Ubiquitin variant modulators of scf e3 ligases and their uses

Abstract

The invention provides ubiquitin variants that specifically bind to SCF E3 ligases, and use of these variants to modulate the activity of SCF E3 ligases.

What is claimed is: 
     
         1 . A ubiquitin variant (Ubv) polypeptide comprising one or more amino acid substitution in one or more region of a ubiquitin polypeptide (SEQ ID NO:1), wherein the region is selected from the group consisting of:
 (a) region 1 (amino acids 2-14 of SEQ ID NO:1) wherein the polypeptide comprises the structure:   X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14      
       wherein 
       X 2  is selected from the group consisting of H and Q; 
       X 4  is selected from the group consisting of F and L; 
       X 8  is selected from the group consisting of G and L; 
       X 9  is selected from the group consisting of A, S, and T; 
       X 10  is selected from the group consisting of G, R, and V; 
       X 11  is selected from the group consisting of K and T; 
       X 12  is selected from the group consisting of A, G, N, and T; 
       X 14  is selected from the group consisting of I and T; 
       any X not specified optionally has the amino acid sequence of the corresponding position in SEQ ID NO:1; and the polypeptide optionally comprising 1-4 additional amino acids;
 (b) region 2 (amino acids 42-49 of SEQ ID NO:1) wherein the polypeptide comprises the structure: 
 X 42 -X 43 -X 44 -X- 45 -X 46 -X 47 -X 48 -X 49    
 
       wherein 
       X 42  is selected from the group consisting of I, R, T, and V; 
       X 44  is selected from the group consisting of I, L, and V; 
       X 46  is selected from the group consisting of A, R, S, and Y; 
       X 47  is selected from the group consisting of G, H, K, and R; 
       X 48  is selected from the group consisting of K, M, and R; 
       X 49  is selected from the group consisting of K, L, P, Q, R, and V; and 
       any X not specified optionally has the amino acid sequence of the corresponding position in SEQ ID NO:1; and
 (c) region 3 (amino acids 62-78 of SEQ ID NO:1) wherein the polypeptide comprises the structure: 
 X 62 -X 63 -X 64 -X 65 -X 66 -X 67 -X 68 -X 69 -X 70 -X 71 -X 72 -X 73 -X 74 -X 75 -X 76 -X 77 -X 78    
 
       wherein 
       X 62  is selected from the group consisting of E, H, and Q; 
       X 63  is selected from the group consisting of K and R; 
       X 66  is selected from the group consisting of S and T; 
       X 68  is selected from the group consisting of H, Q, and R; 
       X 70  is selected from the group consisting of L and V; 
       X 71  is selected from the group consisting of L, M, V, W, and Y; 
       X 72  is selected from the group consisting of F, I, L, R, and V; 
       X 73  is selected from the group consisting of F, L, and V; 
       X 74  is selected from the group consisting of G, L, P, R, and S; 
       X 75  is selected from F, G, K, R, and S; 
       X 76  is selected from the group consisting of A, E, G, L, N, R, and V; 
       X 77  is selected from the group consisting of E, G, R, and T, or is absent; 
       X 78  is selected from the group consisting of A, G, P, and S, or is absent; and 
       wherein X 34  is selected from D, E, and G, and any X not specified in said Ubv polypeptide optionally has the amino acid sequence of the corresponding position in SEQ ID NO:1; or 
       a fragment thereof, wherein the sequence of said Ubv polypeptide does not consist of SEQ ID NO:1. 
     
     
         2 . The Ubv polypeptide of  claim 1 , comprising a substitution in one or more position selected from the group consisting of X 8 , X 11 , and X 73  of the amino acid sequence of SEQ ID NO:1. 
     
     
         3 . The Ubv polypeptide of  claim 2 , comprising one or more substitution selected from L8G, K11T, and L73F. 
     
     
         4 . The Ubv polypeptide of  claim 2  or  3 , further comprising a substitution in position X 42  and/or X 68 . 
     
     
         5 . The Ubv polypeptide of  claim 4 , wherein said substitution in position X 42  and/or X 68  is R42I and/or H68R. 
     
     
         6 . The Ubv polypeptide of any one of  claims 2 - 5 , comprising each of said substitutions and further comprising an amino acid substitution in one or more position selected from the group consisting of X 9 , X 10 , X 12 , X 46 , X 47 , X 49 , X 62 , X 63 , X 72 , X 76 , X 77 , and X 78 . 
     
