one embodiment, the compound is used to treat the damaging effects of subarachnoid hemorrhage.

[0101] In one embodiment, the present invention relates to a method for resisting and / or reducing the damaging effects of excitotoxicity on the brain or spinal cord of an individual, the method comprising the step of administering to the individual an effective amount of a compound as defined herein to resist and / or reduce the damaging effects.

[0102] In one embodiment, the present invention relates to a method for treating, alleviating, or delaying the development of a disease mediated by excitotoxicity, comprising administering a compound as defined herein to a human individual suffering from or at risk of the disease.

[0103] In one embodiment, the present invention relates to a method of treating, suppressing, or delaying at least one sign or symptom of an excitotoxic-mediated disorder in an individual, comprising administering a compound as defined herein to an individual suffering from the disorder or having risk factors associated with the disorder. In one embodiment, the disorder is stroke or traumatic CNS injury. In one embodiment, the excitotoxic-related disorder is ischemic or traumatic CNS injury to the CNS / CNS / CNS.

[0104] In one embodiment, the present invention relates to a method for reducing the damaging effects of stroke on an individual suffering from stroke, comprising administering to the individual an effective amount of a compound as defined herein to reduce the damaging effects of stroke.

[0105] As used in this article, "stroke" is a general term referring to a condition caused by the blockage or hemorrhage of one or more blood vessels supplying the brain, leading to cell death. "Ischemic stroke," as used in this article, refers to a stroke caused by the blockage of one or more blood vessels supplying the brain. Types of ischemic stroke include, for example, embolic stroke, psychogenic stroke, thrombotic stroke, large vessel thrombosis, lacunar infarction, arterial-arterial stroke, and cryptogenic stroke. "Cerebral ischemia" is a condition in which an artery blockage restricts the delivery of oxygen-rich blood to the brain, causing brain tissue damage. Cerebral ischemia is sometimes referred to as cerebral ischemia or cerebrovascular ischemia.

[0106] As used in this article, "hemorrhagic stroke" refers to a stroke caused by bleeding from one or more blood vessels supplying the brain. Types of hemorrhagic stroke include, for example, subdural stroke, intraparenchymal stroke, epidural stroke, and subarachnoid stroke.

[0107] In one embodiment, the disease that can be treated by the compounds of the present invention is ischemic or traumatic CNS injury. In another embodiment, the present invention relates to a method for reducing the damaging effects of traumatic or ischemic injury to an individual's brain or spinal cord, the method comprising treating the individual with a compound as defined herein to achieve such reduction.

[0108] In one embodiment, the present invention relates to a method for inhibiting cerebral ischemia caused by endovascular surgery, comprising administering a compound as defined herein to an individual undergoing endovascular surgery in a manner that can effectively inhibit cerebral ischemia.

[0109] In one embodiment, the present invention relates to a method for suppressing ischemic injury caused by endovascular surgery, diagnostic angiography, or carotid artery stenting for the treatment of aneurysms, comprising administering an effective formulation of a compound as defined herein to an individual undergoing endovascular surgery or diagnostic angiography for the treatment of aneurysms.

[0110] In one embodiment, the invention relates to compounds as defined herein for inhibiting ischemic injury caused by neurosurgery. In one embodiment, the neurosurgery is diagnostic angiography of the brain or endovascular surgery to treat an aneurysm.

[0111] In some embodiments, the compound is administered in combination with reperfusion therapy. In one embodiment, the compound and the reperfusion system are administered to the individual simultaneously, sequentially, or individually.

[0112] As used herein, the term "reperfusion therapy" refers to a medical treatment that restores blood flow through or around a blocked artery. Reperfusion therapy includes both pharmaceutical and mechanical reperfusion. These pharmaceuticals may be thrombolytic agents or fibrinolytic agents used in a process known as thrombolysis. In some embodiments, reperfusion therapy is performed by administering a thrombolytic agent (e.g., a plasminogen activator, such as tPA).

[0113] In one embodiment, the compound is compound 8 (AB144), and the compound is administered in combination with a plasminogen activator (e.g., tPA).

[0114] In some embodiments, reperfusion therapy is mechanical reperfusion, including surgery. The surgery performed may be a minimally invasive endovascular procedure.

[0115] Mechanical reperfusion devices include intra-arterial catheters, balloons, stents, and various clot retrieval devices.

[0116] In one embodiment, the compound is administered in combination with a thrombolytic agent, and the compound and the thrombolytic agent are administered to the individual simultaneously, sequentially, or individually.

[0117] In one embodiment, the present invention relates to a method for treating the damaging effects of ischemia on the central nervous system, comprising: a) Administering compounds as defined herein to individuals suffering from or at risk of ischemia, and b) Perform reperfusion therapy on the individual. Among them, compounds and reperfusion therapy treat the effects of ischemia on the individual's central nervous system.

[0118] In one embodiment, the present invention relates to the use of compounds as defined herein for treating the damaging effects of ischemia on the central nervous system of individuals suffering from or at risk of ischemia, wherein reperfusion therapy is performed on the individual, and the compounds and reperfusion therapy treat the damaging effects of ischemia on the individual's central nervous system.

[0119] In one embodiment, the method further includes administering a thrombolytic agent to an individual simultaneously, sequentially, or individually.

[0120] In one embodiment, the invention relates to a multi-part kit comprising at least two individual unit dose forms (A) and (B), wherein (A) Contains compounds as defined herein; and (B) Contains thrombolytic agents.

[0121] In a pattern, as defined herein, a multipart kit is used to treat the ischemic effects on the central nervous system, wherein (A) and (B) are administered to the individual simultaneously, sequentially, or individually.

[0122] In one embodiment, the present invention relates to the therapeutic effects of compounds as defined herein on subarachnoid hemorrhage. As used herein, the term "subarachnoid hemorrhage" refers to a bleeding condition in the subarachnoid space.

[0123] In one embodiment, the present invention relates to a method for treating subarachnoid hemorrhage in an individual, comprising administering to the individual suffering from subarachnoid hemorrhage a compound as defined herein, wherein the development of neurocognitive deficits in the individual is inhibited.

[0124] In one embodiment, the present invention relates to a method for inhibiting the development of neurological or neurocognitive deficits in an individual with subarachnoid hemorrhage, comprising administering to an individual with subarachnoid hemorrhage a compound as defined herein, wherein the development of the individual's neurological or neurocognitive deficits is inhibited.

[0125] Other known neurological disorders unrelated to excitotoxicity that can be treated with the compounds of this invention include anxiety and pain. Dimeric ligands targeting PSD-95 are under preclinical / clinical evaluation for the treatment of chronic pain and ischemic stroke (Andreasen et al., Neuropharmacol, 2013, 67, 193-200; Bach et al., PNAS USA, 2012, 109, 3317-3322). In one embodiment, the compounds as defined herein are used to treat or prevent pain.

[0126] In one embodiment, such as the mammalian system mentioned herein, for example, humans. [synthesis] []

[0127] The compounds of the present invention as defined herein can be manufactured by a method comprising the following general steps: a) Synthesize peptides P1 / P2 as defined herein; b) Dimerize the peptide in a) using an OPEG n or NPEG n linker, where 'n' represents the number of PEG moieties; c) For example, by using an automated peptide synthesizer to attach a CPP tag to the N-position of an NPEG linker.

[0128] In one embodiment, the compound is manufactured by a method comprising the following general steps: a) Provide Ns-NPEG diacid linkers; b) Prepare peptide P1 / P2 using Fmoc-based solid-phase peptide synthesis; c) Using an Ns-NPEG diacid linker to dimerize the peptide P1 / P2 deprotected by the Fmoc group, forming a linker-dimer conjugate; and d) For example, by using an automated peptide synthesizer to link CPP to the N of the NPEG linker of the linker-dimer conjugate.

[0129] In one embodiment, the compound of the present invention is synthesized as described below.

[0130] Ns-NPEG diacid linker:

[0131] The 'Ns-NPEG dicarboxylic acid linker' is a structure in which the nitrogen of the linker is protected by an o-nitrobenzenesulfonyl (Ns) protecting group, and the terminus of the NPEG linker contains a carboxylic acid. This chemical reagent or structural unit is used to dimerize the two peptide moieties P1 and P2.

[0132] The o-nitrobenzenesulfonyl (Ns) protected NPEG linkage system is generated in the solid phase or in solution.

[0133] Solid-phase processes typically begin with loading a solid support suitable for solid-phase peptide synthesis (e.g., 2-chlorotriphenylmethyl chloride resin) and Fmoc-NH-PEG-CH2CH2COOH using an organic solvent (e.g., DCM, DMF, ACN, THF) and a base (e.g., DIPEA, DBU, collidine, NMM) suitable for a particular resin.

[0134] The Fmoc group can be removed in a suitable solvent (e.g., DMF, DCM, ACN, THF) by using a base (e.g., piperidine, dimethylamine, morpholine, hexahydropyrazine, dicyclohexylamine, DMAP).

[0135] The o-nitrobenzenesulfonyl chloride can be coupled to a free amine using an alkali (e.g., DIPEA, DBU, Colin base, NMM) and a suitable solvent (e.g., THF, DCM) to obtain Ns-NH-PEG-CH2CH2COO- resin.

[0136] The second part of the linker product can be attached to the linker part of the resin using Mitsunobu chemistry. The resin is treated with triphenylphosphine, HO-PEG-CH2CH2COO tBu, solvent, and esters or amides of azodicarbonate (e.g., diisopropyl azodicarbonate, DIAD; diethyl azodicarbonate, DEAD; 1,1'-(azodicarbonyl)-dipiperidine, ADDP).

[0137] The final Ns-NPEG diacid linker is obtained by treating the resin with an acid (e.g., trifluoroacetic acid (TFA)).

[0138] A solution-phase process can be implemented by protecting the amine group of NH₂-PEG-CH₂CH₂COO tBu with Ns, followed by photoelongation chemistry in solution using triphenylphosphine and DIAD, DEAD, or ADDP or similar reagents, HO-PEG-CH₂CH₂COO tBu, and a suitable solvent (THF, DCM). The final Ns-protected NPEG-linker is then obtained by treatment with an acid (e.g., TFA).

[0139] Peptide synthesis: This involves solid-phase peptide synthesis based on Fmoc, using a solid support (e.g., 2-chlorotriphenylmethyl chloride resin or Wang resin), Fmoc-protected amino acids, a base, and a coupling reagent (e.g., HBTU). N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)ureonium hexafluorophosphate Peptide sequences can be synthesized using [ ]], O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylureonium hexafluorophosphate [HATU], PyBOB, DIC / HOBt) and solvents. For alternative coupling reagents, activated esters of Fmoc-protected amino acids (e.g., pentafluorophenyl, succinimide) can be used.

