EP4633680A1

EP4633680A1 - Thyclotide peptide conjugates with cell permeability and inhibitory activity

Info

Publication number: EP4633680A1
Application number: EP23828316.2A
Authority: EP
Inventors: Daniel H. Appella; Hongchao Zheng; Victor CLAUSSE; Harsha C. AMARASEKARA
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 2022-12-13
Filing date: 2023-12-07
Publication date: 2025-10-22
Also published as: WO2024129497A1

Abstract

Disclosed are thyclotide-peptide conjugates of the formula (I): Tn-L-P (I), wherein T is a thyclotide unit of the formula:, wherein the thyclotide units are optionally intercepted by segments of polypeptide which can be the same as or different from P, n is about 5 to about 25, L is a linker, P is a polypeptide, and B is a nucleobase or a heterocyclyl moiety. Also disclosed are pharmaceutical compositions and methods of treating diseases such as cancers by administering an effective amount of a thyclotide-peptide conjugate.

Description

Leydig 769584 HHS E-033-2023-0-PC-01 1 THYCLOTIDE PEPTIDE CONJUGATES WITH CELL PERMEABILITY AND INHIBITORY ACTIVITY STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0001] This invention was made with Government support under Grant Number DK03113 awarded by the Intramural Research Program of NIDDK. The Government has certain rights in this invention. CROSS-REFERENCE TO A RELATED APPLICATION [0002] This patent application claims the benefit of U.S. Provisional Patent Application No.63/387131, filed December 13, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY [0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 2,175 Byte XML file named “769584.xml,” created on December 6, 2023. BACKGROUND OF THE INVENTION [0004] Peptides are oligomeric molecules consisting of amino acids found in proteins as well as other non-natural amino acids. Peptides are often developed as inhibitors of enzymes and protein-protein interactions with a view to develop cure for many kinds of diseases and disorders including cancers. However, peptides typically are poor drugs as they do not penetrate cell membranes and therefore have low bioactivity in vivo. This limitation often prevents peptides from being developed into a therapeutic drug. [0005] Since peptides often display very good biological activities, many strategies have been examined to improve the cellular uptake of peptides, such as cyclization of a linear peptide into a macrocyclic ring, methylation of selected amide bonds within the peptide sequence, and introduction of a hydrocarbon staple that links sidechains within the peptide. None of these approaches guarantees cellular uptake of a peptide, but there are selected cases where cellular uptake may be improved when using one or more of these strategies (Methods Mol. Biol., 2015, v.1266, p.29-53). The subset of peptides that are able to cross a cell Leydig 769584 HHS E-033-2023-0-PC-01 2 membrane commonly have a high number of charged amino acids, namely lysine and arginine, in their sequences. These cell penetrating peptides (CPPs) are typically partial sequences of proteins that are found in the venoms of snakes, scorpions, and spiders. Therefore, CPPs often have some degree of toxicity which limits their development into a therapeutic drug. (J Pep. Sci., 2021, v.27, e3300). [0006] In 2016, Petros and coworkers reported the development of an eight residue peptide that is a good inhibitor of the enzyme SETD8 in vitro. (ACS Med Chem Lett, 2016, v.7, 1102-1106). This enzyme is a histone lysine methyltransferase, and overexpression of SETD8 has been observed in a number of different cancers. Therefore, inhibitors of SETD8 may be promising for future therapeutics to treat cancer patients. No cell based activity or in vivo activity was reported for the Petros peptide. The development of other small molecule inhibitors of SETD8 have not proceeded successfully as the most potent small molecule inhibitors have only modest activity. [0007] The foregoing shows that there exists an unmet need for drug molecules embodying peptides that can penetrate cell membranes and, as a result, be effective in treating diseases and disorders, in particular cancers. BRIEF SUMMARY OF THE INVENTION [0008] The present invention provides a thyclotide-peptide conjugate of the formula (I): Tn-L-P (I), wherein T is a thyclotide unit of the formula: , wherein the configuration of the tetrahydrofuranyl ring the two nitrogen atoms can be (R,R) and/or (S,S), and wherein the thyclotide units are optionally intercepted by segments of polypeptide which can be the same as or different from P, n is about 5 to about 25, L is a linker, P is a polypeptide, and B is a nucleobase or a heterocyclyl moiety, or a pharmaceutically acceptable salt thereof. [0009] The present invention further provides pharmaceutical compositions containing one or more of the thyclotides, as well as method of treating diseases and disorders in a human or animal by the administration of one or more of these conjugates. Leydig 769584 HHS E-033-2023-0-PC-01 3 [0010] The thyclotide peptide of the present invention show reduced or no aggregation and advantageously penetrate the cell membranes, and therefore have advantageous bioavailability, which results in efficacious treatment of diseases many of which have been considered intractable heretofore. These and other advantages of the invention are disclosed herein. