WO2023056368A1 - Cyclic peptides as non-hormonal male contraceptive agents and methods of use thereof - Google Patents

Cyclic peptides as non-hormonal male contraceptive agents and methods of use thereof Download PDF

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WO2023056368A1
WO2023056368A1 PCT/US2022/077278 US2022077278W WO2023056368A1 WO 2023056368 A1 WO2023056368 A1 WO 2023056368A1 US 2022077278 W US2022077278 W US 2022077278W WO 2023056368 A1 WO2023056368 A1 WO 2023056368A1
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arg
gly
seq
cyclo
phe
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PCT/US2022/077278
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French (fr)
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Maria L. DUFAU
Murugananth Kumar RAJU
Raghuveer KAVARTHAPU
Rajakumar ANBAZHAGAN
Sergio A. HASSAN
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2023056368A1 publication Critical patent/WO2023056368A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/16—Masculine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • cyclic peptides for inhibiting phosphorylation of GRTH.
  • cyclic peptides comprise the following formula: X 1 -X 2 -linker1-X 3 -X 4 -X 5 -X 6 -linker2, in which X 1 is amino acid residue F; X 2 is an amino acid residue selected from Q, A, I, V, and L; linker1 comprises one or two linker molecules individually selected from amino acid residue G, amino acid residue P, and a non-peptidic spacer molecule; X 3 is an amino acid residue selected from A, S, N, Q, C, and M; X 4 is an amino acid residue selected from F, R, H, K, S, Y, Q, and N; X 5 is an amino acid residue selected from R, H, and K; X 6 is an amino acid residue selected from R
  • linker2 may comprise 1 or 2 linker molecules.
  • X 1 may be replaced with an amino acid residue selected from Q, A, I, V, and L; and X 2 is replaced with F.
  • the non-peptidic spacer molecule is polyethylene glycol (PEG) or polyvinyl alcohol (PVA).
  • PEG polyethylene glycol
  • PVA polyvinyl alcohol
  • a cyclic peptide is provided having the formula: cyclo( 1 FAGXXXG 7 - AEEAc), where each X is individually chosen from any basic amino acid, optionally arginine (R), lysine (K), or histidine (H).
  • each X is arginine (R).
  • a cyclic peptide may comprise the following structure: Cyclic Peptide 0 (PEP0) (SEQ ID NO: 3, AEEAc) .
  • a cyclic peptide may comprise the following structure: Cyclic Peptide 1 (PEP1) (SEQ ID NO: 7, AEEAc) .
  • a cyclic peptide may comprise the following structure: Cyclic Peptide 2 (PEP2) (SEQ ID NO: 8, AEEAc) .
  • Non-limiting examples of cyclic peptides according to the present disclosure comprise: cyclo-(Phe- ⁇ -Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 3); cyclo-(Phe- ⁇ -Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 3); cyclo-(Phe- ⁇ -Ala-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 4); cyclo-(Phe- ⁇ -Ala-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 4); cyclo-( ⁇ -Phe-Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 5); cyclo-( ⁇ -Phe-Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID
  • the PEG molecule(s) in the exemplary cyclic peptides listed above is replaced by a linker molecule, for example, (2-(2-aminoethoxy)ethoxy)acetic acid (AEEAc), for (PEG2), a combination of PEG and Gly, or an intercalations of Pro residues.
  • AEEAc (2-(2-aminoethoxy)ethoxy)acetic acid
  • PEG2 (2-(2-aminoethoxy)ethoxy)acetic acid
  • one or more of the amino acid residues in the cyclic peptide are omitted or, alternatively, replaced with one or more non-peptidic spacer molecules, optionally polyethylene glycol (PEG), polyvinyl alcohol, or (2-(2-aminoethoxy)ethoxy)acetic acid (AEEAc).
  • one or more side chains are attached at one or more glycine residue within the cyclic peptide.
  • one or more amino acid residues within the cyclic peptide are methylated.
  • the cyclic peptide is linked through head-to-tail cyclization.
  • the cyclic peptide inhibits gonadotropin regulated testicular helicase (GRTH) phosphorylation.
  • a composition is also provided comprising one or more cyclic peptide of the present disclosure.
  • the composition comprises 1 mg to 1 g of the cyclic peptide.
  • the composition is a pharmaceutical composition.
  • the composition further comprises diluents, binders, stabilizers, buffers, salts, solvents, preservatives, or combinations thereof.
  • the composition is formulated for oral administration.
  • a composition for inhibiting spermatogenesis comprising an effective amount of one or more cyclic peptide of the present disclosure.
  • a composition for inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation comprising an effective amount of one or more cyclic peptide of the present disclosure is provided.
  • An oral contraceptive comprising one or more cyclic peptide of the present disclosure is also provided.
  • a method of inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation comprising administering an effective amount of one or more cyclic peptides of the present disclosure or a composition thereof.
  • the method comprises administering an effective amount of one or more cyclic peptides of the present disclosure or a composition thereof to a subject.
  • a method of inhibiting spermatogenesis is also provided comprising administering an effective amount of one or more cyclic peptide of the present disclosure or a composition thereof to a subject.
  • the subject may be a mammal, preferably a human.
  • the human is a male human.
  • administration of the cyclic peptide(s) or composition thereof is oral administration.
  • FIG. 1A-1G show computational design of GRTH-binding cyclic peptides.
  • FIG. 1A shows electrostatic potential on the solvent-accessible surface of GRTH at the PKA-binding interface (dark gray: positive potential; medium gray: negative potential; white: neutral); the circle indicates location of T239.
  • FIG. 1B shows GRTH surface representation (same orientation as in panel 1A with the single amino-acid analogs hotspots indicated (each point corresponds to the location of the C ⁇ atom in the corresponding residue); T239 , basic residues in dark grey (R242 is occluded but indicated by the arrow); acidic residues in lighter grey.
  • FIG. 1A shows electrostatic potential on the solvent-accessible surface of GRTH at the PKA-binding interface (dark gray: positive potential; medium gray: negative potential; white: neutral); the circle indicates location of T239.
  • FIG. 1B shows GRTH surface representation (same orientation as in panel 1A with the single amino-a
  • 1C shows pharmacophore derived from the hotspot distribution of functional groups used here (two others can be derived; cf. SI).
  • F Phe
  • Q Gln
  • b basic
  • a acidic
  • hyd nonpolar/hydrophobic
  • pol net-neutral polar
  • Circles containing more than one symbol means that any of the indicated groups can be used.
  • Solid lines indicate a single peptide bond; dashed lines, one or more (indicated by the adjacent numbers); double arrow indicates allowed permutation.
  • FIG. 1D shows computational prediction of PEPx conformations in solution; each structure is a representative member of a conformational family. Only non-hydrogen atoms shown; the backbone RMSD (including PEG2) between any two conformers is in the 1.5–3 ⁇ range.
  • FIG. 1E shows electrostatic potential (mostly positive; darker grey) on the solvent-accessible surface of two representative PEPx conformers shown in panel 1D and 1F.
  • FIG. 1F shows superposition of the PEPx conformers upon binding to the GRTH/PKA interface (circle indicates the position of T239; GRTH orientation and grey scale same as in panel 1A; only one GRTH surface shown for clarity.
  • FIG. 1G shows utual structural adaptation upon binding enable PEPx to find its way into and expand a shallow crevice on the GRTH surface; solvent-accessible surface of PEPx indicated by arrows; only three conformers are shown to illustrate.
  • FIG. 2 shows immunofluorescence images showing the degree of entry of the PEP1, PEP2, CP1, CP2 and CP3 inside the cytoplasm and nucleus of COS-1 cells stably expressing GRTH. DAPI was used counter stain .
  • FIGS. 3A-3F show effect of cyclic and control peptides on GRTH phosphorylation in COS-1 stable cells expressing GRTH.
  • Cyclic peptides PEP1, PEP2, and PEP0 FIGS. 3A, 3B, and 3C
  • CP1, CP2, and CP3 FIGS. 3D, 3E, and 3F
  • ⁇ -actin was used as a negative control.
  • the protein band intensities of pGRTH and ⁇ -actin from three independent experiments (mean ⁇ SEM) were measured as indicated in the methods section. Values were normalized by ⁇ -actin.
  • FIGS. 4A-4C show time-dependent inhibitory effect of cyclic peptides on pGRTH expression in COS-1 stable cells. Cyclic peptides PEP0, PEP1, and PEP2 (100 ⁇ M) showed a time- dependent response on the expression of pGRTH ( FIGS. 4A, 4B, and 4C, respectively).
  • the pGRTH was identified using custom made GRTH phospho-specific antibody (Raju et al., Sci. Rep. 2019;9: 6705. ) .
  • ⁇ -actin was used as a negative control.
  • FIGS. 5A and 5B show inhibition of pGRTH expression by cyclic peptides in seminiferous tubules.
  • FIG. 5A indicates PEP1, PEP2 and CP1, CP2 and CP3 showed effective internalization in the seminiferous tubules.
  • FIG. 5B shows a Western blot of seminiferous tubules exposed to 100 ⁇ M of the respective peptide showed a significant decrease in the PEP1 and PEP2 treated samples when compared those untreated samples or exposed to control peptides.
  • FIGS. 6A and 6B show thermal shift plots (CETSA) showing melting curve profiles of cyclic and control peptides.
  • FIG. 6A Western blots showing denaturation pattern of non-pGRTH protein upon treatment with CP2, PEP1 and PEP2.
  • FIG. 7A-7E show quantitative FRET images on the live COS-1 cells.
  • FIG. 7A shows FRET images for acceptor photobleaching in COS-1 cells transiently expressed with mCherry- GRTH and exposed to 15 ⁇ M of cyclic peptides (PEP1, PEP2, and CP2). Areas were marked and images were acquired before and after the mCherry (acceptor) photobleaching.
  • the cyclic peptides (PEP1 and PEP2) attached with FITC (donor) showed a significant increase in the intensity after the photobleaching while control peptide (CP2) did not show any increase in the intensity of donor.
  • FIG. 7A shows FRET images for acceptor photobleaching in COS-1 cells transiently expressed with mCherry- GRTH and exposed to 15 ⁇ M of cyclic peptides (PEP1, PEP2, and CP2). Areas were marked and images were acquired before and after the mCherry (acceptor) photobleaching.
  • FIG. 7B shows that the intensity of donor and acceptor before and after photobleaching for the PEP1, PEP2, and CP2 were represented in the graph.
  • FIG. 7C shows images of COS-1 cells expressing mCherry-GRTH with PEP1, PEP2 and CP2 and the respective FRET efficiency images.
  • FIG. 7D indicates that the acceptor photobleaching experiment shown in a plot gave a FRET efficiency of 22 % for PEP1, 20 % for PEP2 and 5 % for CP2 however the FRET efficiency values change considerably for different locations in the marked regions.
  • FIG. 7C shows images of COS-1 cells expressing mCherry-GRTH with PEP1, PEP2 and CP2 and the respective FRET efficiency images.
  • FIG. 7D indicates that the acceptor photobleaching experiment shown in a plot gave a FRET efficiency of 22 % for PEP1, 20 % for PEP2 and 5 % for CP2 however the FRET efficiency values change considerably for different locations in the marked regions.
  • FIGS. 8A-8C show the effect of cyclic peptides on pGRTH with or without PKA induction: Western blot analysis was performed to validate the expression of phospho-GRTH in control (C), PKA induction only (PKA), 100 ⁇ M cyclic peptide treatment, PEP1 or PEP2 or CP2, and both PKA and cyclic peptide. ⁇ -actin was used as an internal control.
  • FIG. 9 shows a schematic “pipeline” flowchart of the computational method used for designing cyclic peptides that bind to shallow protein surfaces.
  • Molecular dynamics (MD) simulations are used to obtain the set of N conformational substates of the protein interface.
  • the binding modes and populations of a series of amino-acid side-chain analogs are obtained with the TaRt-cMC method; the resulting density maps, representing all the possible specific and non-specific binding modes of each analog, are then used to derive one or more pharmacophores; for a given pharmacophore, a series of S amino acid sequences can be proposed.
  • the corresponding linear peptide is subjected to free Langevin dynamics (LD) simulations, and P conformations with tail-to-head distances smaller than a preset cutoff are collected.
  • LD Langevin dynamics
  • FIGS. 10A-10F show graphs of cell viability percent vs. concentration of cyclic peptide reflecting the cytotoxic effect of PEP1 (FIG. 10A), PEP2 (FIG.
  • FIG. 11A shows results from an ex vivo organ testis culture study indicating a significant decrease in the phospho-GRTH in 24 and 48 hours of PEP2 treatment. This decrease indicates that cyclic peptide treatments are effective in inhibiting phosphorylation of GRTH.
  • FIG. 12 shows a plot of the presence of PEP1 in various tissues including significantly in the serum, testis and minimally in the liver and kidney from an in vivo study of bioavailability of PEP1 in mice.
  • CETSA Cellular Thermal Shift Assay COS 1 - CV-1 (Simian) in Origin, and carrying the SV40 genetic material (Fibroblast-like cell lines derived from monkey kidney tissue) CP1 - Control peptide 1 (Linear) CP2 - Control peptide 2 (Cyclic) CP3 - Control peptide 3 (Cyclic) FITC - Fluorescein isothiocyanate FRET - Fluorescence resonance energy transfer GRTH - Gonadotropin-Regulated Testicular RNA Helicase IC50 - Half-maximal inhibitory concentration PEPx - Cyclic Peptide x (basic scaffold) PEP0 - Cyclic Peptide 0 PEP1 - Cyclic Peptide 1 PEP2 - Cyclic Peptide 2 DDX19 - DEAD-Box Helicase 19B pGRTH - Phospho Gonadotropin-Reg
  • GRTH is expressed in rat, mouse, and human testis and acts as a post-transcriptional regulator of genes essential for the completion of spermatogenesis (Tang et al., J. Biol. Chem. 1999;274:37932 ⁇ 37940; Tsai-Morris et al., Proc. Natl. Acad. Sci. U.S.A. 2004;101:6373 ⁇ 6378; Dufau and Tsai-Morris Mol. Hum. Reprod. 2007;13:887 ⁇ 892, Dufau and Kavarthapu Front. Endocrinol. 2019;10:No. 576 ).
  • GRTH Knockout mice are sterile and lack spermatozoa due to failure of elongation of round spermatids during spermiogenesis (Tsai-Morris et al., 2004).
  • GRTH a 56 kDa non-phosphorylated form, predominantly found in the nucleus where it interacts with chromosomal region maintenance 1 protein (CRM1) and participates in mRNA transport to cytoplasmic sites
  • CCM1 chromosomal region maintenance 1 protein
  • pGRTH 61 kDa phosphorylated GRTH species which is present exclusively in the cytosol and found to reside in Chromatoid Bodies (Sato et al., 2010 Anbazhagan et al., 2020) and to be associated with polyribosomes where it may be involved in translation of germ cell-specific genes (Dufau and Tsai-Morris, 2007).
  • GRTH knock-in mice model carrying the human GRTH gene mutation R242H are sterile, lack sperm due to arrest at step 8 of round spermatids, and display complete loss of pGRTH, revealing the functional relevance of pGRTH (Kavarthapu et al., Hum. Mol. Genet. 2019;28:2561 ⁇ 2572 ).
  • the inventors identified residue T239, structurally adjacent to the patient mutant at R242 as the GRTH phosphorylation site.
  • cyclic peptides consist of amino acids (typically, ⁇ 10 aa) linked together to form a macrocyclic ring structure. Many biologically active cyclic peptides are linked through head-to-tail cyclization, where an amide bond is formed between the amino and carboxy termini of each end.
  • cyclic peptides show better biological activity than linear peptides because of their conformational rigidity, and their small ring structure provides resistance towards proteolytic degradation (Joo, Biomol. Ther.
  • cyclic peptides have larger surface areas, leading to higher affinity and selectivity of protein targets (Choi and Joo, Biomol. Ther. 2020;28:18 ⁇ 24 ).
  • the major challenge of cyclic peptides as therapeutics is the poor membrane permeability (Tarek et al., Biophys. J. 2003;85:2287 ⁇ 2298 ).
  • Cyclic peptides can be categorized based on their physicochemical properties or conformations.
  • Several cyclic peptides containing L-amino acids were developed and synthesized to investigate their cell-penetrating properties and examine their role as molecular transporters (Park et al., Mol. Pharm. 2019;16:3727 ⁇ 3743 ).
  • Cyclic peptides rich in arginine residues can be effectively delivered into mammalian cells (Qian et al., ACS Chem. Biol. 2013;8:423 ⁇ 431 ).
  • L-2-naphthylalanine in the peptides facilitates plasma-membrane binding and internalization.
  • a cyclic peptide containing arginine and tryptophan was conjugated to a drug, showing its potential use as an intracellular drug delivery platform (Nasrolahi Shirazi et al., Mol. Pharm.2013;10:488 ⁇ 499 ).
  • Arginine-rich cyclic peptides were shown to display higher structural rigidity and enhanced transduction efficiency (Lötig-Tünnemann et al., Nat. Commun.
  • cyclic peptides are provided herein with the following pharmacophore features (cf. Fig. 1C): an amino acid sequence containing at most 10 amino acid residues.
  • the amino acid sequence may comprise between about 6 and 9 amino acid residues, e.g., 8 amino acid residues.
  • the peptides comprise an amino acid residue (number 1) F; followed by an amino acid residue (number 2) selected from Q, A, I, V, and L; followed by a linker with a maximum length equivalent to 2 amino acids or 2 PEG (polyethylene glycol) molecules (see compositions below) or 2 PVA (polyvinyl alcohol) molecules; followed by an amino acid residue selected from either A or a small polar (non-charged) amino acid selected from S, N, Q, C, and M; followed by either F, or a basic amino acid selected from R, K, and H, or by a polar amino acid selected from S, N, Y, and Q; followed by a basic amino acid residue selected from R, K, and H; followed by a basic amino acid residue selected from R, K, and H; followed by a second linker of a maximum length equivalent to 3 amino acids or 3 PEG molecules (see possible compositions below).
  • amino acid residue number 1 and amino acid residue number 2 may be reversed (e.g., amino acid residue number 1 is selected from Q, A, I, V, and L and amino acid residue number 2 is F).
  • the linkers themselves may comprise the amino acids G or P or non-peptidic spacer molecules, e.g., PEG or PEG2 (2-(2-aminoethoxy)ethoxy)acetic acid (AEEAc).
  • AEEAc 2-(2-aminoethoxy)ethoxyacetic acid
  • the cyclic peptide should ideally contain fewer than ten residues (or being of similar length if non-peptidic spacers, e.g., PEG, are used to separate functional groups), (ii) incorporate local moieties that facilitate penetration into the cell membrane, and (iii) have enough structural flexibility in solution to adapt to the shallow, flexible surface upon binding.
  • the pharmacophore described above may be a preferred framework, but any of the positions may be modified.
  • a cyclic peptide of the disclosure has the formula: cyclo( 1 FAGXXXG 7 -AEEAc), in which each X is individually chosen from any basic amino acid, e.g., arginine (R), lysine (K), or histidine (H). (SEQ ID NO: 1, AEEAc).
  • cyclo( 1 FAGRRRG 7 -AEEAc) cyclo( 1 FAGRRRG 7 -AEEAc), referred to as PEPx (SEQ ID NO: 2, AEEAc).
  • all amino acid residues of the cyclic peptides are in the L configuration.
  • one or more amino acid residues of the cyclic peptides are in the D configuration.
  • the cyclic peptide can have one or more side chains attached thereto, for example at one or more glycine residue.
  • Any suitable side chain (e.g., Pro or Gly in the linkers) can be selected so long as GRTH binding is maintained.
  • Certain structural flexibility is desirable to allow local adaptation to the GRTH/PKA interface and may be controlled by changing the length of the linker (e.g., flexibility may be reduced with Gly2 instead of PEG2).
  • the molecular surface representation of the conformers in solution (two shown in FIG. 1E) reveals a common feature: a pronged cationic structure with basic residues on one end of the ring and a hydrophobic region on the other, giving the peptide the cationic/amphipathic character desirable for membrane translocation and cytosolic localization.
  • a linker of any length may be used.
  • Suitable linkers may be chosen based on a number of factors, so long as GRTH binding is maintained. Length can be expressed in units, where, for example, a unit corresponds to one or more of a monomer residue and an amino acid residue. Any suitable monomer or amino acid or combination thereof can be employed. Examples of monomers and amino acids include ethylene glycol, vinyl acetate, glycine, and proline.
  • a linker can comprise a homopolymer or a copolymer. In some embodiments, the linker can be comprised of polyglycine, polyethylene glycol, polyproline, or polyvinyl alcohol, or any combination thereof.
  • the linker can be comprised of (2-(2- aminoethoxy)ethoxy)acetic acid (AEEAc). In some embodiments, the linker comprises two units of length at most. In some embodiments, the linker comprises three units of length at most. [0067] In some embodiments, the cyclic peptide may be modified to increase bioavailability. Any known methods may be used to increase bioavailability including but not limited to, methylation of one or more amino acid residues on the cyclic peptide.
  • cyclic peptides provided by the disclosure is provided below, which incorporate relatively minor side-chain modifications to PEPx (SEQ ID NO: 2): PEP0 (SEQ ID NO: 3, AEEAc) replaces A2 by L-2-naphthylalanine, whereas PEP1 (SEQ ID NO: 7, AEEAc) and PEP2 (SEQ ID NO: 8, AEEAc) replace G7 by L- and D-Lys(FITC), respectively.
  • Cyclic Peptide 0 (PEP0) (SEQ ID NO: 3, AEEAc):
  • Cyclic Peptide 1 (PEP1) (SEQ ID NO: 7, AEEAc):
  • Cyclic Peptide 2 (PEP2) (SEQ ID NO: 8, AEEAc): [0072]
  • the basic scaffold (PEPx) (SEQ ID NO: 2) comprises only naturally occurring amino acids, is cationic and amphipathic, comprises aromatic and hydrophobic residues, and is rich in arginine residues.
  • Computer simulations of PEPx and its minimally-modified PEP0 showed that both peptides adopt a family of conformations in solution at physiological conditions, possibly in fast interconversion; all the conformers display the basic pharmacophoric features.
  • the inventors introduced some structural flexibility through the length and chemical nature of the spacer (in this case, PEG2), which allows the peptide to adapt to the shallow interface.
  • AEEAc may be used as a spacer in the cyclic peptide.
  • Computer simulations showed that all the conformers bind to the expected interface, although with significant structural variations. This binding-mode variability was expected and, within limits, is a desirable feature as it allows the peptide to find the most favorable interactions, albeit at an entropic price.
  • a cyclic peptide may comprise an amino acid sequence comprising the following formula: X 1 -X 2 -linker1-X 3 -X 4 -X 5 -X 6 -linker2, wherein X 1 is amino acid residue F; X 2 is an amino acid residue selected from Q, A, I, V, and L; linker1 comprises one or two linker molecules individually selected from amino acid residue G, amino acid residue P, or a non-peptidic spacer molecule; X 3 is an amino acid residue selected from A, S, N, Q, C, and M; X 4 is an amino acid residue selected from F, R, H, K, S, Y, Q, and N; X 5 is an amino acid residue selected from R, H, and K; X 6 is an amino acid residue selected from R, H, and K and linker2 comprises one, two, or three linker molecules individually selected from amino acid residue G, amino acid residue P, or a non-peptidic spacer molecule.
