BINDING INHIBITORS OF THE BETA. TRANSDUCIN REPEAT-CONTAINING PROTEIN
20190106460 ยท 2019-04-11
Inventors
- Mark Bradley (Edinburgh, GB)
- Jeffrey George Andrew Walton (Edinburgh, GB)
- Sunay Vijaykumar Chankeshwara (Edinburgh, GB)
- Mazen Sleiman (Edinburgh, GB)
- George S. Baillie (Glasgow, GB)
- Lucien Gibson (Glasgow, GB)
Cpc classification
G01N2500/04
PHYSICS
C12Y603/02019
CHEMISTRY; METALLURGY
C07K7/64
CHEMISTRY; METALLURGY
C07K14/00
CHEMISTRY; METALLURGY
International classification
C07K14/00
CHEMISTRY; METALLURGY
C07K7/64
CHEMISTRY; METALLURGY
C12N9/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to compounds which bind to Beta Trans-ducin repeat-containing protein (TrCP), and modulate the activity of TrCP. In particular, the invention relates to compounds which demonstrate optimised binding to PTrCP. The invention also relates to pharmaceutical compositions comprising such compounds and the use of such compounds as medicaments, specifically for the treatment of disorders associated with aberrant protein degradation, such as cancer. The preferred binding inhibitors are peptides derived from the motive DSGXXS, e.g. DEGFWE, DDGFWD and Succinyl-EGFWE.
Claims
1.-107. (canceled)
108. A compound of formula IAAA, ##STR00184## wherein X.sup.1 is aspartyl ##STR00185## R.sup.4 is independently CO.sub.2H, CH.sub.2CO.sub.2H or OP(O)(OH).sub.2; R.sup.A3 is selected from the group consisting of F, Cl, Br, I, OH, O(C.sub.1-C.sub.10 alkyl), C.sub.1-C.sub.10 alkyl, NO.sub.2; R.sup.3 is ##STR00186## wherein R.sup.A4 is selected from the group consisting of H, F, Cl, Br, I, OH, O(C.sub.1-C.sub.10 alkyl), CN, NO.sub.2, CF.sub.3, OCF.sub.3, CO.sub.2H, C.sub.1-C.sub.10 alkyl, NH.sub.2, NH(C.sub.1-C.sub.10 alkyl) and N(C.sub.1-C.sub.10 alkyl); R.sup.N1 is selected from a group consisting of H, C.sub.1-C.sub.10 alkyl and aryl; R.sup.N2 is selected from a group consisting of R.sup.N1, (CH.sub.2).sub.0-10-(Z.sup.7).sub.0-1-A.sup.a, (CH.sub.2O).sub.0-10CH.sub.2-(Z.sup.7).sub.0-1-A.sup.a, (CH.sub.2CH.sub.2O).sub.1-10-CH.sub.2CH.sub.3, (CH.sub.2CH.sub.2O).sub.1-10-(CH.sub.2).sub.1-3-(Z.sup.7).sub.0-1-A.sup.a; wherein Z.sup.7 is (C=0); A.sup.a is OH, NH.sub.2, C(O)NH.sub.2, a cholesterol derivative, a chain of one or more non-naturally occurring amino acids, or a chain of one or more naturally occurring amino acids, or a chain of a mixture of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids; wherein the one or more non-naturally occurring or naturally occurring amino acids are independently selected from the group consisting of L-amino acids, D-amino acids and aza-amino acids; wherein CG is a capping group; wherein when the compound of formula IAAA comprises an amino/amine group, said amino/amine group may be optionally capped, by replacement of an H atom with a capping group; wherein CG and/or the optionally capping group is selected from the group consisting of: ##STR00187## wherein R.sup.cg is selected from the group consisting of H, F, Cl, Br, I, OH, O(C.sub.1-C.sub.10 alkyl), CN, NO.sub.2, CF.sub.3, OCF.sub.3, CO.sub.2H, NH.sub.2, NH(C.sub.1-C.sub.2 alkyl), N(C.sub.1-C.sub.2 alkyl).sub.2, C.sub.1-C.sub.10 alkyl, aryl and heteroaryl.
109. The compound of claim 108, wherein R.sup.4 is independently CO.sub.2H or CH.sub.2CO.sub.2H and R.sup.A3 is selected from the group consisting of F, Cl, Br, I.