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [0011] FIG.1 illustrates the structures of (S,S) and (R,R) thyclotides in accordance with an aspect of the invention. [0012] FIG.2A-2C display FACS (fluorescence activated cell sorting analysis) data of three replicate experiments, showing that the thyclotide-peptide conjugate molecule 3 was able to enter cells while the control peptide (molecule 1) and aegPNA-peptide conjugate (molecule 2) could not. FIG.2D displays the statistical analysis of the mean FACS cell uptake data. [0013] FIG.3A-3C display fluorescence microscopy data showing that the thyclotide- peptide conjugate 3 is able to enter the nuclei of cells. [0014] FIG.4 displays a series of Western blots that use an antibody to detect a methylated form of the protein p53 (p53K382Me) showing that the thyclotide-peptide conjugate 6 was able to inhibit the SETD8 enzyme in cells while the peptide along (molecule 4) had no activity. [0015] FIG.5 displays the change in cell proliferation as a function of concentration of thyclotide-peptide conjugate 6. [0016] FIG.6 depicts the HPLC chromatogram of peptide 1. [0017] FIG.7 depicts the mass spectrum of peptide 1. [0018] FIG.8 depicts the HPLC chromatogram of thyclotide-peptide conjugate 2. [0019] FIG.9 depicts the mass spectrum of thyclotide-peptide conjugate 2. [0020] FIG.10 depicts the HPLC chromatogram of thyclotide-peptide conjugate 3. [0021] FIG.11 depicts the mass spectrum of thyclotide-peptide conjugate 3. [0022] FIG.12 depicts the HPLC chromatogram of peptide 4. [0023] FIG.13 depicts the mass spectrum of peptide 4. [0024] FIG.14 depicts the HPLC chromatogram of thyclotide-peptide conjugate 5. [0025] FIG.15 depicts the mass spectrum of thyclotide-peptide conjugate 5. [0026] FIG.16 depicts the HPLC chromatogram of thyclotide-peptide conjugate 6. Leydig 769584 HHS E-033-2023-0-PC-01 4 [0027] FIG.17 depicts the mass of thyclotide-peptide conjugate 6. [0028] FIG.18 depicts the HPLC chromatogram of peptide 7. [0029] FIG.19 depicts the mass spectrum of peptide 7. [0030] FIG.20 depicts the structures of peptide 1. [0031] FIG.21 depicts the structure of thyclotide-peptide 2. The molecule is shown split into two parts at the vertical dashed line for the sake of illustration. [0032] FIG.22 depicts the structure of thyclotide-peptide 3. The molecule is shown split into two parts at the vertical dashed line for the sake of illustration. [0033] FIG.23 depicts the structure of peptide 4. [0034] FIG.24 depicts the structure of thyclotide-peptide 5. The molecule is shown split into two parts at the vertical dashed line for the sake of illustration. [0035] FIG.25 depicts the structure of thyclotide-peptide 6. The molecule is shown split into two parts at the vertical dashed line for the sake of illustration. [0036] FIG.26 depicts the structure of peptide 7. [0037] FIG.27 depicts the structures of peptide 8. The molecule is shown split into two parts at the vertical dashed line for the sake of illustration. [0038] FIG.28 depicts the structures of peptide 9. The molecule is shown split into two parts at the vertical dashed line for the sake of illustration. [0039] FIG.29 depicts the FACS data for peptide 8. [0040] FIG.30 depicts the Western blot data for peptide 8, showing that the peptide suppresses the activity of the target enzyme, Setd8, in SK-N-AS cells which are derived from neuroblastoma. DETAILED DESCRIPTION OF THE INVENTION [0041] The present invention provides thyclotide-peptide conjugates represented by the formula (I): T_n-L-P (I), wherein T is a thyclotide unit of the formula: , wherein the configuration of the tetrahydrofuranyl ring carbon nitrogen atoms can be (R,R) and/or (S,S), and wherein the thyclotide units are optionally intercepted by segments of polypeptide which can be the same as or different from P, Leydig 769584 HHS E-033-2023-0-PC-01 5 n is about 5 to about 25, L is a linker, P is a polypeptide, and B is a nucleobase or a heterocyclyl moiety, or a pharmaceutically acceptable salt thereof. [0042] Preparation and characterization of some thyclotides are reported in WO 2021/211786 A1. [0043] In an aspect of the present invention, n of the thyclotide-peptide conjugate or salt can be from about 8 to about 20, for example, about 10 to about 15. [0044] The nucleobase B of the thyclotide-peptide conjugate or salt can be any of adenosine, guanosine, cytosine, thymine, and uracil. Numerous nucleobases of non-natural nucleotides are known in the art. Some non-limiting examples of nucleobases of non-natural nucleotides include, for example, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, and pseudouridine. A readily available reference for modified RNAs and nucleobases and nucleotides contained therein is available. See Cantara, W.A. et al., Nucleic Acids Research, Vol.39, Database issue D195– D201 (2011) and references described therein. [0045] The term “heterocyclyl”, “heterocycle”, or “heterocyclic” as used herein, means a monocyclic heterocyclic or a bicyclic heterocyclic group. The monocyclic heterocyclic is a three-, four-, five-, six- or seven-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, N(H) and S. The three- or four- membered ring can contain zero or one double bond and a heteroatom selected from the group consisting of O, N, N(H) and S. The five-membered ring can contain zero or one double bond, and one, two or three heteroatoms selected from the group consisting of O, N, N(H) and S. The six-membered ring can contain zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N, N(H) and S. The seven- membered ring can contain zero, one, two, or three double bonds and one, two or three heteroatoms selected from the group consisting of O, N, N(H) and S. The monocyclic heterocycle can be unsubstituted or substituted and is connected to the parent molecular moiety through any substitutable carbon atom or any substitutable nitrogen atom contained within the monocyclic heterocycle. [0046] Examples of heterocyclyl groups include pyridyl, piperidinyl, piperazinyl, pyrazinyl, pyrolyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolidinyl, furanyl, Leydig 769584 HHS E-033-2023-0-PC-01 6 tetrahydrofuranyl, thiophenyl, purinyl, pyrimidinyl, thiazolyl, thiazolidinyl, thiazolinyl, oxazolyl, triazolyl, tetrazolyl, tetrazinyl, benzoxazolyl, morpholinyl, thiomorpholinyl, quinolinyl, and