Targeting deregulated Wnt signaling in cancer using stabilized alpha-helices of BCL-9
The invention provides structurally-constrained peptides by hydrocarbon stapling of a BCL9 HD2 helix for use as a therapeutic agent. The invention further provides methods and kits for use of the structurally-constrained peptide of the instant invention. The invention is based, at least in part, on the results provided herein demonstrating that hydrocarbon stapled helical peptides display excellent proteolytic, acid, and thermal stability, restore the native helical structure of the peptide, possess superior pharmacokinetic properties compared to the corresponding unmodified peptides, and are highly effective in binding to β-catenin in vitro, in cellulo, and in vivo, disrupting the BCL-9/β-catenin interaction, and thereby interfering with deregulated Wnt/β-catenin signaling for therapeutic benefit in a variety of human diseases including human cancer.
1. A structurally constrained peptide of an HD2 domain of BCL9 (BCL9-HD2), comprising at least one hydrocarbon staple, wherein the hydrocarbon staple is positioned so as to link a first BCL9-HD2 amino acid selected from the group consisting of Leu-351, Ser-352, Gln-353, Glu-354, Gln-355, His-358, Arg-359, Arg-361, Ser-362, Leu-363, Thr-365, Leu-366, Ile-369, Gln-370, Met-372, Leu-373, and Phe-374 with a second BCL9-HD2 amino acid selected from the group consisting of Leu-351, Ser-352, Gln-353, Glu-354, Gln-355, Leu-356, Glu-357, His-358, Arg-359, Glu-360, Arg-361, Ser-362, Leu-363, Thr-365, Leu-366, Arg-367, Asp-368, Ile-369, Gln-370, Arg-371, Met-372, Leu-373, and Phe-374.
2. The structurally constrained peptide of claim 1 , wherein the peptide comprises an interacting face comprised of amino acids that interact with β-catenin, wherein the interacting face comprises BCL9 residues Gln-355, His-358, Arg-359, Ser-362, Leu-363, Leu-366, Ile-369, Gln-370, Leu-373, and Phe-374.
3. The structurally constrained peptide of claim 2 , wherein the interacting face represents a single face of an α-helix.
4. The structurally constrained peptide of claim 3 , wherein the single face of a helix comprises one, two, three, or four adjacent stacked columns of amino acids, wherein the stacked columns of amino acids are defined by positions a, d, and g; positions b and e; or positions c and f; in an alpha helix having 3.6 amino acids per turn wherein the amino acids are consecutively and serially assigned positions a-g; and positions a and d; positions b and e; or positions c and fin a 3 10 helix having 3 amino acids per turn wherein the amino acids are consecutively and serially assigned positions a-f; or homologues thereof.
5. The structurally constrained peptide of claim 1 , wherein the hydrocarbon staple is between one or more natural or non-natural amino acids.
6. The peptide of claim 5 , wherein the hydrocarbon staple is formed by an olefin metathesis reaction.
7. The peptide of claim 5 , wherein the non-natural amino acids are selected from the following:
8. The structurally constrained peptide of claim 1 , comprising 1 to 5 staples within the BCL9 HD2 peptide.
9. The structurally constrained peptide of claim 8 , wherein one staple is located at the following positions within the BCL9 HD2 peptide: a) i, i+4; b) i, i+7; and c) i, i+3.
10. The structurally constrained peptide of claim 8 , wherein another staple or stitch is located at the following positions within the BCL9 HD2 peptide: a) i, i+4; b) i+7; and c) i, i+3.
11. The structurally constrained peptide of claim 8 , wherein any other staples or stitches are located at the following positions within the BCL9 HD2 peptide: a) i, i+4; b) i, i+7; and c) i, i+3.