110. The compound of claim 108, wherein R.sup.4 is independently CO.sub.2H or CH.sub.2CO.sub.2H; R.sup.A3 is selected from the group consisting of F, Cl, Br, I; and R.sup.3 is ##STR00188##
111. The compound of claim 108, wherein R.sup.4 is independently CH.sub.2CO.sub.2H; R.sup.A3 is selected from the group consisting of F, Cl, Br, I; and R.sup.3 is ##STR00189##
112. The compound of claim 108 wherein R.sup.4 is independently CH.sub.2CO.sub.2H, R.sup.A3 is F, and R.sup.3 is ##STR00190##
113. The compound of claim 108, wherein the CG and/or the optional capping group is selected from the group consisting of: ##STR00191##
114. The compound of claim 108, wherein the CG and/or the optional capping group is selected from List 1: ##STR00192## ##STR00193##
115. The compound of claim 108, wherein R.sup.A3 is selected from the group consisting of F, OH and NO.sub.2.
116. The compound of claim 108, wherein R.sup.A3 is F at one or more of positions 2, 3, 4.
117. The compound of claim 108, wherein R.sup.A3 is Cl at one or more of positions 2, 3, 4.
118. A pharmaceutical composition comprising the compound of claim 108.
119. A method of treating a disease associated with aberrant protein degradation comprising administering the compound of claim 108.
120. A diagnostic kit comprising the compound of claim 108.
Description
BRIEF DESCRIPTION OF FIGURES
[0594]
[0595] Reagents and Conditions: a) Rink amide linker (3 equiv), oxyma (3 equiv), DIC (3 equiv), 0.1 M in DMF, 30 min; b) 20% piperidine in DMF (25 min); c) Amino acid (3 equiv), HBTU (3 equiv), DIPEA (6 equiv) 0.1 M in DMF, 40 min; d) TsCl (5 equiv), DMAP (0.1 equiv), DIPEA (10 equiv), 0.1 M in DMF, 40 min; e) TFA, 5% TIS, 5% DCM, 3h.
[0596]
[0597] Reagents and Conditions: a) Rink amide linker (3 equiv), oxyma (3 equiv), DIC (3 equiv), 0.1 M in DMF, 30 min; b) 20% piperidine in DMF (25 min); c) Amino acid (3 equiv), HBTU (3 equiv), DIPEA (6 equiv) 0.1 M in DMF, 40 min; d) TsCl (5 equiv), DMAP (0.1 equiv), DIPEA (10 equiv), 0.1 M in DMF, 40 min; e) 2% Hydrazine in DMF (615 mins); f) BzCl (5 equiv), DMAP (0.1 equiv), DIPEA (10 equiv), 0.1 M in DMF, 40 min; g) TFA, 5% TIS, 5% DCM, 3h.
[0598]
[0599]
[0600]
[0601]
[0602]
[0603]
[0604]
[0605]
[0606]
[0607] Peptides are numbered according to Table 11: DSGIFE (SEQ ID NO: 119); DpSGIFE (SEQ ID NO: 12); and succ-EGFFE (SEQ ID NO: 57).
[0608]
[0609]
[0610]
[0611]
[0612]
[0613]
[0614]
[0615]
[0616]
[0617]
[0618]
[0619]
[0620]
[0621]
EXAMPLES
Experimental Procedures
[0622] The following experimental conditions were used throughout the examples unless other details are provided.
[0623] General Conditions for the Solid-Phase Synthesis
[0624] All the coupling reactions were carried out at room temperature if no specifications are given. Solid-phase synthesis was performed manually using Isolute filtration reservoirs as the reaction vessel, fitted with polyethylene frits (Argonaut Technologies Inc). Amino acids are Fmoc protected at the N terminus, with suitable acid labile protecting groups on the side chains. For C-terminal modified peptides Fmoc-Lys(Dde)-OH was used to allow selective modification of the Lys side chain. Each coupling step of the synthesis was assessed for completion using either the Kaiser test for primary amines, or the chloranil test for secondary amines.