isoquinolinyl, preferably tetrazolyl. [0047] Any of the heterocyclyl group can be optionally substituted with one or more substituents, which can be electron withdrawing and/or electron donating or hydrophilic and/or hydrophilic. [0048] Examples of substituents on the heterocyclyl groups include halo, hydroxy, carboxy, phosphoryl, phosphonyl, phosphono C1-C6 alkyl, carboxy C1-C6 alkyl, dicarboxy C1-C6 alkyl, dicarboxy halo C1-C6 alkyl, sulfonyl, cyano, nitro, alkoxy, alkylthio, acyl, acyloxy, thioacyl, acylthio, aryloxy, amino, aminoalkyl, alkylamino, dialkylamino, trialkylamino, guanidino, aldehydo, ureido, aminocarbonyl, a cationic group, and an anionic group. [0049] The linker L of the thyclotide-peptide conjugate or salt can be a linear or cyclic linker. In an aspect, the linker is a linear linker. Examples of linear linkers include linkers comprising an alkylenedioxy group, an amido group, or both alkylenedioxy and amido groups. Examples of cyclic linkers include linkers comprising a cyclic aliphatic linker, a cyclic aromatic linker, and/or a cyclic alicyclic linker. [0050] As will be readily apparent to the ordinarily skilled person, the thyclotide unit has two chiral carbon atoms at positions 3 and 4 of the tetrahydrofuran ring: B O . [0051] The tetrahydrofuran the two chiral centers can form four diastereomeric pairs: (3R,4R), (3R,4S), (3S,4R), and (3S,4S). As drawn, the monomeric subunit has the (3R,4R) configuration. When the monomer has the (3R,4R) configuration, the monomer induces a right-handed helix into the PNA, which matches the right-handed helicity of DNA and RNA, thereby allowing for complex formation (by Watson-Crick binding) of the conjugate and DNA or RNA. [0052] In an aspect, P of the thyclotide-peptide conjugate or salt is of the formula: Leydig 769584 HHS E-033-2023-0-PC-01 7 , wherein R is a side the peptide can be anywhere from 1 amino acid to up to about 30 amino acids, preferably from about 3 to about 25 amino acids, more preferably from about 5 to about 20 amino acids, and particularly preferably from about 8 to about 18 amino acids. [0053] An example of a thyclotide-peptide conjugate is 6, which is of the formula: . [0054] Further examples of the thyclotide-peptide conjugate according to formula (I) include compounds 8 and 9 having the following structures:

Leydig 769584 HHS E-033-2023-0-PC-01 8 wherein the vertical dashed lines indicate the point of break for illustration purposes. [0055] The present invention further provides a pharmaceutical composition comprising the thyclotide-peptide conjugate or salt as described above and a pharmaceutically acceptable carrier. [0056] Examples of anticancer peptides are known in the art, for example, Mellitin (active against human and leukemic cells), Polybia-MP1 (active against human prostate cancer cells and bladder cancer cells), NRC-03 and NRC-07 (active against human breast Leydig 769584 HHS E-033-2023-0-PC-01 9 cancer cells and murine mammary carcinoma cells), D-peptides A, B, C, and D (active against human lung, cervix, glioma cancer cell, and mouse myeloma cells), Magainin 2 (active against human bladder cancer cells), Gomesin (active against human colon, breast, and cervix adenocarcinoma cells), SVS-1 (active against human epidermis, leukemia, and various carcinomas), Lactoferrici B (active against human fibrosarcoma), LL-37 (active against human squamous cell carcinoma and leukemic cells), LTX-315 (active against human osteosarcoma), and Pardaxin (active against human squamous cell carcinoma cell). Any of these peptides or segments thereof, or combinations thereof, can be employed to make the thyclotide-peptide conjugate of the present invention. [0057] The thyclotide-peptide conjugates of the present invention can be made by any suitable methods known to those skilled in the art, for example, by the use of “click chemistry” based “click reactions.” Examples of click reactions include cycloadditions such as 1,3-dipolar cycloadditions and hetero-Diels-Alder cycloadditions; nucleophilic ring opening reactions which involve openings of strained heterocyclic electrophiles such as aziridines, epoxides, cyclic sulfates, aziridinium ions, and episulfonium ions; and carbonyl chemistry of the non-aldol type such as the formation of ureas, thioureas, hydrazones, oxime ethers, and aromatic heterocycles; and additions to carbon-carbon multiple bonds including epoxidations, aziridinations, dihydroxylations, sulfonyl halide additions, nitrosyl halide additions, and certain Michael additions. Cycloadditions include Cu1 catalyzed Huisgen 1,3- dipolar cycloadditions of azides and alkynes as illustrated herein: . to include non- toxic salts synthesized from the parent conjugate which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these conjugates with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p.1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977). Leydig 769584 HHS E-033-2023-0-PC-01 10 [0059] Suitable bases for forming salts include inorganic bases such as alkali and alkaline earth metal bases, such as those containing metallic cations such as sodium, potassium, magnesium, calcium and the like. Non-limiting examples of suitable bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate. Suitable acids for forming salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p- toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, maleic acid, tartaric acid, fatty acids, long chain fatty acids, and the like. Preferred pharmaceutically acceptable salts of inventive conjugates having an acidic moiety include sodium and potassium salts. Preferred pharmaceutically acceptable salts of inventive conjugates having a basic moiety (such as a dimethylaminoalkyl group) include hydrochloride and hydrobromide salts. The conjugates of the present invention