Peptidomimetic macrocycles

Abstract

Provided herein are peptidomimetic macrocycles containing amino acid sequences with at least two modified amino acids that form an intramolecular cross-link that can help to stabilize a secondary structure of the amino acid sequence. Suitable sequences for stabilization include those with homology to the p53 protein. These sequences can bind to the MDM2 and/or MDMX proteins. Also provided herein are methods of using such macrocycles for the treatment of diseases and disorders, such as cancers or other disorders characterized by a low level or low activity of a p53 protein or high level of activity of a MDM2 and/or MDMX protein.

1 .- 102 . (canceled) 
     
     
         103 . A method of screening a peptidomimetic macrocycle by affinity-selection mass spectrometry, comprising:
 (a) mixing the peptidomimetic macrocycle and human homolog of mouse double minute 2 (hMDM2) protein in a solution;   (b) clarifying the solution by centrifugation;   (c) incubating the solution at elevated temperature; and   (d) analyzed by size-exclusion chromatography-liquid chromatograph mass spectrometry (LC-MS),   thereby determining the affinity of the peptidomimetic macrocycle to the hMDM2 protein.   
     
     
         104 . The method of  claim 103 , wherein the solution comprises phosphate-buffered saline (PBS) 
     
     
         105 . The method of  claim 104 , wherein the solution further comprises DMSO. 
     
     
         106 . The method of  claim 103 , wherein the hMDM3 protein is added in two batches. 
     
     
         107 . The method of  claim 103 , wherein the centrifugation is conducted at 10,000 g for 10 minutes. 
     
     
         108 . A method of screening a peptidomimetic macrocycle by protein-ligand Kd titration, comprising:
 (a) preparing serially diluted stock solutions of the peptidomimetic macrocycle;   (b) clarifying the stock solution by centrifugation to afford supernatants;   (c) adding human homolog of mouse double minute 2 (hMDM2) protein to the supernatants; and   (d) analyzed by size-exclusion chromatography-liquid chromatograph mass spectrometry (LC-MS),   thereby determining binding and affinity of the peptidomimetic macrocycle to the hMDM2 protein.   
     
     
         109 . The method of  claim 108 , wherein the solution comprises phosphate-buffered saline (PBS) 
     
     
         110 . The method of  claim 109 , wherein the solution further comprises DMSO. 
     
     
         111 . The method of  claim 108 , wherein the hMDM3 protein is added in two batches. 
     
     
         112 . The method of  claim 108 , wherein the centrifugation is conducted at 10,000 g for 10 minutes. 
     
     
         113 . A method of preparing a capsule, comprising:
 (a) preparing a peptidomimetic macrocycle;   (b) combining the peptidomimetic macrocycle with a pharmaceutically acceptable carrier to afford a mixture; and   (c) encapsulating the mixture.   
     
     
         114 . The method of  claim 113 , wherein the mixture further comprises a diluent. 
     
     
         115 . The method of  claim 113 , wherein the mixture further comprises a flavoring agent. 
     
     
         116 . The method of  claim 113 , wherein the mixture further comprises a binder. 
     
     
         117 . The method of  claim 113 , wherein the mixture further comprises a disintegrating agent. 
     
     
         118 . The method of  claim 113 , wherein the mixture further comprises a preservative. 
     
     
         119 . The method of  claim 113 , wherein the peptidomimetic macrocycle comprising an amino acid sequence with at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ IP NOs: 10-457, or pharmaceutically acceptable salt thereof; and wherein the peptidomimetic macrocycle has a Formula: 
       
         
           
           
               
               
           
         
         wherein: 
         each of Xaa 3 , Xaa 5 , Xaa 6 , Xaa 7 , Xaa 8 , Xaa 9 , and Xaa 10  is independently an amino acid, wherein at least three of Xaa 3 , Xaa 5 , Xaa 6 , Xaa 7 , Xaa 8 , Xaa 9 , and Xaa 10  are the same amino acids as the amino acid at the corresponding position of the sequence Phe 3 -X 4 -His 5 -Tyr 6 -Trp 7 -Ala 8 -Gln 9 -Leu 10 -X 11 -Ser 12 (SEQ ID NO: 8) or Phe 3 -X 4 -Glu 5 -Tyr 6 -Trp 7 -Ala 8 -Gln 9 -Leu 10 /Cba 10 -X 11 -Ala 12 (SEQ ID NO: 9), wherein each X 4  and X 11  is independently an amino acid: 
         each D is independently an amino acid; 
         each E is independently an amino acid selected from the group consisting of Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), and Ser (serine); 
         R 1  and R 2  are independently —H, alkyl, alkenyl, alkyenyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-; or forms a macrocycle-forming linker L′ connected to the alpha position of one of said D or E amino acids; 
         L and L′ are independently a macrocycle-forming linker; 
         each R 5  is independently halogen, alkyl. —OR 6 , —N(R 6 ) 2 , —SR 6 , —SOR 6 , SO 2 R 6 , —CO 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; 
         each R 6  is independently —H, alkyl, alkenyl, alkenyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; 
         R 7  is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue; 
         R 8  is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue; 
         v is an integer from 1-10; 
         and w is an integer from 3-10.