  • X 1 may be replaced with an amino acid residue selected from Q, A, I, V, and L; and wherein X 2 may be replaced with F.
  • the cyclic peptide comprises two linkers (linker1 and linker2).
  • the first linker (linker1) comprises one or two linker molecules, preferably one linker molecule.
  • the second linker (linker2) comprises one or two or three linker molecules, preferably 1 or 2 linker molecules, more preferably 2 linker molecules.
  • the linker molecule(s) are individually selected from amino acid residue G, amino acid residue P, or a non-peptidic spacer molecule.
  • the non- peptidic spacer molecule is polyethylene glycol (PEG) or polyvinyl alcohol (PVA).
  • linkers comprising one or more non-peptidic spacer molecule(s) include PEG1 (one PEG molecule), PEG2 (two PEG molecules, e.g., a dimer; i.e., AEEAc), or PEG3 (three PEG molecules, e.g., a PEG trimer).
  • linkers comprising one or more non-peptidic spacer molecule(s) include PVA1 (one PVA molecule), PVA2 (two PVA molecules, e.g., a dimer), or PVA3 (three PVA molecules, e.g., a PVA trimer).
  • Copolymers of PEG and PVA can also be employed, for example, combinations and permutations of PEG and PVA adding up to two or three total molecules in a given linker.
  • dimers and trimers can include PEG1-PVA1, PVA1-PEG1, PEG2- PVA1, PVA1-PEG2, PVA2-PEG1, PEG1-PVA2, PEG1-PVA1-PEG1, or PVA1-PEG-PVA1, or any combination thereof.
  • the maximum length of linker 1 is equivalent to 2 amino acids, 2 PEG molecules, or 2 PVA molecules.
  • the maximum length of linker 2 is equivalent to 3 amino acids, 3 PEG molecules, or 3 PVA molecules.
  • the combined length of the two linkers should be selected so that the maximum length of the cyclic peptide does not exceed 10 amino acids in length.
  • cyclic peptides are listed below: cyclo-(Phe- ⁇ -Gly-Arg-Arg-Arg-Gly-PEG2) [corresponds to PEP0 where AEEAc is replaced by PEG2] (SEQ ID NO: 3) cyclo-(Phe- ⁇ -Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 3) cyclo-(Phe- ⁇ -Ala-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 4) cyclo-(Phe- ⁇ -Ala-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 4) cyclo-( ⁇ -Phe-Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 5) cyclo-( ⁇ -Phe-Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 5) cyclo-( ⁇ -Phe-Ala-Arg-Arg-Arg-Arg-Arg-Gly
  • PEG1 is one PEG molecule
  • PEG2 is two PEG molecules
  • PEG3 is three PEG molecules.
  • each PEG molecule can be replaced by another linker molecule, e.g., (2- (2-aminoethoxy)ethoxy)acetic acid (AEEAc); additional linkers can also be formed by a combination of PEG and Gly or include intercalations of Pro to change the local flexibility for improved binding affinity or specificity.
  • Polyvinyl alcohol (PVA) can be used in place of or in combination with PEG [0078]
  • ⁇ is L-2-naphthylalanine.
  • each of the above exemplary cyclic peptides can be replaced with A, Q or F. In some embodiments, ⁇ can be replaced with Q or F.
  • each instance of Arg can be replaced with Lys or His (e.g., any basic amino acid can be present in any combination).
  • each of the above exemplary cyclic peptides may comprise one or more modifications to improve bioavailability. In some embodiments, the cyclic peptide is methylated at one or more positions.
  • the cyclic peptide comprises one or more non-natural amino acid analogs, for example, replacing one or more Arg with a corresponding non-natural amino acid analog.
  • Methods of inhibiting GRTH phosphorylation [0081] The cyclic peptides described herein effectively block the phosphorylation of GRTH. Thus, the cyclic peptides described herein may be used to inhibit GRTH phosphorylation.
  • a method of inhibiting GRTH phosphorylation is provided in which GRTH is contacted with a cyclic peptide described herein. The method of inhibiting GRTH phosphorylation is an in vitro method. The method of inhibiting GRTH phosphorylation is an in vivo method.
  • CP1 may still bind to GRTH at the PKA-binding surface, even when not conforming to the pharmacophore as well as PEP1 does, the observed differences are more likely related to the reduced CP1 entry into the cells.
  • control cyclic peptides CP2 SEQ ID NO: 46
  • CP3 SEQ ID NO: 47
  • Both controls lack essential pharmacophore features relevant for GRTH binding at the interface, although our fluorescence study shows that both peptides competently reached intracellular sites.
  • Peptidyl-prolyl isomerase which specifically binds to phosphorylated Ser/Thr-Pro motifs to regulate post-translational modification, was effectively blocked by cyclic peptides containing Nal and rich in Arg residues (Liu et al., J. Med. Chem. 2010;53:2494 ⁇ 2501 ).
  • a different, separate effect may be at play in the blockade of GRTH phosphorylation by PEP0, PEP1, and PEP2.
  • PEP0 which is obtained from PEP1 upon removing the Lys(FITC) side chain, also showed similar effects as PEP1, confirming that the presence of the bulky fluorophore did not interfere with binding.
  • a significant reduction of pGRTH was observed at 8 h and 16 h upon treatment with PEP1 and PEP2 of COS-1 cells stably expressing GRTH.
  • a reduction in the expression of phospho-GRTH upon exposure of PEP1 and PEP2 of COS-1 cells was observed, even when COS1 cells stably expressing GRTH were transiently transfected with PKA catalytic subunit, indicating competitive binding of cyclic peptides at the GRTH/PKA site in the presence of PKA.
  • FRET acceptor photobleaching was performed to confirm the interaction of PEP1 and PEP2 with GRTH.
  • An increase in the fluorescence intensity of donor FITC was observed in the photobleached region of acceptor mCherry indicating FRET has occurred due to the proximity of donor FITC and acceptor mCherry.
  • a method of inhibiting spermatogenesis may comprise administering a cyclic peptide described herein to a subject.
  • a subject can be any sperm- producing animal.
  • the subject is a mammal.
  • the mammal may be a primate.
  • the mammal may be a human, cat, dog, horse, sheep, goat, or a pig.
  • GRTH/DDX25 is a DEAD-box RNA-helicase essential for the completion of spermatogenesis. Previous studies by the inventors indicated that blocking the GRTH phospho-site or perturbing the GRTH/PKA interface could provide an avenue for developing a non- hormonal male contraceptive.
  • the cyclic peptides described herein were rationally designed and synthesized as therapeutic agents.
  • the cyclic peptides showed effective delivery into COS-1 and germ cells and a dose-dependent inhibitory effect on GRTH phosphorylation.
  • the cyclic peptides inhibit GRTH phosphorylation in the presence of PKA, and binding to the helicase resulted in thermal stabilization of non-phospho-GRTH.
  • Increased efficiency in FRET assay revealed their interaction with GRTH. Cyclic peptide exposure of cultures from mice seminiferous tubules resulted in significant inhibition of phospho-GRTH. These cyclic peptides did not exhibit toxicity.
  • cyclic peptides e.g., PEP0, PEP1, PEP2
  • PEP1 and PEP2 revealed by FITC, showed internalization into COS-1 cells.
  • a dose-dependent inhibitory effect on GRTH phosphorylation was observed in COS-1 stable cell line expressing GRTH, with significant reduction in pGRTH protein.
  • CETSA showed PEP1 and PEP2 binding resulting in thermal stabilization of the soluble non-pGRTH protein.
  • compositions may further comprise at least one of any suitable auxiliaries including, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof.
  • suitable auxiliaries are preferred. Examples and methods of preparing such sterile solutions are well known in the art.
  • Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the cyclic peptides described herein.
  • compositions described herein can also include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, terra-, and oligosaccharides; derivatized sugars including but not limited to alditols, aldonic acids, esterified sugars, or combinations thereof; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination in ranges of 1-99.99% by weight or volume.
  • proteins es, amino acids, lipids, and carbohydrates
  • carbohydrates e.g., sugars, including monosaccharides, di-, tri-, terra-, and oligosaccharides; derivatized sugars including but not limited to alditols, aldonic acids, esterified sugars, or combinations thereof; and polysaccharides or sugar polymers
  • Exemplary protein excipients include serum albumin including but not limited to human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, or combinations thereof.
  • Representative amino acid components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, or combinations thereof.
  • Carbohydrate excipients suitable for use in the compositions described herein include monosaccharides including but not limited to fructose, maltose, galactose, glucose, D-mannose, sorbose, or combinations thereof; disaccharides, including but not limited to lactose, sucrose, trehalose, cellobiose, or combinations thereof; polysaccharides, including but not limited to raffinose, melezitose, maltodextrins, dextrans, starches, or combinations thereof; and alditols, including but not limited to mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol or combinations thereof.
  • monosaccharides including but not limited to fructose, maltose, galactose, glucose, D-mannose, sorbose, or combinations thereof
  • compositions described herein further can comprise, but is not limited to pharmaceutically acceptable carriers including but not limited to coloring agents, emulsifying agents, suspending agents, ethanol, EDTA, citrate buffer, flavoring, and water.
  • pharmaceutically acceptable carriers including but not limited to coloring agents, emulsifying agents, suspending agents, ethanol, EDTA, citrate buffer, flavoring, and water.
  • Chelators including but not limited to EDTA and EGTA can optionally be added to the pharmaceutical compositions to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the pharmaceutical composition. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the composition to aggregate.
  • composition described herein also can comprise the preservatives methylparaben (also known as 4-hydroxybenzoic acid methyl ester, methyl p-hydroxybenzoate; or METHYL CHEMOSEPT), ethylparaben (also known as 4-hydroxybenzoic acid ethyl ester; ethyl phydroxybenzoate; or ETHYL PARASEPT), propylparaben (also known as 4-hydroxybenzoic acid propyl ester; propyl p-hydroxybenzoate; NIPASOL; or PROPYL CHEMOSEPT) and/or butylparaben (also known as 4-hydroxybenzoic acid propyl ester; propyl p-hydroxybenzoate; or BUTYL CHEMOSEPT).
  • methylparaben also known as 4-hydroxybenzoic acid methyl ester, methyl p-hydroxybenzoate; or METHYL CHEMOSEPT
  • ethylparaben also known as 4-hydroxybenzoic acid
  • Emulsifiers that may be used in the compositions described herein include, but are not limited to ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the pharmaceutical compositions comprising the cyclic peptides described herein can also include a buffer or a pH adjusting agent.
  • the buffer is a salt prepared from an organic acid or base.
  • Representative buffers include organic acid salts including but not limited to salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
  • compositions described herein can include polymeric excipients/additives including but not limited to polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, including but not limited to hydroxypropyl- ⁇ -cyclodextrin), polyethylene glycols, flavoring agents, anti-microbial agents, sweeteners, antioxidants, anti-static agents, surfactants (e.g., polysorbates including but not limited to “Tween® 20” and “Tween® 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA or EGTA).
  • polyvinylpyrrolidones e.g., ficolls (a polymeric sugar)
  • dextrates e.g., cyclodextrins, including but not limited to hydroxypropyl- ⁇ -cyclod
  • excipients and/or additives suitable for use in the present invention are known in the art.
  • excipients e.g., isotonicity agents, buffers, antioxidants, preservative enhancers
  • An isotonicity agent including but not limited to glycerin, is commonly used at known concentrations.
  • a physiologically tolerated buffer is preferably added to provide improved pH control. Suitable buffers include phosphate buffers, sodium phosphate and phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the cyclic peptides disclosed herein can also be administered in the form of liposomes.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can comprise, in addition to the cyclic peptides described herein, stabilizers, preservatives, excipients, or combinations thereof.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
  • Liposomes, methods of making and methods of use are described in U.S. Patent Nos. 4,089,8091 (process for the preparation of liposomes), 4,233,871 (methods regarding biologically active materials in lipid vesicles), 4,438,052 (process for producing mixed micelles), 4,485,054 (large multilamellar vesicles), 4,532,089 (giant-sized liposomes and methods thereof), 4,897,269 (liposomal drug delivery system), 5,820,880 (liposomal formulations), and so forth.
  • cyclic peptides described herein can be solubilized or suspended in a preconcentrate (before dilutions with a diluent), added to the preconcentrate prior to dilution, added to the diluted preconcentrate, or added to a diluent prior to mixing with the preconcentrate.
  • the cyclic peptides described herein can also be co-administered as part of an independent dosage form, for therapeutic effect.
  • the cyclic peptides described herein can be present in a first, solubilized amount, and a second, non-solubilized (suspended) amount.
  • the pharmaceutical compositions described herein can also comprise suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the cyclic peptides described herein into preparations that can be administered to animals, as described herein.
  • compositions described herein can also include additional therapeutic agents including but not limited to, but not limited to hydrophilic drugs, hydrophobic drugs, hydrophilic macromolecules, cytokines, peptidomimetics, peptides, proteins, toxoids, sera, antibodies, vaccines, nucleosides, nucleotides, nucleoside analogs, genetic materials and/or combinations thereof.
  • additional therapeutic agent can be solubilized or suspended in a preconcentrate (before dilutions with a diluent), added to the preconcentrate prior to dilution, added to the diluted preconcentrate, or added to a diluent prior to mixing with the preconcentrate.
  • the additional therapeutic agent can also be co-administered as part of an independent dosage form, for therapeutic effect.
  • the additional therapeutic agent(s) can be present in a first, solubilized amount, and a second, non-solubilized (suspended) amount.
  • Such additional therapeutic agent(s) can be any agent(s) having therapeutic or other value when administered to an animal, particularly to a mammal, including but not limited to drugs, nutrients, and diagnostic agents.
  • Dosages [0107]
  • the pharmaceutical compositions described herein are administered to a subject in a manner known in the art. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • cyclic peptides described herein may be present in any suitable amount within the pharmaceutical compositions described herein. Those of skill in the art can readily determine suitable concentrations of cyclic peptides described herein to include in the pharmaceutical compositions depending on various factors including dosage and route of administration. Pharmaceutical compositions useful in the present invention can comprise a quantity of a cyclic peptides described herein in an amount effective to be active as a contraceptive.
  • the cyclic peptides described herein may be present in the pharmaceutical composition in an amount of at least 0.1 mg/mL, at least 0.5 mg/mL, at least 1 mg/mL, at least 1.5 mg/mL, at least 2 mg/mL, at least 5 mg/mL, at least 10 mg/mL or at least 15 mg/mL.
  • the cyclic peptides described herein may be present in the pharmaceutical composition in an amount from about 0.1 mg/mL to about 100 mg/mL, from about 0.5 mg/mL to about 100 mg/mL, from about 1 mg/mL to about 100 mg/mL, from about 1.5 mg/mL to about 100 mg/mL, from about 2 mg/mL to about 100 mg/mL, from about 5 mg/mL to about 100 mg/mL, from about 10 mg/mL to about 100 mg/mL, from about 15 mg/mL to about 100 mg/mL, from about 20 mg/mL to about 100 mg/mL, or from about 50 mg/mL to about 100 mg/mL.
  • the cyclic peptides described herein may be present in the pharmaceutical composition in an amount from about 0.1 mg/mL to about 50 mg/mL, from about 0.1 mg/mL to about 25 mg/mL, from about 0.1 mg/mL to about 20 mg/mL, from about 0.1 mg/mL to about 15 mg/mL, from about 0.1 mg/mL to about 10 mg/mL, from about 0.1 mg/mL to about 5 mg/mL, from about 0.1 mg/mL to about 3 mg/mL, from about 0.1 mg/mL to about 2 mg/mL, from about 0.1 mg/mL to about 1.5 mg/mL, or from about 0.1 mg/mL to about 1 mg/mL.
  • the cyclic peptides described herein may be present in the pharmaceutical composition in an amount of about 0.1 mg/mL, about 0.5 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 50 mg/mL or about 100 mg/mL.
  • the compositions described herein may comprise between about 1 mg and 250 mg, 100 mg and 500 mg, 250 mg and 750 mg, 500 mg and 1,000 mg of the cyclic peptides described herein.
  • compositions described herein may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115
  • compositions described herein may comprise between about 1 ⁇ g and 250 ⁇ g, 100 ⁇ g and 500 ⁇ g, 250 ⁇ g and 750 ⁇ g, 500 ⁇ g and 1,000 ⁇ g of the cyclic peptides described herein.
  • compositions described herein may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115
  • the pharmaceutical compositions may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily. Doses maybe administered for one week, one month, or over the course of several months, 3, 6, 9 or 12 months, or intervals known in the art and determined to be clinically relevant. Doses may be continued throughout the life of the patient, or discontinues when clinical judgment warrants.
  • the daily dosage of the formulations may be varied over a wide range from about 0.0001 to about 1,000 mg per patient, per day. The range may more particularly be from about 0.001 mg/kg to 10 mg/kg of body weight per day, about 0.1-100 mg, about 1.0-50 mg or about 1.0-20 mg per day for adults (at about 60 kg).
  • the dosages may be about 0.5-10 mg/kg per day, about 1.0-5.0 mg/kg per day, 5.0-10 mg/kg per day, or equivalent doses as determine by a practitioner, to achieve a serum concentration that is clinically relevant.
  • treatment of humans or animals can be provided as a one- time or periodic dosage of the cyclic peptides described herein 0.0001 to about 1,000 mg per patient, per day.
  • the range may more particularly be from about 0.001 mg/kg to 10 mg/kg of body weight per day, about 0.1-100 mg, about 1.0-50 mg or about 1.0-20 mg per day for adults (at about 60 kg).
  • the dosages may be about 0.5-10 mg/kg per day, about 1.0-5.0 mg/kg per day, 5.0-10 mg/kg per day or equivalent doses as determine by a practitioner, to achieve a serum concentration that is clinically relevant.
  • Humans or other animals can be provided as a one-time or periodic dosage of the cyclic peptides described herein 1 ⁇ g to about 1,000 mg per patient, per day. The range may more particularly be from about 1 ⁇ g/kg to 10 ⁇ g/kg of body weight per day, about 1-100 ⁇ g, about 1.0-50 ⁇ g or about 1.0-20 ⁇ g per day for adults (at about 60 kg).
  • the dosages may be about 0.5-10 ⁇ g/kg per day, about 1.0-5.0 ⁇ g/kg per day, 5.0-10 ⁇ g/kg per day or equivalent doses as determine by a practitioner, to achieve a serum concentration that is clinically relevant.
  • the pharmaceutical compositions described herein may be administered at least once a week over the course of several weeks.
  • the pharmaceutical compositions may be administered at least once a day over several weeks to several months to several years.
  • the pharmaceutical compositions may be administered daily over a period of several days, several weeks, several months, or several years, until no longer needed or desired.
  • Routes of Administration [0121] Routes of administration and dosages of effective amounts of the pharmaceutical compositions comprising the cyclic peptides are described herein.
  • the cyclic peptides described herein can be administered in combination with other pharmaceutical agents in a variety of protocols for effective contraception.
  • the present disclosure further relates to the administration of at least one of the cyclic peptides described herein by the following routes, including, but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, iontophoretic means
  • compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may comprise 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 which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition with only the addition of the sterile liquid carrier, water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. [0124] For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally comprise suitable preservatives are employed when intravenous, administration is desired.
  • the pharmaceutical compositions may be administered parenterally via injection of a pharmaceutical composition comprising cyclic peptides described herein dissolved in an inert liquid carrier.
  • parenteral includes, but is not limited to, subcutaneous injections, intravenous, intramuscular, intraperitoneal injections, or infusion techniques.
  • Acceptable liquid carriers include, vegetable oils including but not limited to peanut oil, cotton seed oil, sesame oil or combinations thereof, as well as organic solvents including but not limited to solketal, glycerol formal.
  • the pharmaceutical compositions may be prepared by dissolving or suspending cyclic peptides described herein in the liquid carrier such that the final formulation contains from about 0.005% to 30% by weight of a cyclic peptide described herein.
  • the cyclic peptides described herein may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier including but not limited to ethanol, glycerol, water or combinations thereof.
  • suitable binders include, without limitation, starch; gelatin; natural sugars including but not limited to glucose or beta-lactose; corn sweeteners; natural and synthetic gums including but not limited to acacia, tragacanth, or sodium alginate, carboxymethylcellulose; polyethylene glycol; waxes or combinations thereof.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, or combinations thereof.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, or combinations thereof.
  • the pharmaceutical formulation also optionally comprising a sweetener.
  • Sweeteners include but are not limited to sucrose, fructose, sodium saccharin, sucralose (SPLENDA®), sorbitol, mannitol, aspartame, sodium cyclamate, and combinations thereof.
  • Aqueous suspensions, emulsions and/or elixirs for oral administration can be combined with various sweetening agents, flavoring agents, including but not limited to, but not limited to orange or lemon flavors, coloring agents, including but not limited to dye stuffs, natural coloring agents or pigments, in addition to the diluents including but not limited to water, glycerin and various combinations.
  • compositions described herein suitable for oral administration may be presented as discrete units including but not limited to capsules, dragées, cachets or tablets each comprising a predetermined amount of the cyclic peptides described herein; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion, and as a bolus.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the cyclic peptides described herein in a free-flowing form including but not limited to a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered cyclic peptides described herein moistened with an inert liquid diluent. The tablets may be optionally coated or scored and may be formulated so as to provide a slow or controlled release of the cyclic peptides described herein therein.
  • compositions comprising cyclic peptides described herein may be incorporated into biodegradable polymers allowing for sustained release of the cyclic peptides described herein.
  • biodegradable polymers and their uses are described in detail in Brem et al., 74 J. NEUROSURG. 441-46 (1991).
  • sustained-release compositions include semipermeable matrices of solid hydrophobic polymers comprising a cyclic peptide described herein, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (including poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers including but not limited to the LUPRON DEPOT® (Tap Pharmaceuticals, Inc., Chicago, Ill.) (injectable microspheres composed of lactic acid glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • LUPRON DEPOT® Tap Pharmaceuticals, Inc., Chicago, Ill.
  • cyclic peptides described herein can be delivered over prolonged periods of time, for periods of one week to one year from a single administration.
  • Certain medical devices may be employed to provide a continuous intermittent or on demand dosing of a patient.
  • the devices may be a pump of diffusion apparatus, or other device containing a reservoir of drug and optionally diagnostic or monitoring components to regulate the delivery of the drug.
  • Various slow-release, depot or implant dosage forms can be utilized.
  • a dosage form can comprise a pharmaceutically acceptable non-toxic salt of the cyclic peptides described herein that has a low degree of solubility in body fluids, (a) an acid addition salt with a polybasic acid including but not limited to phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, or combinations thereof; (b) a salt with a polyvalent metal cation including but not limited to zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, combinations thereof, or with an organic cation formed from e.g., N,N’-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b) e.g., a zinc tannate salt.
  • a polybasic acid
  • the cyclic peptides described herein or a relatively insoluble salt can be formulated in a gel, an aluminum monostearate gel with, e.g., sesame oil, suitable for injection.
  • Salts include, but are not limited to, zinc salts, zinc tannate salts, pamoate salts, or combinations thereof.
  • Another type of slow-release depot formulation for injection would comprise a salt of the cyclic peptides described herein dispersed or encapsulated in a slow degrading, non- toxic, non-antigenic polymer including but not limited to a polylactic acid/polyglycolic acid polymer, including the formulations as described in U.S. Patent No. 3,773,919.
  • the cyclic peptides described herein or relatively insoluble salts thereof including but not limited to those described above can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional slow-release, depot or implant formulations, e.g., gas or liquid liposomes are known in the literature. See, e.g., U.S. Patent No. 5,770,222.