[0625] Coupling the Linker to the Resin
[0626] Aminomethyl PS resin (loading 1.23 mmol/g, 0.30 g, 0.369 mmol) in a 6 mL reaction vessel was swollen for 5 minutes in DCM (3 mL), then washed with DCM (33 mL). To a solution of Rink amide linker (598 mg, 1.11 mmol) in DMF (3.69 mL) was added oxyma (157 mg, 1.11 mmol) and the solution shaken for 10 minutes. DIC (173 uL, 1.11 mmol) was added and the solution shaken for 2 minutes. The mixture was added to the resin and shaken for 30 minutes. The resin was filtered and washed with DMF (34 mL), DCM (34 mL) and MeOH (34 mL). Kaiser test negative. The resin was washed with Et.sub.2O (34 mL) and dried under vacuum for storage.
[0627] Coupling of Amino Acids/Spacer
[0628] Resin (0.049 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (1.5 mL) and filtered. A solution of 20% piperidine in DMF (1.5 mL) was added, the vessel was shaken for 5 mins and the resin was filtered and washed with DMF (31.5 mL) and DCM (31.5 mL). Piperidine deprotection and washing cycle was repeated and the resin was dried under vacuum, Kaiser test positive. To a solution of the appropriate amino acid/spacer (0.15 mmol, 3 equiv) in DMF (0.49 mL) was added HBTU (0.15 mmol, 3 equiv) and the solution shaken for 2 minutes. DIPEA (0.30 mmol, 6 equiv) was added and the solution shaken for 1 minute. The mixture was added to the resin and shaken for 40 minutes. The resin was filtered and washed with DMF (31.5 mL), DCM (31.5 mL) and MeOH (31.5 mL). Kaiser test negative, otherwise treatment of activated amino acid repeated.
[0629] Coupling to N-Alkylated Amino Acids
[0630] Resin (0.049 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (1.5 mL) and filtered. A solution of 20% piperidine in DMF (1.5 mL) was added, the vessel was shaken for 5 mins and the resin was filtered and washed with DMF (31.5 mL) and DCM (31.5 mL). Piperidine deprotection and washing cycle was repeated and the resin was dried under vacuum, Choranil test positive. To a solution of the appropriate amino acid (0.15 mmol, 3 equiv) in DMF (0.49 mL) was added oxyma (0.15 mmol, 3 equiv) and the solution shaken for 10 minutes. DIC (0.15 mmol, 3 equiv) was added and the solution shaken for 2 minutes. The mixture was added to the resin and heated in a microwave at 60 C. for 20 minutes. The mixture was then shaken for an additional 20 minutes. The resin was filtered and washed with DMF (34 mL), DCM (34 mL) and MeOH (34 mL). Chloranil test negative, otherwise treatment of activated amino acid repeated.
[0631] Example of the N-Terminus Capping
[0632] Resin (0.049 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (1.5 mL) and filtered. A solution of 20% piperidine in DMF (1.5 mL) was added, the vessel was shaken for 5 mins and the resin was filtered and washed with DMF (31.5 mL) and DCM (31.5 mL). Piperidine addition and washing cycle was repeated and the resin was dried under vacuum, Kaiser test positive. To a solution of 4-toluenesulfonyl chloride (0.25 mmol, 5 equiv) in DCM:DMF (1:1, 0.49 mL) was added DMAP (0.005 mmol, 0.1 equiv) and the solution shaken for 2 minutes. DIPEA (0.50 mmol, 10 equiv) was added and the solution shaken for 1 minute. The mixture was added to the resin and shaken for 40 minutes. The resin was filtered and washed with DMF (31.5 mL), DCM (31.5 mL) and MeOH (31.5 mL). Kaiser test negative, otherwise treatment of with the capping group was repeated.
[0633] Example of the C-Terminus Capping
[0634] After N-terminus capping, resin (0.049 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (1.5 mL) and filtered. A solution of 2% hydrazine monohydrate in DMF (1.5 mL) was added, the vessel was shaken for 15 mins and the resin was filtered and washed with DMF (31.5 mL) and DCM (31.5 mL). Hydrazine addition and washing cycle was repeated (5) and the resin was dried under vacuum, Kaiser test positive. To a solution of benzoyl chloride (0.25 mmol, 5 equiv) in DCM:DMF (1:1, 0.49 mL) was added DMAP (0.005 mmol, 0.1 equiv) and the solution shaken for 2 minutes. DIPEA (0.50 mmol, 10 equiv) was added and the solution shaken for 1 minute. The mixture was added to the resin and shaken for 40 minutes. The resin was filtered and washed with DMF (31.5 mL), DCM (31.5 mL) and MeOH (31.5 mL). Kaiser test negative, otherwise treatment of with the capping group was repeated.