[0140] Dimerization: Using an Ns-NPEG diacid linker, a resin-based dimerization process is employed. This involves repeatedly treating the resin with a substoichiometric amount (e.g., 1 / 6) of the Ns-NPEG diacid linker, a base, a coupling agent, and a suitable solvent (e.g., DMF, DCM, THF) to dimerize the resin-bound peptides that have lost their Fmoc protecting groups. For alternative coupling agents, activated esters of the Ns-NPEG linker can be used.

[0141] The dimerization process can also be carried out in solution using an activated ester of the Ns-NPEG linker (e.g., pentafluorophenyl, succinimide) and 1-hydroxy-7-azabenzotriazole (HOAt) or hydroxybenzotriazole (HOBt), along with a peptide with appropriate side chain protection (e.g., tert-butyl), in a solvent (e.g., ACN, DMF, DCM, THF). Alternatively, solution dimerization can be carried out using an Ns-NPEG diacid linker, coupling reagents (e.g., HBTU, HATU, etc.), a base, and a solvent.

[0142] The Ns group was removed by mercaptoethanol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or by sodium thiophene.

[0143] To link CPP to the NH portion of the NPEG linker in a linker-dimer conjugate: First, an amino acid is coupled to the NH portion of the NPEG linker, and then CPP is synthesized following the standard (manual / automated) Fmoc SPPS on the resin.

[0144] The ester protecting groups can be removed by stirring the pyrolysis products in an alkaline aqueous solution (such as NaOH, LiOH) and acetonitrile, and then acidifying them with TFA or HCl.

[0145] The final compound of the present invention is obtained by freeze-drying and purification by HPLC or similar chromatographic methods.

[0146] In another embodiment, the synthesis of the compounds of the present invention is carried out as outlined in Example 1. [entry] [] 1. A compound comprising a first peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO: 53), wherein... a. X 2 is a group composed of E and S; and b. X 3 is a group composed of L, T, V, R, and D. P1 is linked to the cell-penetrating peptide (CPP). 2. A compound comprising a first peptide (P1) linked to a second peptide (P2) by a linker, wherein P1 and P2 comprise or consist of an amino acid sequence selected from or composed of the group consisting of: KETLV (SEQ ID NO: 2), KETTV (SEQ ID NO: 3), KETVV (SEQ ID NO: 4), KETRV (SEQ ID NO: 5), ISTDV (SEQ ID NO: 6), and VETVV (SEQ ID NO: 7). 3. Compounds such as those in item 2, wherein P1 and P2 are linked via linkers to cell-penetrating peptides (CPP) or albumin-binding sites, thereby increasing the pathway for the peptide to cross membranes or the blood-brain barrier. 4. The compound of any of the preceding items, wherein the CP 50 value of the compound is not greater than 250 µM, for example not greater than 200 µM, for example not greater than 150 µM, for example not greater than 100 µM, for example not greater than 80 µM, for example not greater than 70 µM, for example not greater than 60 µM, for example not greater than 50 µM, for example not greater than 40 µM, for example not greater than 30 µM, for example not greater than 20 µM, for example not greater than 15 µM, for example not greater than 10 µM, for example not greater than 5 µM. 5. The compound of any of the preceding entries, wherein the half-life of the compound in the cytoplasmic stability analysis is at least 10 min, for example at least 30 min, for example at least 1 h, for example at least 2 h, for example at least 3 h, for example at least 4 h, for example at least 5 h, for example at least 6 h, for example at least 7 h, for example at least 8 h, for example at least 9 h, for example at least 10 h, for example at least 15 h, for example at least 20 h, for example at least 30 h. 6. The compound of any of the preceding items, wherein the Ki value of the compound relative to PDZ1-2 of PSD-95 is not greater than 100 nM, for example not greater than 80 nM, for example not greater than 70 nM, for example not greater than 60 nM, for example not greater than 50 nM, for example not greater than 40 nM, for example not greater than 30 nM, for example not greater than 20 nM, for example not greater than 10 nM. 7. The compound of any of the preceding entries, wherein CPP is a poly-1-arginine peptide (poly-Arg). 8. The compound of Item 9, wherein the poly-Arg system consists of 5 to 10 l-arginine residues, for example 7 to 10 L-arginine residues, for example 8 or 9 l-arginine residues. 9. The compound of entry 9, wherein the poly-Arg contains or is composed of the amino acid sequence RRRRRRRRR (SEQ ID NO: 12). 10. The compound of any one of items 1 to 6, wherein the CPP is 1-TAT (YGRKKRRQRRR, SEQ ID NO: 8). 11. The compound of any one of items 1 to 6, wherein the CPP is d-TAT (yGrkkrrqrrr, SEQ ID NO: 9). 12. The compound of any one of items 1 to 6, wherein CPP is mTAT (rRrGrKkRr, SEQ ID NO: 10). 13. The compound of any one of items 1 to 6, wherein the CPP is riTAT (rrrqrrkkr, SEQ ID NO: 11). 14. The compound of any one of items 1 to 6, wherein CPP is d-Arg 9 (rrrrrrrrr, SEQ ID NO:13). 15. The compound of any one of items 1 to 6, wherein CPP is l-Pen (RQIKIWFQNRRMKWKK, SEQ ID NO: 14). 16. The compound of any one of items 1 to 6, wherein CPP is d-Pen (rqikiwfqnrrmkwkk, SEQ ID NO: 15). 17. The compound of any one of items 1 to 6, wherein the CPP is 1-DPV3 (RKKRRRESRKKRRRES, SEQ ID NO: 17). 18. The compound of any one of items 1 to 6, wherein CPP is 1-pVEC (LLIILRRRIRKQAHAHSK, SEQ ID NO: 18). 19. The compound of any one of items 1 to 6, wherein the CPP is 1-MAP (KLALKLALKALKAALKLA, SEQ ID NO: 16). 20. The compound of any one of items 1 to 6, wherein CPP is 1-TP2 (PLIYLRLLRGQF, SEQ ID NO: 19). 21. The compound of any one of items 1 to 6, wherein the CPP is d-TP2 (pliylrllrGqf, SEQ ID NO: 20). 22. The compound of any one of items 1 to 6, wherein the CPP is MiniAp4 (|(Dap)KAPETALD|, SEQ ID NO: 21). 23. A compound of any of items 1 to 6, wherein CPP is CPP12 (|Ff(Nal2)RrRrQ|GABA-K, SEQ ID NO: 22). 24. The compound of any of the preceding entries, wherein the amino acid sequence X 2TX 3V (SEQ ID NO: 53) is the C-terminus of P 1. 25. A compound of any of the preceding entries, wherein P1 consists of 4 to 10 amino acid residues, for example 5 amino acid residues, for example 6 amino acid residues. 26. A compound of any of the preceding entries, wherein P1 Contains or consists of the sequence X 1X 2TX 3V (SEQ ID NO: 54), wherein a. X 1 is a group composed of K, V, and I; b. X 2 is a group composed of E and S; and c. X 3 is a group composed of L, T, V, R and D. 27. The compound of any of the preceding entries, wherein P1 comprises or consists of KETLV (SEQ ID NO: 2), KETTV (SEQ ID NO: 3), KETVV (SEQ ID NO: 4), KETRV (SEQ ID NO: 5), ISTDV (SEQ ID NO: 6), VETVV (SEQ ID NO: 7), and IEDDV (SEQ ID NO: 1). 28. A compound of any of the preceding entries, wherein the compound comprises a second peptide (P2) comprising or consisting of the amino acid sequence X5TX6V (SEQ ID NO: 55), wherein... a. X 5 selects a group composed of E and S; and b. X 6 is a group composed of L, T, V, R and D. 29. The compound of any of the preceding entries, wherein the amino acid sequence X 5TX 6V (SEQ ID NO: 55) is the C-terminus of P 2. 30. A compound of any of the preceding entries, wherein P2 consists of 4 to 10 amino acid residues, for example 5 amino acid residues, for example 6 amino acid residues. 31. A compound of any of the preceding entries, wherein P2 comprises or consists of the sequence X4X5TX6V (SEQ ID NO: 56), wherein a. X 4 is a group composed of K, V, and I; b. X 5 selects a group composed of E and S; and c. X 6 is a group composed of L, T, V, R and D. 32. The compound of any of the preceding entries, wherein P2 comprises or is composed of an amino acid sequence selected from or consisting of the group consisting of: KETLV (SEQ ID NO: 2), KETTV (SEQ ID NO: 3), KETVV (SEQ ID NO: 4), KETRV (SEQ ID NO: 5), ISTDV (SEQ ID NO: 6), VETVV (SEQ ID NO: 7) and IEDDV (SEQ ID NO: 1). 33. Compounds of any of the preceding entries, wherein P1 and P2 are identical. 34. A compound of any of the preceding entries, wherein P1 and P2 are bound to the linker via their N-terminus. 35. A compound of any of the preceding entries, wherein CPP (e.g., poly-Arg) is linked to P1 via a linker. 36. The compound of any of the preceding entries, wherein CPP (e.g., poly-Arg) is linked to P1 and P2 via a linker. 37. A compound of any of the preceding entries, wherein CPP (e.g., poly-Arg) is attached to the linker via its C-terminus, and P1 and P2 are attached to the linker via their N-terminus, and the compound has the following general structure of formula (I): . Formula (I) 38. A compound of any of the preceding entries, wherein CPP is poly-Arg and the compound has the following general structure of formula (II): . Equation (II) 39. A compound of any of the preceding entries, wherein the linker comprises one or more PEG units. 40. A compound such as 39, wherein at least one oxygen atom of one of the PEG units is substituted with a nitrogen atom to obtain NPEG. 41. A compound as described in any of the preceding entries, wherein the linker comprises an NPEG unit and a CPP (e.g., poly-Arg) is attached to the nitrogen atom of the linker by a amide bond. 42. A compound of any of the preceding entries, wherein the compound has the general structure of formula (III): Equation (III) in p is an integer from 0 to 10; and q is an integer from 0 to 10. 43. A compound of any of the preceding entries, wherein the compound has the general structure of formula (IV): Formula (IV) in p is an integer from 0 to 10; and q is an integer from 0 to 10. 44. A compound of any of the preceding entries, wherein the compound has the general structure of formula (V): Formula (V) in X 1 is a group composed of K, V, and I; X 2 is a group composed of E and S; and X 3 can be freely chosen from groups composed of L, T, V, R, and D; X 4 is a group composed of K, V, and I; X 5 is a group composed of E and S; X 6 can be freely chosen from groups composed of L, T, V, R, and D; p is an integer from 0 to 10; and q is an integer from 0 to 10. 45. A compound of any of the preceding entries, wherein the compound has the general structure of formula (VI): Formula (VI) in X 1 is a group composed of K, V, and I; X 2 is a group composed of E and S; and X 3 can be freely chosen from groups composed of L, T, V, R, and D; X 4 is a group composed of K, V, and I; X 5 is a group composed of E and S; X 6 can be freely chosen from groups composed of L, T, V, R, and D; p is an integer from 0 to 10; and q is an integer from 0 to 10. 46. ​​A compound of any of the preceding entries, where p = q. 47. A compound of any of the preceding entries, wherein p > q. 48. A compound of any of the preceding entries, where p < q. 49. A compound of any of the preceding entries, wherein the sum of p and q is an integer between 1 and 20. 50. The compound of any of the preceding entries, wherein the number p of the ethylene glycol moiety is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ethylene glycol moieties. 51. The compound of any of the preceding entries, wherein the amount p of the ethylene glycol moiety is from 0 to 4. 52. The compound of any of the preceding entries, wherein the number q of the ethylene glycol moiety is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ethylene glycol moieties. 53. The compound of any of the preceding entries, wherein the amount q of the ethylene glycol portion is between 0 and 4. 54. A compound of any of the preceding entries, wherein the total number of ethylene glycol portions p + q is between 2 and 12, for example 2, 4, 6, 8, 10, or 12. 55. For any of the preceding entries, the total number of ethylene glycol portions p + q is 4. 56. A compound of any of the preceding entries, wherein p-system 2 and q-system 2. 57. A compound of any of the preceding entries, wherein the compound has the general structure of formula (VII): . Equation (VII) 58. A compound of any of the preceding entries, wherein the compound has the general structure of formula (VIII): . Formula (VIII) 59. A compound of any of the preceding entries, wherein the compound has the general structure of formula (IX): . Formula (IX) 60. Compounds of any of the preceding entries, wherein the compounds are selected from the group consisting of formulas (X) to (XVI): Formula (X) Formula (XI) Formula (XII) Formula (XIII) Formula (XIV) Formula (XV) Formula (XVI) 61. A compound of any of the preceding entries, wherein the N-terminus of CPP is acetylated. 62. A compound of any of entries 1 to 60, wherein the N-terminus of the CPP is bound to a chloroalkyl tag (CA), having the following structure: . 63. A compound of any of entries 1 to 60, wherein the N-terminus of CPP is methylated. 64. A compound of any of entries 1 to 60, wherein the N-terminus of CPP is methylated. 65. The compound of any of the preceding items is in the form of a pharmaceutically acceptable salt or prodrug of that compound. 66. A compound comprising a. A first peptide (P1), comprising or consisting of the amino acid sequence X 2TX 3V (SEQ ID NO: 53), wherein X 2 is a group composed of E and S; X 3 can be freely chosen from groups composed of L, T, V, R, and D; b. A second peptide (P2) comprising or consisting of the amino acid sequence X 5TX 6V (SEQ ID NO: 55), wherein X 5 is a group composed of E and S; X 6 is a group composed of L, T, V, R, and D; and c. Cell-penetrating peptide (CPP) The CPP is attached to the linker via its C-terminus, and P1 and P2 are attached to the linker via their N-terminus, and the compound has the general structure of formula (I): Formula (I) Furthermore, the compounds in this compound have a CP 50 value of no more than 20 µM. 67. The compound of entry 66, wherein P1 comprises or consists of the sequence X1X2TX3V (SEQ ID NO: 54), wherein X 1 is a group composed of K, V, and I; X 2 is a group composed of E and S; and X 3 can be freely chosen from groups composed of L, T, V, R, and D; And P2 contains or consists of the sequence X 4X 5TX 6V (SEQ ID NO: 56), wherein X 4 is a group composed of K, V, and I; X 5 selects a group composed of E and S; and X 6 is a group composed of L, T, V, R and D. 68. A compound of any of the items 66 or 67, wherein P1 and / or P2 are composed of 4 to 10 amino acid residues, for example 5 amino acid residues. 69. A compound of any of entries 66 to 68, wherein X3 and X6 are selected from the group consisting of L, T, V and R. 70. The compound of any one of entries 66 to 68, wherein P1 and P2 are composed of an amino acid sequence selected from the group consisting of: KETLV (SEQ ID NO: 2), KETTV (SEQ ID NO: 3), KETVV (SEQ ID NO: 4), KETRV (SEQ ID NO: 5), ISTDV (SEQ ID NO: 6) and VETVV (SEQ ID NO: 7). 71. A compound of any of entries 66 to 70, wherein the linker comprises one or more PEG units, wherein at least one oxygen atom of one PEG unit is substituted with a nitrogen atom to obtain NPEG, and wherein CPP is linked to the nitrogen atom of the linker by a amide bond, and the compound has the general structure of formula (III): Equation (III) in p is an integer from 0 to 10; and q is an integer from 0 to 10. 72. A compound of any of entries 66 to 72, wherein CPP is selected from the group consisting of: poly-L-arginine peptide (poly-Arg); D-TAT (yGrkkrrqrrr, SEQ ID NO: 9), L-Pen (RQIKIWFQNRRMKWKK, SEQ ID NO: 14); D-Pen (rqikiwfqnrrmkwkk, SEQ ID NO: 15); L-DPV3 (RKKRRRESRKKRRRES, SEQ ID NO: 17); L-pVEC (LLIILRRRIRKQAHAHSK, SEQ ID NO: 18); L-MAP (KLALKLALKALKAALKLA, SEQ ID NO: 16); L-TP2 (PLIYLRLLRGQF, SEQ ID NO: 19); MiniAp4 (|(Dap)KAPETALD|, SEQ ID NO: 19). 21); and CPP12 (|Ff(Nal2)RrRrQ|GABA-K, SEQ ID NO: 22). 73. A compound of any of entries 66 to 72, wherein the CPP comprises a poly-L-arginine peptide (poly-Arg), such as a poly-Arg consisting of 5 to 10 L-arginine residues, such as 8 or 9 L-arginine residues, or consisting thereof, such as wherein the poly-Arg comprises or consists of the amino acid sequence RRRRRRRRR (SEQ ID NO: 12). 74. A compound of any of entries 66 to 73, wherein the compound has the general structure of formula (VI): Formula (VI) in X 1 is a group composed of K, V, and I; X 2 is a group composed of E and S; and X 3 can be freely chosen from groups composed of L, T, V, R, and D; X 4 is a group composed of K, V, and I; X 5 is a group composed of E and S; X 6 can be freely chosen from groups composed of L, T, V, R, and D; p is an integer from 0 to 10; and q is an integer from 0 to 10. 75. A compound of any of the items 66 to 74, wherein p-system 2 and q-system 2. 76. A compound of any of entries 66 to 75, wherein the N-terminus of the CPP is acetylated or wherein the N-terminus of the CPP is bound to a chloroalkyl tag (CA), having the following structure: . 77. The compound of any of the preceding entries, wherein the compound is a PSD_95 inhibitor. 78. A pharmaceutical composition comprising any of the compounds listed in the preceding entries. 79. A compound of any of the preceding entries, used as a medicine. 80. A compound of any of the items 1 to 76, used for the treatment, prevention, relief of excitotoxic-related diseases and / or delay of their development. 81. Compounds used in item 80, wherein the disease associated with excitotoxicity is stroke. 82. The compound used in item 80, wherein the disease associated with its excitotoxicity is ischemic stroke. 83. Compounds used in item 80, in which the excitotoxicity-related disease is cerebral ischemia. 84. The compound used in item 80, wherein the disease associated with its excitotoxicity is acute ischemic stroke. 85. Compounds used in item 80, wherein the excitotoxicity-related disease is subarachnoid hemorrhage. 86. A compound of any of the items 1 to 76, used for the treatment or prevention of pain. 87. A multi-part kit comprising at least two individual unit dose forms (A) and (B), wherein (A) Contains compounds as described in any of entries 1 to 76; and (B) Contains thrombolytic agents. 88. A multipart kit as described in item 87, which is used to treat, prevent, alleviate, and / or delay the development of excitotoxic-related diseases and / or pain, wherein (A) and (B) are administered to an individual simultaneously, sequentially, or individually. [Example] [] [Example] [1] [:synthesis] []