12. A structurally constrained peptide of an HD2 domain of BCL9 (BCL9-HD2) comprising at least one hydrocarbon staple, wherein one hydrocarbon staple is located at the following positions within the BCL9 HD2 peptide:
i, i + 4 single staples:
X SQE X LEHRERSLQTLRDIQRBLF
(SEQ ID NO: 8)
L X QEQ X EHRERSLQTLRDIQRBLF
(SEQ ID NO: 9)
LS X EQL X HRERSLQTLRDIQRBLF
(SEQ ID NO: 10)
LSQ X QLE X RERSLQTLRDIQRBLF
(SEQ ID NO: 11)
LSQE X LEH X ERSLQTLRDIQRBLF
(SEQ ID NO: 12)
LSQEQ X EHR X RSLQTLRDIQRBLF
(SEQ ID NO: 13)
LSQEQL X HRE X SLQTLRDIQRBLF
(SEQ ID NO: 14)
LSQEQLE X RER X LQTLRDIQRBLF
(SEQ ID NO: 15)
LSQEQLEH X ERS X QTLRDIQRBLF
(SEQ ID NO: 16)
LSQEQLEHR X RSL X TLRDIQRBLF
(SEQ ID NO: 17)
LSQEQLEHRE X SLQ X LRDIQRBLF
(SEQ ID NO: 18)
LSQEQLEHRER X LQT X RDIQRBLF
(SEQ ID NO: 19)
LSQEQLEHRERS X QTL X DIQRBLF
(SEQ ID NO: 20)
LSQEQLEHRERSL X TLR X IQRBLF
(SEQ ID NO: 21)
LSQEQLEHRERSLQ X LRD X QRBLF
(SEQ ID NO: 22)
LSQEQLEHRERSLQT X RDI X RBLF
(SEQ ID NO: 23)
LSQEQLEHRERSLQTL X DIQ X BLF
(SEQ ID NO: 24)
LSQEQLEHRERSLQTLR X IQR X LF
(SEQ ID NO: 25)
LSQEQLEHRERSLQTLRD X QRB X F
(SEQ ID NO: 26)
LSQEQLEHRERSLQTLRDI X RBL X
(SEQ ID NO: 27)
i, i + 7 staples:
X SQEQLE X RERSLQTLRDIQRBLF
(SEQ ID NO: 28)
L X QEQLEH X ERSLQTLRDIQRBLF
(SEQ ID NO: 29)
LS X EQLEHR X RSLQTLRDIQRBLF
(SEQ ID NO: 30)
LSQ X QLEHRE X SLQTLRDIQRBLF
(SEQ ID NO: 31)
LSQE X LEHRER X LQTLRDIQRBLF
(SEQ ID NO: 32)
LSQEQ X EHRERS X QTLRDIQRBLF
(SEQ ID NO: 33)
LSQEQL X HRERSL X TLRDIQRBLF
(SEQ ID NO: 34)
LSQEQLE X RERSLQ X LRDIQRBLF
(SEQ ID NO: 35)
LSQEQLEH X ERSLQT X RDIQRBLF
(SEQ ID NO: 36)
LSQEQLEHR X RSLQTL X DIQRBLF
(SEQ ID NO: 37)
LSQEQLEHRE X SLQTLR X IQRBLF
(SEQ ID NO: 38)
LSQEQLEHRER X LQTLRD X QRBLF
(SEQ ID NO: 39)
LSQEQLEHRERS X QTLRDI X RBLF
(SEQ ID NO: 40)
LSQEQLEHRERSL X TLRDIQ X BLF
(SEQ ID NO: 41)
LSQEQLEHRERSLQ X LRDIQR X LF
(SEQ ID NO: 42)
LSQEQLEHRERSLQT X RDIQRB X F
(SEQ ID NO: 43)
LSQEQLEHRERSLQTL X DIQRBL X
(SEQ ID NO: 44)
i, i + 3 single staples:
X SQ X QLEHRERSLQTLRDIQRBLF
(SEQ ID NO: 45)
L X QE X LEHRERSLQTLRDIQRBLF
(SEQ ID NO: 46)
LS X EQ X EHRERSLQTLRDIQRBLF
(SEQ ID NO: 47)
LSQE X LE X RERSLQTLRDIQRBLF
(SEQ ID NO: 48)
LSQEQ X EH X ERSLQTLRDIQRBLF
(SEQ ID NO: 49)
LSQEQL X HR X RSLQTLRDIQRBLF
(SEQ ID NO: 50)
LSQEQLE X RE X SLQTLRDIQRBLF
(SEQ ID NO: 51)
LSQEQLEH X ER X LQTLRDIQRBLF
(SEQ ID NO: 52)
LSQEQLEHR X RS X QTLRDIQRBLF
(SEQ ID NO: 53)
LSQEQLEHRE X SL X TLRDIQRBLF
(SEQ ID NO: 54)
LSQEQLEHRER X LQ X LRDIQRBLF
(SEQ ID NO: 55)
LSQEQLEHRERS X QT X RDIQRBLF
(SEQ ID NO: 56)
LSQEQLEHRERSL X TL X DIQRBLF
(SEQ ID NO: 57)
LSQEQLEHRERSLQ X LR X IQRBLF
(SEQ ID NO: 58)
LSQEQLEHRERSLQT X RD X QRBLF
(SEQ ID NO: 59)
LSQEQLEHRERSLQTL X DI X RBLF
(SEQ ID NO: 60)
LSQEQLEHRERSLQTLR X IQ X BLF
(SEQ ID NO: 61)
LSQEQLEHRERSLQTLRD X QR X LF
(SEQ ID NO: 62)
LSQEQLEHRERSLQTLRDI X RB X F
(SEQ ID NO: 63)
LSQEQLEHRERSLQTLRDIQ X BL X
(SEQ ID NO: 64),
wherein X is any amino acid and further identifies the amino acid residues which are linked by a hydrocarbon staple.