[0635] Characterization of selected examples:
TABLE-US-00001 ID (SEQ ID RT NO:) Structure MS (mins) UBP001 DpSGIFE-NH.sub.2 744.1.sup.a 4.272.sup.e (12) UBP002 Suc-EGFFE-NH.sub.2 725.2.sup.a 5.107.sup.e (57) UBP003 Suc-EGF(2F)F(4NO.sub.2)E-NH.sub.2 788.2.sup.a 4.989.sup.e (58) UBP004 Suc-EGF(3F)F(4NO.sub.2)E-NH.sub.2 788.2.sup.a 5.073.sup.e (58) UBP005 Suc-EGF(4F)F(4NO.sub.2)E-NH.sub.2 788.2.sup.a 5.088.sup.e (58) UBP006 Suc-EGF(2F)Y(Me)E-NH.sub.2 773.2.sup.a 5.023.sup.e (59) UBP007 Suc-EGYFE-NH.sub.2 741.1.sup.a 4.500.sup.e (60) UBP008 Mal-EGF(3F)F(4NO.sub.2)E-NH.sub.2 786.1.sup.a 3.739.sup.e (61) UBP009 Suc-EGY1NalE-NH.sub.2 791.2.sup.a 5.222.sup.e (62) UBP010 Suc-EGF(3F)1NalE-NH.sub.2 793.0.sup.a 5.783.sup.e (62) UBP011 Suc-EGF(4NO.sub.2)1NalE-NH.sub.2 820.0.sup.a 5.775.sup.e (62) UBP012 Suc-QGF(3F)F(4NO.sub.2)E-NH.sub.2 787.0.sup.a 4.924.sup.e (63) UBP013 Suc-EGYF(4NO.sub.2)E-NH.sub.2 786.1.sup.a 4.422.sup.e (64) UBP014 Suc-EGF(3F)WE-NH.sub.2 781.9.sup.a 4.597.sup.e (65) UBP015 Suc-EGF(3F)HE-NH.sub.2 733.0.sup.a 3.257.sup.e (65) UBP016 Ac-dEGF(3F)1NalE-NH.sub.2 850.0.sup.a 5.705.sup.e UBP017 Ac-dEGF(3F)WE-NH.sub.2 838.9.sup.a 4.976.sup.e UBP018 Bz-dEGF(3F)WE-NH.sub.2 901.0.sup.a 5.514.sup.e UBP019 Et(CO)-dEGF(3F)WE-NH.sub.2 853.0.sup.a 5.167.sup.e UBP020 MeO(CO)-dEGF(3F)WE-NH.sub.2 855.1.sup.a 5.138.sup.e UBP021 Ts-dEGF(3F)WE-NH.sub.2 951.2.sup.a 5.800.sup.e UBP022 4-(MeO)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 967.2.sup.a 5.690.sup.e UBP023 EtO(CO)-dEGF(3F)WE-NH.sub.2 896.2.sup.a 5.362.sup.e UBP024 Ts-DEGF(3F)WE-NH.sub.2 951.2.sup.a 5.673.sup.e (66) UBP025 Ts-dDGF(3F)WE-NH.sub.2 937.2.sup.a 5.745.sup.e UBP026 4-(MeO)-PhSO.sub.2-DEGF(3F)WE-NH.sub.2 967.2.sup.a 5.534.sup.e (67) UBP027 Ts-dEGF(3F)WD-NH.sub.2 937.2.sup.a 5.770.sup.e UBP028 Ts-dDGF(3F)WD-NH.sub.2 923.2.sup.a 5.729.sup.e UBP029 3,4-(MeO).sub.2-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 997.2.sup.a 5.495.sup.e UBP030 4-(BuO)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1009.4.sup.a 6.404.sup.e UBP031 2-NaphthylSO.sub.2-dEGF(3F)WE-NH.sub.2 987.2.sup.a 6.010.sup.e UBP032 Ts-dEGF(3F)WE(Me)-NH.sub.2 956.2.sup.a 5.891.sup.e UBP033 Ts-dEGF(3Cl)WE(Me)-NH.sub.2 981.2.sup.a 5.992.sup.e UBP034 Ts-dEGF(3Cl)WE-NH.sub.2 967.2.sup.a 6.086.sup.e UBP035 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-kkkkkkkkk-NH.sub.2 2236.2.sup.b 3.547.sup.e UBP036 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(Ahx-Chol)-NH.sub.2 1773.4.sup.d 7.970.sup.f UBP037 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCOC.sub.17H.sub.35)-NH.sub.2 1499.0.sup.c 10.398.sup.f UBP038 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCOC.sub.19H.sub.39)-NH.sub.2 1527.2.sup.c 11.247.sup.f UBP039 4-(t-Bu)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1017.0.sup.c 9.502.sup.g UBP040 4-(i-Pr)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1003.1.sup.a 9.688.sup.g UBP041 4-(Pr)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1003.1.sup.c 9.471.sup.g UBP042 4-(Br)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1039.2.sup.c 8.589.sup.g UBP043 4-(Br)-2-(CH.sub.3)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1053.0.sup.c 8.932.sup.g UBP044 2-Naph-SO.sub.2-dEGF(3F)WE-NH.sub.2 1011.2.sup.c 8.924.sup.g UBP045 4-(OCF.sub.3)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1045.0.sup.c 9.289.sup.g UBP046 4-(Br)-3-(CF.sub.3)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1107.0.sup.c 9.866.sup.g UBP047 