[0147] As previously described, the compound is a combination of synthetic resins. [2] to [7] (OPEG 4-KETLV, OPEG 4-KETTV, OPEG 4-KETVV, OPEG 4-KETRV, OPEG 4-ISTDV and OPEG 4-VETVV): Bach et al., Angew. Chem. Int. Ed., 2009, 48, 9685.

[0148] As previously described by Bach et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 3317, NPEG 4-IETDV, NPEG 4-KETLV, NPEG 4-KETTV, NPEG 4-KETVV, NPEG 4-KETRV, NPEG 4-ISTDV, and NPEG 4-VETVV are synthesized resins bound together. O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethylureonium hexafluorophosphate (HCTU) is used as the coupling agent to bind linear CPP. The first C-terminal amino acids (Fmoc-Ala-OH, Fmoc-l-Arg(Pbf)-OH, Fmoc-d-Arg(Pbf)-OH, Fmoc-Asp(OAll)-OH, Fmoc-l-Lys(Boc)-OH, Fmoc-d-Lys(Boc)-OH, Fmoc-l-Lys(Ns)-OH, Fmoc-l-Phe-OH, Fmoc-d-Phe-OH, Fmoc-l-Ser(tBu)-OH) of (l-TAT, dTAT, mTAT, riTAT, polyR, d-polyR, l-Pen, d-Pen, l-pVEC, l-MAP, l-DPV3, l-TP2, d-TP2, MiniAp4, CPP12) are artificially coupled to the nitrogen of the NPEG4 linker three times. CPP was then synthesized using an automated peptide synthesizer (Prelude X, Gyros Protein Technologies, US). For synthesis using Prelude X, all reagents were prepared as solutions in DMF: Fmoc-protected amino acids (0.2 M), HCTU (0.4 M), and DIPEA (1.0 M). Sequence extension was achieved using the following protocol: deprotection (2 × 2 min, rt, 350 rpm shaking) and coupling (3 × 10 min, 50 °C, 350 rpm shaking). Triple coupling of the amino acids was performed using a 5-fold excess of the Fmoc-amino acid / HCTU / DIPEA (1:1:2.5) mixture compared to the resin loading. Peptides containing cyclic CPP (MiniAp4 and CPP12) were treated with 20 eq. PhSiH3 and 0.2 eq. Pd(PPh3)4 in dichloromethane (DCM) under nitrogen for 2 × 15 min to remove allyl and allyloxycarbonyl (alloc) protecting groups. The resin was then washed with DCM and subsequently with dimethylformamide (DMF).Cyclation of the Asp / Dap side chain with the C-terminus of Glu and the N-terminus of Phe was achieved by treating the peptide with 2 eq. (benzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate (PyBOP) and 2 eq. DIPEA in DMF at room temperature for 16 h, followed by washing with DMF. After successful synthesis of the CPP-tagged dimer peptide, the resin was divided into two portions. The N-terminus of one portion of the resin-bound peptide was capped twice for 10 min each time using a mixture of DMF:acetic anhydride:DIPEA (8:1.5:0.5). The N-terminus of the other portion of the resin-bound peptide was functionalized with a chloroalkyl tag (CA). The CA tag was coupled to the N-terminal nitrogen group of the CPP-tagged peptide using a mixture of CA:PyBOP:DIPEA in DMF (3:3:10) for 16 h. In the case of peptides containing CPP12, prior to CA tag coupling, the o-nitrobenzenesulfonyl (Ns) was removed by adding 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.5 mmol) to DMF (2 mL) and mercaptoethanol (0.5 mmol) to DMF (2 mL) followed by shaking for 30 min. After flow washing in DMF, the mercaptoethanol / DBU treatment was repeated four times. After a final flow wash in DMF, the CA tag was coupled to the side chain nitrogen of the Lys residue.

[0149] The peptide compound was prepared using TFA / triisopropylsilane / 2,2′-(diethyloxy)diethanethiol (DODT) / H₂O (90 / 2.5 / 2.5). [2] to

[75] The peptide ligands were lysed from anhydrous resin for 2 h, then filtered, evaporated, precipitated with ice-cold ether, lyophilized, and purified by preparative RP-HPLC. The final peptide ligands were characterized by LC-MS for molecular weight determination and UPLC (214 nm) for purity (>95%).

[0150] The synthesis described above can also be carried out on a scale of 40 mmol or up to 100 mmol to obtain multi-gram formulations of the peptide compounds reported in this paper.

[0151] The peptide compounds described in this article exhibit excellent solubility properties, reaching concentrations of 25 mg / mL and higher.

[0152] This example demonstrates that ligands can be synthesized, purified, and obtained in their pure form. [Example] [2] [:right] [PSD-95] [Of] [PDZ1-2] [Measurement of Affinity] []

[0153] Compounds were measured using in vitro fluorescence polarization (FP) analysis as described by Bach et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 3317. [1] to

[75] Binding affinity of PDZ1-2 to PSD-95. First, saturation binding curves were obtained to determine the KD value of the interaction between the fluorescent dimer probe and PDZ1-2 of PSD-95. Incremental concentrations of PDZ1-2 (0.015–30 nM) were added to a constant concentration of probe (0.5 nM). The fluorescence polarization (FP) of the samples was measured at excitation / emission wavelengths of 635 / 670 nm, and the generated FP values ​​were fitted to a single-site binding model using the software GraphPad Prism. Then, the affinity of unlabeled peptides for PSD-95 PDZ12 was evaluated by heterocompetitive binding analysis, in which the peptides to be tested were added in increments (0.4–960 nM, final concentration) to a fixed amount of PSD-95 PDZ12 (4 nM) and probe (0.5 nM) under the same conditions as the saturation binding experiments. The FP values ​​were fitted to a site competition (variable slope) model in GraphPad prism. The resulting IC50 values ​​were then converted to Ki values ​​as previously described by Nikolovska-Coleska et al., Anal. Biochem., 2004, 332, 261.

[0154] This example illustrates a method for determining the affinity of the PDZ1-2 ligand bound to PSD-95. The results are presented in Tables 4 to 7 and Figures 1 to 4. In summary, the tested compounds exhibited affinity (Ki values) in the low nanomor concentration range comparable to compound 1. [surface] [4.] Binding affinity (K i) is presented as mean ± SEM (Fig. 1). [Compound] [ligand] [CPP] [CPP] [Sequence] [, K , ] [, , ] [[nM]] [1] OPEG 4-(IETDV) 2 - - 13.7 ± 1.6 [2] OPEG 4-(KETLV) 2 - - 11.​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The data is represented as n ≥ 3. [] [surface] [5.] The binding affinity (Ki) of the dimer ligands fused to L-TAT and L-Arg 9 is shown as mean ± SEM (Fig. 2). [Compound] [ligand] [CPP] [CPP] [sequence] [, K , ] [, i , ] [[nM]] [8] NPEG 4-(IETDV) 2 l-TAT YGRKKRRQRRR- 07.2 ± 1.5 [9] NPEG 4-(IETDV) 2 l-TAT Ac-YGRKKRRQRRR- 12.5 ± 1.1

[11] NPEG 4-(KETLV) 2 l-TAT Ac-YGRKKRRQRRR- 05.4 ± 0.9

[13] NPEG 4-(KETTV) 2 l-TAT Ac-YGRKKRRQRRR- 06.5 ± 0.7

[15] NPEG 4-(KETVV) 2 l-TAT Ac-YGRKKRRQRRR- 02.1 ± 0.2

[17] NPEG 4-(KETRV) 2 l-TAT Ac-YGRKKRRQRRR- 06.6 ± 0.6

[19] NPEG 4-(ISTDV) 2 l-TAT Ac-YGRKKRRQRRR- 36.1 ± 1.8

[21] NPEG 4-(VETVV) 2 l-TAT Ac-YGRKKRRQRRR- 15.8 ± 1.6

[35] NPEG 4-(IETDV) 2 l -Arg 9 Ac-RRRRRRRRR- 16.0 ± 0.6

[23] NPEG 4-(KETLV) 2 l -Arg 9 Ac-RRRRRRRRR- 07.8 ± 0.4

[25] NPEG 4-(KETTV) 2 l -Arg 9 Ac-RRRRRRRRR- 05.6 ± 0.8

[27] NPEG 4-(KETVV) 2 l -Arg 9 Ac-RRRRRRRRR- 06.3 ± 0.7

[29] NPEG 4-(KETRV) 2 l-Arg 9 Ac-RRRRRRRRR- 10.1 ± 1.3

[31] NPEG 4-(ISTDV) 2 l-Arg 9 Ac-RRRRRRRRR- 62.3 ± 2.4

[33] NPEG 4-(VETVV) 2 l-Arg 9 Ac-RRRRRRRRR- 19.9 ± 2.8 The data is represented as n ≥ 3; Ac = N-terminal acetylation. [surface] [6]. The binding affinity (Ki) of NPEG 4-(IETDV) 2 dimer ligands fused to different CPP tags is shown as mean ± SEM (Fig. 3). [Compound] [ligand] [CPP] [CPP] [sequence] [, K , , i , [nM] ] [8] NPEG 4-(IETDV) 2 l-TAT YGRKKRRQRRR- 07.2 ± 1.5 [9] NPEG 4-(IETDV) 2 l-TAT Ac-YGRKKRRQRRR- 12.5 ± 1.1

[38] NPEG 4-(IETDV) 2 d-TAT Aj-yGrkkrrqrrr- 11.8 ± 0.8

[40] NPEG 4-(IETDV) 2 mTAT Ac-rRrGrKkRr- 11.9 ± 2.7

[42] NPEG 4-(IETDV) 2 riTAT Ac-rrrqrrkkr- 14.4 ± 0.6

[35] NPEG 4-(IETDV) 2 l -Arg 9 Ac-RRRRRRRRR- 11.0 ± 1.3

[44] NPEG 4-(IETDV) 2 d -Arg 9 Ac-rrrrrrrrr- 08.6 ± 0.4

[46] NPEG 4-(IETDV) 2 l-Pen Ac-RQIKIWFQNRRMKWKK- 11.3 ± 1.9

[48] NPEG 4-(IETDV) 2 d-Pen Ac-thinfqnrrmkwkk- 07.8 ± 0.7

[50] NPEG 4-(IETDV) 2 l-DPV3 Ac-RKKRRRESRKKRRRES- 16.6 ± 3.2

[52] NPEG 4-(IETDV) 2 l-pVEC Ac-LLIILRRRIRKQAHAHSK- 13.3 ± 2.2

[54] NPEG 4-(IETDV) 2 l -MAP Ac-KLALCLALKALKAALKLA- 22.4 ± 0.8

[56] NPEG 4-(IETDV) 2 l-TP2 Ac-PLYLRLLRGQF- 12.1 ± 1.3

[58] NPEG 4-(IETDV) 2 d-TP2 Ac-pliylrllrGqf- 11.9 ± 0.7

[60] NPEG 4-(IETDV) 2 MiniAp4 Ac-|(Dap)CAPETALD|- 12.1 ± 1.3

[62] NPEG 4-(IETDV) 2 CPP12 |Ff(Nal2)RrRrQ|GABA-K- 12.5 ± 1.1 The data is represented as n ≥ 3; Ac = N-terminal acetylation. [] [surface] [7.] The binding affinity (Ki) of the NPEG 4-(KETLV) 2 dimer ligand fused to the polyArg CPP tag is shown as mean ± SEM (Fig. 4). [Chemicals] [thing] [ligand] [CPP] [CPP] [sequence] [, K , ] [, i , ] [[nM]]

[67] NPEG 4-(KETLV) 2 l-Arg 7 Ac-RRRRRRR- 5.8 ± 0.6

[69] NPEG 4-(KETLV) 2 l-Arg 6 Ac-RRRRRR- 8.6 ± 0.3

[71] NPEG 4-(KETLV) 2 l-Arg 5 Ac-RRRRR- 10.9 ± 1.3

[72] NPEG 4-(KETLV) 2 l-Arg 4 Ac-RRRR- 11.1 ± 0.9 The data is represented as n ≥ 3; Ac = N-terminal acetylation. [] [Example] [3] [In vitro stability of ligands on plasma] []

[0155] The compound was determined by incubating 100 µM ligands in phosphate-buffered saline (PBS) supplemented with cytoplasm (5 ug / mL) at 37°C for 0–1440 minutes. [8] to

[62] In vitro cytoplasmic stability. Ligands were extracted from 80 µL of analytical matrix by treating with 80 µL of 50% acetonitrile (ACN) at selected time points during incubation. Samples were filtered and analyzed by UPLC to determine the amount of remaining ligands. LC-MS analysis was performed to confirm ligand integrity and identify cleavage sites.