     
         7 . The Ubv polypeptide of  claim 6 , wherein said substitution in one or more position selected from the group consisting of X 9 , X 10 , X 12 , X 46 , X 47 , X 49 , X 62 , X 63 , X 72 , X 76 , X 77 , and X 78 , is one or more of the following substitutions T9A, G10R, T12A, A46S, G47R, Q49L, Q62H, K63R, R72I, R76N, G77E, and G78S. 
     
     
         8 . The Ubv polypeptide of  claim 7 , comprising each of said substitutions (Fw7.1). 
     
     
         9 . The Ubv polypeptide of  claim 3 , comprising each of said substitutions and the following substitutions:
 (a) G10V, T12N, T14I, R42T, Q62E, T66S, H68R, R74G, R76L, 77T, and 78A (Fw7.2);   (b) Q2H, F4L, G10V, R42I, I44V, A46Y, H68Q, V70L, R74L, 77R, and 78P (Fw7.3); or   (c) T9A, T12N, R42I, A46S, Q49L, Q62H, K63R, H68R, R72I, R76N, 77E, and 78A (Fw7.4).   
     
     
         10 . A Ubv polypeptide comprising one or more amino acid substitution selected from the group consisting of A12G, I42R or V, L49R, H62Q, R63K, and G75R in the amino acid sequence of Fw7.1, and 
       wherein amino acids 77 and 78 are optional. 
     
     
         11 . The Ubv polypeptide of  claim 10 , comprising the following substitutions: A10G, I42V, L49R, H62Q, R63K, I72V, R74G, G75R, and G76R (Fw7.5), and wherein amino acids 77 and 78 are optional. 
     
     
         12 . The Ubv polypeptide of  claim 10 , comprising the following substitutions:
 (a) A10G, L49R, H62Q, R63K, R74P, and G75S (Fw7.6);   (b) A10G, I42R, S46A, R47G, K48R, L49R, H62Q, R63K, L71Y, I72V, and G75R (Fw7.7);   (c) I42R, S46A, R47G, H62Q, R63K, L71W, I72F, F73L, G75F, and G76V (Fw7.8);   (d) A10G, L49R, H62Q, R63K, L71M, I72V, R74S, and G75K (Fw7.9);   (e) A9T, A10G, I42V, I44L, L49V, H62Q, R63K, L71W, I72L, F73L, and G75R (Fw7.10);   (f) E36G, I42V, R47K, L49R, H62Q, R63K, and G75R (Fw7.11);   (g) I42R, S46A, R47G, L49R, H62Q, R63K, I72L, G75R, and G76E (Fw7.12);   (h) A9S, A10G, E36G, I44L, L49R, H62Q, R63K, R74S, and G76A (Fw7.13);   (i) E36D, I42V, I44L, S46R, L49K, H62Q, R63K, V70L, L71W, F73V, and G75R (Fw7.14);   (j) A10G, R47H, K48M, L49R, H62Q, R63K, and R74S (Fw.7.15);   (k) A10G, E36G, I44L, L49P, H62Q, R63K, L71V, I72V, and G75R (Fw7.16);   (l) A10G, E36G, I42R, S46A, R47G, L49R, H62Q, R63K, and T66S (Fw7.17); and   (m) I42R, L49R, H62Q, R63K, I72L, and R74P (Fw7.18);   wherein amino acids 77 and 78 are optional.   
     
     
         13 . A Ubv polypeptide comprising the amino acid sequence of Fw11.1 (SEQ ID NO:20), optionally comprising 1-5 substitutions. 
     