4-(CF.sub.3)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1029.2.sup.c 9.319.sup.g UBP048 2,4-(Cl).sub.2-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1029.0.sup.c 9.437.sup.g UBP049 2,4-(Br).sub.2-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1117.0.sup.c 9.042.sup.g UBP050 3,5-(CH.sub.3).sub.2-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 989.0.sup.c 8.959.sup.g UBP051 4-(Br)-2-(OCF.sub.3)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1123.0.sup.c 9.499.sup.g UBP052 4-(I)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1096.5.sup.c 11.312.sup.g UBP053 4-(Cl)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 995.2.sup.c 10.944.sup.g UBP054 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(t-Bu)-Ph))-NH.sub.2 1392.5.sup.c 10.163.sup.g UBP055 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-2-Naph)-NH.sub.2 1386.3.sup.c 9.834.sup.g UBP056 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(2,4,6-(Me).sub.3-Ph))-NH.sub.2 1378.5.sup.c 9.313.sup.g UBP057 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(Me)-Ph))-NH.sub.2 1350.2.sup.c 9.462.sup.g UBP058 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(Br)-Ph))-NH.sub.2 1414.3.sup.c 9.497.sup.g UBP059 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHSO.sub.2-(4-(Br)-Ph))-NH.sub.2 1450.2.sup.c 9.903.sup.g UBP060 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(Cl)-Ph))-NH.sub.2 1370.3.sup.c 9.289.sup.g UBP061 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCOPh)-NH.sub.2 1335.6.sup.c 10.507.sup.g UBP062 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(3,5-(Cl).sub.2-Ph))-NH.sub.2 1380.0.sup.a 10.035.sup.g UBP063 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(CH.sub.2).sub.4CH.sub.3)-NH.sub.2 1330.6.sup.c 9.493.sup.g UBP064 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(CF.sub.3)-Ph))-NH.sub.2 1380.3.sup.a 9.874.sup.g UBP065 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCOO-Ph)-NH.sub.2 1352.3.sup.c 9.575.sup.g UBP066 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(OMe)-Ph))-NH.sub.2 1366.2.sup.c 8.875.sup.g UBP067 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCONH-Ph)-NH.sub.2 1327.4.sup.a 9.389.sup.g UBP068 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-CH.sub.2CH(CH.sub.3).sub.2)-NH.sub.2 1316.0.sup.c 8.316.sup.g UBP069 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHOCO-1-Naph)-NH.sub.2 1378.2.sup.a 10.118.sup.g UBP070 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(Cl)-2,6(F).sub.2-Ph))-NH.sub.2 1406.5.sup.c 10.815.sup.g UBP071 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(Me.sub.2N)-Ph))-NH.sub.2 1379.3.sup.c 8.889.sup.g UBP072 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHSO.sub.2-(4-(i-Pr)-Ph))-NH.sub.2 1414.0.sup.c 10.271.sup.g UBP073 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHSO.sub.2Ph)-NH.sub.2 1348.2.sup.a 8.877.sup.g UBP074 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHSO.sub.2-(4-(n-Pr)-Ph)-NH.sub.2 1414.2.sup.c 10.269.sup.g UBP075 4-(t-Bu)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(t-Bu)Ph))-NH.sub.2 1394.3.sup.a 7.138.sup.e UBP076 4-(t-Bu)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2-Naphth))-NH.sub.2 1388.2.sup.a 6.922.sup.e UBP077 4-(t-Bu)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2,4,6-(Me).sub.3-Ph))-NH.sub.2 1380.3.sup.a 6.911.sup.e UBP078 4-(t-Bu)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(Me)Ph))-NH.sub.2 1352.3.sup.a 6.802.sup.e UBP079 