[0156] This example illustrates a method for determining the in vitro cytoplasmic stability of the PDZ1-2 ligand bound to PSD-95. The results are presented in Tables 8 and 9 and Figures 5 and 6. In summary, the tested compounds containing CPP L-Arg 9 (SEQ ID NO 12) exhibited in vitro cytoplasmic half-lives comparable to those containing D-amino acids or macrocyclic CPPs (Figure 6). Surprisingly, the half-life of the compound containing L-Arg 9 was significantly better than that of the compound containing L-TAT (SEQ ID NO 8) (Figures 5 and 6). [surface] [8]. In vitro stability of cytoplasmic plasma by NPEG 4-dimer ligands fused to L-TAT and L-Arg 9, half-life time is shown as mean ± SEM (Fig. 5). [Compound] [ligand] [CPP] [CPP] [sequence] [THE , 1 / 2 , [name] ] [8] NPEG 4-(IETDV) 2 l-TAT YGRKKRRQRRR- 543 ± 060 [9] NPEG 4-(IETDV) 2 l-TAT Ac-YGRKKRRQRRR- 0229 ± 129

[11] NPEG 4-(KETLV) 2 l-TAT Ac-YGRKKRRQRRR- 0346 ± 105

[13] NPEG 4-(KETTV) 2 l-TAT Ac-YGRKKRRQRRR- 0570 ± 180

[15] NPEG 4-(KETVV) 2 l-TAT Ac-YGRKKRRQRRR- 0385 ± 130

[17] NPEG 4-(KETRV) 2 l-TAT Ac-YGRKKRRQRRR- 0385 ± 130

[19] NPEG 4-(ISTDV) 2 l-TAT Ac-YGRKKRRQRRR- 0534 ± 040

[21] NPEG 4-(VETVV) 2 l-TAT Ac-YGRKKRRQRRR- 0532 ± 041

[35] NPEG 4-(IETDV) 2 l -Arg 9 Ac-RRRRRRRRR- 1492 ± 114

[23] NPEG 4-(KETLV) 2 l -Arg 9 Ac-RRRRRRRRR- 2618 ± 276

[25] NPEG 4-(KETTV) 2 l -Arg 9 Ac-RRRRRRRRR- 1152 ± 203

[27] NPEG 4-(KETVV) 2 l -Arg 9 Ac-RRRRRRRRR- 1530 ± 578

[29] NPEG 4-(KETRV) 2 l -Arg 9 Ac-RRRRRRRRR- 1003 ± 171

[31] NPEG 4-(ISTDV) 2 l -Arg 9 Ac-RRRRRRRRR- 1405 ± 124

[33] NPEG 4-(VETVV) 2 l-Arg 9 Ac-RRRRRRRRR- 0479 ± 077 The data is represented as n ≥ 3; Ac = N-terminal acetylation. [surface] [9.] In vitro cytoplasmic stability of NPEG 4-(IETDV) 2 dimer ligands fused to different CPP tags, half-life times are presented as mean ± SEM (Fig. 6). [Compound] [ligand] [CPP] [CPP] [sequence] [T, 1 / 2 , [min] ] [8] NPEG 4-(IETDV) 2 l-TAT YGRKKRRQRRR- 0543 ± 060 [9] NPEG 4-(IETDV) 2 l-TAT Ac-YGRKKRRQRRR- 0229 ± 129

[38] NPEG 4-(IETDV) 2 d-TAT Ac-ygrkkrrqrrr- 1440 ± 000

[40] NPEG 4-(IETDV) 2 mTAT Ac-rRrGrKkRr- 1440 ± 000

[42] NPEG 4-(IETDV) 2 riTAT Ac-rrrqrrkkr- 1440 ± 000

[35] NPEG 4-(IETDV) 2 l -Arg 9 Ac-RRRRRRRRR- 1492 ± 114

[44] NPEG 4-(IETDV) 2 d -Arg 9 Ac-rrrrrrrrr- 1440 ± 000

[46] NPEG 4-(IETDV) 2 l-Pen Ac-RQIKIWFQNRRMKWKK- 0094 ± 008

[48] NPEG 4-(IETDV) 2 D-Pen Ac-thinfqnrrmkwkk- 1440 ± 000

[50] NPEG 4-(IETDV) 2 l-DPV3 Ac-RKKRRRESRKKRRRES- 0169 ± 020

[52] NPEG 4-(IETDV) 2 l-pVEC Ac-LLIILRRRIRKQAHAHSK- 0049 ± 009

[54] NPEG 4-(IETDV) 2 l-MAP Ac-KLALKLALKALKAALKLA- 0013 ± 004

[56] NPEG 4-(IETDV) 2 l-TP2 Ac-PLIYLRLLRGQF- 0910 ± 000

[58] NPEG 4-(IETDV) 2 d-TP2 Ac-pliylrllrGqf- 1440 ± 000

[60] NPEG 4-(IETDV) 2 MiniAp4 Ac-|(Dap)KAPETALD|- 1440 ± 000

[62] NPEG 4-(IETDV) 2 CPP12 |Ff(Nal2)RrRrQ|GABA-K- 1440 ± 000 The data is represented as n ≥ 3; Ac = N-terminal acetylation. [] [Example] [4] [Determination of ligand membrane permeability and cellular uptake] []

[0157] Compounds were determined in HeLa cells that stably expressed HaloGFP only in the cytoplasm.

[10] to

[75] Membrane permeability. Cells were seeded at a density of 40,000 cells / well one day prior to the experiment. After removing the growth medium and replacing it with 100 µL of Opti-MEM, 25 µL of a serially diluted ligand in Opti-MEM was added to the cells (constant DMSO concentration), and the plate was incubated at 37°C and 5% CO2 for 4 h. The contents of the wells were removed, and the cells were washed with fresh Opti-MEM for 15 min. After removing the wash, the cells were incubated with TAMRA-CA (5 µM) for 15 min. After removing the tracking solution, the cells were washed with Opti-MEM for 30 min. After removing the wash, the cells were trypsinized, resuspended in PBS (2% FBS), and analyzed using a benchtop flow cytometry. Fluorescence intensity data obtained from control wells without ligand and without TAMRA-CA were normalized and plotted as dose-response curves. The reported CP 50 value represents half-maximal red fluorescence, which is the opposite of the ligand’s cell penetration.

[0158] This example illustrates a method for determining the membrane permeability and cellular uptake of CA-tagged compounds bound to PSD-95 PDZ1-2. Results are presented in Tables 10-12 and Figures 7-9. In summary, the tested compounds exhibited significantly different cellular uptake efficiencies, highly dependent on the CPP used. Surprisingly, the CPP tags L-Arg 9 (SEQ ID NO: 12) and L-pVEC (SEQ ID NO: 18) produced the lowest CP 50 values ​​(Figure 8). At the dimeric ligand end, L-Arg 9 further promoted highly efficient cellular uptake with CP 50 values ​​below 1 µM (Figure 7), indicating excellent BBB penetration and intracellular delivery. Furthermore, the L-Arg 9 CPP tag could be truncated to 7, 6, 5, or 4 arginines (see Table 12) without losing its cellular permeability properties (Figure 9). [surface] [10.] Membrane permeability and cellular uptake of NPEG 4 dimer ligands fused to L-TAT and L-Arg 9, CP 50 is shown as mean ± SEM (Fig. 7). [Compound] [ligand] [CPP] [CPP] [sequence] [CP,50 [µM]

[10] NPEG 4-(IETDV) 2 l-TAT CA-YGRKKRRQRRR- 55.1 ± 3.0

[12] NPEG 4-(KETLV) 2 l-TAT CA-YGRKKRRQRRR- 72.5 ± 4.4

[14] NPEG 4-(KETTV) 2 l-TAT CA-YGRKKRRQRRR- 78.4 ± 3.9

[16] NPEG 4-(KETVV) 2 l-TAT CA-YGRKKRRQRRR- 81.8 ± 1.2

[18] NPEG 4-(KETRV) 2 l-TAT CA-YGRKKRRQRRR- 47.7 ± 1.0

[20] NPEG 4-(ISTDV) 2 l-TAT CA-YGRKKRRQRRR- 41.9 ± 1.6

[22] NPEG 4-(VETVV) 2 l-TAT CA-YGRKKRRQRRR- 86.1 ± 4.5

[36] NPEG 4-(IETDV) 2 l-Arg 9 CA-RRRRRRRR- 03.25 ± 0.19

[24] NPEG 4-(KETLV) 2 l-Arg 9 CA-RRRRRRRR- 00.78 ± 0.05

[26] NPEG 4-(KETTV) 2 l-Arg 9 CA-RRRRRRRR- 00.74 ± 0.09

[28] NPEG 4-(KETVV) 2 l-Arg 9 CA-RRRRRRRR- 00.79 ± 0.06

[30] NPEG 4-(KETRV) 2 l-Arg 9 CA-RRRRRRRR- 00.68 ± 0.05

[32] NPEG 4-(ISTDV) 2 l-Arg 9 CA-RRRRRRRR- 01.05 ± 0.15

[34] NPEG 4-(VETVV) 2 l-Arg 9 CA-RRRRRRRR- 00.84 ± 0.15 The data is represented as n ≥ 3; Ac = N-terminal acetylation. [surface] [11.] Membrane permeability and cellular uptake of NPEG 4-(IETDV) 2 dimer ligands fused to different CPP tags, CP 50 is shown as mean ± SEM (Fig. 8). [Compound] [ligand] [CPP] [CPP] [sequence] [CP, 50 , [µM] ]