containing an acidic or basic moiety are useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof. [0060] It should be recognized that the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. [0061] It is further understood that the above conjugates and salts may form solvates, or exist in a substantially uncomplexed form, such as the anhydrous form. As used herein, the term “solvate” refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice. When the solvent incorporated in the solvate is water, the molecular complex is called a hydrate. Pharmaceutically acceptable solvates include hydrates, alcoholates such as ethanolates, acetonitrilates and the like. These conjugates can also exist in polymorphic forms. [0062] The conjugates and salts thereof of the present invention can be prepared by any suitable method, in particular as disclosed herein. [0063] The present invention further provides a pharmaceutical composition comprising the thyclotide-peptide conjugate or salt and a pharmaceutically acceptable carrier. It is preferred that the pharmaceutically acceptable carrier be one that is chemically inert to the active conjugates and one that has no detrimental side effects or toxicity under the conditions of use. Leydig 769584 HHS E-033-2023-0-PC-01 11 [0064] The choice of carrier will be determined in part by the particular conjugate or salt of the present invention chosen, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, nasal, pulmonary, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intratumoral, topical, rectal, and vaginal administration are merely exemplary and are in no way limiting. [0065] The pharmaceutical composition can be administered parenterally, such as intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration that comprise a solution or suspension of the inventive conjugate or salt dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous isotonic sterile injection solutions. [0066] Overall, the requirements for effective pharmaceutical carriers for parenteral compositions are well known to those of ordinary skill in the art. Such solutions can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The conjugate or salt of the present invention may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. [0067] Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Leydig 769584 HHS E-033-2023-0-PC-01 12 [0068] Suitable soaps for use in formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof. [0069] The parenteral formulations can contain preservatives and buffers. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. [0070] Topical formulations, including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the invention for application to skin. Topically applied compositions are generally in the form of liquids, creams, pastes, lotions and gels. Topical administration includes application to the oral mucosa, which includes the oral cavity, oral epithelium, palate, gingival, and the nasal mucosa. In some aspects, the composition contains at least one active component and a suitable vehicle or carrier. It may also contain other components, such as an anti-irritant. The carrier can be a liquid, solid or semi-solid. In aspects, the composition is an aqueous solution. Alternatively, the composition can be a dispersion, emulsion, gel, lotion or cream vehicle for the various components. In one aspect, the primary vehicle is water or a biocompatible solvent that is substantially neutral or that has been rendered substantially neutral. The liquid vehicle can include other materials, such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain Leydig 769584 HHS E-033-2023-0-PC-01 13 the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved in water or a biocompatible solvent prior to use to form a solution that is substantially neutral or that has been rendered substantially neutral and that can then be applied to the target site. In aspects of the invention, the vehicle for topical application to the skin can include water, buffered solutions, various alcohols, glycols such as glycerin, lipid materials such as fatty acids, mineral oils, phosphoglycerides, collagen, gelatin and silicone based materials. [0071] Formulations suitable for oral administration can consist of (a) liquid solutions, such as a therapeutically effective amount of the inventive conjugate or salt thereof dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules, (c) powders, (d) suspensions in an appropriate liquid, and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art. [0072] The conjugate or salt of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. The conjugates or salts are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of active conjugate are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such surfactants are the esters or partial esters of fatty acids Leydig 769584 HHS E-033-2023-0-PC-01 14 containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25%-5%. The balance of the composition is ordinarily propellant. A carrier can also be included as desired, such as lecithin for intranasal delivery. These aerosol formulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations may be used to spray mucosa. [0073] Additionally, the conjugate or salt of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. [0074] It will be appreciated by one of ordinary skill in the art that, in addition to the above described pharmaceutical compositions, the conjugate or salt of the present invention may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes serve to target the conjugates to a particular tissue, such as lymphoid tissue or cancerous hepatic cells. Liposomes can also be used to increase the half-life of the inventive conjugate. Liposomes useful in the present invention include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the active agent