4-(t-Bu)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(Br)Ph))-NH.sub.2 1418.2.sup.a 6.917.sup.e UBP080 4-(i-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(t-Bu)Ph))-NH.sub.2 1380.4.sup.a 7.037.sup.e UBP081 4-(i-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2-Naphth))-NH.sub.2 1374.2.sup.a 6.813.sup.e UBP082 4-(i-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2,4,6-(Me).sub.3Ph))-NH.sub.2 1366.3.sup.a 6.786.sup.e UBP083 4-(i-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO((4-(Br)Ph))-NH.sub.2 1402.2.sup.a 6.815.sup.e UBP084 4-(n-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(tBu)Ph))-NH.sub.2 1380.3.sup.a 7.175.sup.e UBP085 4-(n-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2-Naphth))-NH.sub.2 1374.3.sup.a 6.853.sup.e UBP086 4-(n-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO((2,4,6-(Me).sub.3Ph))-NH.sub.2 1366.3.sup.a 6.826.sup.e UBP087 4-(n-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(Me)Ph))-NH.sub.2 1338.3.sup.a 6.735.sup.e UBP088 4-(n-Pr)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(Br)Ph))-NH.sub.2 1404.2.sup.a 6.858.sup.e UBP089 4-(Br)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(tBu)Ph))-NH.sub.2 1418.2.sup.a 7.047.sup.e UBP090 4-(Br)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2-Naphth))-NH.sub.2 1412.2.sup.a 6.848.sup.e UBP091 4-(Br)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2,4,6-(Me).sub.3Ph))-NH.sub.2 1404.2.sup.a 6.814.sup.e UBP092 4-(Br)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(Me)Ph))-NH.sub.2 1376.1.sup.a 6.732.sup.e UBP093 4-(Br)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(Br)Ph))-NH.sub.2 1442.1.sup.a 6.866.sup.e UBP094 4-(Br)-2-(Me)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(tBu)Ph))-NH.sub.2 1432.2.sup.a 7.036.sup.e UBP095 4-(Br)-2-(Me)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2-Naphth))-NH.sub.2 1426.3.sup.a 6.831.sup.e UBP096 4-(Br)-2-(Me)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(2,4,6-(Me).sub.3Ph))-NH.sub.2 1418.2.sup.a 6.805.sup.e UBP097 4-(Br)-2-(Me)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(Me)Ph))-NH.sub.2 1390.2.sup.a 6.696.sup.e UBP098 4-(Br)-2-(Me)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO(4-(Br)Ph))-NH.sub.2 1452.0.sup.a 6.824.sup.e .sup.aMass identified as [M H].sup.; .sup.bMass identified as [M + H].sup.+; .sup.cMass identified as [M + Na].sup.+; .sup.dMass identified as [M + K].sup.+; HPLC analysis preformed using a Supleco Discovery C18 5 cm 4.6 mm, 5 m column, samples analysed by ELSD, 220 nM and 254 nM, conditions used where .sup.e5% to 95% MeOH (+0.1% formic acid) in H.sub.2O (+0.1% formic acid) over 6 minutes, 3 minute hold, then 1 minute at 5% MeOH (+0.1% formic acid); .sup.f5% to 95% MeCN (+0.1% formic acid) in H.sub.2O (+0.1% formic acid) over 10 minutes, 4 minute hold, then 1 minute at 5% MeCN (+0.1% formic acid); .sup.g5% to 95% MeOH (+0.1% formic acid) in H.sub.2O (+0.1% formic acid) over 10 minutes, 4 minute hold, then 1 minute at 5% MeOH (+0.1% formic acid);
[0636] Cleavage from Resin
[0637] Resin (0.0.49 mmol) in a 3 mL reaction vessel was swollen for 5 minutes in DCM (2 mL) and filtered. A solution of TFA:TIS:DCM (90:5:5 0.49 mL) was added, and the vessel was shaken for 3 h. The resin was removed by filtration, and ice-cold Et.sub.2O (10 mL) was added to the filtrate. The resultant solid was pelleted by centrifuge, and the solvent removed by decantation. Solid was dried under vacuum.