[10] NPEG 4-(IETDV) 2 l-TAT CA-YGRKKRRQRRR- 55.1 ± 3.0

[39] NPEG 4-(IETDV) 2 d-TAT CA-yGrkkrrqrrr- 10.7 ± 0.7

[41] NPEG 4-(IETDV) 2 mTAT CA-rRrGrKkRr- 180 ± 8.2

[43] NPEG 4-(IETDV) 2 riTAT CA-rrrqrrkkr- 221 ± 36

[36] NPEG 4-(IETDV) 2 l-Arg 9 CA-RRRRRRRRR- 03.3 ± 0.2

[45] NPEG 4-(IETDV) 2 d -Arg 9 CA-rrrrrrrrr- 161 ± 25

[47] NPEG 4-(IETDV) 2 l-Pen CA-RQIKIWFQNRRMKWKK- 05.0 ± 0.4

[49] NPEG 4-(IETDV) 2 d-Pen CA-thinfqnrrmkwkk- 18.8 ± 1.5

[51] NPEG 4-(IETDV) 2 l-DPV3 CA-RKKRRRESRKKRRRES- 03.2 ± 0.5

[53] NPEG 4-(IETDV) 2 l-pVEC CA-LLILLRRRIRKQAHAHSK- 01.7 ± 0.1

[55] NPEG 4-(IETDV) 2 l -MAP CA-KLALKLALKALKAALKLA- 03.7 ± 0.3

[57] NPEG 4-(IETDV) 2 l-TP2 CA-PLIYLRLLRGQF- 17.9 ± 0.3

[59] NPEG 4-(IETDV) 2 d-TP2 CA-pliylrllrGqf- 67.2 ± 5.3

[61] NPEG 4-(IETDV) 2 MiniAp4 CA-|(Dap)KAPETALD|- 13.2 ± 0.4

[63] NPEG 4-(IETDV) 2 CPP12 |Ff(Nal2)RrRrQ|GABA-K(CA-) 11.2 ± 0.7 The data is represented as n ≥ 3; Ac = N-terminal acetylation. [] [surface] [12.] Membrane permeability and cellular uptake of NPEG 4-(IETDV) 2 and NPEG 4-(KETLV) 2 dimer ligands fused to polyArg CPP tags, CP 50 is shown as mean ± SEM (Fig. 9). [Compound] [ligand] [CPP] [CPP] [sequence] [CP, 50 , [µM] ]

[64] NPEG 4-(IETDV) 2 l-Arg 7 CA-RRRRRRR- 1.66 ± 0.43

[65] NPEG 4-(IETDV) 2 l -Arg 5 CA-RRRRR- 1.17 ± 0.37

[66] NPEG 4-(IETDV) 2 l -Arg 3 CA-RRR- 3.71 ± 0.30

[68] NPEG 4-(KETLV) 2 l -Arg 7 CA-RRRRRRR- 0.58 ± 0.09

[70] NPEG 4-(KETLV) 2 l -Arg 6 CA-RRRRRR- 0.51 ± 0.08

[72] NPEG 4-(KETLV) 2 l -Arg 5 CA-RRRRR- 0.66 ± 0.14

[74] NPEG 4-(KETLV) 2 l -Arg 4 CA-RRRR- 0.37 ± 0.08

[75] NPEG 4-(KETLV) 2 l -Arg 3 CA-RRR- 7.36 ± 0.31 If n ≥ 3;Ac = N. [] [Example] [5] Determination of in vitro plasma stability []

[0159] The compounds were determined by the following methods [8] [twenty three],

[35]

[67]

[69]

[71] and

[73] In vitro plasma stability: 100 µM ligands were incubated in undiluted pooled human plasma at 37°C for 1440 min. At selected time points during incubation, ligands were extracted from the analytical matrix by adding the analytical matrix to 6M urea and then precipitating with 20% TCA in acetone. After overnight incubation, samples were filtered and analyzed by UPLC to determine the amount of remaining ligands. LC-MS analysis was performed to confirm ligand integrity and identify cleavage sites.

[0160] This example illustrates a method for determining the in vitro plasma stability of the PDZ1-2 ligand bound to PSD-95. The results are presented in Table 13 and Figure 10. In summary, the L-Arg 9CPP tag can be truncated to 7, 6, 5, or 4 arginines (see Table 13) without significantly reducing the stability of human plasma. [surface] [13.] In vitro plasma stability of NPEG 4-(KETLV) 2 dimer ligands fused to the polyArg CPP tag, half-life time is presented as mean ± SEM (Fig. 10). [] [Compound] [ligand] [CPP] [CPP] [sequence] [T, 1 / 2 , [min] ] [8] NPEG 4-(IETDV) 2 l-TAT YGRKKRRQRRR- 1440 ± 0

[35] NPEG 4-(IETDV) 2 l-Arg 9 Ac-RRRRRRRRR- 1440 ± 0

[23] NPEG 4-(KETLV) 2 l-Arg 9 Ac-RRRRRRRRR- 1440 ± 0

[67] NPEG 4-(KETLV) 2 l-Arg 7 Ac-RRRRRRR- 1440 ± 0

[69] NPEG 4-(KETLV) 2 l-Arg 6 Ac-RRRRRR- 1440 ± 0

[71] NPEG 4-(KETLV) 2 l-Arg 5 Ac-RRRRR- 1266 ± 138

[73] NPEG 4-(KETLV) 2 l-Arg 4 Ac-RRRR- 912 ± 216 Data are expressed as n ≥ 3; Ac = N-terminal acetylation. [] [Example] [6] [:] [iv] [Determination of brain function in vivo after injection] []

[0161] Female Balb / c mice (n=3 per group) were administered the compound of interest at 18 nmol / g body weight using isotonic saline as a medium. Blood concentrations at selected time points following intravenous injection were obtained in group 1 (max. 4 h post-injection). Blood and brain concentrations were measured at three different time points in groups 2 through 4. Animals were then terminated at selected time points following intravenous injection, and relevant tissues were collected and analyzed using appropriate methods (LC-MS / MS).

[0162] This example illustrates a method for determining the in vivo brain level of the PDZ1-2 ligand bound to PSD-95. Additionally, the blood-to-brain ratio and related tissue targeting efficiency are measured to inform the compound's ability to cross the blood-brain barrier and reach its target in the brain. [Example] [7] [:use] [pMCAO] [Mouse model assay for stroke-induced damage] [In vivo] [Neuroprotective effect.] []

[0163] For experimental and surgical details, see Bach et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 3317. In short, this study measured the degree of ischemic infarction in a C57BL / 6J mouse model of permanent middle cerebral artery occlusion (pMCAO) in a randomized, double-blind, placebo-controlled trial. Following anesthesia and arterial occlusion, the animals were administered drugs 30 minutes post-surgery. Animals surviving 6 hours post-surgery were terminated, and the brain was carefully removed. The brain was fixed and sectioned, and infarct volume analysis was performed to calculate the total infarct volume.