  • Other examples include provision of the cyclic peptides described herein to be administered by sustained release delivery system containing a biodegradable composition.
  • the biodegradable composition may be composed of a biodegradable, water-coagulable, non-polymeric material and a biocompatible, non-toxic organic solvent that is miscible to dispersible in an aqueous medium.
  • the delivery system may be implanted at an implant site causing the solvent to dissipate, disperse or leach from the composition into surrounding tissue fluid through a resulting microporous matrix.
  • implant site is meant to include a site, in or on which the non-polymeric composition is applied. Implantation or implant site can also include the incorporation of the pharmaceutical composition comprising at least one of the cyclic peptides described herein with a solid device. The pharmaceutical composition can be incorporated into a coating on a stent that is implanted into a subject. Additionally, other solid or biodegradable materials can be used as a substrate on which the pharmaceutical composition is applied.
  • biodegradable means that the non-polymeric material and/or matrix of the implant will degrade over time by the action of enzymes, by simple or enzymatically catalyzed hydrolytic action and/or by other similar mechanisms in the human body.
  • bioerodible it is meant that the implant matrix will erode or degrade over time due, at least in part, to contact with substances found in the surrounding tissue fluids, cellular action,.
  • bioabsorbable it is meant that the non- polymeric matrix will be broken down and absorbed within the human body, by a cell, a tissue.
  • Non-polymeric materials that can be used in the composition generally are those that are biocompatible, substantially insoluble in water and body fluids, and biodegradable and/or bioerodible.
  • the non-polymeric material is capable of being at least partially solubilized in a water- soluble organic solvent.
  • the non-polymeric materials are also capable of coagulating or solidifying to form a solid implant matrix.
  • the non-polymeric material is combined with a compatible and suitable organic solvent to form a composition that has the desired consistency ranging from watery to viscous to a spreadable putty or paste.
  • Suitable organic solvents are those that are biocompatible, pharmaceutically-acceptable, and will at least partially dissolve the non-polymeric material.
  • the organic solvent has a solubility in water ranging from miscible to dispersible.
  • a pore-forming agent can be included in the composition to generate additional pores in the implant matrix.
  • the pore-forming agent can be any organic or inorganic, pharmaceutically-acceptable substance that is substantially soluble in water or body fluid, and will dissipate from the coagulating non-polymeric material and/or the solid matrix of the implant into surrounding body fluid at the implant site.
  • the cyclic peptides described herein are capable of providing a local or systemic biological, physiological or therapeutic effect in the body of an animal.
  • the cyclic peptides described herein may be soluble or dispersible in the non-polymeric composition to form a homogeneous mixture, and upon implantation, becomes incorporated into the implant matrix. As the solid matrix degrades over time, the cyclic peptides described herein are capable of being released from the matrix into the adjacent tissue fluid, and to the pertinent body tissue or organ, either adjacent to or distant from the implant site, preferably at a controlled rate.
  • the release of the cyclic peptides described herein from the matrix may be varied by the solubility of the cyclic peptides described herein in an aqueous medium, the distribution of the cyclic peptides described herein within the matrix, the size, shape, porosity, and solubility and biodegradability of the solid matrix. See e.g., U.S. Patent No. 5,888,533.
  • the amounts and concentrations of ingredients in the composition administered to the patient will generally be effective to accomplish the task intended.
  • the cyclic peptides described herein may be administered by bioactive agent delivery systems containing microparticles suspended in a polymer matrix.
  • the microparticles may be microcapsules, microspheres or nanospheres currently known in the art.
  • the microparticles should be capable of being entrained intact within a polymer that is or becomes a gel once inside a biological environment.
  • the microparticles can be biodegradable or nonbiodegradable.
  • Many microencapsulation techniques used to incorporate a bioactive agent into a microparticle carrier are taught in the art. See e.g., U.S. Patent Nos. 4,652,441; 5,100,669; 4,438,253; and 5,665,428.
  • a preferred polymeric matrix will be biodegradable and exhibit water solubility at low temperature and will undergo reversible thermal gelation at physiological mammalian body temperatures.
  • the polymeric matrix is capable of releasing the substance entrained within its matrix over time and in a controlled manner.
  • the polymers are gradually degraded by enzymatic or non- enzymatic hydrolysis in aqueous or physiological environments. See e.g., U.S. Patent No. 6,287,588.
  • Methods of preparing various pharmaceutical compositions with a certain amount of active ingredients are known, or will be apparent in light of this disclosure, to those skilled in the art. Methods of preparing said pharmaceutical compositions can incorporate other suitable pharmaceutical excipients and their formulations are known in the art.
  • Methods of preparing the pharmaceutical preparations described herein are manufactured in a manner that is known, including conventional mixing, dissolving, or lyophilizing processes.
  • liquid pharmaceutical preparations can be obtained by combining the cyclic peptides described herein with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary.
  • a method of administering pharmaceutically effective amounts of the cyclic peptides described herein to a patient in need thereof can be determined empirically, or by standards currently recognized in the medical arts.
  • the agents can be administered to a patient as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents of the compositions described herein will be decided within the scope of sound medical judgment by the attending physician.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. It is well within the skill of the art to start doses of the agents at levels lower than those employed to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.
  • This pattern is significant and may function as recognition sites for specific, high-affinity binding of cyclic peptides.
  • One possible pharmacophore chosen here as the basis for the design of all the peptides is shown in FIG. 1C. Although some leeway exists in the choice of residues matching the pharmacophore, some parameters are imposed a priori, which restrict the options: (i) the cyclic peptide should ideally contain fewer than ten residues (or being of similar length if nonpeptidic spacers, e.g., PEG, are used to separate functional groups), (ii) incorporate local moieties that facilitate penetration into the cell membrane, (iii) accommodate a fluorescence probe for in vitro monitoring of cellular localization without disrupting GRTH binding, and (iv) have enough structural flexibility in solution to adapt to the shallow, flexible surface upon binding.
  • Certain structural flexibility is desirable to allow local adaptation to the interface and may be controlled by changing the length of the linker (e.g., flexibility may be reduced with Gly 2 instead of PEG2; not tested).
  • the molecular surface representation of the conformers in solution (two shown in FIG. 1E) reveals a common feature: a pronged cationic structure with basic residues on one end of the ring and a hydrophobic region on the other, giving the peptide the cationic/amphipathic character desirable for membrane translocation and cytosolic localization.
  • analysis of the predicted binding-modes shows that all the conformers are attracted to GRTH at or near the PKA-binding interface, although with significant structural variability (FIG. 1F) and mutual adaptation upon binding (FIG. 1G).
  • the inventors synthesized the set of cyclic peptides shown in Table 1, which incorporate relatively minor side-chain modifications to PEPx (SEQ ID NO: 2): PEP0 (SEQ ID NO: 3, AEEAc) replaces A2 by L-2-naphthylalanine, whereas PEP1 (SEQ ID NO: 7, AEEAc) and PEP2 (SEQ ID NO: 8, AEEAc) replace G7 by L- and D-Lys(FITC), respectively (the inventors tested both configurations because of the uncertain effects of the conjugated fluorophore size on GRTH binding).
  • CP1 linear peptide
  • PEP1 SEQ ID NO: 7, AEEAc
  • CP3 inversion of charge at the polar end of the ring
  • CP3 reduction in the size of the hydrophobic/nonpolar groups at the nonpolar end
  • Table 1 Peptides used in the experiments Molecular modeling and simulations [0150] The computational method for designing cyclic peptides targeting shallow protein surfaces is described below.
  • the inventors started with the derivation of a pharmacophore and the design of a series of cyclic peptides consistent with both the pharmacophore and a set of chemical features known to facilitate membrane crossing, followed by the prediction of binding modes and the selection of candidates for synthesis and in vitro testing.
  • the structure of GRTH/DDX25 domain 1 was obtained by homology with DDX19 (Raju et al., 2019); a recent model obtained from an AI- based approach (AlphaFold) yielded similar structures, with no conformational differences at the PKA-binding interface, including the side-chain orientations of relevant residues (Jumper et al., Nature 2021, 596, 583 ).
  • the inventors performed molecular dynamics (MD) simulations to obtain a family of conformers (substates) of GRTH in an aqueous solution using the TIP3P water model and the all-atom CHARMM forcefield (Brooks et. al. J. Comput. Chem. 2009 Jul 30;30(10):1545-1614) (c36 version) at constant temperature (37 °C) and pressure (1 atm), with protonation states at neutral pH.
  • MD molecular dynamics
  • Clustering analysis of GRTH residues at the GRTH/PKA interface yielded 13 distinct substates, each a potential target of one or more cyclic peptides.
  • MC simulated annealing Monte Carlo
  • the inventors first identified GRTH “hotspots” for high-affinity binding of single amino-acid side-chain analogs to the GRTH/PKA interface. Based on the binding patterns, the inventors generated a proposed general pharmacophore. Once a specific amino acid sequence is selected for a cyclic peptide candidate, a multi-stage sampling method is used for tail-to-head cyclization.
  • Structural clustering of the resulting ensemble produced a set of peptide conformers that were subsequently used for binding prediction.
  • the GRTH-cyclic peptide binding modes were obtained by simulated annealing MC followed by structural relaxation through MD simulations.
  • the method combines MC, MD, and Langevin Dynamics (LD) simulations in both implicit (Hassan and Steinbach, J. Phys. Chem. B 2011;115:14668 ⁇ 14682 ; Hassan et al., Proteins 2003;51:109 ⁇ 125) and explicit solvent models, with a technique developed previously to predict strong, weak, and ultraweak protein/peptide associations (Cardone et al., J. Phys. Chem.
  • the binding modes and populations of a series of amino-acid side-chain analogs are obtained with the TaRt-cMC method; the resulting density maps, representing all the possible specific and non-specific binding modes of each analog, are then used to derive one or more pharmacophores; for a given pharmacophore, a series of S amino acid sequences can be proposed.
  • the corresponding linear peptide is subjected to free Langevin dynamics (LD) simulations, and P conformations with tail-to-head distances smaller than a preset cutoff are collected.
  • LD Langevin dynamics
  • Each of the P structures is fully cyclized through the gradual application of a force between the peptide termini over the course of LD simulation, producing M distinct cyclic peptide structures.
  • Each of the M structures is relaxed through MD simulations at the desired thermodynamic conditions.
  • a conformational-bias MC simulation is performed to find all the peptide/protein binding modes at the interface. This stage involved full relaxation of the complexes through MD simulations.
  • the inventors first identified all the conformational substates of the isolated DDX25 protein in an aqueous solution at thermodynamic equilibrium 1. For this, a 100-ns molecular dynamics (MD) simulation is carried out at 35 °C and 1 atm using the TIP3P water model at neural pH with the all-atom CHARMM forcefield (version c36). (Brooks et al., 2009) The trajectory is then analyzed based on the heavy-atom-RMSD of residues within a 1-nm distance from the C ⁇ of T239. Clustering analysis with a threshold of 1.5 ⁇ yielded thirteen distinct conformational families.
  • MD molecular dynamics
  • TaRt-cMC Temporal-annealing/Residue-titration canonical Monte Carlo
  • the method is conceptually similar to the chemical-potential simulated-annealing grand canonical MC method but avoids ligand-ligand interactions during binding.
  • a single analog is introduced into a rigid spherical cavity centered at Ca of T239; the cavity radius, here 1 nm, is such that it encloses the interface.
  • a rigid-body simulated annealing MC simulation is then performed, from 1000 °C to 35 °C in a 15-step logarithmic schedule, using only roto-translations of the analog to identify binding modes at 35 °C.
  • the SCP implicit solvent model used here has been parameterized based on experimental hydration energies of this series of analogs. (Hassan et al., J. Phys. Chem. B 2000;104(27):6478-6489). With the first analog fixed in its most stable (highest-population) binding mode, a second analog of the same type is introduced and its binding modes and populations calculated.
  • a third identical analog is introduced with the previous two fixed in their most stable modes, and the process is repeated n times until no binding is observed for the n-th analog of this type.
  • the same process is carried out for each analog type and each protein substate.
  • Each map is calculated from the spatial distribution of the analog center of mass and represents specific and non-specific binding to the interface. Comparison of the maps shows distinct binding patterns, with acidic, basic, net-neutral polar, and nonpolar (hydrophobic) analogs tending to occupy specific nitches on the protein surface; the physicochemical features of each analog determine the details of the binding modes.
  • the maps can then be variously combined to derive one or more pharmacophores. Based on this, the inventors created the pharmacophore shown in FIG.
  • the inventors replaced the termini by dummy groups: -C (x) H-NH 2 at the N- terminus, where C (x) is the C ⁇ of the first residue, and -O (y) -CO-NH-C (z) H 3 at the C-terminus, where O (y) is the oxygen of the last ethylene glycol.
  • the atoms in the -NH 2 and -NH-C (z) H 3 groups have no charge or size, serving only as geometric reference points.
  • the closure protocol consists of gradually superimposing C (x) on C (z) and N on N during an LD simulation at 35 °C in the SCP implicit solvent.
  • the peptides are subsequently relaxed through MD simulations in an explicit solvent at 35 °C and 1 atm using the all-atom CHARMM forcefield 5.
  • Advanced MD techniques can be used at this stage to sample the configurational space more thoroughly or to evaluate the kinetics of interconversions (see Damjanovic et al., Chem. Rev. 2021;121(4):2292-2324 ).
  • the conformations at the end of the simulations are taken as the cyclic peptide structures in an aqueous solution at the given thermodynamic conditions (FIG. 1D).
  • the inventors predicted the protein-peptide binding modes at the interface 6.
  • the inventors used the conformational-bias MC technique for protein-protein and protein-peptide binding prediction, described in detail elsewhere. (Cardone et al., 2015; Cardone et al., 2013). The search is restricted to the interface of interest, reducing the computational cost significantly.
  • COS-1 cells were obtained from ATCC® CRL-1650TM and cultured in a T75 flask at 37 °C with 5% CO 2 , containing Dulbecco Modified Eagle Medium (DMEM) high glucose, GlutaMax TM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Inc., Lawrenceville, GA) and 1 ⁇ Antibiotic-Antimycotic (#15240062, Thermo Fisher Scientific).
  • DMEM Dulbecco Modified Eagle Medium
  • GlutaMax TM Thermo Fisher Scientific
  • FBS fetal bovine serum
  • FBS Atlanta Biologicals, Inc., Lawrenceville, GA
  • Antibiotic-Antimycotic #15240062
  • the full-length human GRTH cDNA fragment (GenBank Acc # AF155140) was cloned into pcDNATM3.1/V5-His A (Thermo Fisher Scientific, Waltham, MA) at KpnI and XbaI restriction sites, and the sequence was confirmed.
  • This GRTH plasmid construct and COS-1 cells were used to generate a stable cell line expressing human GRTH.
  • the expression of pGRTH in a western blot was assessed using a custom-made affinity-purified phospho-site-specific GRTH polyclonal antibody raised in rabbit to the peptide sequence (CKLIDL[pT239]KIRV) of human GRTH.
  • Open reading frame (ORF) primers with restriction sites ApaI and AgeI (Fw: (SEQ ID NO: 43) and Rv: (SEQ ID NO: 44)) were designed to amplify the mCherry fragment from the commercially ordered plasmid p27-IRES-mCherry (Addgene, MA).
  • the mCherry fragment containing the restriction sites and the human GRTH plasmid was digested using the same restriction enzymes, ApaI and AgeI.
  • the digested mCherry fragment and human GRTH plasmid were purified and kept for ligation at 4 °C for 16 hr.
  • the ligation mixture was transformed into DH5 ⁇ Competent Cells, and positive cells containing plasmid with human GRTH and mCherry were selected and purified.
  • the expression of mCherry was checked by transfecting the plasmid expressing mCherry-GRTH into COS-1 cells. After 24 hr transfection, 20 ⁇ M of PEP1, PEP2, CP1, CP2, and CP3 were added into the COS-1 cells for 4h, and the images were photographed using EVOS M5000 Imaging System (Thermo Fisher Scientific).
  • COS-1 cells were stably transfected with a human GRTH-V5-HIS vector, essentially as previously described (Villar et al., Mol. Cell. Biol. 2012;32:1566 ⁇ 1580 ). Briefly, following the determination of the optimal concentration of the antibiotic G418 (Geneticin), COS-1 cells were transfected with human GRTH-V5-His construct using Lipofectamine 2000 (Invitrogen) and cultured in DMEM-high glucose media in the presence of 10% FBS. Cells expressing GRTH were selected and cultured for 2 weeks in the presence of Geneticin to obtain stable transfected cells. Single clones were expanded and checked for pGRTH expression.
  • Protein extracts were prepared and checked for pGRTH expression using phospho-specific GRTH antibody.
  • Treatment of COS-1 cells with cyclic peptides [0161] Cyclic peptides (PEP1 and PEP2) with FITC tag and control peptides (CP1, CP2, and CP3) with FITC tag, and a cyclic peptide without FITC (PEP0) were designed and synthesized commercially (LifeTein, USA). The purity of all the peptides used in this study are > 95%, as determined by HPLC and mass spectrometry. The obtained lyophilized peptides were dissolved in molecular biology grade water and used for the experiments.
  • peptides were added to the stable cell cultures of expressing GRTH (See Table 1). Different concentrations (5, 20, 60, and 100 ⁇ M) of peptides (both the cyclic peptides and control) were added into each well of a full-grown ( ⁇ 90% confluency) 6-well culture plate seeded at a density of 0.3x10 6 cells/well. The cell culture media containing the respective peptide was kept for up to 16 hr. Cells exposed to the peptides were washed with 1 ⁇ PBS and harvested using Trypsin-EDTA. Protein extracts were prepared in the presence of 1 ⁇ protease phosphatase inhibitor.
  • pGRTH protein extracts were prepared at different times of exposure to the peptides (4, 8, 16, and 24 hr). Western blots were performed using 30 ⁇ g of protein from each sample and the expression of pGRTH was revealed using phospho- specific GRTH antibody described below.
  • ⁇ -Actin Antibody (1:2000; sc-69879) and secondary goat anti-mouse HRP-IgG (1:5000; sc-2055) were purchased from Santa Cruz Biotechnology and used for immunoblotting. All the membranes were blocked with 5% milk protein dissolved in 1 ⁇ PBS in 0.1 % Tween 20 (PBST) following standard blot transfer and western blot protocols as recommended by the manufacturers. All the antibodies were diluted in 5% milk protein in PBST. The membranes were developed using TanonTM High-sig ECL Western Blotting Substrate solution according to the manufacturer’s recommendations. Band intensities were detected using an iBright chemiluminescence imaging system (Thermo Fisher Scientific) and quantified with ImageJ Software.
  • PBST 0.1 % Tween 20
  • Antibodies used in this study are a custom-made affinity-purified phospho-site specific GRTH polyclonal antibody raised in rabbit to the peptide sequence (CKLIDL[pT239]KIRV) (1:2000) (Raju et al., 2019) and a GRTH rabbit polyclonal antibody (Sheng et al., J. Biol. Chem. 2006;281:35048 ⁇ 35056) which recognizes the non-phospho of GRTH (non-phospho, 56 kDa). Cytotoxicity analysis of cyclic peptides [0164] MTT cell cytotoxicity assay was performed to determine the IC 50 values of cyclic and control peptides used in this study.
  • the cell viability was not significantly altered at higher concentrations of cyclic peptides.
  • the IC 50 value for each peptide was calculated from the dose- response plot and data fit with a linear regression.
  • the IC 50 values of PEP0, PEP1, PEP2, CP1, CP2, and CP3 were 392 ⁇ M, 312.30 ⁇ M, 333.06 ⁇ M, 314.06 ⁇ M, 173.77 ⁇ M, and 256.66 ⁇ M, respectively. Higher IC 50 values indicate less toxicity and hence the maximum concentration of cyclic peptides for the experiments were fixed as 100 ⁇ M.
  • Cell cytotoxicity assay was performed to determine cytotoxic levels (IC50) of the peptides using in vitro CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega).
  • COS-1 stable cells expressing GRTH were seeded in a 96-well cell culture plate at a seeding density of 0.01x10 6 cells/well and kept for 16 hr.
  • PEP0, PEP1, PEP2, CP1, CP2, and CP3 at different concentrations (10, 50, 100, 250, and 500 ⁇ M) were added into the cell culture media and incubated for 24 hr at 37 °C supplemented by 5% CO 2 .
  • EXAMPLE 3 Effective delivery of cyclic peptides into COS-1 cells
  • the PEP1, PEP2, CP1, CP2, and CP3 tagged with FITC were utilized to assess the effective delivery in COS-1 cells.
  • stable COS-1 cells expressing GRTH were incubated with the respective cyclic peptides for 4 h, and DAPI was used as counter stain.
  • Immunofluorescence microscopy to monitor effective delivery of cyclic peptides
  • COS-1 stable cells expressing GRTH were seeded on the sterile cover slips kept inside the 6-well cell culture plate at a concentration of 0.3x10 6 /well.
  • the plate was kept overnight at 37 °C at 5% CO 2 to allow the cells to adhere to the coverslip.
  • Peptides PEP1, PEP2, CP1, CP2, and CP3 at a concentration of 20 ⁇ M were added to each well.
  • coverslips were carefully taken out from the cell culture plate and washed gently with 1 ⁇ PBS.
  • the cells were further fixed with 50 % and 100 % methanol for 1 min each and mounted inversely on a microscopic slide using ProLongTM Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific).
  • EXAMPLE 4 Dose-dependent effect on GRTH phosphorylation upon treatment with cyclic peptides [0169]
  • overexpression of pGRTH in COS-1 stable cells using a phospho-specific GRTH antibody is shown (FIG. 3).
  • Addition of cyclic peptides PEP0, PEP1, and PEP2 to stable cells expressing pGRTH showed a dose-dependent inhibitory response on phosphorylation of GRTH (FIGS. 3A-3C).
  • a significant dose-dependent decrease on pGRTH protein was observed in the concentration range 20-100 ⁇ M (FIGS. 3A-3C). Cyclic peptides showed a maximum inhibition at 100 ⁇ M concentration.
  • Seminiferous tubule culture from adult mice Seminiferous tubules from the testes of adult mice (90 days old) were used for the in vitro culture experiments. Testes were decapsulated and dispersed with sterile forceps in a petri-dish containing ice-cold medium 199 (Cat. No. 11150059; Thermo Fisher Scientific) with 0.1% BSA for 10 min. The dispersed seminiferous tubules were segmented and placed into the 24-well cell culture plate containing medium 199 and incubated in CO 2 incubator. After 4h, 100 ⁇ M of cyclic peptides were added and incubated for 16 hr.
  • Cell debris was removed from the mixture by centrifugation at 10,000 rpm for 5 min at 4 °C and the supernatant was taken for the experiment.
  • the cell lysates were diluted in lysis buffer and 40 ⁇ M of either PEP1, PEP2, or CP2 were added to each tube and kept under rotation for 16 hours at 4 °C. Then, the tubes were centrifuged at 10000 rpm for 3 min.
  • Each cell lysates treated with different peptides were divided into 100 ⁇ L aliquots and heated individually at different temperatures (40-70 °C) for 3 mins in a Veriti thermal cycler (Applied Biosystems) followed by cooling for 3 mins at room temperature.