     
         14 . The Ubv polypeptide of  claim 13 , comprising the following substitutions:
 (a) S12Y, S14T, and N17H (Fw11.2; SEQ ID NO:21);   (b) S14T and Y15F (Fw11.3; SEQ ID NO:22);   (c) S12Y (Fw11.4; SEQ ID NO:23);   (d) S14T (Fw11.5; SEQ ID NO:24);   (e) S14T, Y15F, and N17D (Fw11.6; SEQ ID NO:25);   (f) S14T and N17H (Fw11.7; SEQ ID NO:26);   (g) S12Y and S14T (Fw11.8; SEQ ID NO:27);   (h) Y15F (Fw11.9; SEQ ID NO:28);   (i) N17H (Fw11.10; SEQ ID NO:29);   (j) S14N and Y15F (Fw11.11; SEQ ID NO:30);   (k) N17D (Fw11.12; SEQ ID NO:31);   (l) S12H and N17Y (Fw11.13; SEQ ID NO:32);   (m) S12Y and S15F (Fw11.14; SEQ ID NO:33);   (n) K11R and S14T (Fw11.15; SEQ ID NO:34);   (o) S12T (Fw11.16; SEQ ID NO:35); and   (p) S12A (Fw11.17; SEQ ID NO:36).   
     
     
         15 . A UbV polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs:2-36, or a variant thereof comprising a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:2-36, or a fragment thereof. 
     
     
         16 . A nucleic acid molecule encoding a Ubv polypeptide of any one of  claims 1 - 15 . 
     
     
         17 . A recombinant expression vector comprising a nucleic acid molecule of  claim 16 . 
     
     
         18 . A host cell comprising a nucleic acid molecule of  claim 16  or a recombinant expression vector of  claim 17 . 
     
     
         19 . A method for obtaining a ubiquitin variant polypeptide that binds to a Skp1-F-box protein complex, the method comprising randomizing sequences in region 1, region 2, and/or region 3 of ubiquitin to create a library of variants, and screening the library of variants for binding to the F-box protein of said Skp1-F-box protein complex, or a fragment thereof, optionally wherein region 1 comprises up to 8 amino acids in addition to those present in ubiquitin. 
     
     
         20 . The method of  claim 19 , wherein the F-box protein comprises Fbw7 or Fbw11, or a fragment thereof. 
     
     
         21 . A method of modulating the activity of an Skp1-Cul1-F-box (SCF) E3 ligase in a cell, the method comprising contacting the cell with an agent that alters binding of Cul1 to a complex comprising Skp1 and an F-box protein in the cell. 
     
     
         22 . The method of  claim 21 , wherein said agent decreases the activity of said SCFE3 ligase. 
     
     
         23 . The method of  claim 20 , wherein said agent decreases or inhibits binding of Cul1 to said complex comprising Skp1 and an F-box protein. 
     
     
         24 . The method of any one of  claims 21 - 23 , wherein said agent comprises a Ubv polypeptide, a nucleic acid molecule encoding a Ubv polypeptide, or a fragment thereof. 
     
     
         25 . The method of any one of  claims 21 - 24 , wherein said agent has specificity for a particular SCF E3 ligase. 
     
     
         26 . The method of any one of  claims 21 - 24 , wherein said agent is active against more than one SCF E3 ligase. 
     
     
         27 . The method of any one of  claims 21 - 26 , wherein said SCF E3 ligase comprises Fbw7 or Fbw11. 
     
     
         28 . The method of  claim 24 , wherein said Ubv polypeptide is a Ubv polypeptide of any one of  claims 1  to  15 , or said nucleic acid molecule is a nucleic acid molecule of  claim 16 . 
     
     
         29 . The method of any one of  claims 21 - 28 , wherein said cell is a cancer cell. 
     
     
         30 . The method of  claim 29 , wherein said cancer cell is comprised within a subject having cancer. 
     
     
         31 . A method of treating cancer in a subject, the method comprising modulating the activity of an Skp1-Cul1-F-box (SCF) E3 ligase in a cell of the subject according to the method of any one of  claims 21 - 30 . 
     
     
         32 . A method of identifying an agent that modulates the activity of an SCF E3 ligase in a cell, the method comprising contacting a cell expressing an SCF E3 ligase with a candidate agent, and determining whether said agent affects the binding of Cul1 to a complex comprising Skp1 and an F-box protein. 
     
     
         33 . The method of  claim 32 , wherein said cell further expresses a Ubv polypeptide. 
     
     
         34 . The method of  claim 32  or  33 , wherein said candidate agent comprises a small molecule compound. 
     
     
         35 . The method of any one of  claims 32 - 34 , wherein said determining is carried out using an immunoprecipitation assay. 
     
     
         36 . The method of any one of  claims 33 - 35 , wherein said Ubv polypeptide is a Ubv polypeptide of any one of  claims 1 - 14 .