13. A structurally constrained peptide of an HD2 domain of BCL9 (BCL9-HD2) comprising two hydrocarbon staples, wherein two hydrocarbon staples are located at the following positions within the BCL9 HD2 peptide:
i, i + 3 double staples:
X SQ X QLEHRERSLQTLRDIQ X BL X
(SEQ ID NO: 65)
X SQ X QLEHRERSLQTLRDI X RB X F
(SEQ ID NO: 66)
X SQ X QLEHRERSLQTLRD X QRB X F
(SEQ ID NO: 67)
i, i + 4 double staples:
X SQE X LEHRERSLQTLRDI X RBL X
(SEQ ID NO: 68)
X SQE X LEHRERSLQTLRD X QRB X F
(SEQ ID NO: 69)
X SQE X LEHRERSLQTL X DIQR X LF
(SEQ ID NO: 70)
i, i + 7 double staples:
X SQEQLE X RERSLQTL X DIQRBL X
(SEQ ID NO: 71)
X SQEQLE X RERSLQT X RDIQRB X F
(SEQ ID NO: 72)
X SQEQLE X RERSLQ X LRDIQR X LF
(SEQ ID NO: 73).
14. A structurally constrained peptide of an HD2 domain of BCL9 (BCL9-HD2) comprising at least one hydrocarbon staple, wherein the at least one hydrocarbon staples is located at any of the following positions within the BCL9 HD9 domain:
Mixed i, i + 4; i, i + 3; and
i, i + 7 double staples:
X SQE X LEHRERSLQTL X DIQRBL X
(SEQ ID NO: 74)
X SQE X LEHRERSLQT X RDIQRB X F
(SEQ ID NO: 75)
X SQE X LEHRERSLQ X LRDIQR X LF
(SEQ ID NO: 76)
X SQE X LEHRERSLQTLRDIQ X BL X
(SEQ ID NO: 77)
X SQE X LEHRERSLQTLRDI X RB X F
(SEQ ID NO: 78)
X SQE X LEHRERSLQTLRD X QR X LF
(SEQ ID NO: 79)
X SQEQLE X RERSLQTLRDI X RBL X
(SEQ ID NO: 80)
X SQEQLE X RERSLQTLRD X QRB X F
(SEQ ID NO: 81)
X SQEQLE X RERSLQTLR X IQR X LF
(SEQ ID NO: 82)
X SQEQLE X RERSLQTLRDIQ X BL X
(SEQ ID NO: 83)
X SQEQLE X RERSLQTLRDI X RB X F
(SEQ ID NO: 84)
X SQEQLE X RERSLQTLRD X QR X LF
(SEQ ID NO: 85)
X SQ X QLEHRERSLQTLRDI X RBL X
(SEQ ID NO: 86)
X SQ X QLEHRERSLQTLRD X QRB X F
(SEQ ID NO: 87)
X SQ X QLEHRERSLQTLR X IQR X LF
(SEQ ID NO: 88)
X SQ X QLEHRERSLQTL X DIQRBL X
(SEQ ID NO: 89)
XSQ X QLEHRERSLQT X RDIQRB X F
(SEQ ID NO: 90)
X SQ X QLEHRERSLQ X LRDIQR X LF
(SEQ ID NO: 91)
Sequential i, i + 4 staples:
X SQE X LEH X ERSLQTLRDIQRBLF
(SEQ ID NO: 92)
L X QEQ X EHR X RSLQTLRDIQRBLF
(SEQ ID NO: 93)
LS X EQL X HRE X SLQTLRDIQRBLF
(SEQ ID NO: 94)
LSQ X QLE X RER X LQTLRDIQRBLF
(SEQ ID NO: 95)
LSQE X LEH X ERS X QTLRDIQRBLF
(SEQ ID NO: 96)
LSQEQ X EHR X RSL X TLRDIQRBLF
(SEQ ID NO: 97)