  • FRET microscopy and acceptor photobleaching [0177] COS-1 cells expressing mCherry cultured in a 35-mm glass-bottom dish were exposed to 15 ⁇ M of PEP1, PEP2, and CP2 for 16 hr and subjected to live-cell imaging. mCherry was excited at 561 nm, and the emission detected at 570 and 620 nm, whereas FITC was excited at 488 nm, and detection was from 500 to 550 nm. FRET channel was acquired with an excitation at 488 nm and a 570 and 620 nm emission filter.
  • PEP1 and PEP2 showed effective cellular intake when compared to CP1, CP2, and CP3. Acceptor photobleaching method was carried out to observe the intracellular FRET. After photobleaching of the acceptor (mCherry-GRTH), the inventors observed an increase in fluorescence intensity of the donor FITC (PEP1 and PEP2) in the bleached regions, as shown in FIGS. 7A and 7B.
  • the enhanced emission of donor FITC indicates that the donor in the excited state transferred energy to the acceptor mCherry in the ground state, hence the observed increase in the donor intensity. This is accomplished only when donor and acceptor are in close proximity, indicating interaction of the cyclic peptide (PEP1 or PEP2) with the GRTH protein. Conversely, the presence of CP2 (donor FITC) in the photobleached region did not show any increase in fluorescence intensity, indicating no interaction between CP2 and GRTH protein (FIGS. 7A and 7B). In addition, the increase in the donor FITC fluorescence signal was measured to calculate the FRET efficiency. Selected regions in the bleached sections were taken for the calculation of FRET efficiency (FIG. 7C).
  • EXAMPLE 9 Effect of cyclic peptides on pGRTH with or without PKA induction [0179]
  • the transfection of PKA in the stable cells increased the phosphorylation of GRTH.
  • the increase in phosphorylation of GRTH was significantly reduced/inhibited in the presence of 100 ⁇ M concentration of PEP1 and PEP2 with or without the PKA induction.
  • the reduced expression of pGRTH could indicate the competitive binding of cyclic peptides (PEP1 and PEP2) at the PKA site in the presence of PKA.
  • PKA ⁇ (3 ⁇ g/well) were transfected into the respective groups, while for equalization, empty plasmids were transfected into the other groups and cultured further for 24 hr.
  • cyclic peptides PEP1, PEP2, and CP2 at a concentration of 100 ⁇ M each, were added into the respective wells and kept at 37 °C, 5% CO 2 overnight.
  • Protein samples were prepared and run in a PAGE gel for western blotting and developed the blot for phospho-GRTH using phospho-specific GRTH antibody (1: 1000 dilution) as mentioned earlier.
  • Statistical analysis [0181] Data are presented as the mean ⁇ SEM of three independent experiments.
  • Example 10 Ex vivo study of cyclic peptide efficacy in testis culture [0183] Ex vivo (organ culture) experiments were performed to check the efficacy of the cyclic peptides. The efficacy of the cyclic peptides in organ testis culture in inhibiting the phosphorylation of the target protein (GRTH/DDX25) was studied as a prelude to in vivo experiments in mice.
  • agarose (1.5%) blocks were prepared in aseptic conditions and immersed for two hours in a cell culture medium containing ⁇ -MEM with 5% KO-serum.
  • cyclic peptide PEP2 150 ⁇ M was dissolved in the medium before adding the agarose blocks.
  • Testis was dissected out from a 60-day old C57BL/6J mouse.
  • the outer layer (tunica albuginea) was removed and then placed on the agarose blocks soaked with the cell culture medium with or without the PEP2 present.
  • Control and PEP2 treated testis samples were taken from the culture after 24 and 48 hours of treatments.
  • Protein samples were prepared and run in an SDS-gel. After band separation, the bands were transferred into a nitrocellulose membrane for the detection of phospho-GRTH using phospho-specific GRTH antibody.
  • the results showed a significant decrease in the phospho-GRTH in 24 and 48 hours of PEP2 treatment, as indicated in FIGS. 11A and 11B.
  • Example 11 In vivo study of bioavailability of cyclic peptide in mice
  • mice 60 days old
  • mice were injected intraperitoneally with 2.5 mg of PEP1 dissolved in 100 ⁇ L of saline and for sham control only saline (100 ⁇ l) was injected.
  • blood was collected by cardiac puncture, and testis, liver and kidney were removed for the fluorometric analysis.
  • Tissue extracts were prepared for the analysis. Serum was obtained through centrifugation.
  • Presence of FITC in the samples was measured in a Glomax reader.
  • Example 12 Pre-clinical studies of cyclic peptide in mice [0186] The study and results described in Example 11 can serve as the basis for additional in vivo studies in mice and other animals.
  • PEP1 can be exchanged, for example, for PEP0 (cyclic peptide without the FITC tag) as a primary bioactive druggable peptide for long term treatments in mice for 30-45 days.
  • Such studies can be based on efficacy to inhibit GRTH phosphorylation.
  • the peptide’s effectiveness in blocking GRTH phosphorylation in the testis and the morphological changes induced in the testis, sperm count, and sperm motility can be assessed.
  • the fertility and mating behavior of male mice during extended periods (for example, 4-5 weeks) of treatment is studied.
  • Such studies can determine if the degree of azoospermia or low sperm count, and the reversibility of such effects upon cessation of the treatment.
  • Example 13 Studies of cyclic peptide efficacy in population control of nuisance animals [0188]
  • the study and results described in Example 11 and results from the potential studies described in Example 12 can serve as the basis for field studies for population control of nuisance animals.
  • Such field studies can include studies directed again pest wildlife such as rodents, rabbits, and deer.
  • animals can include, for example, animals that carry disease, as well as animals that cause environmental, agriculture, and/or property damage.
  • Test and control groups can be carried out at different locations that are sufficiently geographically isolated from one another, but that otherwise have similar environments. Outcomes can be judged based on litter sizes and general population trends, as well as such factors as male secondary sexual characteristics and mating behavior. More direct testing such as blood and urine samples can also be employed. Administration of a cyclic peptide to male animals of the target species and subsequent decreases in such factors can indicate efficacy of the treatment in population control.
  • Example 14 Studies of cyclic peptide efficacy in animal husbandry [0189]
  • the study and results described in Example 11 and results from the potential studies described in Example 12 can serve as the basis for studies of breeding management in animal husbandry.
  • breeding domestic or wild animals whether for pet animals, agricultural animals, zoo animals, or endangered species, there is a desire to control breeding to prevent mating that could lead to undesired characteristics or interbreeding, as well as timing breeding for a desired seasonal gestation and birth.
  • Animals can include, for example, dogs, horses, cattle, sheep, pigs, and goats.
  • Test and control groups can be carried out in accordance with standard veterinary pre-clinical and clinical studies modified as appropriate for cyclic peptide used.
  • Outcomes can be judged based on preventing or limiting undesired mating, as well as such factors as male secondary sexual characteristics and mating behavior. More direct testing such as blood and urine samples can also be employed. Administration of a cyclic peptide to target male animals and subsequent decreases in mating and other such factors can indicate efficacy of the treatment in the context of animal husbandry. [0190] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above.

Abstract

Cyclic peptides capable of inhibiting Gonadotropin Regulated Testicular Helicase (GRTH) phosphorylation are provided. The cyclic peptides showed effective delivery into COS-1 and germ cells and a dose-dependent inhibitory effect on GRTH phosphorylation. The cyclic peptides inhibit GRTH phosphorylation in the presence of PKA, and binding to the helicase resulted in thermal stabilization of non-phospho-GRTH. Increased efficiency in FRET assay revealed their interaction with GRTH. Cyclic peptide exposure of cultures from mice seminiferous tubules resulted in significant inhibition of phospho-GRTH. These cyclic peptides did not exhibit toxicity.

Description

CYCLIC PEPTIDES AS NON-HORMONAL MALE CONTRACEPTIVE AGENTS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This international application claims the benefit of U.S. Provisional Patent Application No. 63/250,665, filed on September 30, 2021, which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE (.TXT) [0002] Pursuant to the EFS-Web legal framework and 37 C.F.R. § 1.821-825 (see M.P.E.P. § 2442.03(a)), a Sequence Listing in the form of an ST.26-compliant XML file (entitled “Sequence_Listing_3000091-020977.XML” created on September 27, 2022, and 80,636 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference. BACKGROUND OF THE INVENTION [0003] Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. GRTH phosphorylation is required for activity. Inhibition of phosphorylation may be sufficient to inhibit GRTH activity and interfere with spermatogenesis. [0004] There exists a need for non-hormonal male contraceptives. BRIEF SUMMARY OF THE INVENTION [0005] This disclosure provides cyclic peptides for inhibiting phosphorylation of GRTH. [0006] In an embodiment, cyclic peptides comprise the following formula: X1-X2-linker1-X3-X4-X5-X6-linker2, in which X1 is amino acid residue F; X2 is an amino acid residue selected from Q, A, I, V, and L; linker1 comprises one or two linker molecules individually selected from amino acid residue G, amino acid residue P, and a non-peptidic spacer molecule; X3 is an amino acid residue selected from A, S, N, Q, C, and M; X4 is an amino acid residue selected from F, R, H, K, S, Y, Q, and N; X5 is an amino acid residue selected from R, H, and K; X6 is an amino acid residue selected from R, H, and K; and linker2 comprises one or two or three linker molecules individually selected from amino acid residue G, amino acid residue P, and a non-peptidic spacer molecule. [0007] In an embodiment, linker2 may comprise 1 or 2 linker molecules. [0008] In an embodiment, X1 may be replaced with an amino acid residue selected from Q, A, I, V, and L; and X2 is replaced with F. [0009] In an embodiment, the non-peptidic spacer molecule is polyethylene glycol (PEG) or polyvinyl alcohol (PVA). [0010] In an embodiment, a cyclic peptide is provided having the formula: cyclo(1FAGXXXG7- AEEAc), where each X is individually chosen from any basic amino acid, optionally arginine (R), lysine (K), or histidine (H). [0011] In an embodiment, each X is arginine (R).
[0012] In an embodiment, a cyclic peptide may comprise the following structure: Cyclic Peptide 0 (PEP0) (SEQ ID NO: 3, AEEAc)
Figure imgf000004_0001
.
[0013] In an embodiment, a cyclic peptide may comprise the following structure: Cyclic Peptide 1 (PEP1) (SEQ ID NO: 7, AEEAc)
Figure imgf000005_0001
.
[0014] In an embodiment, a cyclic peptide may comprise the following structure: Cyclic Peptide 2 (PEP2) (SEQ ID NO: 8, AEEAc)
Figure imgf000006_0001
. [0015] Non-limiting examples of cyclic peptides according to the present disclosure comprise: cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 3); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 3); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 4); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 4); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 5); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 5); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 6); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 6); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 7); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 7); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 8); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 8); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-PEG3) (SEQ ID NO: 9); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-PEG2) (SEQ ID NO: 9); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 10); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 10); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 11); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 11); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-PEG3) (SEQ ID NO: 12); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-PEG2) (SEQ ID NO: 12); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 13); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 13); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 14); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 14); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-PEG3) (SEQ ID NO: 15); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-PEG2) (SEQ ID NO: 15); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 16); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 16); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 17); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 17); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-PEG3) (SEQ ID NO: 18); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-PEG2) (SEQ ID NO: 18); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 19); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 20); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 21); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 22); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 23); cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 24); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 25); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 26); cyclo-(Phe-ɸ-Ala-Arg-Arg-rg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 27); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 28); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 29); cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 30); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 31); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 32); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 33); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 34); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 35); cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 36); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 37); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 38); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 39); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 40); cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 41); or cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 42); in which ɸ is L-2-naphthylalanine, Q, A, or F (preferably L-2-naphthylalanine), and PEG1 is one PEG molecule, PEG2 is two PEG molecules, and PEG3 is three PEG molecules. [0016] In an embodiment, the PEG molecule(s) in the exemplary cyclic peptides listed above is replaced by a linker molecule, for example, (2-(2-aminoethoxy)ethoxy)acetic acid (AEEAc), for (PEG2), a combination of PEG and Gly, or an intercalations of Pro residues. [0017] In an embodiment, ɸ is L-2-naphthylalanine. In an embodiment, ɸ is Q or F. In an embodiment, ɸ is A, Q, or F. [0018] In an embodiment, one or more of the amino acid residues in the cyclic peptide are omitted or, alternatively, replaced with one or more non-peptidic spacer molecules, optionally polyethylene glycol (PEG), polyvinyl alcohol, or (2-(2-aminoethoxy)ethoxy)acetic acid (AEEAc). [0019] In an embodiment, one or more side chains are attached at one or more glycine residue within the cyclic peptide. [0020] In an embodiment, one or more amino acid residues within the cyclic peptide are methylated. [0021] In an embodiment, the cyclic peptide is linked through head-to-tail cyclization. [0022] In an embodiment, the cyclic peptide inhibits gonadotropin regulated testicular helicase (GRTH) phosphorylation. [0023] A composition is also provided comprising one or more cyclic peptide of the present disclosure. [0024] In an embodiment, the composition comprises 1 mg to 1 g of the cyclic peptide. [0025] In an embodiment, the composition is a pharmaceutical composition. [0026] In an embodiment, the composition further comprises diluents, binders, stabilizers, buffers, salts, solvents, preservatives, or combinations thereof. [0027] In an embodiment, the composition is formulated for oral administration. [0028] In an embodiment, a composition for inhibiting spermatogenesis comprising an effective amount of one or more cyclic peptide of the present disclosure is provided. [0029] In an embodiment, a composition for inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation comprising an effective amount of one or more cyclic peptide of the present disclosure is provided. [0030] An oral contraceptive comprising one or more cyclic peptide of the present disclosure is also provided. [0031] A method of inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation is provided comprising administering an effective amount of one or more cyclic peptides of the present disclosure or a composition thereof. In an embodiment, the method comprises administering an effective amount of one or more cyclic peptides of the present disclosure or a composition thereof to a subject. [0032] A method of inhibiting spermatogenesis is also provided comprising administering an effective amount of one or more cyclic peptide of the present disclosure or a composition thereof to a subject. [0033] In the methods provided herein, the subject may be a mammal, preferably a human. In an embodiment, the human is a male human. [0034] In an embodiment, administration of the cyclic peptide(s) or composition thereof is oral administration. [0035] The use of one or more cyclic peptide of the present disclosure in the manufacture of a medicament for inhibiting spermatogenesis in a subject is also provided. [0036] The use of one or more cyclic peptide of the present disclosure in the manufacture of a medicament for inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation in a subject is also provided. BRIEF DESCRIPTION OF THE DRAWINGS [0037] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements. [0038] FIGS. 1A-1G show computational design of GRTH-binding cyclic peptides. FIG. 1A shows electrostatic potential on the solvent-accessible surface of GRTH at the PKA-binding interface (dark gray: positive potential; medium gray: negative potential; white: neutral); the circle indicates location of T239. FIG. 1B shows GRTH surface representation (same orientation as in panel 1A with the single amino-acid analogs hotspots indicated (each point corresponds to the location of the Cβ atom in the corresponding residue); T239 , basic residues in dark grey (R242 is occluded but indicated by the arrow); acidic residues in lighter grey. FIG. 1C shows pharmacophore derived from the hotspot distribution of functional groups used here (two others can be derived; cf. SI). F: Phe; Q: Gln; b (basic): Arg+, His+, or Lys+; a (acidic): Glu– or Asp–; hyd (nonpolar/hydrophobic): Ala, Ile, Val, or Leu; pol (net-neutral polar): Gln or Asn. Circles containing more than one symbol means that any of the indicated groups can be used. Solid lines indicate a single peptide bond; dashed lines, one or more (indicated by the adjacent numbers); double arrow indicates allowed permutation. FIG. 1D shows computational prediction of PEPx conformations in solution; each structure is a representative member of a conformational family. Only non-hydrogen atoms shown; the backbone RMSD (including PEG2) between any two conformers is in the 1.5–3 Å range. FIG. 1E shows electrostatic potential (mostly positive; darker grey) on the solvent-accessible surface of two representative PEPx conformers shown in panel 1D and 1F. FIG. 1F shows superposition of the PEPx conformers upon binding to the GRTH/PKA interface (circle indicates the position of T239; GRTH orientation and grey scale same as in panel 1A; only one GRTH surface shown for clarity. All the conformers find favorable interactions with the protein, albeit with significant variability in binding modes due to the flexibility of both PEPx and protein surface. FIG. 1G shows utual structural adaptation upon binding enable PEPx to find its way into and expand a shallow crevice on the GRTH surface; solvent-accessible surface of PEPx indicated by arrows; only three conformers are shown to illustrate. [0039] FIG. 2 shows immunofluorescence images showing the degree of entry of the PEP1, PEP2, CP1, CP2 and CP3 inside the cytoplasm and nucleus of COS-1 cells stably expressing GRTH. DAPI was used counter stain . [0040] FIGS. 3A-3F show effect of cyclic and control peptides on GRTH phosphorylation in COS-1 stable cells expressing GRTH. Cyclic peptides PEP1, PEP2, and PEP0 ( FIGS. 3A, 3B, and 3C) showed a dose dependent (5, 20, 60, and 100 μM) inhibitory response while control peptides CP1, CP2, and CP3 ( FIGS. 3D, 3E, and 3F) showed no effect on GRTH phosphorylation. β-actin was used as a negative control. The protein band intensities of pGRTH and β-actin from three independent experiments (mean ± SEM) were measured as indicated in the methods section. Values were normalized by β-actin. * P < 0.05 indicates statistically significant. [0041] FIGS. 4A-4C show time-dependent inhibitory effect of cyclic peptides on pGRTH expression in COS-1 stable cells. Cyclic peptides PEP0, PEP1, and PEP2 (100 μM) showed a time- dependent response on the expression of pGRTH ( FIGS. 4A, 4B, and 4C, respectively). The pGRTH was identified using custom made GRTH phospho-specific antibody (Raju et al., Sci. Rep. 2019;9: 6705. ) . β-actin was used as a negative control. The protein band intensities of pGRTH and β-actin from three independent experiments (mean ± SEM) are shown. (*, P < 0.05) indicates statistical different from control. [0042] FIGS. 5A and 5B show inhibition of pGRTH expression by cyclic peptides in seminiferous tubules. FIG. 5A indicates PEP1, PEP2 and CP1, CP2 and CP3 showed effective internalization in the seminiferous tubules. FIG. 5B shows a Western blot of seminiferous tubules exposed to 100 μM of the respective peptide showed a significant decrease in the PEP1 and PEP2 treated samples when compared those untreated samples or exposed to control peptides. Band intensities were calculated for three independent experiments (n=3) and the values were plotted in the form of graph. All data represent mean ± SEM (*, P < 0.05). [0043] FIGS. 6A and 6B show thermal shift plots (CETSA) showing melting curve profiles of cyclic and control peptides. FIG. 6A Western blots showing denaturation pattern of non-pGRTH protein upon treatment with CP2, PEP1 and PEP2. FIG. 6B shows that band intensities were calculated for three independent experiments (n=3) and the values were plotted in the form of graph. The sample at temperature 40 °C was taken as an initial control for all the samples. All data represent mean ± SEM. [0044] FIGS. 7A-7E show quantitative FRET images on the live COS-1 cells. FIG. 7A shows FRET images for acceptor photobleaching in COS-1 cells transiently expressed with mCherry- GRTH and exposed to 15 μM of cyclic peptides (PEP1, PEP2, and CP2). Areas were marked and images were acquired before and after the mCherry (acceptor) photobleaching. The cyclic peptides (PEP1 and PEP2) attached with FITC (donor) showed a significant increase in the intensity after the photobleaching while control peptide (CP2) did not show any increase in the intensity of donor. FIG. 7B shows that the intensity of donor and acceptor before and after photobleaching for the PEP1, PEP2, and CP2 were represented in the graph. FIG. 7C shows images of COS-1 cells expressing mCherry-GRTH with PEP1, PEP2 and CP2 and the respective FRET efficiency images. FIG. 7D indicates that the acceptor photobleaching experiment shown in a plot gave a FRET efficiency of 22 % for PEP1, 20 % for PEP2 and 5 % for CP2 however the FRET efficiency values change considerably for different locations in the marked regions. FIG. 7E shows mean values (Mean ± SEM) of FRET efficiency % of mCherry-GRTH exposed to 15 μM of PEP1, PEP2 and CP2 obtained through the acceptor photobleaching experiment. [0045] FIGS. 8A-8C show the effect of cyclic peptides on pGRTH with or without PKA induction: Western blot analysis was performed to validate the expression of phospho-GRTH in control (C), PKA induction only (PKA), 100 μM cyclic peptide treatment, PEP1 or PEP2 or CP2, and both PKA and cyclic peptide. β-actin was used as an internal control. The protein band intensities of pGRTH (61 kDa) from three independent experiments (mean ± SEM) were measured using ImageJ software and normalized with the β-actin. All data represent mean ± SEM. * P < 0.05. [0046] FIG. 9 shows a schematic “pipeline” flowchart of the computational method used for designing cyclic peptides that bind to shallow protein surfaces. (1) Molecular dynamics (MD) simulations are used to obtain the set of N conformational substates of the protein interface. (2) For each of the N substates, the binding modes and populations of a series of amino-acid side-chain analogs are obtained with the TaRt-cMC method; the resulting density maps, representing all the possible specific and non-specific binding modes of each analog, are then used to derive one or more pharmacophores; for a given pharmacophore, a series of S amino acid sequences can be proposed. (3) Once a specific sequence is chosen, the corresponding linear peptide is subjected to free Langevin dynamics (LD) simulations, and P conformations with tail-to-head distances smaller than a preset cutoff are collected. (4) Each of the P structures is fully cyclized through the gradual application of a force between the peptide termini over the course of LD simulation, producing M distinct cyclic peptide structures. (5) Each of the M structures is relaxed through MD simulations at the desired thermodynamic conditions. (6) A conformational-bias MC simulation is performed for each of the M peptide structures and each of the N protein structures to find the peptide/protein binding modes at the interface. This stage involved full relaxation of the complexes through MD simulations in explicit solvent. [0047] FIGS. 10A-10F show graphs of cell viability percent vs. concentration of cyclic peptide reflecting the cytotoxic effect of PEP1 (FIG. 10A), PEP2 (FIG. 10B), PEP0 (FIG. 10C), CP1 (FIG. 10D), CP2 (FIG. 10E), and CP3 (FIG. 10F), respectively, with various concentrations (10 μM to 500 μM) to which stable cells expressing GRTH were exposed. Relative cell viability (%) was determined by taking the OD value of treatment divided by control and multiplied by 100. [0048] FIG. 11A shows results from an ex vivo organ testis culture study indicating a significant decrease in the phospho-GRTH in 24 and 48 hours of PEP2 treatment. This decrease indicates that cyclic peptide treatments are effective in inhibiting phosphorylation of GRTH. [0049] FIG. 11B shows results from an ex vivo organ testis culture study in which protein samples were prepared and run on an SDS-gel. After band separation, the bands were transferred into a nitrocellulose membrane for the detection of phospho-GRTH using phospho-specific GRTH antibody. [0050] FIG. 12 shows a plot of the presence of PEP1 in various tissues including significantly in the serum, testis and minimally in the liver and kidney from an in vivo study of bioavailability of PEP1 in mice. DETAILED DESCRIPTION OF THE INVENTION [0051] Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims. Definitions [0052] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Preferred methods and compositions are described, although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention. [0053] The following abbreviations are used throughout the disclosure: CETSA - Cellular Thermal Shift Assay COS 1 - CV-1 (Simian) in Origin, and carrying the SV40 genetic material (Fibroblast-like cell lines derived from monkey kidney tissue) CP1 - Control peptide 1 (Linear) CP2 - Control peptide 2 (Cyclic) CP3 - Control peptide 3 (Cyclic) FITC - Fluorescein isothiocyanate FRET - Fluorescence resonance energy transfer GRTH - Gonadotropin-Regulated Testicular RNA Helicase IC50 - Half-maximal inhibitory concentration PEPx - Cyclic Peptide x (basic scaffold) PEP0 - Cyclic Peptide 0 PEP1 - Cyclic Peptide 1 PEP2 - Cyclic Peptide 2 DDX19 - DEAD-Box Helicase 19B pGRTH - Phospho Gonadotropin-Regulated Testicular RNA Helicase PKA - Protein Kinase A ROI - Regions of interest Cyclic Peptide GRTH Inhibitors [0054] Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) is a testis-specific member of the DEAD-box family of RNA helicases expressed in Leydig and germ cells of the male gonad and found to have an essential role in spermatid development during spermatogenesis. GRTH is expressed in rat, mouse, and human testis and acts as a post-transcriptional regulator of genes essential for the completion of spermatogenesis (Tang et al., J. Biol. Chem. 1999;274:37932−37940; Tsai-Morris et al., Proc. Natl. Acad. Sci. U.S.A. 2004;101:6373−6378; Dufau and Tsai-Morris Mol. Hum. Reprod. 