to be delivered is incorporated as part of a liposome, alone or in conjunction with a suitable chemotherapeutic agent. Thus, liposomes filled with a desired inventive conjugate or salt thereof, can be directed to the site of a specific tissue type, hepatic cells, for example, where the liposomes then deliver the selected compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, for example, liposome size and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980), and U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369. For targeting to the cells of a particular tissue type, a ligand to be incorporated into the liposome can include, for example, antibodies or fragments thereof Leydig 769584 HHS E-033-2023-0-PC-01 15 specific for cell surface determinants of the tissue type. A liposome suspension containing a conjugate or salt of the present invention may be administered intravenously, locally, topically, etc. in a dose that varies according to the mode of administration, the agent being delivered, and the stage of disease being treated. Encapsulation formulations including liposome formulations are well suited for use in oral administration of the inventive oligomer, optionally further comprising permeation enhancers such as sodium caprate (see, e.g., van Putten, M. et al, Mol Ther Nucleic Acids, Nov; 3(11): e211 (2014). [0075] The conjugates or salts thereof can be used in any suitable dose. Suitable doses and dosage regimens can be determined by conventional range finding techniques. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose. Thereafter, the dosage is increased by small increments until optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. In proper doses and with suitable administration of certain conjugates, the present invention provides for a wide range of responses. Typically, the dosages range from about 0.001 to about 1000 mg/kg body weight of the animal being treated/day. For example, in aspects, the conjugates or salts may be administered from about 100 mg/kg to about 300 mg/kg, from about 120 mg/kg to about 280 mg/kg, from about 140 mg/kg to about 260 mg/kg, from about 150 mg/kg to about 250 mg/kg, from about 160 mg/kg to about 240 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. [0076] The present invention further provides a method of treating an animal afflicted with a disease or disorder comprising administering to the animal an effective amount of a thyclotide-peptide conjugate or salt or a pharmaceutical composition as described herein. In an aspect, the thyclotide-peptide conjugate or salt thereof is administered intravenously or subcutaneously. [0077] In an aspect of the invention, the disease or disorder is cancer. Any suitable can be treated. In particular, the cancer is selected from the group consisting of leukemia, adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, brain cancer, basal cancer of the skin, bile duct cancer, Ewing Sarcoma, bladder cancer, lung cancer, breast cancer, gastrointestinal cancer, central nervous system cancer, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasm, colorectal cancer, T-cell lymphoma, uterine cancer, esophageal cancer, head and neck cancer, Leydig 769584 HHS E-033-2023-0-PC-01 16 eye cancer, gallbladder cancer, stomach germ cell tumor, hepatocellular cancer, Hodgkin’s lymphoma, pancreatic tumor, kidney cancer, liver cancer, melanoma, mouth cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, thyroid cancer, parathyroid cancer, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, skin cancer, vaginal cancer, and combinations thereof, and more particularly neuroblastoma. [0078] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLE 1 [0079] This example illustrates methods of preparation of peptides, thyclotides, peptide- PNA conjugates and thyclotide-peptide conjugates in accordance with aspects of the invention. [0080] Reagents and Materials: All 9-fluorenylmethoxycarbonyl (Fmoc)-PNA monomers were purchased from PolyOrg, Inc. (Leominster, MA, USA). Acetonitrile, Fmoc-Lys(Boc)-OH, acetic anhydride (Ac2O), pyridine, dichloromethane (DCM), 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIPEA), diethyl ether (Et2O), N,N-dimethylformamide (DMF), Kaiser test reagents, m-cresol, N-methyl-2-pyrrolidinone (NMP), piperidine, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Fmoc-Val-OH and Fmoc-Leu-OH were purchased from Advanced ChemTech (Louisville, KY, USA). Fmoc-his(trityl)-OH, Fmoc-Nle-OH, and Fmoc- Lys(Boc)-OH were purchased from Sigma-Aldrich (St Louis, MO, USA). High purity water (18 MΩ) was generated from a Millipore (Billerica, MA, USA) Milli-Q water system. Rink Amide ProTide resin (LL, 100–200 mesh, 0.19 mmol/g) was purchased from CEM corporation (Matthews, NC, USA). NHS-Fluorescein (5/6-carboxyfluorescein succinimidyl ester, mixed isomer) was purchased from ThermoFisher Scientific (Waltham, MA, USA). Fmoc-Arg(Pbf)-OH and Fmoc-8-amino-3,6-dioxaoctanoic acid were purchased from Peptides International, Inc. (Louisville, KY, USA). All Fmoc-protected thyclotide monomers were prepared based on a procedure reported in a manuscript by our group (Clausse V.; Zheng, H.; Amarasekara, H.; Kruhlak, M.; Appella, D. H. Nucleic Acids Res. 2022, 50, 10839–10856). Leydig 769584 HHS E-033-2023-0-PC-01 17 [0081] General Method for Peptide and thyclotide Conjugate Synthesis: Peptides, peptide-PNA conjugates, thyclotide-peptide conjugates, and thyclotides were prepared on 5 μmol scale using Fmoc-solid phase peptide synthesis protocols on an Intavis Multipep CF with HATU [O-(7-Azabenzotriazolyl-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate] as the amide-forming reagent. All peptides, peptide-PNA conjugates, peptide-thyclotide conjugates, and thyclotides were synthesized on Rink Amide ProTide resin. [0082] Labeling of Peptide (or Thyclotide-Peptide