[0164] This example illustrates a method for determining the in vivo neuroprotective effect of PDZ1-2 ligands bound to PSD-95. The readout parameter represents the infarct volume in the compound treatment group compared to the placebo control group. [sequence] [] [SEQ ID NO] [Sequence] [Annotation] 1 IETDV 2 KETLV 3 KETTV 4 KETVV 5 KETRV 6 ISTDV [[ID=۳۸]] 7 VETVV 8 YGRKKRRQRRR l-TAT 9 yGrkkrrqrrr D-TAT 10 rRrGrKkRr mTAT 11 rrrqrrkkr riTAT 12 RRRRRRRRR l-Arg 9 13 rrrrrrrrr d-Arg 9 14 RQIKIWFQNRRMKWKK l-Pen 15 rqikiwfqnrrmkwkk d-Pen 16 KLALKLALKALKAALKLA l-MAP 17 RKKRRRESRKKRRRES l-DPV3 18 LLIILRRRIRKQAHAHSK l-pVEC 19 PLIYLRLLRGQF l-TP2 20 pliylrllrGqf d-TP2 twenty one |(Dap)KAPETALD| MiniAp4 Leptylation of the side chains via D and Dap Dap-based 1-2,3-diaminopropionic acid twenty two |Ff(Nal2)RrRrQ|GABA-K CPP12 via head-to-tail endolane amination of Q and F Na12-series l-2-naphthylalanine twenty three Ac-YGRKKRRQRRR- l-TAT N-terminal acetylation twenty four CA-YGRKKRRQRRR- l-TAT The N-terminus binds to a chloroalkane (CA). 25 Ac-RRRRRRRRR- l-Arg 9 N-terminal acetylation 26 CA-RRRRRRRRR- l-Arg 9 The N-terminus binds to a chloroalkane (CA). 27 Ac-ygrkkrrqrrr- d-TAT N-terminal acetylation 28 CA-ygrkkrrqrrr- d-TAT The N-terminus binds to a chloroalkane (CA). 29 Ac-rRrGrKkRr- mTAT N-terminal acetylation 30 CA-rRrGrKkRr- mTAT The N-terminus binds to a chloroalkane (CA). 31 Ac-rrrqrrkkr- riTAT N-terminal acetylation 32 CA-rrrqrrkkr- riTAT The N-terminus binds to a chloroalkane (CA). 33 Ac-rrrrrrrrr- d-Arg 9 N-terminal acetylation 34 CA-rrrrrrrrr- d-Arg 9 The N-terminus binds to a chloroalkane (CA). 35 Ac-RQIKIWFQNRRMKWKK- l-Pen N-terminal acetylation 36 CA-RQIKIWFQNRRMKWKK- l-Pen The N-terminus binds to a chloroalkane (CA). 37 Ac-rqikiwfqnrrmkwkk- d-Pen N-terminal acetylation 38 CA-rqikiwfqnrrmkwkk- d-Pen The N-terminus binds to a chloroalkane (CA). 39 Ac-RKKRRRESRKKRRRES- l-DPV3 N-terminal acetylation 40 CA-RKKRRRESRKKRRRES- l-DPV3 The N-terminus binds to a chloroalkane (CA). 41 Ac-LLIILRRRIRKQAHAHSK- l-pVEC N-terminal acetylation 42 CA-LLIILRRRIRKQAHAHSK- l-pVEC The N-terminus binds to a chloroalkane (CA). 43 Ac-KLALKLALKALKAALKLA- l-MAP N-terminal acetylation 44 CA-KLALKLALKALKAALKLA- l-MAP The N-terminus binds to a chloroalkane (CA). 45 Ac-PLIYLRLLRGQF- l-TP2 N-terminal acetylation 46 CA-PLIYLRLLRGQF- l-TP2 The N-terminus binds to a chloroalkane (CA). 47 Ac-pliylrllrGqf- N-terminal acetylation d-TP2 N-terminal acetylation 48 CA-pliylrllrGqf- d-TP2 The N-terminus binds to a chloroalkane (CA). 49 Ac-|(Dap)KAPETALD|- N-terminal acetylation MiniAp4 N-terminal acetylation 50 CA-|(Dap)KAPETALD|- MiniAp4 N-terminus binds to chloroalkane 51 |Ff(Nal2)RrRrQ|GABA-K- CPP12 K's side chain is unadorned. 52 |Ff(Nal2)RrRrQ|GABA-K(CA-) CPP12 amines formed by the binding of chloroalkanes (CA) to the side chain of K 53 X 2TX 3V X2 Series E or S X 3 series L, T, V, R or D 54 X 1X 2TX 3V X 1 series K, V or I X2 Series E or S X 3 series L, T, V, R or D 55 X 5TX 6V X5 Series E or S X 6 series L, T, V, R or D 56 X 4X 5TX 6V X4 series K, V or I X5 Series E or S X 6 series L, T, V, R or D 57 RRRRRRRR l-Arg 8 58 RRRRRRR l-Arg 7 59 RRRRRR l-Arg 6 60 RRRRR l-Arg 5 61 RRRR l-Arg 4 62 Ac-RRRRRRRR- l-Arg 8 N-terminal acetylation 63 Ac-RRRRRRR- l-Arg 7 N-terminal acetylation 64 Ac-RRRRRR- l-Arg 6 N-terminal acetylation 65 Ac-RRRRR- l-Arg 5 N-terminal acetylation 66 Ac-RRRR- l-Arg 4 N-terminal acetylation 67 CA-RRRRRRRR- l-Arg 8 The N-terminus binds to a chloroalkane (CA). 68 CA-RRRRRRR- l-Arg 7 The N-terminus binds to a chloroalkane (CA). 69 CA-RRRRRR- l-Arg 6 The N-terminus binds to a chloroalkane (CA). 70 CA-RRRRR- l-Arg 5 The N-terminus binds to a chloroalkane (CA). 71 CA-RRRR- l-Arg 4 The N-terminus binds to a chloroalkane (CA). [References] [] Aarts, M., Liu, Y., Liu, L., Besshoh, S., Arundine, M., Gurd, JW, Wang, YT, Salter, MW, Tymianski, M., 2002. Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science 298, 846-850. JT Andreasen, A. Bach, M. Gynther, A. Nasser, J. Mogensen, K. Strømgaard, DS Pickering, UCCB01-125, a dimeric inhibitor of PSD-95, reduces inflammatory pain without disrupting cognitive or motor performance: comparison with the NMDA receptor antagonist MK-801, Neuropharmacology, 67, 193-200 (2013). Bach, A., Chi, C.N., Pang, G.F., Olsen, L., Kristensen, A.S., Jemth, P., Strømgaard, K., Design and synthesis of highly potent and plasma-stable dimeric inhibitors of the PSD-95-NMDA receptor interaction. Angew. Chem. Int. Ed. 48, 9685-9689 (2009). Bach, A., Clausen, B.H., Møller, M., Vestergaard, B., Chi, C.N., Round, A., Sørensen, P.L., Nissen, K.B., Kastrup, J.S., Gajhede, M., Jemth, P., Kristensen, A.S., Lundström, P., Lambertsen, K.L., Strømgaard, K., A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage. Proc. Natl. Acad. Sci. U.S.A. 109, 3317-3322 (2012). Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S., Snyder, S.H., 1991. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. Natl. Acad. Sci. U. S. A 88, 6368-6371. Deprey, K.; Kritzer, J. A., 2020. Quantitative measurement of cytosolic penetration using the chloroalkane penetration assay. Method. Enzymol. 641, 277-309. Hill et al. 2020. Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke (ESCAPE-NA1): A multicentre, double-blind, randomised controlled trial. Lancet 395, 878-887. Huang, Z., Huang, P.L., Panahian, N., Dalkara, T., Fishman, M.C., Moskowitz, M.A., 1994. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265, 1883-1885. Kornau, H. C., Schenker, L. T., Kennedy, M. B., Seeburg, P. H., 1995. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269, 1737-1740. Kucharz, K., Rasmussen, I.S., Bach, A., Strømgaard, K., Lauritzen, M., PSD-95 uncoupling from NMDA receptors by Tat-N-dimer ameliorates neuronal depolarization in cortical spreading depression. J. Cereb. Blood Flow Metab. 37, 1820-1828 (2017). Mayor-Nunez, D., Ji, Z., Sun, X., Teves, L., Garman, J. D., Tymianski, M., 2021. Plasmin-resistant PSD-95 inhibitors resolve effect-modifying drug-drug interactions between alteplase and nerinetide in acute stroke, Sci. Transl. Med. 13, eabb1498. Peraro, L.; Deprey, K. L.; Moser, M. K.; Zou, Z.; Ball, H. L.; Levine, B.; Kritzer, J. A., 2018. Cell Penetration Profiling Using the Chloroalkane Penetration Assay. J. Am. Chem. Soc. 140, 11360-11369. Sattler, R., Xiong, Z., Lu, W.Y., Hafner, M., MacDonald, J.F., Tymianski, M., 1999. Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science 284, 1845-1848.

[0165] None

[0166] <![CDATA[ <110> University of Copenhagen <![CDATA[ <120> PSD-95 inhibitors and their uses <![CDATA[ <140> TW 111117679]]> <![CDATA[ <141> 2022-05-11 <![CDATA[ <150> EP 21173587.3 <![CDATA[ <151> 2021-05-12 <![CDATA[ <160> 71 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <400> 1]]> Ile Glu Thr Asp Val 1 5 <![CDATA[ <210> 2]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <400> 2]]> Lys Glu Thr Leu Val 1 5 <![CDATA[ <210> 3]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <400> 3]]> Lys Glu Thr Thr Val 1 5 <![CDATA[ <210> 4]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <400> 4]]> Lys Glu Thr Val Val 1 5 <![CDATA[ <210> 5]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <400> 5]]> Lys Glu Thr Arg Val 1 5 <![CDATA[ <210> 6]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <400> 6]]> Ile Ser Thr Asp Val 1 5 <![CDATA[ <210> 7]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <400> 7]]> Val Glu Thr Val Val 1 5 <![CDATA[ <210> 8]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> L-TAT]]> <![CDATA[ <400> 8]]> Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <![CDATA[ <210> 9]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> D-TAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 9]]> Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <![CDATA[ <210> 10]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> mTAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (5)..(5)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (7)..(7)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (9)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 10]]> Arg Arg Arg Gly Arg Lys Lys Arg Arg 1 5 <![CDATA[ <210> 11]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> riTAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 11]]> Arg Arg Arg Gln Arg Arg Lys Lys Arg 1 5 <![CDATA[ <210> 12]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> L-Arg9]]> <![CDATA[ <400> 12]]> Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 13]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D-Arg9]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 13]]> Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 14]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> L-Pen]]> <![CDATA[ <400> 14]]> Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <![CDATA[ <210> 15]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> D-Pen]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 15]]> Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <![CDATA[ <210> 16]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(19)]]> <![CDATA[ <223> L-MAP <![CDATA[ <400> 16]]> Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala <![CDATA[ <210> 17]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> L-DPV3]]> <![CDATA[ <400> 17]]> Arg Lys Lys Arg Arg Arg Glu Ser Arg Lys Lys Arg Arg Arg Glu Ser 1 5 10 15 <![CDATA[ <210> 18]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(18)]]> <![CDATA[ <223> L-pVEC]]> <![CDATA[ <400> 18]]> Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys <![CDATA[ <210> 19]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> L-TP2]]> <![CDATA[ <400> 19]]> Pro Leu Ile Tyr Leu Arg Leu Leu Arg Gly Gln Phe 1 5 10 <![CDATA[ <210> 20]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> D-TP2]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 20]]> Pro Leu Ile Tyr Leu Arg Leu Leu Arg Gly Gln Phe 1 5 10 <![CDATA[ <210> 21]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> MiniAp4]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> X-series Dap]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> [Through the side chains of D and Dap to macrolide amylation]]> <![CDATA[ <400> 21]]> Xaa Lys Ala Pro Glu Thr Ala Leu Asp 1 5 <![CDATA[ <210> 22]]> <![CDATA[ <211> 13]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(13)]]> <![CDATA[ <223> CPP12]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(8)]]> <![CDATA[ <223> Through head-to-tail endonerization of F and Q]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (2)..(2)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> X-series Nal2]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (5)..(5)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (7)..