2007;13:887−892, Dufau and Kavarthapu Front. Endocrinol. 2019;10:No. 576 ). GRTH Knockout mice are sterile and lack spermatozoa due to failure of elongation of round spermatids during spermiogenesis (Tsai-Morris et al., 2004). There are two species of GRTH, a 56 kDa non-phosphorylated form, predominantly found in the nucleus where it interacts with chromosomal region maintenance 1 protein (CRM1) and participates in mRNA transport to cytoplasmic sites, and a 61 kDa phosphorylated GRTH (pGRTH) species which is present exclusively in the cytosol and found to reside in Chromatoid Bodies (Sato et al., 2010 Anbazhagan et al., 2020) and to be associated with polyribosomes where it may be involved in translation of germ cell-specific genes (Dufau and Tsai-Morris, 2007). Transient expression of GRTH cDNA in the COS-1 cells showed a similar pattern of expression as observed in mice (Tsai-Morris et al., 2007). Our previous studies found a missense mutation of arginine to histidine at position 242 (R242H) in exon 8 of GRTH in 5.8% of Japanese patients with azoospermia. In vitro experiments using GRTH (R242H) mutant plasmid in COS-1 cells resulted in the loss of pGRTH form with preservation of the non-phospho GRTH species (Tsai-Morris et al., 2007). The inventors showed that GRTH knock-in (KI) mice model carrying the human GRTH gene mutation R242H are sterile, lack sperm due to arrest at step 8 of round spermatids, and display complete loss of pGRTH, revealing the functional relevance of pGRTH (Kavarthapu et al., Hum. Mol. Genet. 2019;28:2561−2572 ). [0055] The inventors identified residue T239, structurally adjacent to the patient mutant at R242 as the GRTH phosphorylation site. Molecular modeling and biochemical studies allowed us to determine the role of R242 and other relevant solvent-exposed residues at the GRTH/PKA interface that controls the phosphorylation status of the protein (Raju et al., 2019). These include residues E165, K240, and D237, which are all structurally distant from the conserved motifs involved in the ATPase and helicase activities. Site-directed mutagenesis of these residues resulted in marked reduction or abolition of pGRTH species. Computer simulations showed that relevant disruptions of intramolecular H-bonds at the GRTH/PKA interface lead to modest but consequential structural changes that can affect the PKAα catalytic efficiency. Computational modeling also showed that the surface topography of the GRTH/PKA interface is relatively shallow, lacking the desirable deep crevices typically targeted as potential binding pockets for small drug-like molecules. Cyclic peptides are an alternative for such (non-druggable) surfaces, and the GRTH/PKA interface appears to have unique features that could be exploited for selectivity, specificity, and high-affinity binding. A cyclic peptide that binds at or near the phospho-site can potentially perturb PKA activity or block the phosphorylation of GRTH, thereby inhibiting spermatogenesis. [0056] In their simplest form, cyclic peptides consist of amino acids (typically, ≤ 10 aa) linked together to form a macrocyclic ring structure. Many biologically active cyclic peptides are linked through head-to-tail cyclization, where an amide bond is formed between the amino and carboxy termini of each end. There are several advantages of using cyclic peptides for the development of therapeutics (Zorzi et al., Curr. Opin. Chem. Biol. 2017;38:24−29). First, cyclic peptides show better biological activity than linear peptides because of their conformational rigidity, and their small ring structure provides resistance towards proteolytic degradation (Joo, Biomol. Ther. 2012;20:19−26; Dougherty, Patrick et al., Chem. Rev. 2019;119:10241−10287 ). Also, cyclic peptides have larger surface areas, leading to higher affinity and selectivity of protein targets (Choi and Joo, Biomol. Ther. 2020;28:18−24 ). Second, the toxicity of cyclic peptides is less compared to the other drugs, as their degradation products are amino acids. However, the major challenge of cyclic peptides as therapeutics is the poor membrane permeability (Tarek et al., Biophys. J. 2003;85:2287−2298 ). Thus, a great deal of effort has been devoted to designing cell-penetrating cyclic peptides for efficient delivery into cells. Cyclic peptides can be categorized based on their physicochemical properties or conformations. Several cyclic peptides containing L-amino acids were developed and synthesized to investigate their cell-penetrating properties and examine their role as molecular transporters (Park et al., Mol. Pharm. 2019;16:3727−3743 ). Cyclic peptides rich in arginine residues can be effectively delivered into mammalian cells (Qian et al., ACS Chem. Biol. 2013;8:423−431 ). The addition of L-2-naphthylalanine in the peptides facilitates plasma-membrane binding and internalization. In another study, a cyclic peptide containing arginine and tryptophan was conjugated to a drug, showing its potential use as an intracellular drug delivery platform (Nasrolahi Shirazi et al., Mol. Pharm.2013;10:488−499 ). Arginine-rich cyclic peptides were shown to display higher structural rigidity and enhanced transduction efficiency (Lättig-Tünnemann et al., Nat. Commun. 2011;2:453 ); this study also suggested that cyclic peptides have higher cellular uptake efficiency compared to their linear forms indicating that the cyclization strategy could be employed for the delivery of various potential drug candidates. [0057] Considering these facts and the structural features of the GRTH surrounding the exposed site at T239, the inventors rationally designed cyclic peptides that meet the above parameters, for example, effective ingress into cells and binding to the target protein at the expected binding interface. The inventors evaluated the efficacy of cyclic peptides using a FITC tag to show that they effectively enter COS-1 cells and mice seminiferous tubules. CETSA and FRET assays were performed to assess the binding ability of the cyclic peptide to GRTH protein in COS-1 cells. Further, the efficiency of the cyclic peptide binding to the PKA binding site was assessed in the presence of PKA. Treatment of both cyclic peptides in COS-1 stable cells expressing GRTH and seminiferous tubules endogenously expressing GRTH showed a significant reduction in pGRTH species indicating their effectiveness in preventing phosphorylation of GRTH by PKA. [0058] Although other possible pharmacophores may exist with the desired function of preventing GRTH phosphorylation, the pharmacophore used as the basis for the design of the cyclic peptides is described herein. In an aspect, cyclic peptides are provided herein with the following pharmacophore features (cf. Fig. 1C): an amino acid sequence containing at most 10 amino acid residues. For example, the amino acid sequence may comprise between about 6 and 9 amino acid residues, e.g., 8 amino acid residues. The peptides comprise an amino acid residue (number 1) F; followed by an amino acid residue (number 2) selected from Q, A, I, V, and L; followed by a linker with a maximum length equivalent to 2 amino acids or 2 PEG (polyethylene glycol) molecules (see compositions below) or 2 PVA (polyvinyl alcohol) molecules; followed by an amino acid residue selected from either A or a small polar (non-charged) amino acid selected from S, N, Q, C, and M; followed by either F, or a basic amino acid selected from R, K, and H, or by a polar amino acid selected from S, N, Y, and Q; followed by a basic amino acid residue selected from R, K, and H; followed by a basic amino acid residue selected from R, K, and H; followed by a second linker of a maximum length equivalent to 3 amino acids or 3 PEG molecules (see possible compositions below). The order of amino acid residue number 1 and amino acid residue number 2 may be reversed (e.g., amino acid residue number 1 is selected from Q, A, I, V, and L and amino acid residue number 2 is F). The linkers themselves may comprise the amino acids G or P or non-peptidic spacer molecules, e.g., PEG or PEG2 (2-(2-aminoethoxy)ethoxy)acetic acid (AEEAc). [0059] This pharmacophore is also illustrated in FIG. 1C. Although some leeway exists in the choice of residues matching the pharmacophore, some parameters are imposed a priori, which restrict the options: (i) the cyclic peptide should ideally contain fewer than ten residues (or being of similar length if non-peptidic spacers, e.g., PEG, are used to separate functional groups), (ii) incorporate local moieties that facilitate penetration into the cell membrane, and (iii) have enough structural flexibility in solution to adapt to the shallow, flexible surface upon binding. [0060] The pharmacophore described above may be a preferred framework, but any of the positions may be modified. For example, removing one X could be compensated by a modification of a nearby X, to, for example, provide for better cyclization or other reasons (e.g., to compensate for size or to accommodate methylation for oral bioavailability). [0061] In some embodiments, a cyclic peptide of the disclosure has the formula: cyclo(1FAGXXXG7-AEEAc), in which each X is individually chosen from any basic amino acid, e.g., arginine (R), lysine (K), or histidine (H). (SEQ ID NO: 1, AEEAc). [0062] One of the simplest cyclic peptides that fits the pharmacophore and accommodates the stringent conditions is cyclo(1FAGRRRG7-AEEAc), referred to as PEPx (SEQ ID NO: 2, AEEAc). [0063] In some embodiments, all amino acid residues of the cyclic peptides are in the L configuration. In some embodiments, one or more amino acid residues of the cyclic peptides are in the D configuration. [0064] In some embodiments, the cyclic peptide can have one or more side chains attached thereto, for example at one or more glycine residue. Any suitable side chain (e.g., Pro or Gly in the linkers) can be selected so long as GRTH binding is maintained. [0065] Certain structural flexibility is desirable to allow local adaptation to the GRTH/PKA interface and may be controlled by changing the length of the linker (e.g., flexibility may be reduced with Gly2 instead of PEG2). The molecular surface representation of the conformers in solution (two shown in FIG. 1E) reveals a common feature: a pronged cationic structure with basic residues on one end of the ring and a hydrophobic region on the other, giving the peptide the cationic/amphipathic character desirable for membrane translocation and cytosolic localization. [0066] In some embodiments, a linker of any length may be used. Suitable linkers may be chosen based on a number of factors, so long as GRTH binding is maintained. Length can be expressed in units, where, for example, a unit corresponds to one or more of a monomer residue and an amino acid residue. Any suitable monomer or amino acid or combination thereof can be employed. Examples of monomers and amino acids include ethylene glycol, vinyl acetate, glycine, and proline. A linker can comprise a homopolymer or a copolymer. In some embodiments, the linker can be comprised of polyglycine, polyethylene glycol, polyproline, or polyvinyl alcohol, or any combination thereof. In some embodiments, the linker can be comprised of (2-(2- aminoethoxy)ethoxy)acetic acid (AEEAc). In some embodiments, the linker comprises two units of length at most. In some embodiments, the linker comprises three units of length at most. [0067] In some embodiments, the cyclic peptide may be modified to increase bioavailability. Any known methods may be used to increase bioavailability including but not limited to, methylation of one or more amino acid residues on the cyclic peptide. [0068] A set of non-limiting examples of cyclic peptides provided by the disclosure is provided below, which incorporate relatively minor side-chain modifications to PEPx (SEQ ID NO: 2): PEP0 (SEQ ID NO: 3, AEEAc) replaces A2 by L-2-naphthylalanine, whereas PEP1 (SEQ ID NO: 7, AEEAc) and PEP2 (SEQ ID NO: 8, AEEAc) replace G7 by L- and D-Lys(FITC), respectively. [0069] Cyclic Peptide 0 (PEP0) (SEQ ID NO: 3, AEEAc):
Figure imgf000019_0001
[0070] Cyclic Peptide 1 (PEP1) (SEQ ID NO: 7, AEEAc):
Figure imgf000020_0001
[0071] Cyclic Peptide 2 (PEP2) (SEQ ID NO: 8, AEEAc):
Figure imgf000021_0001
[0072] The basic scaffold (PEPx) (SEQ ID NO: 2) comprises only naturally occurring amino acids, is cationic and amphipathic, comprises aromatic and hydrophobic residues, and is rich in arginine residues. Computer simulations of PEPx and its minimally-modified PEP0 showed that both peptides adopt a family of conformations in solution at physiological conditions, possibly in fast interconversion; all the conformers display the basic pharmacophoric features. The inventors introduced some structural flexibility through the length and chemical nature of the spacer (in this case, PEG2), which allows the peptide to adapt to the shallow interface. Further, AEEAc may be used as a spacer in the cyclic peptide. Computer simulations showed that all the conformers bind to the expected interface, although with significant structural variations. This binding-mode variability was expected and, within limits, is a desirable feature as it allows the peptide to find the most favorable interactions, albeit at an entropic price. [0073] A cyclic peptide may comprise an amino acid sequence comprising the following formula: X1-X2-linker1-X3-X4-X5-X6-linker2, wherein X1 is amino acid residue F; X2 is an amino acid residue selected from Q, A, I, V, and L; linker1 comprises one or two linker molecules individually selected from amino acid residue G, amino acid residue P, or a non-peptidic spacer molecule; X3 is an amino acid residue selected from A, S, N, Q, C, and M; X4 is an amino acid residue selected from F, R, H, K, S, Y, Q, and N; X5 is an amino acid residue selected from R, H, and K; X6 is an amino acid residue selected from R, H, and K and linker2 comprises one, two, or three linker molecules individually selected from amino acid residue G, amino acid residue P, or a non-peptidic spacer molecule. [0074] In the cyclic peptide, X1 may be replaced with an amino acid residue selected from Q, A, I, V, and L; and wherein X2 may be replaced with F. [0075] The cyclic peptide comprises two linkers (linker1 and linker2). The first linker (linker1) comprises one or two linker molecules, preferably one linker molecule. The second linker (linker2) comprises one or two or three linker molecules, preferably 1 or 2 linker molecules, more preferably 2 linker molecules. For each linker, the linker molecule(s) are individually selected from amino acid residue G, amino acid residue P, or a non-peptidic spacer molecule. In some embodiments, the non- peptidic spacer molecule is polyethylene glycol (PEG) or polyvinyl alcohol (PVA). Examples of linkers comprising one or more non-peptidic spacer molecule(s) include PEG1 (one PEG molecule), PEG2 (two PEG molecules, e.g., a dimer; i.e., AEEAc), or PEG3 (three PEG molecules, e.g., a PEG trimer). Further examples of linkers comprising one or more non-peptidic spacer molecule(s) include PVA1 (one PVA molecule), PVA2 (two PVA molecules, e.g., a dimer), or PVA3 (three PVA molecules, e.g., a PVA trimer). Copolymers of PEG and PVA can also be employed, for example, combinations and permutations of PEG and PVA adding up to two or three total molecules in a given linker. Examples of such dimers and trimers can include PEG1-PVA1, PVA1-PEG1, PEG2- PVA1, PVA1-PEG2, PVA2-PEG1, PEG1-PVA2, PEG1-PVA1-PEG1, or PVA1-PEG-PVA1, or any combination thereof. The maximum length of linker 1 is equivalent to 2 amino acids, 2 PEG molecules, or 2 PVA molecules. The maximum length of linker 2 is equivalent to 3 amino acids, 3 PEG molecules, or 3 PVA molecules. The combined length of the two linkers should be selected so that the maximum length of the cyclic peptide does not exceed 10 amino acids in length. [0076] Additional non-limiting examples of cyclic peptides are listed below: cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-PEG2) [corresponds to PEP0 where AEEAc is replaced by PEG2] (SEQ ID NO: 3) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 3) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 4) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 4) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 5) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 5) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 6) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 6) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-PEG2) [corresponds to PEP1 where AEEAc is replaced by PEG2] (SEQ ID NO: 7) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 7) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG2) [corresponds to PEP2 where AEEAc is replaced by PEG2] (SEQ ID NO: 8) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 8) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-PEG3) (SEQ ID NO: 9) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-PEG2) (SEQ ID NO: 9) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 10) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 10) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 11) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 11) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-PEG3) (SEQ ID NO: 12) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-PEG2) (SEQ ID NO: 12) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 13) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 13) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 14) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 14) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-PEG3) (SEQ ID NO: 15) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-PEG2) (SEQ ID NO: 15) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 16) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 16) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 17) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 17) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-PEG3) (SEQ ID NO: 18) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-PEG2) (SEQ ID NO: 18) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 19) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 20) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 21) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 22) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 23) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 24) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 25) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 26) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 27) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 28) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 29) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 30) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 31) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 32) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 33) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 34) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 35) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 36) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 37) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 38) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 39) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 40) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 41) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 42) [0077] In the above exemplary cyclic peptides, PEG is polyethylene glycol. PEG1 is one PEG molecule, PEG2 is two PEG molecules, and PEG3 is three PEG molecules. In each of the above exemplary cyclic peptides, each PEG molecule can be replaced by another linker molecule, e.g., (2- (2-aminoethoxy)ethoxy)acetic acid (AEEAc); additional linkers can also be formed by a combination of PEG and Gly or include intercalations of Pro to change the local flexibility for improved binding affinity or specificity. Polyvinyl alcohol (PVA) can be used in place of or in combination with PEG [0078] In the above exemplary cyclic peptides, ɸ is L-2-naphthylalanine. In each of the above exemplary cyclic peptides, ɸ can be replaced with A, Q or F. In some embodiments, ɸ can be replaced with Q or F. [0079] Also, in each of the above exemplary cyclic peptides, each instance of Arg can be replaced with Lys or His (e.g., any basic amino acid can be present in any combination). [0080] As described above, each of the above exemplary cyclic peptides may comprise one or more modifications to improve bioavailability. In some embodiments, the cyclic peptide is methylated at one or more positions. In some embodiments, the cyclic peptide comprises one or more non-natural amino acid analogs, for example, replacing one or more Arg with a corresponding non-natural amino acid analog. Methods of inhibiting GRTH phosphorylation [0081] The cyclic peptides described herein effectively block the phosphorylation of GRTH. Thus, the cyclic peptides described herein may be used to inhibit GRTH phosphorylation. A method of inhibiting GRTH phosphorylation is provided in which GRTH is contacted with a cyclic peptide described herein. The method of inhibiting GRTH phosphorylation is an in vitro method. The method of inhibiting GRTH phosphorylation is an in vivo method. [0082] The effects of the cyclic peptides described herein on GRTH phosphorylation were evaluated in living cells. To monitor its cytosolic localization, G7 was chosen as the position for the fluorophore. Lys(FITC) conjugation in either the L (PEP1) or D (PEP2) configurations was chosen, although smaller, more conspicuous probes are possible (Qian et al., 2013). The inventors show herein that PEP1 and PEP2 contain all the relevant characteristics that enable effective and easy entry into COS-1 cells and seminiferous tubules. PEP0 is expected to have similar binding features, based on computer simulations and its biological inhibitory function on GRTH phosphorylation. In contrast, the linear peptide CP1 (SEQ ID NO: 45), which has the same amino acid sequence as PEP1 (SEQ ID NO: 7, AEEAc), failed to enter the cells, supporting that cyclization enhances membrane crossing. [0083] The treatment/exposure of PEP1 and PEP2 to COS-1 cells stably expressing GRTH abolished the expression of pGRTH in vitro significantly, indicating the effective abolition of GRTH phosphorylation by endogenous PKA and supporting the therapeutic nature of these cyclic peptides; in contrast, the linear control peptide CP1 did not have any effects on the expression of pGRTH in COS-1 cells. Although CP1 may still bind to GRTH at the PKA-binding surface, even when not conforming to the pharmacophore as well as PEP1 does, the observed differences are more likely related to the reduced CP1 entry into the cells. In contrast, control cyclic peptides CP2 (SEQ ID NO: 46), rich in acidic residues Glu, and CP3 (SEQ ID NO: 47), which lacks the aromatic side chains of Phe and Nal, did not have any inhibitory effect on the phosphorylation of GRTH despite their surprisingly adequate access to the cells. Both controls lack essential pharmacophore features relevant for GRTH binding at the interface, although our fluorescence study shows that both peptides competently reached intracellular sites. [0084] Peptidyl-prolyl isomerase, which specifically binds to phosphorylated Ser/Thr-Pro motifs to regulate post-translational modification, was effectively blocked by cyclic peptides containing Nal and rich in Arg residues (Liu et al., J. Med. Chem. 2010;53:2494−2501 ). Although different considerations have dictated the inclusion of Nal and Arg residues in PEP1 and PEP2, namely, the binding to the GRTH interface and the concomitant structural perturbation affecting PKA efficacy, a different, separate effect may be at play in the blockade of GRTH phosphorylation by PEP0, PEP1, and PEP2. Moreover, PEP0, which is obtained from PEP1 upon removing the Lys(FITC) side chain, also showed similar effects as PEP1, confirming that the presence of the bulky fluorophore did not interfere with binding. A significant reduction of pGRTH was observed at 8 h and 16 h upon treatment with PEP1 and PEP2 of COS-1 cells stably expressing GRTH. Additionally, a reduction in the expression of phospho-GRTH upon exposure of PEP1 and PEP2 of COS-1 cells was observed, even when COS1 cells stably expressing GRTH were transiently transfected with PKA catalytic subunit, indicating competitive binding of cyclic peptides at the GRTH/PKA site in the presence of PKA. Having established the inhibitory effect of PEP1 and PEP2 on GRTH phosphorylation, a deeper analysis of the peptide-protein interactions and binding stability was performed. This is exemplified in CETSA studies described below, where thermal stabilization of the GRTH protein was effectively retained by PEP1 or PEP2 treatment when compared to CP2. The thermal stabilization of proteins by a peptide was successfully demonstrated in recent studies to validate cyclization and binding stability (Almqvist et al., Nat. Commun;2016:7:11040 ; Tetley et al., J. Biol. Chem. 2020;295:2866−2884 ). [0085] The inventors found that changes in thermal stability resulted in the presence of soluble protein at a higher temperature. After the heating step, the presence of non-phospho GRTH in solution at 50°C and 55°C upon the exposure of PEP1 and PEP2 indicated that there was protein- ligand interaction, while this was not observed with CP2 due to its inability to bind GRTH. The peptides were also evaluated for their cytotoxic effects. The integrity of the plasma membrane can be disrupted by the entry of molecules, possibly causing cytotoxicity, leading to apoptosis and cell death. This process depends on the specific interactions of the molecule with the membrane and is difficult to predict (Yang and Hinner, Methods Mol. Biol. 2015;1266:29−53 ; Galluzzi et al., Cell Death Differ. 