LSQEQL X HRE X SLQ X LRDIQRBLF
(SEQ ID NO: 98)
LSQEQLE X RER X LQT X RDIQRBLF
(SEQ ID NO: 99)
LSQEQLEH X ERS X QTL X DIQRBLF
(SEQ ID NO: 100)
LSQEQLEHR X RSL X TLR X IQRBLF
(SEQ ID NO: 101)
LSQEQLEHRE X SLQ X LRD X QRBLF
(SEQ ID NO: 102)
LSQEQLEHRER X LQT X RDI X RBLF
(SEQ ID NO: 103)
LSQEQLEHRERS X QTL X DIQ X BLF
(SEQ ID NO: 104)
LSQEQLEHRERSL X TLR X IQR X LF
(SEQ ID NO: 105)
LSQEQLEHRERSLQ X LRD X QRB X F
(SEQ ID NO: 106)
LSQEQLEHRERSLQT X RDI X RBL X
(SEQ ID NO: 107)
Sequential i, i + 3 staples:
X SQ X QL X HRERSLQTLRDIQRBLF
(SEQ ID NO: 108)
Sequential i, i + 7 staples:
NSQEQLE X RERSLQ X LRDIQRBLF
(SEQ ID NO: 109)
Mixed sequential staples:
X SQ X QLE X RERSLQTLRDIQRBLF
(SEQ ID NO: 110)
X SQ X QLEHRE X SLQTLRDIQRBLF
(SEQ ID NO: 111)
X SQE X LE X RERSLQTLRDIQRBLF
(SEQ ID NO: 112)
X SQE X LEHRER X LQTLRDIQRBLF
(SEQ ID NO: 113)
X SQEQLE X RE X SLQTLRDIQRBLF
(SEQ ID NO: 114)
X SQEQLE X RER X LQTLRDIQRBLF.
(SEQ ID NO: 115).
15. The structurally constrained peptide of claim 12 , further comprising one to four additional hydrocarbon staples.
16. The structurally constrained peptide of claim 15 , wherein the additional hydrocarbon staples are located at any of the following positions within the BCL9 HD2 domain and iterated by staple scanning:
i, i + 4 single staples:
X SQE X LEHRERSLQTLRDIQRBLF
(SEQ ID NO: 8)
L X QEQ X EHRERSLQTLRDIQRBLF
(SEQ ID NO: 9)
LS X EQL X HRERSLQTLRDIQRBLF
(SEQ ID NO: 10)
LSQ X QLE X RERSLQTLRDIQRBLF
(SEQ ID NO: 11)
LSQE X LEH X ERSLQTLRDIQRBLF
(SEQ ID NO: 12)
LSQEQ X EHR X RSLQTLRDIQRBLF
(SEQ ID NO: 13)
LSQEQL X HRE X SLQTLRDIQRBLF
(SEQ ID NO: 14)
LSQEQLE X RER X LQTLRDIQRBLF
(SEQ ID NO: 15)
LSQEQLEH X ERS X QTLRDIQRBLF
(SEQ ID NO: 16)
LSQEQLEHR X RSL X TLRDIQRBLF
(SEQ ID NO: 17)
LSQEQLEHRE X SLQ X LRDIQRBLF
(SEQ ID NO: 18)
LSQEQLEHRER X LQT X RDIQRBLF
(SEQ ID NO: 19)
LSQEQLEHRERS X QTL X DIQRBLF
(SEQ ID NO: 20)
LSQEQLEHRERSL X TLR X IQRBLF
(SEQ ID NO: 21)
LSQEQLEHRERSLQ X LRD X QRBLF
(SEQ ID NO: 22)
LSQEQLEHRERSLQT X RDI X RBLF
(SEQ ID NO: 23)
LSQEQLEHRERSLQTL X DIQ X BLF
(SEQ ID NO: 24)
LSQEQLEHRERSLQTLR X IQR X LF
(SEQ ID NO: 25)
LSQEQLEHRERSLQTLRD X QRB X F
(SEQ ID NO: 26)
LSQEQLEHRERSLQTLRDI X RBL X
(SEQ ID NO: 27)
i, i + 7 staples:
X SQEQLE X RERSLQTLRDIQRBLF
(SEQ ID NO: 28)
L X QEQLEH X ERSLQTLRDIQRBLF
(SEQ ID NO: 29)
LS X EQLEHR X RSLQTLRDIQRBLF
(SEQ ID NO: 30)
LSQ X QLEHRE X SLQTLRDIQRBLF
(SEQ ID NO: 31)
LSQE X LEHRER X LQTLRDIQRBLF
(SEQ ID NO: 32)
LSQEQ X EHRERS X QTLRDIQRBLF
(SEQ ID NO: 33)
LSQEQL X HRERSL X TLRDIQRBLF
(SEQ ID NO: 34)
LSQEQLE X RERSLQ X LRDIQRBLF
(SEQ ID NO: 35)
LSQEQLEH X ERSLQT X RDIQRBLF
(SEQ ID NO: 36)
LSQEQLEHR X RSLQTL X DIQRBLF
(SEQ ID NO: 37)
LSQEQLEHRE X SLQTLR X IQRBLF
(SEQ ID NO: 38)
LSQEQLEHRER X LQTLRD X QRBLF
(SEQ ID NO: 39)
LSQEQLEHRERS X QTLRDI X RBLF
(SEQ ID NO: 40)
LSQEQLEHRERSL X TLRDIQ X BLF
(SEQ ID NO: 41)
LSQEQLEHRERSLQ X LRDIQR X LF
(SEQ ID NO: 42)
LSQEQLEHRERSLQT X RDIQRB X F
(SEQ ID NO: 43)
LSQEQLEHRERSLQTL X DIQRBL X
(SEQ ID NO: 44)
i, i + 3 single staples:
X SQ X QLEHRERSLQTLRDIQRBLF
(SEQ ID NO: 45)
L X QE X LEHRERSLQTLRDIQRBLF
(SEQ ID NO: 46)
LS X EQ X EHRERSLQTLRDIQRBLF
(SEQ ID NO: 47)
LSQE X LE X RERSLQTLRDIQRBLF
(SEQ ID NO: 48)
LSQEQ X EH X ERSLQTLRDIQRBLF
(SEQ ID NO: 49)
LSQEQL X HR X RSLQTLRDIQRBLF
(SEQ ID NO: 50)
LSQEQLE X RE X SLQTLRDIQRBLF
(SEQ ID NO: 51)
LSQEQLEH X ER X LQTLRDIQRBLF
(SEQ ID NO: 52)
LSQEQLEHR X RS X QTLRDIQRBLF
(SEQ ID NO: 53)
LSQEQLEHRE X SL X TLRDIQRBLF
(SEQ ID NO: 54)
LSQEQLEHRER X LQ X LRDIQRBLF
(SEQ ID NO: 55)
LSQEQLEHRERS X QT X RDIQRBLF
(SEQ ID NO: 56)
LSQEQLEHRERSL X TL X DIQRBLF
(SEQ ID NO: 57)
LSQEQLEHRERSLQ X LR X IQRBLF
(SEQ ID NO: 58)
LSQEQLEHRERSLQT X RD X QRBLF
(SEQ ID NO: 59)
LSQEQLEHRERSLQTL X DI X RBLF
(SEQ ID NO: 60)
LSQEQLEHRERSLQTLR X IQ X BLF
(SEQ ID NO: 61)
LSQEQLEHRERSLQTLRD X QR X LF
(SEQ ID NO: 62)
LSQEQLEHRERSLQTLRDI X RB X F
(SEQ ID NO: 63)
LSQEQLEHRERSLQTLRDIQ X BL X
(SEQ ID NO: 64)
i, i + 3 double staples:
X SQ X QLEHRERSLQTLRDIQ X BL X
(SEQ ID NO: 65)
X SQ X QLEHRERSLQTLRDI X RB X F
(SEQ ID NO: 66)
X SQ X QLEHRERSLQTLRD X QRB X F
(SEQ ID NO: 67)
i, i + 4 double staples:
X SQE X LEHRERSLQTLRDI X RBL X
(SEQ ID NO: 68)
X SQE X LEHRERSLQTLRD X QRB X F
(SEQ ID NO: 69)
X SQE X LEHRERSLQTL X DIQR X LF
(SEQ ID NO: 70)
i, i + 7 double staples:
X SQEQLE X RERSLQTL X DIQRBL X
(SEQ ID NO: 71)
X SQEQLE X RERSLQT X RDIQRB X F
(SEQ ID NO: 72)
X SQEQLE X RERSLQ X LRDIQR X LF
(SEQ ID NO: 73).