(7)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 22]]> Phe Phe Xaa Arg Arg Arg Arg Gln Gly Ala Asx Ala Lys 1 5 10 <![CDATA[ <210> 23]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> L-TAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> Acetylation <![CDATA[ <400> 23]]> Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <![CDATA[ <210> 24]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> L-TAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 24]]> Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <![CDATA[ <210> 25]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> L-Arg9]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 25]]> Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 26]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> L-Arg9]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 26]]> Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 27]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> D-TAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 27]]> Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <![CDATA[ <210> 28]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> D-TAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 28]]> Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <![CDATA[ <210> 29]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> mTAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (5)..(5)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (7)..(7)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (9)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 29]]> Arg Arg Arg Gly Arg Lys Lys Arg Arg 1 5 <![CDATA[ <210> 30]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> mTAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (5)..(5)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (7)..(7)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (9)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 30]]> Arg Arg Arg Gly Arg Lys Lys Arg Arg 1 5 <![CDATA[ <210> 31]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> riTAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 31]]> Arg Arg Arg Gln Arg Arg Lys Lys Arg 1 5 <![CDATA[ <210> 32]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> riTAT]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 32]]> Arg Arg Arg Gln Arg Arg Lys Lys Arg 1 5 <![CDATA[ <210> 33]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D-Arg9]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 33]]> Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 34]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D-Arg9]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 34]]> Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 35]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> L-Pen]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 35]]> Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <![CDATA[ <210> 36]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> L-Pen]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 36]]> Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <![CDATA[ <210> 37]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> D-Pen]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 37]]> Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <![CDATA[ <210> 38]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> D-Pen]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 38]]> Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <![CDATA[ <210> 39]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> L-DPV3]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 39]]> Arg Lys Lys Arg Arg Arg Glu Ser Arg Lys Lys Arg Arg Arg Glu Ser 1 5 10 15 <![CDATA[ <210> 40]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(16)]]> <![CDATA[ <223> L-DPV3]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 40]]> Arg Lys Lys Arg Arg Arg Glu Ser Arg Lys Lys Arg Arg Arg Glu Ser 1 5 10 15 <![CDATA[ <210> 41]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(18)]]> <![CDATA[ <223> L-pVEC]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 41]]> Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys <![CDATA[ <210> 42]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(18)]]> <![CDATA[ <223> L-pVEC]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 42]]> Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys <![CDATA[ <210> 43]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(18)]]> <![CDATA[ <223> L-MAP <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 43]]> Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala <![CDATA[ <210> 44]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(18)]]> <![CDATA[ <223> L-MAP <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 44]]> Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala <![CDATA[ <210> 45]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> L-TP2]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(11)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 45]]> Pro Leu Ile Tyr Leu Arg Leu Leu Arg Gly Gln Phe 1 5 10 <![CDATA[ <210> 46]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> L-TP2]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 46]]> Pro Leu Ile Tyr Leu Arg Leu Leu Arg Gly Gln Phe 1 5 10 <![CDATA[ <210> 47]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> D-TP2]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 47]]> Pro Leu Ile Tyr Leu Arg Leu Leu Arg Gly Gln Phe 1 5 10 <![CDATA[ <210> 48]]> <![CDATA[ <211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> D-TP2]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(12)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 48]]> Pro Leu Ile Tyr Leu Arg Leu Leu Arg Gly Gln Phe 1 5 10 <![CDATA[ <210> 49]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> MiniAp4]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> X-series Dap]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 49]]> Xaa Lys Ala Pro Glu Thr Ala Leu Asp 1 5 <![CDATA[ <210> 50]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(9)]]> <![CDATA[ <223> MiniAp4]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> X series Dap]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 50]]> Xaa Lys Ala Pro Glu Thr Ala Leu Asp 1 5 <![CDATA[ <210> 51]]> <![CDATA[ <211> 13]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(13)]]> <![CDATA[ <223> CPP12]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (2)..(2)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> X-series Nal2]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (5)..(5)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (7)..(7)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 51]]> Phe Phe Xaa Arg Arg Arg Arg Gln Gly Ala Asx Ala Lys 1 5 10 <![CDATA[ <210> 52]]> <![CDATA[ <211> 13]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(13)]]> <![CDATA[ <223> CPP12]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (2)..(2)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> X-series Nal2]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (5)..(5)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (7)..(7)]]> <![CDATA[ <223> D amino acids]]> <![CDATA[ <400> 52]]> Phe Phe Xaa Arg Arg Arg Arg Gln Gly Ala Asx Ala Lys 1 5 10 <![CDATA[ <210> 53]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(4)]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> X series E or S]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> X series L, T, V, R or D]]> <![CDATA[ <400> 53]]> Xaa Thr Xaa Val 1 <![CDATA[ <210> 54]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> X series K, V or I]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (2)..(2)]]> <![CDATA[ <223> X series E or S]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (4)..(4)]]> <![CDATA[ <223> X series L, T, V, R or D]]> <![CDATA[ <400> 54]]> Xaa Xaa Thr Xaa Val 1 5 <![CDATA[ <210> 55]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(4)]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> X series E or S]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (3)..(3)]]> <![CDATA[ <223> X series L, T, V, R or D]]> <![CDATA[ <400> 55]]> Xaa Thr Xaa Val 1 <![CDATA[ <210> 56]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> X is K, V, or I. <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (2)..(2)]]> <![CDATA[ <223> X series E or S]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (4)..(4)]]> <![CDATA[ <223> X series L, T, V, R or D]]> <![CDATA[ <400> 56]]> Xaa Xaa Thr Xaa Val 1 5 <![CDATA[ <210> 57]]> <![CDATA[ <211> 8]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(8)]]> <![CDATA[ <223> L-Arg8]]> <![CDATA[ <400> 57]]> Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 58]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(7)]]> <![CDATA[ <223> L-Arg7]]> <![CDATA[ <400> 58]]> Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 59]]> <![CDATA[ <211> 6]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(6)]]> <![CDATA[ <223> L-Arg6]]> <![CDATA[ <400> 59]]> Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 60]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <223> L-Arg5]]> <![CDATA[ <400> 60]]> Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 61]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(4)]]> <![CDATA[ <223> L-Arg4]]> <![CDATA[ <400> 61]]> Arg Arg Arg Arg 1 <![CDATA[ <210> 62]]> <![CDATA[ <211> 8]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(8)]]> <![CDATA[ <223> L-Arg8]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 62]]> Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 63]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(7)]]> <![CDATA[ <223> L-Arg7]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 63]]> Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 64]]> <![CDATA[ <211> 6]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(6)]]> <![CDATA[ <223> L-Arg6]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 64]]> Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 65]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <223> L-Arg5]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 65]]> Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 66]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(4)]]> <![CDATA[ <223> L-Arg4]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> N-terminal acetylation]]> <![CDATA[ <400> 66]]> Arg Arg Arg Arg 1 <![CDATA[ <210> 67]]> <![CDATA[ <211> 8]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(8)]]> <![CDATA[ <223> L-Arg8]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 67]]> Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 68]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(7)]]> <![CDATA[ <223> L-Arg7]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 68]]> Arg Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 69]]> <![CDATA[ <211> 6]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(6)]]> <![CDATA[ <223> L-Arg6]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 69]]> Arg Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 70]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(5)]]> <![CDATA[ <223> L-Arg5]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkyl hydrocarbon (CA). <![CDATA[ <400> 70]]> Arg Arg Arg Arg Arg 1 5 <![CDATA[ <210> 71]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic peptides <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(4)]]> <![CDATA[ <223> L-Arg4]]> <![CDATA[ <220> ]]> <![CDATA[ <221> MISC_FEATURE]]> <![CDATA[ <222> (1)..(1)]]> <![CDATA[ <223> The N-terminus binds to a chloroalkane (CA). <![CDATA[ <400> 71]]> Arg Arg Arg Arg 1