2018;25:486−541). The inventors’ in vitro studies showed that exposure to high concentrations of cyclic peptides displayed limited cytotoxicity with high IC50 values. FRET acceptor photobleaching was performed to confirm the interaction of PEP1 and PEP2 with GRTH. An increase in the fluorescence intensity of donor FITC (PEP1 and PEP2) was observed in the photobleached region of acceptor mCherry indicating FRET has occurred due to the proximity of donor FITC and acceptor mCherry. This was further confirmed by the significant increase in the FRET efficiency for PEP1 and PEP2 when compared to the linear control peptide (CP1) in COS-1 cells. The interaction of a ligand with a protein by the acceptor photobleaching method was demonstrated in several studies (Tavares et al., Biochim. Biophys. Acta 2014;1842:981−991 ; Bao et al., Sci. Rep. 2015;5:10947), showing increases in the donor intensity after photobleaching and FRET efficiency. Methods of inhibiting spermatogenesis [0086] The cyclic peptides described herein may be used to inhibit spermatogenesis. Studies from our laboratory highlighted the essential role of GRTH in the completion of spermatogenesis and established the crucial role of the phospho-form of GRTH in spermatid development during spermiogenesis in mice (Kavarthapu et al., 2019). Thus a method of inhibiting spermatogenesis may comprise administering a cyclic peptide described herein to a subject. A subject can be any sperm- producing animal. The subject is a mammal. The mammal may be a primate. The mammal may be a human, cat, dog, horse, sheep, goat, or a pig. Methods of Use—Non-Hormonal Male Contraceptive [0087] Currently available male contraceptive pills are hormone-based (Yuen et al., Best Pract. Res. Clin. Obstet. Gynaecol. 2020;66:83−94 ). However, a safe, reversible, effective non-hormonal contraceptive is needed. [0088] As described herein, GRTH/DDX25 is a DEAD-box RNA-helicase essential for the completion of spermatogenesis. Previous studies by the inventors indicated that blocking the GRTH phospho-site or perturbing the GRTH/PKA interface could provide an avenue for developing a non- hormonal male contraceptive. The cyclic peptides described herein were rationally designed and synthesized as therapeutic agents. The cyclic peptides showed effective delivery into COS-1 and germ cells and a dose-dependent inhibitory effect on GRTH phosphorylation. The cyclic peptides inhibit GRTH phosphorylation in the presence of PKA, and binding to the helicase resulted in thermal stabilization of non-phospho-GRTH. Increased efficiency in FRET assay revealed their interaction with GRTH. Cyclic peptide exposure of cultures from mice seminiferous tubules resulted in significant inhibition of phospho-GRTH. These cyclic peptides did not exhibit toxicity. Effective delivery and targeted decrease of in vitro expression of phospho-GRTH by cyclic peptides provide a strategy to develop effective compounds as a non-hormonal male contraceptive. [0089] The cyclic peptides (e.g., PEP0, PEP1, PEP2) described herein have been designed and synthesized as therapeutic agents. PEP1 and PEP2 revealed by FITC, showed internalization into COS-1 cells. A dose-dependent inhibitory effect on GRTH phosphorylation was observed in COS-1 stable cell line expressing GRTH, with significant reduction in pGRTH protein. CETSA showed PEP1 and PEP2 binding resulting in thermal stabilization of the soluble non-pGRTH protein. Exposure of cultures from seminiferous tubules with PEP1 and PEP2 resulted in significant inhibition of pGRTH. Similar results were obtained with PEP0 lacking FITC. The present study has shown that the proposed synthetic cyclic peptides can enter the germ cells in the seminiferous tubule and decrease the presence of phospho-GRTH species. Taken together, effective internalization and targeted decrease in the expression of pGRTH by PEP0, PEP1 and PEP2 provide a strategy to develop effective compounds for use as a non-hormonal male contraceptive. [0090] The cyclic peptides or pharmaceutical compositions described herein can be administered to any animal that can experience the beneficial effects thereof. Such animals include mammals, including but not limited to humans, farm animals, pets, and exotic animals. Pharmaceutical Compositions [0091] In addition to the cyclic peptides described herein, the pharmaceutical compositions may further comprise at least one of any suitable auxiliaries including, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. Pharmaceutically acceptable auxiliaries are preferred. Examples and methods of preparing such sterile solutions are well known in the art. Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the cyclic peptides described herein. [0092] Pharmaceutical excipients and additives useful in the compositions described herein can also include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, terra-, and oligosaccharides; derivatized sugars including but not limited to alditols, aldonic acids, esterified sugars, or combinations thereof; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination in ranges of 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin including but not limited to human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, or combinations thereof. Representative amino acid components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, or combinations thereof. [0093] Carbohydrate excipients suitable for use in the compositions described herein include monosaccharides including but not limited to fructose, maltose, galactose, glucose, D-mannose, sorbose, or combinations thereof; disaccharides, including but not limited to lactose, sucrose, trehalose, cellobiose, or combinations thereof; polysaccharides, including but not limited to raffinose, melezitose, maltodextrins, dextrans, starches, or combinations thereof; and alditols, including but not limited to mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol or combinations thereof. [0094] The compositions described herein further can comprise, but is not limited to pharmaceutically acceptable carriers including but not limited to coloring agents, emulsifying agents, suspending agents, ethanol, EDTA, citrate buffer, flavoring, and water. [0095] Chelators including but not limited to EDTA and EGTA can optionally be added to the pharmaceutical compositions to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the pharmaceutical composition. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the composition to aggregate. [0096] The composition described herein also can comprise the preservatives methylparaben (also known as 4-hydroxybenzoic acid methyl ester, methyl p-hydroxybenzoate; or METHYL CHEMOSEPT), ethylparaben (also known as 4-hydroxybenzoic acid ethyl ester; ethyl phydroxybenzoate; or ETHYL PARASEPT), propylparaben (also known as 4-hydroxybenzoic acid propyl ester; propyl p-hydroxybenzoate; NIPASOL; or PROPYL CHEMOSEPT) and/or butylparaben (also known as 4-hydroxybenzoic acid propyl ester; propyl p-hydroxybenzoate; or BUTYL CHEMOSEPT). [0097] Emulsifiers that may be used in the compositions described herein include, but are not limited to ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. [0098] The pharmaceutical compositions comprising the cyclic peptides described herein can also include a buffer or a pH adjusting agent. Typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts including but not limited to salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. [0099] Additionally, the pharmaceutical compositions described herein can include polymeric excipients/additives including but not limited to polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, including but not limited to hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents, anti-microbial agents, sweeteners, antioxidants, anti-static agents, surfactants (e.g., polysorbates including but not limited to “Tween® 20” and “Tween® 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA or EGTA). These and additional known pharmaceutical excipients and/or additives suitable for use in the present invention are known in the art. [0100] Other excipients, e.g., isotonicity agents, buffers, antioxidants, preservative enhancers, can be optionally added to the diluent. An isotonicity agent including but not limited to glycerin, is commonly used at known concentrations. A physiologically tolerated buffer is preferably added to provide improved pH control. Suitable buffers include phosphate buffers, sodium phosphate and phosphate buffered saline (PBS). [0101] The cyclic peptides disclosed herein can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can comprise, in addition to the cyclic peptides described herein, stabilizers, preservatives, excipients, or combinations thereof. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art (see Prescott, ed., METH. CELL BIOL. 14:33 (1976)). Liposomes, methods of making and methods of use are described in U.S. Patent Nos. 4,089,8091 (process for the preparation of liposomes), 4,233,871 (methods regarding biologically active materials in lipid vesicles), 4,438,052 (process for producing mixed micelles), 4,485,054 (large multilamellar vesicles), 4,532,089 (giant-sized liposomes and methods thereof), 4,897,269 (liposomal drug delivery system), 5,820,880 (liposomal formulations), and so forth. [0102] During any of the processes for preparation of the cyclic peptides described herein, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups. The protecting groups may be removed at a convenient subsequent stage using methods known from the art. [0103] The cyclic peptides described herein can be solubilized or suspended in a preconcentrate (before dilutions with a diluent), added to the preconcentrate prior to dilution, added to the diluted preconcentrate, or added to a diluent prior to mixing with the preconcentrate. The cyclic peptides described herein can also be co-administered as part of an independent dosage form, for therapeutic effect. Optionally, the cyclic peptides described herein can be present in a first, solubilized amount, and a second, non-solubilized (suspended) amount. [0104] The pharmaceutical compositions described herein can also comprise suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the cyclic peptides described herein into preparations that can be administered to animals, as described herein. [0105] The pharmaceutical compositions described herein can also include additional therapeutic agents including but not limited to, but not limited to hydrophilic drugs, hydrophobic drugs, hydrophilic macromolecules, cytokines, peptidomimetics, peptides, proteins, toxoids, sera, antibodies, vaccines, nucleosides, nucleotides, nucleoside analogs, genetic materials and/or combinations thereof. [0106] The additional therapeutic agent can be solubilized or suspended in a preconcentrate (before dilutions with a diluent), added to the preconcentrate prior to dilution, added to the diluted preconcentrate, or added to a diluent prior to mixing with the preconcentrate. The additional therapeutic agent can also be co-administered as part of an independent dosage form, for therapeutic effect. Optionally, the additional therapeutic agent(s) can be present in a first, solubilized amount, and a second, non-solubilized (suspended) amount. Such additional therapeutic agent(s) can be any agent(s) having therapeutic or other value when administered to an animal, particularly to a mammal, including but not limited to drugs, nutrients, and diagnostic agents. Dosages [0107] The pharmaceutical compositions described herein are administered to a subject in a manner known in the art. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. [0108] The cyclic peptides described herein may be present in any suitable amount within the pharmaceutical compositions described herein. Those of skill in the art can readily determine suitable concentrations of cyclic peptides described herein to include in the pharmaceutical compositions depending on various factors including dosage and route of administration. Pharmaceutical compositions useful in the present invention can comprise a quantity of a cyclic peptides described herein in an amount effective to be active as a contraceptive. [0109] The cyclic peptides described herein may be present in the pharmaceutical composition in an amount of at least 0.1 mg/mL, at least 0.5 mg/mL, at least 1 mg/mL, at least 1.5 mg/mL, at least 2 mg/mL, at least 5 mg/mL, at least 10 mg/mL or at least 15 mg/mL. [0110] The cyclic peptides described herein may be present in the pharmaceutical composition in an amount from about 0.1 mg/mL to about 100 mg/mL, from about 0.5 mg/mL to about 100 mg/mL, from about 1 mg/mL to about 100 mg/mL, from about 1.5 mg/mL to about 100 mg/mL, from about 2 mg/mL to about 100 mg/mL, from about 5 mg/mL to about 100 mg/mL, from about 10 mg/mL to about 100 mg/mL, from about 15 mg/mL to about 100 mg/mL, from about 20 mg/mL to about 100 mg/mL, or from about 50 mg/mL to about 100 mg/mL. [0111] The cyclic peptides described herein may be present in the pharmaceutical composition in an amount from about 0.1 mg/mL to about 50 mg/mL, from about 0.1 mg/mL to about 25 mg/mL, from about 0.1 mg/mL to about 20 mg/mL, from about 0.1 mg/mL to about 15 mg/mL, from about 0.1 mg/mL to about 10 mg/mL, from about 0.1 mg/mL to about 5 mg/mL, from about 0.1 mg/mL to about 3 mg/mL, from about 0.1 mg/mL to about 2 mg/mL, from about 0.1 mg/mL to about 1.5 mg/mL, or from about 0.1 mg/mL to about 1 mg/mL. [0112] The cyclic peptides described herein may be present in the pharmaceutical composition in an amount of about 0.1 mg/mL, about 0.5 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 50 mg/mL or about 100 mg/mL. [0113] The compositions described herein may comprise between about 1 mg and 250 mg, 100 mg and 500 mg, 250 mg and 750 mg, 500 mg and 1,000 mg of the cyclic peptides described herein. [0114] The compositions described herein may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1,000 mg of cyclic peptides described herein. [0115] The compositions described herein may comprise between about 1 µg and 250 µg, 100 µg and 500 µg, 250 µg and 750 µg, 500 µg and 1,000 µg of the cyclic peptides described herein. [0116] The compositions described herein may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1,000 µg of cyclic peptides described herein. [0117] The pharmaceutical compositions may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily. Doses maybe administered for one week, one month, or over the course of several months, 3, 6, 9 or 12 months, or intervals known in the art and determined to be clinically relevant. Doses may be continued throughout the life of the patient, or discontinues when clinical judgment warrants. The daily dosage of the formulations may be varied over a wide range from about 0.0001 to about 1,000 mg per patient, per day. The range may more particularly be from about 0.001 mg/kg to 10 mg/kg of body weight per day, about 0.1-100 mg, about 1.0-50 mg or about 1.0-20 mg per day for adults (at about 60 kg). Additionally, the dosages may be about 0.5-10 mg/kg per day, about 1.0-5.0 mg/kg per day, 5.0-10 mg/kg per day, or equivalent doses as determine by a practitioner, to achieve a serum concentration that is clinically relevant. [0118] As a non-limiting example, treatment of humans or animals can be provided as a one- time or periodic dosage of the cyclic peptides described herein 0.0001 to about 1,000 mg per patient, per day. The range may more particularly be from about 0.001 mg/kg to 10 mg/kg of body weight per day, about 0.1-100 mg, about 1.0-50 mg or about 1.0-20 mg per day for adults (at about 60 kg). Additionally, the dosages may be about 0.5-10 mg/kg per day, about 1.0-5.0 mg/kg per day, 5.0-10 mg/kg per day or equivalent doses as determine by a practitioner, to achieve a serum concentration that is clinically relevant. [0119] Humans or other animals can be provided as a one-time or periodic dosage of the cyclic peptides described herein 1 µg to about 1,000 mg per patient, per day. The range may more particularly be from about 1 µg/kg to 10 µg/kg of body weight per day, about 1-100 µg, about 1.0-50 µg or about 1.0-20 µg per day for adults (at about 60 kg). Additionally, the dosages may be about 0.5-10 µg/kg per day, about 1.0-5.0 µg/kg per day, 5.0-10 µg/kg per day or equivalent doses as determine by a practitioner, to achieve a serum concentration that is clinically relevant. [0120] Specifically, the pharmaceutical compositions described herein may be administered at least once a week over the course of several weeks. The pharmaceutical compositions may be administered at least once a day over several weeks to several months to several years. The pharmaceutical compositions may be administered daily over a period of several days, several weeks, several months, or several years, until no longer needed or desired. Routes of Administration [0121] Routes of administration and dosages of effective amounts of the pharmaceutical compositions comprising the cyclic peptides are described herein. The cyclic peptides described herein can be administered in combination with other pharmaceutical agents in a variety of protocols for effective contraception. [0122] The present disclosure further relates to the administration of at least one of the cyclic peptides described herein by the following routes, including, but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, iontophoretic means, or transdermal means. A composition formulated for oral administration may comprise the cyclic peptides described herein [0123] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may comprise 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 which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition with only the addition of the sterile liquid carrier, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. [0124] For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally comprise suitable preservatives are employed when intravenous, administration is desired. The pharmaceutical compositions may be administered parenterally via injection of a pharmaceutical composition comprising cyclic peptides described herein dissolved in an inert liquid carrier. The term “parenteral,” as used herein, includes, but is not limited to, subcutaneous injections, intravenous, intramuscular, intraperitoneal injections, or infusion techniques. [0125] Acceptable liquid carriers include, vegetable oils including but not limited to peanut oil, cotton seed oil, sesame oil or combinations thereof, as well as organic solvents including but not limited to solketal, glycerol formal. The pharmaceutical compositions may be prepared by dissolving or suspending cyclic peptides described herein in the liquid carrier such that the final formulation contains from about 0.005% to 30% by weight of a cyclic peptide described herein. [0126] For oral administration in the form of a tablet or capsule, the cyclic peptides described herein may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier including but not limited to ethanol, glycerol, water or combinations thereof. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents may also be incorporated into the mixture. Suitable binders include, without limitation, starch; gelatin; natural sugars including but not limited to glucose or beta-lactose; corn sweeteners; natural and synthetic gums including but not limited to acacia, tragacanth, or sodium alginate, carboxymethylcellulose; polyethylene glycol; waxes or combinations thereof. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, or combinations thereof. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, or combinations thereof. [0127] For oral administration, the pharmaceutical formulation also optionally comprising a sweetener. Sweeteners include but are not limited to sucrose, fructose, sodium saccharin, sucralose (SPLENDA®), sorbitol, mannitol, aspartame, sodium cyclamate, and combinations thereof. [0128] Aqueous suspensions, emulsions and/or elixirs for oral administration can be combined with various sweetening agents, flavoring agents, including but not limited to, but not limited to orange or lemon flavors, coloring agents, including but not limited to dye stuffs, natural coloring agents or pigments, in addition to the diluents including but not limited to water, glycerin and various combinations. [0129] The pharmaceutical compositions described herein suitable for oral administration may be presented as discrete units including but not limited to capsules, dragées, cachets or tablets each comprising a predetermined amount of the cyclic peptides described herein; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion, and as a bolus. [0130] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the cyclic peptides described herein in a free-flowing form including but not limited to a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered cyclic peptides described herein moistened with an inert liquid diluent. The tablets may be optionally coated or scored and may be formulated so as to provide a slow or controlled release of the cyclic peptides described herein therein. [0131] In addition, the pharmaceutical compositions comprising cyclic peptides described herein may be incorporated into biodegradable polymers allowing for sustained release of the cyclic peptides described herein. The biodegradable polymers and their uses are described in detail in Brem et al., 74 J. NEUROSURG. 441-46 (1991). Suitable examples of sustained-release compositions include semipermeable matrices of solid hydrophobic polymers comprising a cyclic peptide described herein, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (including poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers including but not limited to the LUPRON DEPOT® (Tap Pharmaceuticals, Inc., Chicago, Ill.) (injectable microspheres composed of lactic acid glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. [0132] It can be sometimes desirable to deliver the cyclic peptides described herein to the subject over prolonged periods of time, for periods of one week to one year from a single administration. Certain medical devices may be employed to provide a continuous intermittent or on demand dosing of a patient. The devices may be a pump of diffusion apparatus, or other device containing a reservoir of drug and optionally diagnostic or monitoring components to regulate the delivery of the drug. Various slow-release, depot or implant dosage forms can be utilized. A dosage form can comprise a pharmaceutically acceptable non-toxic salt of the cyclic peptides described herein that has a low degree of solubility in body fluids, (a) an acid addition salt with a polybasic acid including but not limited to phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, or combinations thereof; (b) a salt with a polyvalent metal cation including but not limited to zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, combinations thereof, or with an organic cation formed from e.g., N,N’-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b) e.g., a zinc tannate salt. Additionally, the cyclic peptides described herein or a relatively insoluble salt including but not limited to those just described, can be formulated in a gel, an aluminum monostearate gel with, e.g., sesame oil, suitable for injection. Salts include, but are not limited to, zinc salts, zinc tannate salts, pamoate salts, or combinations thereof. Another type of slow-release depot formulation for injection would comprise a salt of the cyclic peptides described herein dispersed or encapsulated in a slow degrading, non- toxic, non-antigenic polymer including but not limited to a polylactic acid/polyglycolic acid polymer, including the formulations as described in U.S. Patent No. 3,773,919. The cyclic peptides described herein or relatively insoluble salts thereof including but not limited to those described above can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional slow-release, depot or implant formulations, e.g., gas or liquid liposomes are known in the literature. See, e.g., U.S. Patent No. 5,770,222. [0133] Other examples include provision of the cyclic peptides described herein to be administered by sustained release delivery system containing a biodegradable composition. The biodegradable composition may be composed of a biodegradable, water-coagulable, non-polymeric material and a biocompatible, non-toxic organic solvent that is miscible to dispersible in an aqueous medium. The delivery system may be implanted at an implant site causing the solvent to dissipate, disperse or leach from the composition into surrounding tissue fluid through a resulting microporous matrix. [0134] The term “implant site” is meant to include a site, in or on which the non-polymeric composition is applied. Implantation or implant site can also include the incorporation of the pharmaceutical composition comprising at least one of the cyclic peptides described herein with a solid device. The pharmaceutical composition can be incorporated into a coating on a stent that is implanted into a subject. Additionally, other solid or biodegradable materials can be used as a substrate on which the pharmaceutical composition is applied. The coated material, comprising the pharmaceutical composition is then implanted, inserted or is adjacent to the subject or patient. The term “biodegradable” means that the non-polymeric material and/or matrix of the implant will degrade over time by the action of enzymes, by simple or enzymatically catalyzed hydrolytic action and/or by other similar mechanisms in the human body. By “bioerodible,” it is meant that the implant matrix will erode or degrade over time due, at least in part, to contact with substances found in the surrounding tissue fluids, cellular action,. By “bioabsorbable,” it is meant that the non- polymeric matrix will be broken down and absorbed within the human body, by a cell, a tissue. [0135] Non-polymeric materials that can be used in the composition generally are those that are biocompatible, substantially insoluble in water and body fluids, and biodegradable and/or bioerodible. The non-polymeric material is capable of being at least partially solubilized in a water- soluble organic solvent. The non-polymeric materials are also capable of coagulating or solidifying to form a solid implant matrix. The non-polymeric material is combined with a compatible and suitable organic solvent to form a composition that has the desired consistency ranging from watery to viscous to a spreadable putty or paste. [0136] Suitable organic solvents are those that are biocompatible, pharmaceutically-acceptable, and will at least partially dissolve the non-polymeric material. The organic solvent has a solubility in water ranging from miscible to dispersible. Optionally, a pore-forming agent can be included in the composition to generate additional pores in the implant matrix. The pore-forming agent can be any organic or inorganic, pharmaceutically-acceptable substance that is substantially soluble in water or body fluid, and will dissipate from the coagulating non-polymeric material and/or the solid matrix of the implant into surrounding body fluid at the implant site. [0137] The cyclic peptides described herein are capable of providing a local or systemic biological, physiological or therapeutic effect in the body of an animal. In formulating some pharmaceutical compositions described herein, the cyclic peptides described herein may be soluble or dispersible in the non-polymeric composition to form a homogeneous mixture, and upon implantation, becomes incorporated into the implant matrix. As the solid matrix degrades over time, the cyclic peptides described herein are capable of being released from the matrix into the adjacent tissue fluid, and to the pertinent body tissue or organ, either adjacent to or distant from the implant site, preferably at a controlled rate. The release of the cyclic peptides described herein from the matrix may be varied by the solubility of the cyclic peptides described herein in an aqueous medium, the distribution of the cyclic peptides described herein within the matrix, the size, shape, porosity, and solubility and biodegradability of the solid matrix. See e.g., U.S. Patent No. 5,888,533. The amounts and concentrations of ingredients in the composition administered to the patient will generally be effective to accomplish the task intended. [0138] The cyclic peptides described herein may be administered by bioactive agent delivery systems containing microparticles suspended in a polymer matrix. The microparticles may be microcapsules, microspheres or nanospheres currently known in the art. The microparticles should be capable of being entrained intact within a polymer that is or becomes a gel once inside a biological environment. The microparticles can be biodegradable or nonbiodegradable. Many microencapsulation techniques used to incorporate a bioactive agent into a microparticle carrier are taught in the art. See e.g., U.S. Patent Nos. 4,652,441; 5,100,669; 4,438,253; and 5,665,428. [0139] A preferred polymeric matrix will be biodegradable and exhibit water solubility at low temperature and will undergo reversible thermal gelation at physiological mammalian body temperatures. The polymeric matrix is capable of releasing the substance entrained within its matrix over time and in a controlled manner. The polymers are gradually degraded by enzymatic or non- enzymatic hydrolysis in aqueous or physiological environments. See e.g., U.S. Patent No. 6,287,588. [0140] Methods of preparing various pharmaceutical compositions with a certain amount of active ingredients are known, or will be apparent in light of this disclosure, to those skilled in the art. Methods of preparing said pharmaceutical compositions can incorporate other suitable pharmaceutical excipients and their formulations are known in the art. [0141] Methods of preparing the pharmaceutical preparations described herein are manufactured in a manner that is known, including conventional mixing, dissolving, or lyophilizing processes. Thus, liquid pharmaceutical preparations can be obtained by combining the cyclic peptides described herein with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary. [0142] One of ordinary skill in the art will appreciate that a method of administering pharmaceutically effective amounts of the cyclic peptides described herein to a patient in need thereof, can be determined empirically, or by standards currently recognized in the medical arts. The agents can be administered to a patient as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents of the compositions described herein will be decided within the scope of sound medical judgment by the attending physician. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. It is well within the skill of the art to start doses of the agents at levels lower than those employed to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved. [0143] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. Further, to the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated can be further modified to incorporate features shown in any of the other embodiments disclosed herein. [0144] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. [0145] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. [0146] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention. EXAMPLES EXAMPLE 1 Computational design of cyclic peptides [0147] Despite the shallowness of the GRTH surface at the PKA-binding interface, there is a clear electrostatic pattern radiating from the center, with a region of negative potential surrounded by a nonpolar transition halo and regions of positive potential further away (FIG. 1A). This pattern is consistent with the hotspots distribution obtained from the single-analog simulations (FIG. 1B): some areas are targeted preferentially by basic groups, others by acidic groups, and others by nonpolar (in this context, hydrophobic) or net-neutral polar groups; other areas are avoided altogether by all the analogs. This pattern is significant and may function as recognition sites for specific, high-affinity binding of cyclic peptides. One possible pharmacophore chosen here as the basis for the design of all the peptides is shown in FIG. 1C. Although some leeway exists in the choice of residues matching the pharmacophore, some parameters are imposed a priori, which restrict the options: (i) the cyclic peptide should ideally contain fewer than ten residues (or being of similar length if nonpeptidic spacers, e.g., PEG, are used to separate functional groups), (ii) incorporate local moieties that facilitate penetration into the cell membrane, (iii) accommodate a fluorescence probe for in vitro monitoring of cellular localization without disrupting GRTH binding, and (iv) have enough structural flexibility in solution to adapt to the shallow, flexible surface upon binding. One of the simplest cyclic peptides that fit the pharmacophore and accommodates these stringent conditions is cyclo(1FAGRRRG7-AEEAc), referred to as PEPx (all residues in the L configuration, unless otherwise stated). [0148] To obtain evidence that this molecular scaffold can provide the basis for the design of GRTH-binding cyclic peptides, the inventors executed a computational multistage cyclization procedure and predicted the binding modes at the interface. The simulations suggest that, in solution, the peptide is quite flexible, with several coexisting conformational families, presumably, in fast interconversion (FIG. 1D). Certain structural flexibility is desirable to allow local adaptation to the interface and may be controlled by changing the length of the linker (e.g., flexibility may be reduced with Gly2 instead of PEG2; not tested). The molecular surface representation of the conformers in solution (two shown in FIG. 1E) reveals a common feature: a pronged cationic structure with basic residues on one end of the ring and a hydrophobic region on the other, giving the peptide the cationic/amphipathic character desirable for membrane translocation and cytosolic localization. Furthermore, analysis of the predicted binding-modes shows that all the conformers are attracted to GRTH at or near the PKA-binding interface, although with significant structural variability (FIG. 1F) and mutual adaptation upon binding (FIG. 1G). Such changes in the local GRTH structure are the desired perturbations hypothesized above as a possible inhibitory mechanism of T239 phosphorylation by PKA, barring a direct competitive binding inhibition of the kinase. Although every motif in the pharmacophore plays a part in binding, and stabilization, not all the groups interact simultaneously with the protein, suggesting potential improvement in both the affinity and specificity. [0149] This computational analysis suggested to the inventors that the PEPx can indeed be used as the basic molecular scaffold for the synthesis of a series of cell-penetrating cyclic peptides. The inventors took advantage of the pharmacophore itself to incorporate some features known to facilitate membrane crossing in vitro. Studies have shown that having 3-4 arginine residues and at least one aromatic or hydrophobic group are common characteristics of cell-penetrating peptides, enabling effective permeabilization into cells (Liu et al., 2010; Qian et al., 2013). Given the uncertainty in the optimal length of the linkers and the local flexibility they introduce, which should ideally be kept within limits for both enthalpic and entropic reasons, the inventors found that either polyglycine or polyethylene glycol of two units in length at most were the most preferred. Based on these considerations, the inventors synthesized the set of cyclic peptides shown in Table 1, which incorporate relatively minor side-chain modifications to PEPx (SEQ ID NO: 2): PEP0 (SEQ ID NO: 3, AEEAc) replaces A2 by L-2-naphthylalanine, whereas PEP1 (SEQ ID NO: 7, AEEAc) and PEP2 (SEQ ID NO: 8, AEEAc) replace G7 by L- and D-Lys(FITC), respectively (the inventors tested both configurations because of the uncertain effects of the conjugated fluorophore size on GRTH binding). As controls, the inventors designed a linear peptide (CP1) (SEQ ID NO: 45) with the same amino acid composition as PEP1 (SEQ ID NO: 7, AEEAc) to prove the role of cyclization, and two additional control cyclic peptides to study the effects of individual moieties: inversion of charge at the polar end of the ring (CP2) (SEQ ID NO: 46) and reduction in the size of the hydrophobic/nonpolar groups at the nonpolar end (CP3) (SEQ ID NO: 47). Table 1. Peptides used in the experiments
Figure imgf000044_0001
Molecular modeling and simulations [0150] The computational method for designing cyclic peptides targeting shallow protein surfaces is described below. The inventors started with the derivation of a pharmacophore and the design of a series of cyclic peptides consistent with both the pharmacophore and a set of chemical features known to facilitate membrane crossing, followed by the prediction of binding modes and the selection of candidates for synthesis and in vitro testing. The structure of GRTH/DDX25 (domain 1) was obtained by homology with DDX19 (Raju et al., 2019); a recent model obtained from an AI- based approach (AlphaFold) yielded similar structures, with no conformational differences at the PKA-binding interface, including the side-chain orientations of relevant residues (Jumper et al., Nature 2021, 596, 583 ). Given the structural uncertainties inherent in this kind of modeling, the inventors performed molecular dynamics (MD) simulations to obtain a family of conformers (substates) of GRTH in an aqueous solution using the TIP3P water model and the all-atom CHARMM forcefield (Brooks et. al. J. Comput. Chem. 2009 Jul 30;30(10):1545-1614) (c36 version) at constant temperature (37 °C) and pressure (1 atm), with protonation states at neutral pH. This procedure avoids potential biases in the derivation of the pharmacophore stemming from the reliance on a single structure. Clustering analysis of GRTH residues at the GRTH/PKA interface yielded 13 distinct substates, each a potential target of one or more cyclic peptides. Using simulated annealing Monte Carlo (MC) simulations combined with a residue-titration protocol, the inventors first identified GRTH “hotspots” for high-affinity binding of single amino-acid side-chain analogs to the GRTH/PKA interface. Based on the binding patterns, the inventors generated a proposed general pharmacophore. Once a specific amino acid sequence is selected for a cyclic peptide candidate, a multi-stage sampling method is used for tail-to-head cyclization. Structural clustering of the resulting ensemble produced a set of peptide conformers that were subsequently used for binding prediction. The GRTH-cyclic peptide binding modes were obtained by simulated annealing MC followed by structural relaxation through MD simulations. The method combines MC, MD, and Langevin Dynamics (LD) simulations in both implicit (Hassan and Steinbach, J. Phys. Chem. B 2011;115:14668−14682 ; Hassan et al., Proteins 2003;51:109−125) and explicit solvent models, with a technique developed previously to predict strong, weak, and ultraweak protein/peptide associations (Cardone et al., J. Phys. Chem. B 2013;117:12360−12374 ; Cardone et al., J. Comput. Chem. 2015;36:983−995 ). Computational Modeling of Cyclic Peptides [0151] A computational method was developed for the design of macrocyclic peptides to target shallow protein surfaces with high affinity, specificity, and selectivity. The general computational pipeline is as follows. (1) Molecular dynamics (MD) simulations are used to obtain the set of N conformational substates of the protein interface. (2) For each of the N substates, the binding modes and populations of a series of amino-acid side-chain analogs are obtained with the TaRt-cMC method; the resulting density maps, representing all the possible specific and non-specific binding modes of each analog, are then used to derive one or more pharmacophores; for a given pharmacophore, a series of S amino acid sequences can be proposed. (3) Once a specific sequence is chosen, the corresponding linear peptide is subjected to free Langevin dynamics (LD) simulations, and P conformations with tail-to-head distances smaller than a preset cutoff are collected. (4) Each of the P structures is fully cyclized through the gradual application of a force between the peptide termini over the course of LD simulation, producing M distinct cyclic peptide structures. (5) Each of the M structures is relaxed through MD simulations at the desired thermodynamic conditions. (6) For each of the M peptide structures and each of the N protein structures, a conformational-bias MC simulation is performed to find all the peptide/protein binding modes at the interface. This stage involved full relaxation of the complexes through MD simulations. [0152] The application of this general computational pipeline to GRTH/DDX25 is described below, and is represented schematically in FIG. 9. [0153] The inventors first identified all the conformational substates of the isolated DDX25 protein in an aqueous solution at thermodynamic equilibrium ①. For this, a 100-ns molecular dynamics (MD) simulation is carried out at 35 °C and 1 atm using the TIP3P water model at neural pH with the all-atom CHARMM forcefield (version c36). (Brooks et al., 2009) The trajectory is then analyzed based on the heavy-atom-RMSD of residues within a 1-nm distance from the Cα of T239. Clustering analysis with a threshold of 1.5 Å yielded thirteen distinct conformational families. The differences in atomic positions among these substates, although modest, can affect the binding modes and affinities of small molecules, so the entire set should be considered in the derivation of pharmacophores. [0154] The inventors then calculated the binding modes of amino-acid side-chain analogs on the DDX25/PKA interface ②. Amino acid side chain analogs used to derive the binding density maps for the derivation of the pharmacophores are shown below. Brackets indicate grouping by similar calculated affinity to the GRTH interface.
Figure imgf000047_0001
[0155] The method, referred to as TaRt-cMC (Temperature-annealing/Residue-titration canonical Monte Carlo), is conceptually similar to the chemical-potential simulated-annealing grand canonical MC method but avoids ligand-ligand interactions during binding. (Guarnieri et al., J. Am. Chem. Soc. 1996;18(35):8493-8494 ; Kulp et al., PLoS One. 2017;12(8):e0183327 ). For each protein conformer, a single analog is introduced into a rigid spherical cavity centered at Ca of T239; the cavity radius, here 1 nm, is such that it encloses the interface. A rigid-body simulated annealing MC simulation is then performed, from 1000 °C to 35 °C in a 15-step logarithmic schedule, using only roto-translations of the analog to identify binding modes at 35 °C. The SCP implicit solvent model used here has been parameterized based on experimental hydration energies of this series of analogs. (Hassan et al., J. Phys. Chem. B 2000;104(27):6478-6489). With the first analog fixed in its most stable (highest-population) binding mode, a second analog of the same type is introduced and its binding modes and populations calculated. Then, a third identical analog is introduced with the previous two fixed in their most stable modes, and the process is repeated n times until no binding is observed for the n-th analog of this type. The same process is carried out for each analog type and each protein substate. Each map is calculated from the spatial distribution of the analog center of mass and represents specific and non-specific binding to the interface. Comparison of the maps shows distinct binding patterns, with acidic, basic, net-neutral polar, and nonpolar (hydrophobic) analogs tending to occupy specific nitches on the protein surface; the physicochemical features of each analog determine the details of the binding modes. The maps can then be variously combined to derive one or more pharmacophores. Based on this, the inventors created the pharmacophore shown in FIG. 1C, from which a series of peptide sequences with the potential to bind the DDX25 interface and inhibit phosphorylation can be derived. [0156] Once a sequence is selected, the following steps involve cyclization. Ideally, the cyclization algorithm should mimic, as closely as possible, the experimental protocol during synthesis, but the inventors used a practical approach for tail-to-head cyclization. The linear peptide, with standard –NH3 + and –CO2 – termini, is first subjected to an extensive Langevin dynamics (LD) simulation (here, 100-ns long) at 35 °C using the all-atom CHARMM force field and the SCP solvent model ③. Structures with a tail-to-head distance (measured between the N and C terminal atoms) shorter than a cutoff rc (here, 4 Å) are collected, and conformational families identified using the backbone-RMSD as a clustering criterion with 1.5-Å threshold. For the peptides considered here, this process produced 20-50 distinct conformers. Because the conformers are obtained during free dynamics, they are structurally relaxed, and complete closure can be attained by applying a gentle force to the termini ④. The inventors replaced the termini by dummy groups: -C(x)H-NH2 at the N- terminus, where C(x) is the Cα of the first residue, and -O(y)-CO-NH-C(z)H3 at the C-terminus, where O(y) is the oxygen of the last ethylene glycol. The atoms in the -NH2 and -NH-C(z)H3 groups have no charge or size, serving only as geometric reference points. The closure protocol consists of gradually superimposing C(x) on C(z) and N on N during an LD simulation at 35 °C in the SCP implicit solvent. This is done by adding a harmonic potential k1d1 2+k2d2 2 to the all-atom CHARMM force field, where d1 and d2 are the distances between the corresponding atoms, and k1 = k2 are increased at an exponential rate in 100 steps from 0 to 103 kcal mol-1 Å-2 over 25 ns. The structural families are then identified using the same clustering criterion, producing a smaller subset of conformers, in our case 8-10, depending on the sequence. [0157] The closed peptides are then curated by removing the dummy groups and fussing the termini through covalent bonds. The peptides are subsequently relaxed through MD simulations in an explicit solvent at 35 °C and 1 atm using the all-atom CHARMM forcefield ⑤. Advanced MD techniques can be used at this stage to sample the configurational space more thoroughly or to evaluate the kinetics of interconversions (see Damjanovic et al., Chem. Rev. 2021;121(4):2292-2324 ). The conformations at the end of the simulations (snapshots) are taken as the cyclic peptide structures in an aqueous solution at the given thermodynamic conditions (FIG. 1D). In the final stage, the inventors predicted the protein-peptide binding modes at the interface ⑥. The inventors used the conformational-bias MC technique for protein-protein and protein-peptide binding prediction, described in detail elsewhere. (Cardone et al., 2015; Cardone et al., 2013). The search is restricted to the interface of interest, reducing the computational cost significantly. EXAMPLE 2 Cell culture and plasmid preparation [0158] COS-1 cells were obtained from ATCC® CRL-1650™ and cultured in a T75 flask at 37 °C with 5% CO2, containing Dulbecco Modified Eagle Medium (DMEM) high glucose, GlutaMaxTM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Inc., Lawrenceville, GA) and 1× Antibiotic-Antimycotic (#15240062, Thermo Fisher Scientific). The full-length human GRTH cDNA fragment (GenBank Acc # AF155140) was cloned into pcDNA™3.1/V5-His A (Thermo Fisher Scientific, Waltham, MA) at KpnI and XbaI restriction sites, and the sequence was confirmed. This GRTH plasmid construct and COS-1 cells were used to generate a stable cell line expressing human GRTH. The expression of pGRTH in a western blot was assessed using a custom-made affinity-purified phospho-site-specific GRTH polyclonal antibody raised in rabbit to the peptide sequence (CKLIDL[pT239]KIRV) of human GRTH. Expression of mCherry-GRTH plasmid in COS-1 cells [0159] Open reading frame (ORF) primers with restriction sites ApaI and AgeI (Fw:
Figure imgf000049_0001
(SEQ ID NO: 43) and Rv:
Figure imgf000049_0002
(SEQ ID NO: 44)) were designed to amplify the mCherry fragment from the commercially ordered plasmid p27-IRES-mCherry (Addgene, MA). The mCherry fragment containing the restriction sites and the human GRTH plasmid (generated previously) was digested using the same restriction enzymes, ApaI and AgeI. After gel run, the digested mCherry fragment and human GRTH plasmid were purified and kept for ligation at 4 °C for 16 hr. The ligation mixture was transformed into DH5α Competent Cells, and positive cells containing plasmid with human GRTH and mCherry were selected and purified. The expression of mCherry was checked by transfecting the plasmid expressing mCherry-GRTH into COS-1 cells. After 24 hr transfection, 20 μM of PEP1, PEP2, CP1, CP2, and CP3 were added into the COS-1 cells for 4h, and the images were photographed using EVOS M5000 Imaging System (Thermo Fisher Scientific). Generation of GRTH COS-1 stable cell line [0160] COS-1 cells were stably transfected with a human GRTH-V5-HIS vector, essentially as previously described (Villar et al., Mol. Cell. Biol. 2012;32:1566−1580 ). Briefly, following the determination of the optimal concentration of the antibiotic G418 (Geneticin), COS-1 cells were transfected with human GRTH-V5-His construct using Lipofectamine 2000 (Invitrogen) and cultured in DMEM-high glucose media in the presence of 10% FBS. Cells expressing GRTH were selected and cultured for 2 weeks in the presence of Geneticin to obtain stable transfected cells. Single clones were expanded and checked for pGRTH expression. Protein extracts were prepared and checked for pGRTH expression using phospho-specific GRTH antibody. Treatment of COS-1 cells with cyclic peptides [0161] Cyclic peptides (PEP1 and PEP2) with FITC tag and control peptides (CP1, CP2, and CP3) with FITC tag, and a cyclic peptide without FITC (PEP0) were designed and synthesized commercially (LifeTein, USA). The purity of all the peptides used in this study are > 95%, as determined by HPLC and mass spectrometry. The obtained lyophilized peptides were dissolved in molecular biology grade water and used for the experiments. All the peptides were added to the stable cell cultures of expressing GRTH (See Table 1). Different concentrations (5, 20, 60, and 100 μM) of peptides (both the cyclic peptides and control) were added into each well of a full-grown (~90% confluency) 6-well culture plate seeded at a density of 0.3x106 cells/well. The cell culture media containing the respective peptide was kept for up to 16 hr. Cells exposed to the peptides were washed with 1× PBS and harvested using Trypsin-EDTA. Protein extracts were prepared in the presence of 1× protease phosphatase inhibitor. Similarly, the expression of pGRTH was also analyzed during different times of exposure of cells to the individual peptides to determine the effectiveness of the peptides (time and concentration) in blocking GRTH phosphorylation of cells stably expressing GRTH. Since in initial studies cyclic peptides 1 and 2 at a concentration of 100 μM showed effective decrease in pGRTH in cells exposed for 16 hr, protein extracts were prepared at different times of exposure to the peptides (4, 8, 16, and 24 hr). Western blots were performed using 30μg of protein from each sample and the expression of pGRTH was revealed using phospho- specific GRTH antibody described below. Western blots [0162] All the protein extracts were resolved in NuPage Novex Bis-Tris 4-12% polyacrylamide gels with NuPAGE MOPS SDS running buffer (Life Technologies). After the gels were run, proteins were transferred to nitrocellulose membranes using the iBlot2 blotting system (Life Technologies). For detection of pGRTH, the custom-made phospho-specific GRTH antibody (1:2000) described below was used and the secondary antibody (1:5000) used is a bovine anti-rabbit HRP-IgG (sc-2374) (Thermo Fisher Scientific). β-Actin Antibody (1:2000; sc-69879) and secondary goat anti-mouse HRP-IgG (1:5000; sc-2055) were purchased from Santa Cruz Biotechnology and used for immunoblotting. All the membranes were blocked with 5% milk protein dissolved in 1× PBS in 0.1 % Tween 20 (PBST) following standard blot transfer and western blot protocols as recommended by the manufacturers. All the antibodies were diluted in 5% milk protein in PBST. The membranes were developed using Tanon™ High-sig ECL Western Blotting Substrate solution according to the manufacturer’s recommendations. Band intensities were detected using an iBright chemiluminescence imaging system (Thermo Fisher Scientific) and quantified with ImageJ Software. [0163] Antibodies used in this study are a custom-made affinity-purified phospho-site specific GRTH polyclonal antibody raised in rabbit to the peptide sequence (CKLIDL[pT239]KIRV) (1:2000) (Raju et al., 2019) and a GRTH rabbit polyclonal antibody (Sheng et al., J. Biol. Chem. 2006;281:35048−35056) which recognizes the non-phospho of GRTH (non-phospho, 56 kDa). Cytotoxicity analysis of cyclic peptides [0164] MTT cell cytotoxicity assay was performed to determine the IC50 values of cyclic and control peptides used in this study. The cell viability was not significantly altered at higher concentrations of cyclic peptides. The IC50 value for each peptide was calculated from the dose- response plot and data fit with a linear regression. The IC50 values of PEP0, PEP1, PEP2, CP1, CP2, and CP3 were 392 μM, 312.30 μM, 333.06 μM, 314.06 μM, 173.77 μM, and 256.66μM, respectively. Higher IC50 values indicate less toxicity and hence the maximum concentration of cyclic peptides for the experiments were fixed as 100 μM. Cell cytotoxicity assay [0165] Cell cytotoxicity assay was performed to determine cytotoxic levels (IC50) of the peptides using in vitro CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega). COS-1 stable cells expressing GRTH were seeded in a 96-well cell culture plate at a seeding density of 0.01x106 cells/well and kept for 16 hr. PEP0, PEP1, PEP2, CP1, CP2, and CP3 at different concentrations (10, 50, 100, 250, and 500 μM) were added into the cell culture media and incubated for 24 hr at 37 °C supplemented by 5% CO2. After incubation, the plates were removed from the wells and 15 μL of dye solution was added to each well and further incubated for 4 hr in the humidified chamber at 37 °C, 5% CO2 atmosphere. The reaction was stopped by adding the stop solution supplied with the kit and the color development was recorded using TriStar² LB 942 Multimode Microplate Reader at 570 nm. Cell viability percentage was calculated by taking the OD value of treatment divided by control and multiplied by 100. The IC50 value for each peptide was calculated from the dose-response plot (concentration of peptide on the x-axis and % cell availability on the y-axis) and data fit with linear regression. Results of cytotoxic assays are shown in FIGS. 10A-10F. EXAMPLE 3 Effective delivery of cyclic peptides into COS-1 cells [0166] The PEP1, PEP2, CP1, CP2, and CP3 tagged with FITC were utilized to assess the effective delivery in COS-1 cells. To determine the uptake of the cyclic peptides, stable COS-1 cells expressing GRTH were incubated with the respective cyclic peptides for 4 h, and DAPI was used as counter stain. Immunofluorescence microscopy to monitor effective delivery of cyclic peptides [0167] COS-1 stable cells expressing GRTH were seeded on the sterile cover slips kept inside the 6-well cell culture plate at a concentration of 0.3x106/well. The plate was kept overnight at 37 °C at 5% CO2 to allow the cells to adhere to the coverslip. Peptides