[0638] The experimental scheme for solid phase synthesis is shown in
[0639] Fluorescence Polarization Screening of TrCP
[0640] Assay Components
[0641] 0.035 M TrCP (tag cleaved and complexed with Skp1)
[0642] 10 nM fluorescein-RHDpSGLDpSMKD (SEQ ID NO:68)
[0643] 50 mM Hepes pH 7.5
[0644] 50 mM NaCl
[0645] 1 mM DTT
[0646] 0.1 mg/ml BSA (Bovine Serum Albumin)
[0647] 50 M compound (in DMSO)
[0648] Assay Protocol
[0649] Assay components (without compound) were premixed in a microcentrifuge tube and incubated for 1 hour to ensure equilibrium was achieved. Each compound was then added to one tube, mixed by vortexing, and then dispensed into 3 wells of a black 384-well plate and incubated for 30 minutes. Fluorescence polarization was then read (excitation 485 nM, emission 530 nM) using an Analyst-AD from Molecular Devices.
[0650] For dose-response curves to determine Ki, 10 different concentrations of compound were tested at equally spaced intervals. DMSO was added such that final concentration was 2%. Conditions are very tolerant to DMSO. Up to 10% DMSO has been tested previously, with no significant change to Kd values.
[0651] Surface Plasmon Resonance (SPR)
[0652] Experiments were carried out using the Biacore T200 SPR detection system. This system exploits the phenomenon of surface plasmon resonance (SPR) to monitor interactions between molecules. The system involves the attachment of one interacting partner to a surface (an appropriate sensor chip) while the other interacting partner is passed over it in solution. The binding of molecules to the surface generates an SPR response (measured in response units (RU)) that is proportional to the mass and amount of the biomolecule (in this case TrCP/Skp1) bound to the chip. The relative responses obtained are dependent on the concentration of the molecule binding. At RU.sub.maximum, the attached protein's binding sites are saturated. Binding events can be followed in real time and a range of interaction characteristics can be determined including kinetics, specificity of interactions and the concentration of specific molecules in a sample.
[0653] As TrCP was His tagged, an NTA sensor chip was used. This sensor chip has a dextran surface matrix with immobilized nitrilotriacetic acid (NTA) which provides a means of capturing polyHis-tagged proteins through Nickel chelation. It was hoped that addition of Ni.sup.+ would orient the protein in a specific (and hopefully active) manner as it is covalently immobilised via amine coupling. Addition of EDC:NHS (N-ethyl-N-(3-diethylaminopropyl)-carbodiimide:N-hydroxysuccinimide) converts carboxyl groups on the dextran sensor chip surface to succinamide esters which readily form covalent bonds with primary amines. Each chip contains four flow cells (each a separate surface) which means that compounds/peptides can be passed over different forms of TrCP and a reference surface simultaneously. If binding to TrCP is occurring, responses should be the same (accounting for differences in density of surface etc) on each surface.
[0654] Two different TrCP protein complexes were immobilised on to an NTA biacore sensor chip. One included the GSTSkp1 fusion (HisTrCP/GSTSkp1) while the other was immobilised after GST Removal by Thrombin (HisTrCP/Skp1). To ensure that the GST moiety is completely removed from Skp1, TrCP/GSTSkp1 was incubated with thrombin ((10units/mg protein) for at least 16 hours at room temperature in 10mMHEPES 150mMNaCl pH7.4+2 mM CaCl.sub.2. Thrombin and GST were removed from TrCP/Skp1 by buffer exchange through a 50 KDa MWCO vivaspin concentrator.