[0167]

Claims

1. A PSD-95 inhibitor comprising: a. a first peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO: 53), wherein X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R and D; b. a second peptide (P2) comprising or consisting of the amino acid sequence X5TX6V (SEQ ID NO: 55), wherein X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R and D; and c. a cell-penetrating peptide (CPP) selected from the group consisting of: i. a poly-L-arginine peptide (poly-Arg) consisting of 3 to 9 L-arginine residues; ii. yGrkkrrqrrr (SEQ ID NO: 9, D-TAT); iii. RQIKIWFQNRRMKWKK (SEQ ID NO: 14, L-Pen); iv. rqikiwfqnrrmkwkk (SEQ ID NO: 15, D-Pen); v. RKKRRRESRKKRRRES (SEQ ID NO: 17, L-DPV3); vi. LLIILRRRIRKQAHAHSK (SEQ ID NO: 18, L-pVEC); vii. KLALKLALKALKAALKLA (SEQ ID NO: 16, L-MAP); viii. PLIYLRLLRGQF (SEQ ID NO: 19, L-TP2); ix. |(Dap)KAPETALD| (SEQ ID NO: 21, MiniAp4); and x. |Ff(Nal2)RrRrQ|GABA-K (SEQ ID NO: 22, CPP12), The CPP is connected to the linker via its C-terminus, and P1 and P2 are bound to the linker via their N-terminus, and the PSD-95 inhibitor has the general structure of formula (I): Formula (I) or a pharmaceutically acceptable salt thereof.

2. The PSD-95 inhibitor of claim 1, wherein P1 comprises or consists of the sequence X1X2TX3V (SEQ ID NO: 54), wherein X1 is selected from the group consisting of K, V and I; X2 is selected from the group consisting of E and S; and X3 is selected from the group consisting of L, T, V, R and D; and wherein P2 comprises or consists of the sequence X4X5TX6V (SEQ ID NO: 56), wherein X4 is selected from the group consisting of K, V and I; X5 is selected from the group consisting of E and S; and X6 is selected from the group consisting of L, T, V, R and D.

3. The PSD-95 inhibitor of claim 1, wherein P1 and / or P2 are composed of 4 to 10 amino acid residues.

4. The PSD-95 inhibitor of any one of the claims 1 to 3, wherein X3 and X6 are selected from the group consisting of L, T, V and R.

5. A PSD-95 inhibitor as claimed in any one of claims 1 to 3, wherein the linker comprises one or more PEG units, wherein at least one oxygen atom of one of the PEG units is replaced by a nitrogen atom to obtain NPEG, and wherein the CPP is linked to the nitrogen atom of the linker by a amide bond, and the PSD-95 inhibitor has the general structure of formula (III): Formula (III) where p is an integer from 0 to 10; and q is an integer from 0 to 10.

6. The PSD-95 inhibitor of any one of claims 1 to 3, wherein P1 and P2 are composed of the amino acid sequences KETLV (SEQ ID NO: 2), KETTV (SEQ ID NO: 3), KETVV (SEQ ID NO: 4), KETRV (SEQ ID NO: 5), ISTDV (SEQ ID NO: 6), or VETVV (SEQ ID NO: 7).

7. A PSD-95 inhibitor as claimed in any of claims 1 to 3, wherein the PSD-95 inhibitor has a general structure of formula (VI): Formula (VI) wherein X1 is selected from the group consisting of K, V and I; X2 is selected from the group consisting of E and S; and X3 is selected from the group consisting of L, T, V, R and D; X4 is selected from the group consisting of K, V and I; X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R and D; p is an integer from 0 to 10; q is an integer from 0 to 10; and poly-Arg is a poly-L-arginine peptide consisting of 3 to 9 L-arginine residues.

8. The PSD-95 inhibitor of any one of claims 1 to 3, wherein the CPP is RRRRRRRRR (SEQ ID NO: 12), RRRRRRRR (SEQ ID NO: 57), RRRRRRR (SEQ ID NO: 58), RRRRRR (SEQ ID NO: 59), RRRRR (SEQ ID NO: 60), RRRR (SEQ ID NO: 61) or RRR.

9. The PSD-95 inhibitor as requested in item 5, wherein p is 2 and q is 2.

10. The PSD-95 inhibitor as requested in item 7, wherein p is 2 and q is 2.

11. A PSD-95 inhibitor of any one of claims 1 to 3, wherein the N-terminus of the CPP is acetylated or wherein the N-terminus of the CPP is bound to a chloroalkyl tag (CA), having the following structure:

12. The PSD-95 inhibitor of claim 1, wherein the PSD-95 inhibitor has a general structure of formula (XXVIII): Formula (XXVIII) wherein P1, P2 and CPP are: and wherein the N-terminus of the CPP is acetylated as appropriate.

13. The PSD-95 inhibitor of claim 1, wherein the PSD-95 inhibitor is selected from the group consisting of formulas (X) to (XXIII):

14. A pharmaceutical composition comprising a therapeutically effective amount of any one of claims 1 to 13 of a PSD-95 inhibitor.

15. Use of a PSD-95 inhibitor as claimed in any one of claims 1 to 13, wherein the inhibitor is used to prepare a medicament for treating, preventing, alleviating excitotoxic-related diseases and / or delaying their development.

16. As claimed in claim 15, wherein the excitotoxic-related disease is a stroke.

17. As requested in claim 16, wherein the stroke is an acute ischemic stroke or subarachnoid hemorrhage.

Images