PEP1, PEP2, CP1, CP2, and CP3 at a concentration of 20 μM were added to each well. After 4 hr of incubation with respective peptides, coverslips were carefully taken out from the cell culture plate and washed gently with 1× PBS. The cells were further fixed with 50 % and 100 % methanol for 1 min each and mounted inversely on a microscopic slide using ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). The slides were observed under the microscope and cells were microphotographed using Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescence Microscope and Zen pro software. [0168] Compared to CP1 (linear peptide), all the cyclic peptides gained entry into cells and accumulated in both the cytoplasm and nucleus, as shown by intracellular FITC (FIG. 2). However, it is interesting to note that not all the peptides displayed the same uptake efficiency, presumably because of their specific properties, three-dimensional structure, and amino acid compositions. Nevertheless, these initial findings confirmed the efficient delivery of cyclic peptides into mammalian cells, thus deemed suitable for subsequent studies. EXAMPLE 4 Dose-dependent effect on GRTH phosphorylation upon treatment with cyclic peptides [0169] Here, overexpression of pGRTH in COS-1 stable cells using a phospho-specific GRTH antibody is shown (FIG. 3). Addition of cyclic peptides PEP0, PEP1, and PEP2 to stable cells expressing pGRTH showed a dose-dependent inhibitory response on phosphorylation of GRTH (FIGS. 3A-3C). A significant dose-dependent decrease on pGRTH protein was observed in the concentration range 20-100 μM (FIGS. 3A-3C). Cyclic peptides showed a maximum inhibition at 100 μM concentration. Effective inhibition of pGRTH upon PEP0 treatment indicated that Lys(FITC) present in the PEP1 did not interfere or participate in the inhibitory effect of cyclic peptide. On the other hand, although effective internalization was achieved, with control peptides (CP1, CP2, and CP3), no significant changes were observed in the phosphorylation of GRTH in the COS-1 cells (FIGS. 3D-3F). This demonstrated that cyclic peptides (1 and 2) perturb the GRTH protein at the GRTH/PKA interface and inhibit phosphorylation. The linear peptide, despite its sequence identity to PEP1, was unable to inhibit GRTH phosphorylation. Also, the replacement of Arg with Glu and the introduction of two Gly residues in the other cyclic control peptides (CP2 and CP3) did not influence on GRTH phosphorylation. These observations indicate that effective inhibition of GRTH phosphorylation is dependent on the composition of amino acid residues and surface association capability predicted by the pharmacophore and our dynamics simulations. EXAMPLE 5 Time-dependent inhibitory effect of cyclic peptides on the pGRTH expression [0170] Time-dependent analysis was initiated to determine the effective inhibition of PEP0, PEP1, and PEP2. Significant decrease in pGRTH protein was observed at 8 hr and 16 hr of cyclic peptides PEP1 and PEP2 treatments (FIGS. 4A and 4B), whereas PEP0 showed significant decrease of pGRTH at 16 hr and 24 hr time intervals (FIG. 4C). PEP0 and PEP1 showed more effective and sustained inhibition of pGRTH when compared to PEP2, even after 24 hr. These results suggested that PEP0 and PEP1 could act as an effective inhibitor of GRTH phosphorylation. EXAMPLE 6 Inhibitory effect of cyclic peptides on pGRTH in seminiferous tubules [0171] Methods and results of a study of the inhibitory effect of cyclic peptides directly on seminiferous tubules cultures are described below. Preparation of seminiferous tubule culture from adult mice [0172] Seminiferous tubules from the testes of adult mice (90 days old) were used for the in vitro culture experiments. Testes were decapsulated and dispersed with sterile forceps in a petri-dish containing ice-cold medium 199 (Cat. No. 11150059; Thermo Fisher Scientific) with 0.1% BSA for 10 min. The dispersed seminiferous tubules were segmented and placed into the 24-well cell culture plate containing medium 199 and incubated in CO2 incubator. After 4h, 100 μM of cyclic peptides were added and incubated for 16 hr. Later the media was removed, and tubules were washed with 1× PBS. The tubules were kept on a microscopic slide and mounted with SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific) and imaged using EVOS M5000 fluorescent microscope (Thermo Fisher Scientific). Protein samples prepared from treated and untreated seminiferous tubules were run on a PAGE gel and the expression of pGRTH was detected using the phospho- specific GRTH antibody mentioned above. From at least three independent experiments performed, a graph was plotted by calculating the intensity of pGRTH protein band and normalized using β- actin band. [0173] Immunofluorescence images of seminiferous tubules showed effective uptake of the different all five peptides (PEP1, PEP2, CP1, CP2, CP3) after 16 hr of treatment (FIG. 5A). Exposure of PEP1 and PEP2 (100 μM) to seminiferous tubules showed a significant reduction in pGRTH protein by 60% compared to the untreated sample (FIG. 5B). While CP1, CP2, and CP3 did not show any effect on pGRTH expression in seminiferous tubules. This study demonstrates that PEP1 and PEP2 inhibit phosphorylation of GRTH, which is endogenously expressed in the seminiferous tubules, while the controls were ineffective. EXAMPLE 7 Assessment of cyclic peptides interaction to non-phospho GRTH protein interface by CETSA [0174] To assess the binding of cyclic peptides to non-phospho GRTH target protein, PEP1 and PEP2 were added to cell lysates prepared from COS-1 cells expressing GRTH and subjected to heat shock across a range of temperatures, from 40 – 70 °C. CP2 is used as control in this CETSA assay. In the case of CP2, the heat shock effectively denatured all the proteins, including GRTH, indicating that CP2 did not engage in the thermal stabilization of GRTH (FIGS. 6A and 6B). While the addition of PEP1 and PEP2 effectively thermostabilized GRTH significantly in the 50-55 °C temperature range (FIG. 6). The interaction of PEP1 and PEP2 at the higher temperature significantly shifted the thermal shift curve between the temperature 45 – 55 °C when compared to the CP2. Cellular Thermal Shift Assay (CETSA) [0175] CETSA assays were performed as previously described (Almqvist et al., 2016) with few modifications. For the cell lysate CETSA assay, cell lysates were prepared from the stable cells expressing GRTH in the presence of 1× protease phosphatase inhibitor. Cell debris was removed from the mixture by centrifugation at 10,000 rpm for 5 min at 4 °C and the supernatant was taken for the experiment. The cell lysates were diluted in lysis buffer and 40 μM of either PEP1, PEP2, or CP2 were added to each tube and kept under rotation for 16 hours at 4 °C. Then, the tubes were centrifuged at 10000 rpm for 3 min. Each cell lysates treated with different peptides were divided into 100 μL aliquots and heated individually at different temperatures (40-70 °C) for 3 mins in a Veriti thermal cycler (Applied Biosystems) followed by cooling for 3 mins at room temperature. The heated lysate samples were centrifuged at 10,000 rpm for 10 mins at 4 °C to sediment the precipitates from the soluble fractions. The supernatant containing the remaining soluble proteins was run in a NuPAGE™ 4 to 12%, Bis-Tris protein gels and analyzed for the expression of non- phospho GRTH using GRTH rabbit polyclonal antibody (Tang et al., 1999) by western blot. EXAMPLE 8 FRET analysis confirms interactions of cyclic peptides with GRTH protein [0176] The expression of mCherry-GRTH in COS-1 cells showed a wide distribution throughout the cells. FRET microscopy and acceptor photobleaching [0177] COS-1 cells expressing mCherry cultured in a 35-mm glass-bottom dish were exposed to 15 μM of PEP1, PEP2, and CP2 for 16 hr and subjected to live-cell imaging. mCherry was excited at 561 nm, and the emission detected at 570 and 620 nm, whereas FITC was excited at 488 nm, and detection was from 500 to 550 nm. FRET channel was acquired with an excitation at 488 nm and a 570 and 620 nm emission filter. For acceptor photobleaching, steady-state fluorescent images were acquired with an Inverted microscope Zeiss Axio Observer.Z1 with laser scanning unit LSM 780 (Carl Zeiss, Germany) using a 63× oil immersion objective and a 25 mW Argon Diode 405-30. Regions of interest (ROI) were marked in the cells and bleached in the acceptor channel (mCherry) using 30 cycles time series with 10 bleaching iterations with 100 % laser power at 514 nm. Pre- and post-bleach images were obtained in the mCherry, FITC, and FRET channels. ROI was marked in the photobleached region intensity of donor and acceptor, and FRET efficiency percentage was calculated using the Zen 2.1 SP3 software. FRET efficiency percentage was calculated from E % = 100 (I FITC postbleach – I FITC prebleach) / I FITC postbleach), where IFITC is the fluorescence intensity of the FITC. [0178] PEP1 and PEP2 showed effective cellular intake when compared to CP1, CP2, and CP3. Acceptor photobleaching method was carried out to observe the intracellular FRET. After photobleaching of the acceptor (mCherry-GRTH), the inventors observed an increase in fluorescence intensity of the donor FITC (PEP1 and PEP2) in the bleached regions, as shown in FIGS. 7A and 7B. The enhanced emission of donor FITC indicates that the donor in the excited state transferred energy to the acceptor mCherry in the ground state, hence the observed increase in the donor intensity. This is accomplished only when donor and acceptor are in close proximity, indicating interaction of the cyclic peptide (PEP1 or PEP2) with the GRTH protein. Conversely, the presence of CP2 (donor FITC) in the photobleached region did not show any increase in fluorescence intensity, indicating no interaction between CP2 and GRTH protein (FIGS. 7A and 7B). In addition, the increase in the donor FITC fluorescence signal was measured to calculate the FRET efficiency. Selected regions in the bleached sections were taken for the calculation of FRET efficiency (FIG. 7C). The inventors observed a significant four-fold increase in the FRET efficiency for PEP1 and PEP2 when compared to the control peptide CP2 (FIGS. 7D and 7E). EXAMPLE 9 Effect of cyclic peptides on pGRTH with or without PKA induction [0179] The transfection of PKA in the stable cells increased the phosphorylation of GRTH. However, the increase in phosphorylation of GRTH was significantly reduced/inhibited in the presence of 100 μM concentration of PEP1 and PEP2 with or without the PKA induction. The reduced expression of pGRTH could indicate the competitive binding of cyclic peptides (PEP1 and PEP2) at the PKA site in the presence of PKA. In contrast, the presence of CP2 did not have any effects on the expression of pGRTH with or without PKA induction (FIG. 8). Analysis of cyclic peptides on the expression of GRTH with or without PKA transfection [0180] Stable cells expressing GRTH were cultured in a 6-well plate at a seeding density of 0.3x106/well. Plasmid expressing PKA α catalytic subunit (PKAα) was used for the experiment. The wells were categorized into four groups: control, only PKAα, only cyclic peptides, and both PKAα + cyclic peptides. PKAα (3 μg/well) were transfected into the respective groups, while for equalization, empty plasmids were transfected into the other groups and cultured further for 24 hr. After 24 hr transfection, cyclic peptides PEP1, PEP2, and CP2, at a concentration of 100 μM each, were added into the respective wells and kept at 37 °C, 5% CO2 overnight. Protein samples were prepared and run in a PAGE gel for western blotting and developed the blot for phospho-GRTH using phospho-specific GRTH antibody (1: 1000 dilution) as mentioned earlier. Statistical analysis [0181] Data are presented as the mean ± SEM of three independent experiments. Mean values of the data were compared and analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using the Prism software (GraphPad Prism 7.02 Software, San Diego, CA). A probability of P < 0.05 was considered statistically significant. [0182] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention. Example 10 Ex vivo study of cyclic peptide efficacy in testis culture [0183] Ex vivo (organ culture) experiments were performed to check the efficacy of the cyclic peptides. The efficacy of the cyclic peptides in organ testis culture in inhibiting the phosphorylation of the target protein (GRTH/DDX25) was studied as a prelude to in vivo experiments in mice. For developing testis organ culture, agarose (1.5%) blocks were prepared in aseptic conditions and immersed for two hours in a cell culture medium containing α-MEM with 5% KO-serum. For treatments, cyclic peptide PEP2 (150 µM) was dissolved in the medium before adding the agarose blocks. Testis was dissected out from a 60-day old C57BL/6J mouse. The outer layer (tunica albuginea) was removed and then placed on the agarose blocks soaked with the cell culture medium with or without the PEP2 present. Control and PEP2 treated testis samples were taken from the culture after 24 and 48 hours of treatments. Protein samples were prepared and run in an SDS-gel. After band separation, the bands were transferred into a nitrocellulose membrane for the detection of phospho-GRTH using phospho-specific GRTH antibody. The results showed a significant decrease in the phospho-GRTH in 24 and 48 hours of PEP2 treatment, as indicated in FIGS. 11A and 11B. These results indicate that cyclic peptide treatments are effective in inhibiting phosphorylation of GRTH. Example 11 In vivo study of bioavailability of cyclic peptide in mice [0184] In vivo experiments were performed in which the bioavailability of cyclic peptides in mice was determined. Mice (60 days old) were injected intraperitoneally with 2.5 mg of PEP1 dissolved in 100 µL of saline and for sham control only saline (100 µl) was injected. After 30 mins, blood was collected by cardiac puncture, and testis, liver and kidney were removed for the fluorometric analysis. Tissue extracts were prepared for the analysis. Serum was obtained through centrifugation. [0185] Presence of FITC in the samples was measured in a Glomax reader. Known concentration of PEP1 samples were prepared to get the standard values. Standard values were plotted in a graph and the amount of FITC in all the samples were obtained by plotting the OD value in the graph (FIG. 12). Mean value was obtained and ug FITC/ml of sample is represented in the graph. Presence of PEP1 was observed significantly in the serum, testis and minimally in the liver and kidney. These results indicate that PEP1 does reach the testis and support the testis as a therapeutic target of PEP1 and related cyclic peptides. Example 12 Pre-clinical studies of cyclic peptide in mice [0186] The study and results described in Example 11 can serve as the basis for additional in vivo studies in mice and other animals. Such studies have utility in the animals directly and as models for pre-human clinical testing. For example, pre-clinical studies in mice can expanded to test both the bioavailability and efficacy of PEP1 or other cycle peptides. These studies can be performed in target tissue (testis) and its inhibitory effect on GRTH phosphorylation. Dosage and time interval of PEP1 for intraperitoneal (IP) injections in mice is determined. Testicular bioavailability of PEP1 can be performed using confocal imaging and by serum and tissue pharmacokinetic analysis. [0187] PEP1 can be exchanged, for example, for PEP0 (cyclic peptide without the FITC tag) as a primary bioactive druggable peptide for long term treatments in mice for 30-45 days. Such studies can be based on efficacy to inhibit GRTH phosphorylation. The peptide’s effectiveness in blocking GRTH phosphorylation in the testis and the morphological changes induced in the testis, sperm count, and sperm motility can be assessed. The fertility and mating behavior of male mice during extended periods (for example, 4-5 weeks) of treatment is studied. Such studies can determine if the degree of azoospermia or low sperm count, and the reversibility of such effects upon cessation of the treatment. Example 13 Studies of cyclic peptide efficacy in population control of nuisance animals [0188] The study and results described in Example 11 and results from the potential studies described in Example 12 can serve as the basis for field studies for population control of nuisance animals. Such field studies can include studies directed again pest wildlife such as rodents, rabbits, and deer. Such animals can include, for example, animals that carry disease, as well as animals that cause environmental, agriculture, and/or property damage. Test and control groups can be carried out at different locations that are sufficiently geographically isolated from one another, but that otherwise have similar environments. Outcomes can be judged based on litter sizes and general population trends, as well as such factors as male secondary sexual characteristics and mating behavior. More direct testing such as blood and urine samples can also be employed. Administration of a cyclic peptide to male animals of the target species and subsequent decreases in such factors can indicate efficacy of the treatment in population control. Example 14 Studies of cyclic peptide efficacy in animal husbandry [0189] The study and results described in Example 11 and results from the potential studies described in Example 12 can serve as the basis for studies of breeding management in animal husbandry. In breeding domestic or wild animals, whether for pet animals, agricultural animals, zoo animals, or endangered species, there is a desire to control breeding to prevent mating that could lead to undesired characteristics or interbreeding, as well as timing breeding for a desired seasonal gestation and birth. Animals can include, for example, dogs, horses, cattle, sheep, pigs, and goats. Test and control groups can be carried out in accordance with standard veterinary pre-clinical and clinical studies modified as appropriate for cyclic peptide used. Outcomes can be judged based on preventing or limiting undesired mating, as well as such factors as male secondary sexual characteristics and mating behavior. More direct testing such as blood and urine samples can also be employed. Administration of a cyclic peptide to target male animals and subsequent decreases in mating and other such factors can indicate efficacy of the treatment in the context of animal husbandry. [0190] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

Claims

CLAIMS What is claimed is: 1. A cyclic peptide comprising an amino acid sequence comprising the following formula: X1-X2-linker1-X3-X4-X5-X6-linker2, wherein X1 is amino acid residue F; X2 is an amino acid residue selected from Q, A, I, V, and L; linker1 comprises one or two linker molecules individually selected from amino acid residue G, amino acid residue P, and a non-peptidic spacer molecule; X3 is an amino acid residue selected from A, S, N, Q, C, and M; X4 is an amino acid residue selected from F, R, H, K, S, Y, Q, and N; X5 is an amino acid residue selected from R, H, and K; X6 is an amino acid residue selected from R, H, and K; and linker2 comprises one or two or three linker molecules individually selected from amino acid residue G, amino acid residue P, and a non-peptidic spacer molecule.
2. The cyclic peptide of claim 1, wherein the linker2 comprises 1 or 2 linker molecules.
3. The cyclic peptide of claim 1 or 2, wherein X1 is replaced with an amino acid residue selected from Q, A, I, V, and L; and wherein X2 is replaced with F.
4. The cyclic peptide of any one of claims 1-3, wherein the non-peptidic spacer molecule is polyethylene glycol (PEG), or polyvinyl alcohol (PVA), or both.
5. A cyclic peptide having the formula: cyclo(1FAGXXXG7-AEEAc), wherein each X is individually chosen from any basic amino acid, optionally arginine (R), lysine (K), or histidine (H).
6. The cyclic peptide of claim 5, wherein each X is arginine (R).
7. A cyclic peptide comprising the following structure: Cyclic Peptide 0 (PEP0) (SEQ ID NO: 3, AEEAc)
Figure imgf000062_0001
8. A cyclic peptide comprising the following structure: Cyclic Peptide 1 (PEP1) (SEQ ID NO: 7, AEEAc)
Figure imgf000063_0001
.
9. A cyclic peptide comprising the following structure: Cyclic Peptide 2 (PEP2) (SEQ ID NO: 8, AEEAc)
Figure imgf000064_0001
.
10. A cyclic peptide comprising cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 3) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 3) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 4) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 4) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 5) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 5) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-PEG2) (SEQ ID NO: 6) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-PEG1) (SEQ ID NO: 6) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 7) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 7) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 8) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 8) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-PEG3) (SEQ ID NO: 9) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-PEG2) (SEQ ID NO: 9) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 10) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 10) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 11) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 11) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-PEG3) (SEQ ID NO: 12) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-PEG2) (SEQ ID NO: 12) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 13) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 13) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 14) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 14) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-PEG3) (SEQ ID NO: 15) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-PEG2) (SEQ ID NO: 15) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-PEG2) (SEQ ID NO: 16) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-PEG1) (SEQ ID NO: 16) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG2) (SEQ ID NO: 17) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-PEG1) (SEQ ID NO: 17) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-PEG3) (SEQ ID NO: 18) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-PEG2) (SEQ ID NO: 18) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 19) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 20) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 21) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 22) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 23) cyclo-(Phe-ɸ-Gly-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 24) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 25) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 26) cyclo-(Phe-ɸ-Ala-Arg-Arg-rg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 27) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 28) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 29) cyclo-(Phe-ɸ-Ala-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 30) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 31) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 32) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 33) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 34) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 35) cyclo-(ɸ-Phe-Gly-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 36) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-Gly-Gly) (SEQ ID NO: 37) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Lys(FITC)-Gly) (SEQ ID NO: 38) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly-Gly) (SEQ ID NO: 39) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-D-Lys(FITC)-Gly) (SEQ ID NO: 40) cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-Gly-Gly) (SEQ ID NO: 41), or cyclo-(ɸ-Phe-Ala-Arg-Arg-Arg-Gly-Gly) (SEQ ID NO: 42), wherein ɸ is L-2-naphthylalanine, Q, A, or F, and wherein PEG1 is one PEG molecule, PEG2 is two PEG molecules, and PEG3 is three PEG molecules.
11. The cyclic peptide of claim 10, wherein the PEG molecule is replaced by a linker molecule.
12. The method of claim 11, wherein the linker molecule is (2-(2-aminoethoxy)ethoxy)acetic acid (AEEAc), a combination of PEG and Gly, or an intercalations of Pro residues.
13. The cyclic peptide of any one of claims 10-12, wherein ɸ is L-2-naphthylalanine.
14. The cyclic peptide of any one of claims 10-12, wherein ɸ is Q or F.
15. The cyclic peptide of any one of claims 10-12, wherein ɸ is A, Q, or F.
16. The cyclic peptide of any one of claims 1-15, wherein one or more of the amino acid residues are replaced with one or more non-peptidic spacer molecules, optionally polyethylene glycol (PEG), polyvinyl alcohol (PVA), or (2-(2-aminoethoxy)ethoxy)acetic acid (AEEAc).
17. The cyclic peptide of any one of claims 1-16, wherein one or more side chains are attached at one or more glycine residue.
18. The cyclic peptide of any one of claims 1-17, wherein one or more amino acid residues are methylated.
19. The cyclic peptide of any one of claims 1-18, wherein the cyclic peptide is linked through head- to-tail cyclization.
20. The cyclic peptide of any one of claims 1-19, wherein the cyclic peptide inhibits gonadotropin regulated testicular helicase (GRTH) phosphorylation.
21. A composition comprising one or more cyclic peptide of any one of claims 1-20.
22. The composition of claim 21, wherein the composition comprising 1 mg to 1 g of the cyclic peptide.
23. The composition of claim 23 or 24, wherein the composition is a pharmaceutical composition.
24. The composition of claim 23, wherein the composition further comprises diluents, binders, stabilizers, buffers, salts, solvents, preservatives, or combinations thereof.
25. The composition of claim 24 or 25, wherein the composition is formulated for oral administration.
26. An oral contraceptive comprising one or more cyclic peptide of any one of claims 1-20.
27. A method of inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation comprising administering an effective amount of one or more cyclic peptide of any one of claims 1-20.
28. A method of inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation comprising administering an effective amount of the composition any one of claims 21-25 to a subject.
29. A method of inhibiting spermatogenesis comprising administering an effective amount of one or more cyclic peptide of any one of claims 1-20 to a subject.
30. A method of inhibiting spermatogenesis comprising administering effective amount of the composition any one of claims 21-25 to a subject.
31. The method of claims 28-30, wherein the subject is a mammal.
32. The method of claim 31, wherein the mammal is a human.
33. The method of claim 32, wherein the human is a male human.
34. The method of any one of claims 28-33, wherein the administration is oral administration.
35. A composition for inhibiting spermatogenesis comprising an effective amount of one or more cyclic peptide of any one of claims 1-20.
36. A composition for inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation comprising an effective amount of one or more cyclic peptide of any one of claims 1-20.
37. The use of one or more cyclic peptide of any one of claims 1-20 in the manufacture of a medicament for inhibiting spermatogenesis in a subject.
38. The use of one or more cyclic peptide of any one of claims 1-20 in the manufacture of a medicament for inhibiting gonadotropin regulated testicular helicase (GRTH) phosphorylation in a subject.
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