17. The structurally constrained peptide of claim 1 , wherein the amino acid sequence of positions 351 to 374 is selected from the group consisting of:
SEQ ID NO: 1:
BCL9 HD2 domain:
LSQEQLEHRERSLQTLRDIQRMLF
SEQ ID NO: 2:
BCL9 HD2 domain M372B
LSQEQLEHRERSLQTLRDIQRBLF
SEQ ID NO: 3:
SAH-BCL9 A :
LSQEQLEHRERSLQTLR X IQR X LF
SEQ ID NO: 4:
SAH-BCL9 B :
LSQEQLEHRERSL X TLR X IQRBLF
SEQ ID NO: 5:
SAH-BCL9 C :
LSQEQLEHRE X SLQ X LRDIQRBLF
SEQ ID NO: 6:
SAH-BCL9 B (H358D):
LSQEQLE D RERSL X TLR X IQRBLF and
SEQ ID NO: 7:
SAH-BCL9 B (R359E):
LSQEQLEH E ERSL X TLR X IQRBLF.
18. A composition comprising the peptide of claim 1 and a pharmaceutically acceptable carrier.
19. A kit comprising a structurally constrained peptide of claim 1 and instructions for use in treating cancer.
20. A structurally constrained peptide of an HD2 domain of BCL9 (BCL9-HD2) comprising at least one hydrocarbon staple, wherein one hydrocarbon staple is positioned so as to link amino acid residues i and i+3, wherein the structurally constrained peptide is selected from the group consisting of
(SEQ ID NO: 45)
X SQ X QLEHRERSLQTLRDIQRBLF,
(SEQ ID NO: 46)
L X QE X LEHRERSLQTLRDIQRBLF,
(SEQ ID NO: 47)
LS X EQ X EHRERSLQTLRDIQRBLF,
(SEQ ID NO: 48)
LSQE X LE X RERSLQTLRDIQRBLF,
(SEQ ID NO: 49)
LSQEQ X EH X ERSLQTLRDIQRBLF,
(SEQ ID NO: 50)
LSQEQL X HR X RSLQTLRDIQRBLF,
(SEQ ID NO: 51)
LSQEQLE X RE X SLQTLRDIQRBLF,
(SEQ ID NO: 52)
LSQEQLEH X ER X LQTLRDIQRBLF,
(SEQ ID NO: 53)
LSQEQLEHR X RS X QTLRDIQRBLF,
(SEQ ID NO: 54)
LSQEQLEHRE X SL X TLRDIQRBLF,
(SEQ ID NO: 55)
LSQEQLEHRER X LQ X LRDIQRBLF,
(SEQ ID NO: 56)
LSQEQLEHRERS X QT X RDIQRBLF,
(SEQ ID NO: 57)
LSQEQLEHRERSL X TL X DIQRBLF,
(SEQ ID NO: 58)
LSQEQLEHRERSLQ X LR X IQRBLF,
(SEQ ID NO: 59)
LSQEQLEHRERSLQT X RD X QRBLF,
(SEQ ID NO: 60)
LSQEQLEHRERSLQTL X DI X RBLF,
(SEQ ID NO: 61)
LSQEQLEHRERSLQTLR X IQ X BLF,
(SEQ ID NO: 62)
LSQEQLEHRERSLQTLRD X QR X LF,
(SEQ ID NO: 63)
LSQEQLEHRERSLQTLRDI X RB X F, and
(SEQ ID NO: 64)
LSQEQLEHRERSLQTLRDIQ X BL X ,
wherein X is any amino acid and further identifies the amino acid residues which are linked by a hydrocarbon staple.