Compositions and methods for treating endometriosis
An amino acid and sequence technology, applied in drug combinations, pharmaceutical formulations, medical preparations containing active ingredients, etc., can solve the problems of progesterone receptor resistance resistance, low program compliance, and limited curative effect
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Embodiment 2
[0230] Synthesis and cyclization of embodiment 2 peptide
[0231] Peptides containing N-methyl amino acids show increased protease resistance. Furthermore, cyclization of peptides has been shown to increase the binding affinity of ligand peptides.
[0232]All solvents were purchased from Sigma. All peptides except N-Me-L14 were synthesized by manual solid-phase peptide synthesis with preloaded Leu-2-chlorotrityl resin (250 mg, 0.15 mmol; Anaspec). N-Me-L14 was synthesized after loading Fmoc-N-methylleucine (Fmoc=9-fluorenylmethoxycarbonyl; Anaspec) on 2-chlorotrityl resin (Anaspec). Five N-terminal Asn residues were added to improve peptide solubility. By adding FmocN-Me-L14 to 2-chlorotrityl resin (5 equiv; 250 mg, 0.35 mmol) in DMF in the presence of N,N diisopropylethylamine (DIEA; 5 equiv) for 2 hours to complete the loading. Standard couplings were performed with monomer (5 equiv; Novabiochem) in HATU (2 mL, 0.6 mmol; Novabiochem), HOAt (1.2 mmol; Genescript) in DM...
Embodiment 3
[0234] Example 3 Nuclear β-Catenin Inhibition and Wnt Signaling
[0235] Peptides were assessed for their ability to inhibit Wnt signaling. In the case of increasing peptide inhibitor concentration, with Wnt3a (hit (hit) 1: SEQ ID NO:217, hit 2: SEQ ID NO:224, hit 3: SEQ ID NO:228, hit 4: SEQ ID NO: 227, hit 5: SEQ ID NO: 220, hit 6: SEQ ID NO: 168. Hit 7: SEQ ID NO: 226) or stimulated with vehicle control at the Wnt-responsive promoter (TCF / LEF) (Enzo Life Sciences , Farmingdale, NY) mouse 3T3 fibroblasts expressing luciferase ( Figure 2A ). Optimized compounds show EC 50 Values ranged from 500–999 nM, and luciferase levels were reduced to those of unstimulated (-Wnt3a) controls. Luciferase activity is not affected by the addition of scrambled peptides (comprising the same amino acids in a scrambled manner), non-functional peptides (peptides that do not target β-catenin), or non-permeable peptides (peptides without permeability-enhancing features) . The results sho...
Embodiment 4
[0236] Example 4 Peptides Do Not Inhibit Membrane-Bound β-Catenin or Prevent Access to Binding Sites on E-cadherin
[0237] A competitive binding assay was performed ( image 3 ), where biotinylated β-catenin was immobilized on streptavidin plates, and 50 nM of E-cadherin-FC fusion was added in the presence of different amounts of peptide inhibitor (SEQ ID NO:42) things. None of the doses tested inhibited the β-catenin and E-cadherin interaction. The results showed that these peptides did not inhibit membrane-bound β-catenin or prevent access to the binding site on E-cadherin.
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