Conjugate) with NHS-Fluorescein: The resin, in a peptide synthesis vessel, was first washed with 20% piperidine in DMF (2 × 5 mL for 5 min) to deprotect the Fmoc group. The progress of the deprotection was followed by a qualitative Kaiser test. If the test was negative, the resin was resubmitted to additional deprotection. After a positive test for primary amines, the resin was drained and ready for use. To the resin was added a solution of NHS-Fluorescein (47 mg, 100 µmol) in anhydrous DMF (0.4 mL) and DIPEA (30 µL) at room temperature. The suspension was stirred at room temperature overnight. The next day, the resin was washed thoroughly with DMF (4 × 5.0 mL) followed by peptide (or peptide-thyclotide conjugate) cleavage from solid support using standard procedures (see below). [0083] Cleavage and Recovery of Crude Peptide and Thyclotide-Peptide Conjugate from Resin: The resin, in a peptide synthesis vessel, was first washed with 20% piperidine in DMF (2 × 5 mL for 5 min) to deprotect the Fmoc group. The progress of the deprotection was followed by a qualitative Kaiser test. If the test was negative, the resin was resubmitted to additional deprotection. After a positive test for primary amines, the resin was drained and treated with cleavage cocktail (2 mL, 5% m-cresol in TFA) for 1 hour. The cleavage mixture was collected in a glass vial using N2 pressure to drain the vessel. The resin was resubmitted to fresh cleavage cocktail and cleaved for 1 hour, and was drained into the first cleavage fraction. The volatiles were removed by flowing dry N2 over the solution to produce a yellow-brown oil. Approximately 10 mL of Et2O was added to the cleavage oil to create a suspended white precipitate. The suspension was partitioned into five 2 ml microcentrifuge tubes and chilled over dry ice for 10 minutes. The tubes were centrifuged at 12,000 r.p.m. for 40 seconds to produce a white pellet. Et₂O was carefully decanted, leaving the white crude peptide or peptide-thyclotide conjugate solid. Further washing was performed by adding about 1.6 mL of Et₂O to each tube, mixing to resuspend the precipitate, then chilling on dry ice for 5 minutes. Following centrifugation Leydig 769584 HHS E-033-2023-0-PC-01 18 and decanting, the washes were repeated twice without dry ice. After the final wash, the white precipitate was dried by carefully passing a stream of dry N2 over the crude peptides and peptide-thyclotide conjugates. [0084] Purification and Characterization of Crude Peptide (and Thyclotide-Peptide Conjugate): Purification was performed on an Agilent (Santa Clara, CA) 1260 Series RP- HPLC with automatic fraction collection using ultraviolet detection at 260 nm. Waters (Milford, MA, USA) XBridge C18 (10 × 250 mm, 5 μm) column was used in conjunction with Solvents A and B for purification at 45 ^oC. Waters (Milford, MA, USA) XBridge C18 (4.6 × 250 mm, 5 μm) column was used in conjunction with Solvents A and B for purity checking at room temperature. Solvent A was 0.05% TFA in water and Solvent B consisted of 90% acetonitrile in water. Peptide and thyclotide-peptide conjugate HPLC isolates were characterized using electrospray ionization-mass spectrometry on a Waters/Micromass LCT Premier time-of-flight mass spectrometer. The instrument was operated in W-mode at a nominal resolution of 10,000. The electrospray capillary voltage was 2 kV and the sample cone voltage was 60 V. The desolvation temperature was 275 °C and the desolvation gas was N₂ with a flow rate of 300 L h⁻¹. Accurate masses were obtained using the internal reference standard method. The sample was introduced into the mass spectrometer via the direct loop injection method. Deconvolution of multiply charged ions was performed with MaxEnt I. All peptides and thyclotide-peptide conjugates gave molecular ions consistent with the calculated theoretical product values. [0085] HPLC and MS Analyses of Peptides and Thyclotide-Peptide Conjugates HPLC Conditions: The column was equilibrated with 100% A (0% B) for 15 min prior to run, 0% B (100% A) for 2 min, a linear gradient from 0% B (100% A) to 100% B (0% A) over 18 min, 100% B (0% A) for 4 min, and a linear gradient to 0% B (100% A) over 1 min. The flow rate was 1.2 mL/min. EXAMPLE 2 [0086] This example illustrates the molecules prepared and tested and the results obtained in accordance with the present invention. Illustrative molecules are presented in Table 1. Molecules 1, 2, and 3 are designed to test for cell uptake. Each molecule had a fluorescent group (fluorescein (FI)) attached to the end. Molecules 4, 5, and 6 were used to test for biological activity. For the thyclotide and aegPNA portions of molecules 2, 3, 5, 6, and 7, thymine (T) nucleobase was used. Leydig 769584 HHS E-033-2023-0-PC-01 19 Table 1. Mass characterization data for thyclotides, peptide-PNA conjugates and thyclotide-peptide conjugates^a peptides, thyclotides, peptide-PNA conjugates and peptide- entry Calcd. Obserd thyclotide conjugates 1 1 0 0 7 FI = H]²⁺, [M+H+NH₄]²⁺ or triply charged ion [M+3H]³⁺. [0087] The Petros peptide was used as a negative control in molecules 1 and 4. The aegPNA-peptide conjugates are also negative controls, shown as molecules 2 and 5. The (S,S) THF-thyclotide with ten T residues is shown as molecule 7. The (S,S) THF-thyclotide- peptide conjugates are shown in molecule 2 (with the FI) and molecule 6 (which was used to test for its biological activity). [0088] FIG.2A-2D depicts that the SETD8 inhibitor peptide conjugated to a T₁₀- thyclotide showed a significantly better cell uptake than the inhibitor alone in SH-SY5Y neuroblastoma cells. (A) SH-SY5Y cells were either untreated (light gray), or treated with 5 μM of fluorescein-labelled SETD8 peptide inhibitor alone (molecule 1, solid line), aegPNA- inhibitor conjugate (molecule 2, dotted line), or thyclotide-inhibitor conjugate (molecule 3, black) for 3 hours at 37°C. Molecules were directly diluted in complete RPMI cell culture medium with 10% FBS. After 3 hours, cells were washed thrice with PBS, collected with Leydig 769584 HHS E-033-2023-0-PC-01 20 trypsin and analyzed on a FACS Canto II. The three pictures represent three independent replicates of the experiment. (B) Statistical analyses of FACS cell uptake data. Mean of fluorescence intensity was calculated with FlowJo v10 software for all samples, showing that only the peptide inhibitor-thyclotide conjugate (molecule 3) was able to efficiently penetrate cells (N=3). The results from the FACS data showing that the thyclotide-peptide conjugate molecule 3 was able to enter cells while the control peptide (molecule 1) and aegPNA- peptide conjugate (molecule 2) could not. [0089] As shown by FIG.2A-2D, the SETD8 inhibitor peptide conjugated to a T10- thyclotide (molecule 3) can enter the nuclei of SH-SY5Y neuroblastoma cells.3D volume reconstruction of the nuclei of SH-SY5Y cells treated with 5 μM of fluorescein-labelled SETD8 peptide inhibitor-thyclotide conjugate (molecule 3) for 16 hours at 37°C. Cells were fixed with 4% PFA then stained with 2 μg/mL Hoechst 33342 solution in PBS, and mounted on glass slides with Prolong glass mounting medium. Super-resolution images were acquired on a Nikon SoRa spinning disk microscope equipped with a 60x oil immersion objective lens and Photometrics BSI sCMOS camera. Z-stacks were collected using a 0.15 μm step size and images were generated using Imaris software v9.3. Surface negative for Hoechst staining were excluded to reconstruct the nucleus volume and remove all cytoplasmic signal (A). Fluorescein signal corresponding to the inhibitor-thyclotide conjugate (molecule 3) was analyzed only in the previously isolated nuclei surfaces (B) showing the presence of the molecule in the 3D-reconstructed nuclei of the cells. Finally, a single picture of the middle plane of the nuclei was taken to confirm the localization of the thyclotide inside the nuclei (C). [0090] The results in FIG.3A-3C display fluorescence microscopy data showing that the thyclotide-peptide conjugate molecule 3 was able to enter the nuclei of cells. This is important because the SETD8 enzyme is in the nucleus of cells, and therefore any molecule that will inhibit the SETD8 must be able to reach the nucleus. [0091] The data in FIG.4 represent a series of Western blots that used an antibody to detect a methylated form of the protein p53 (p53K382Me). The SETD8 enzyme will methylate the p53 protein at lysine 382. If the SETD8 enzyme is inhibited, then the amount of methylated p53 at lysine 382 will decrease. Treatment of the cells with molecule 6 resulted in a decrease in p53K382Me, while treatment with the peptide alone (molecule 4) showed no decrease in p53K382Me. Leydig 769584 HHS E-033-2023-0-PC-01 21 [0092] FIG.5 showed the following: The SETD8 inhibitor peptide conjugated to a T₁₀- thyclotide (molecule 3) can efficiently decrease SETD8-dependent p53 methylation on lysine 382. SH-SY5Y neuroblastoma cells were either untreated or treated with 5 μM of the SETD8 peptide inhibitor only (molecule 4), or 5 μM of the SETD8 inhibitor peptide conjugated to a T₁₀-thyclotide (molecule 6), for 24, 48, or 72 hours. There is no significant difference between untreated cells and cells treated with the peptide inhibitor (molecule 4) only. However, cells treated with the inhibitor conjugated to the thyclotide (molecule 6) showed significant decrease in p53 methylation in lysine 382, detected with a monoclonal antibody targeting the specific methylation on this amino acid residue. Hence, the thyclotide conjugated to the SETD8 inhibitor (molecule 6) can deliver the peptide inside the nucleus of the cell to allow inhibition of SETD8, preventing the SETD8-dependent methylation of p53. The data shown in FIG.5 reveal that the thyclotide-peptide conjugate molecule 6 was able to inhibit the SETD8 enzyme in cells while the peptide along (molecule 4) had no activity. [0093] FIG.5 depicts the following. The SETD8 inhibitor peptide conjugated to a T₁₀- thyclotide (molecule 6) prevents the proliferation of SH-SY5Y neuroblastoma cells.50,000 SH-SY5Y neuroblastoma cells were seeded in each wells of 24 well-plates and were either untreated or treated with 10 or 20 μM of the SETD8 inhibitor alone (molecule 4) or the inhibitor conjugated to the T10-thyclotide (molecule 6).3 days after the treatment, a CyQUANT proliferation assay was performed, showing that the conjugate (molecule 6) efficiently prevents the proliferation of the neuroblastoma cells, whereas the inhibitor (molecule 4) alone doesn’t show any significant difference compared with untreated cells. The data in Figure 4 show how the thyclotide-peptide conjugate molecule 6 can decrease proliferation of cancer cells while the peptide alone (molecule 4) does not show any effect. The ability to decrease cell proliferation shows there is biological activity that is consistent with the prior data showing that molecule 6 can enter cells, arrive at the nucleus, and inhibit the SETD8 enzyme. [0094] FIG.29 depicts the FACS data for peptide 8. Peak 1 depicts the untreated cell count, peak 2 depicts the cell count when treated with the peptide alone, peak 3 correspond to the cell count when treated with peptide 8, and peak 4 corresponds to the cell count when treated with a THF polynucleotide (6). The sharpness of peak 3 indicates less aggregation of the peptide having tetrazole moieties, which in turn translates to the conclusion that the tetrazole substituted peptide would be more effective as a drug molecule. Leydig 769584 HHS E-033-2023-0-PC-01 22 [0095] FIG.30 depicts the Western blot data for peptide 8, showing that the peptide suppresses the activity of the target enzyme, Setd8, in SK-N-AS cells which are derived from neuroblastoma. The target of methylation of Setd8 is p53, and the lowering of methylation in the + lane compared to the NT (not treated) lane shows that the peptide works for the intended purpose. A similar effect was not seen in the SH-SY5Y cells, which indicates that there could be some cell-to-cell variation. [0096] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0097] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [0098] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as Leydig 769584 HHS E-033-2023-0-PC-01 23 appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 769584 HHS E-033-2023-0-PC-01 24 CLAIM(S): 1. A thyclotide-peptide conjugate of the formula (I): Tn-L-P (I), wherein T is a thyclotide unit of the formula: , wherein the configuration of the tetrahydrofuranyl ring carbon nitrogen atoms can be (R,R) and/or (S,S), and wherein the thyclotide units are optionally intercepted by segments of polypeptide which can be the same as or different from P, n is about 5 to about 25, L is a linker, P is a polypeptide, and B is a nucleobase or a heterocyclyl moiety, or a pharmaceutically acceptable salt thereof. 2. The thyclotide-peptide conjugate or salt of claim 1, wherein n is about 8 to about 20. 3. The thyclotide-peptide conjugate or salt of claim 1 or 2, wherein n is about 10 to about 15. 4. The thyclotide-peptide conjugate or salt of any one of claims 1-3, wherein B is a nucleobase. 5. The thyclotide-peptide conjugate or salt of any one of claims 1-3, wherein B is a heterocyclyl moiety. 6. The thyclotide-peptide conjugate or salt of any one of claims 1-3 or claim 5, wherein the heterocyclyl moiety is selected from the group consisting of pyridyl, piperidinyl, piperazinyl, pyrazinyl, pyrolyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thiophenyl, tetrahydrothiophenyl, purinyl, pyrimidinyl, thiazolyl, thiazolidinyl, thiazolinyl, oxazolyl, triazolyl, tetrazolyl, tetrazinyl, benzoxazolyl, morpholinyl, thiomorpholinyl, quinolinyl, and isoquinolinyl, 7. The thyclotide-peptide conjugate or salt of claim 6, wherein the heterocyclyl moiety is tetrazolyl. Leydig 769584 HHS E-033-2023-0-PC-01 25 8. The thyclotide-peptide or salt of any one of claims 1-7, wherein L is linear or cyclic linker. 9. The thyclotide-peptide conjugate or salt of any one of claims 1-8, wherein L is a linear linker. 10. The thyclotide-peptide conjugate or salt of claim 9, wherein L comprises an alkylenedioxy groups, amido groups, or both alkylenedioxy and amido groups. 11. The thyclotide-peptide conjugate or salt of claim 1-7, wherein L is a cyclic linker. 12. The thyclotide-peptide conjugate or salt of claim 11, wherein the cyclic linker comprises a cyclic aliphatic linker, a cyclic aromatic linker, and/or a cyclic alicyclic linker. 13. The thyclotide-peptide conjugate or salt of any one of claims 1-12, wherein P is of the formula: , wherein R length of the peptide can be anywhere from 1 amino acid to up to 30 amino acids. 14. The thyclotide-peptide conjugate or salt of any one of claims 1-3 or 5-7, wherein the conjugate is of the formula: Leydig 769584 HHS E-033-2023-0-PC-01 26 . 15. The thyclotide-peptide conjugate or salt of any one of claims 1-3 or 5-7, which has the formula: Leydig 769584 HHS E-033-2023-0-PC-01 27 salt of any one of claims 1-15 and a pharmaceutically acceptable carrier. 17. A method of treating an animal afflicted with a disease or disorder comprising administering to the animal an effective amount of a thyclotide-peptide conjugate or salt of any one of claims 1-15 or a pharmaceutical composition of claim 16. 18. The method of claim 17, wherein the thyclotide-peptide conjugate or salt thereof is administered intravenously or subcutaneously. 19. The method of claim 17 or 18, wherein the disease or disorder is cancer. 20. The method of claim 19, wherein the cancer is selected from the group consisting of leukemia, adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, brain cancer, basal cancer of the skin, bile duct cancer, Ewing Sarcoma, bladder cancer, lung cancer, breast cancer, gastrointestinal cancer, central nervous system cancer, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasm, colorectal cancer, T-cell lymphoma, uterine cancer, esophageal cancer, head and neck cancer, eye cancer, gallbladder cancer, stomach cancer, germ cell tumor, hepatocellular cancer, Hodgkin’s lymphoma, pancreatic tumor, kidney cancer, liver cancer, melanoma, mouth cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, thyroid cancer, parathyroid cancer, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, skin cancer, vaginal cancer, and combinations thereof. 21. The method of claim 120, wherein the cancer is neuroblastoma. 22. Use of a thyclotide-peptide conjugate or salt of any one of claims 1-15 or a pharmaceutical composition of claim 16 in the manufacture of a medicament for the treatment of an animal afflicted with a disease or disorder. Leydig 769584 HHS E-033-2023-0-PC-01 28 23. The use of claim 22, wherein the disease or disorder is cancer, preferably one or more selected from the group consisting of leukemia, adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, brain cancer, basal cancer of the skin, bile duct cancer, Ewing Sarcoma, bladder cancer, lung cancer, breast cancer, gastrointestinal cancer, central nervous system cancer, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasm, colorectal cancer, T-cell lymphoma, uterine cancer, esophageal cancer, head and neck cancer, eye cancer, gallbladder cancer, stomach cancer, germ cell tumor, hepatocellular cancer, Hodgkin’s lymphoma, pancreatic tumor, kidney cancer, liver cancer, melanoma, mouth cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, thyroid cancer, parathyroid cancer, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, skin cancer, and vaginal cancer.