[0655] Protein Immobilisation Procedure for NTA Chip
[0656] Ni+ (500 M NiCl) loaded on to surface at a flow rate of 5 l/min for 60s. EDC/NHS (activates dextran carboxylates) loaded at a flow rate of 5 l/min for 240s. Protein ([protein]=100 nM to 1 M) loaded at a flow rate of 10 l/min for 180s. Strip solution (350 M EDTA/1M NaCl) added at flow rate of 10 l/min for 30s (to chelate excess Ni+ and remove non-covalently bound protein e.g. protein oligomers). Quench solution (ethanolamine) at flow rate of 5 l/min for 240s (to deactivate surface molecules on the chip that have not crosslinked protein). The immobilisation buffer used was 10 mM HEPES pH 7.4, 150 mM NaCl,
[0657] GST Capture Procedure (for Immobilisation Via GSTSkp1)
[0658] EDC:NHS was injected at 5 l/min for 4 min to activate surface for amine coupling (this converts carboxyl groups on the surface of the chip to succinamide esters that react with primary amines)
[0659] Anti-GST (60 g/ml). was loaded at 10 l/min for 4 min resulting in an increase in response units of 6730 (Biacore manual states it should result in 7000)
[0660] Ethanolamine was injected at 5 l/min for 5 min to deactivate remaining unreacted esters at surface (quenching).
[0661] Injection of a low concentration of purified GST (from kit) was injected for 3 mins at 5 l/min before running a regeneration cycle with glycine pH2.0 that disrupts the antibody-GST interaction. This step is recommended in the Biacore manual in order to block a minority of high affinity GST binding sites that may prevent regeneration and therefore reloading of fresh GST-protein of interest.
[0662] GSTSkp1/TrCP (0.16 mg/ml) was then loaded at 10 l/min for 4 min resulting in an increase of 1550 RU (2000 RU is about the maximum to expect according to Biacore manual).
[0663] SPR Assay Conditions
[0664] Small molecule/peptide samples to be assayed for binding to TrCP surfaces, were provided as 10 mM stocks in 100% DMSO. All samples were tested in running buffer composed of 10 mM HEPES pH 7.4, 150 mM NaCl, 50 M EDTA, 0.005% p20, and 1% DMSO. Serial dilutions were made using running buffer. Samples were tested over varying concentrations up to a maximum of 100 M. Two methods of measuring the SPR response were employed: single cycle kinetics which measures the response across different concentrations of sample within a single cycle (no regeneration of surface) and a method that measures the response at a given concentration in each cycle and includes a regeneration wash after each sample injection. The regeneration solution used was the same as running buffer, but included 500 mM NaCl. Data was fitted using Biacore T200 evaluation software. KD values calculated from binding curves from both surfaces (with or without the GST moiety) were averaged to produce apparent KD's for each sample tested.
[0665] Biotin Pull Down Assay
[0666] Assay components
[0667] 150 mM NaCl
[0668] 10.01% NP40
[0669] 1 mM DTT
[0670] 0.3 M TrCP (1 g)
[0671] 0.3 M biotinylated IB peptide (KKERLLDDRHDpSGLDpSMKDEE)
[0672] 100 M compound (mabridge library: 50 M)
[0673] Protocol
[0674] TrCP1 and biotinylated peptide were incubated in a volume of 25 l at a final concentration of DMSO of 1% for 30 minutes to achieve equilibrium. Compounds were then added to a final concentration of 100 M and allowed to incubate for an additional 30 minutes. 7.5 l of streptavidin-agarose beads were then added to the reaction mix and allowed to incubate at room temperature for 30 minutes with gentle rocking. Beads were spun down and washed in buffer 3 times and then loaded onto a 10% SDS PAGE gel and visualized by GelCode blue staining.
[0675] TrCP Ubiquitination Assay and Selectivity Assays
[0676] Assay Components
[0677] 0.2 M E1 (Ube1)
[0678] 2 M E2 (UbcH5C)
[0679] 0.25 M E3 (Cul1/Rbx1)
[0680] 0.25 M E3 (-TrCP1/Skp1)
[0681] 12 M Ubiquitin
[0682] 0.5 M Peptide substrate (biotin)
[0683] 10 mM MgCl.sub.2
[0684] 2 mM ATP
[0685] Protocol
[0686] Master mixes were prepared in a 50 mM Herpes buffer at pH 7.5, in 75 mM NaCl and 1 mM DTT without Mg or ATP and peptides were added to a final concentration of 100 M. Reactions were incubated at room temperature for 30 minutes and then Mg/ATP was added to the mix. Reactions were further incubated for an additional 60 minutes and then stopped by adding SDS gel loading buffer and boiled for 5 minutes. Reactions were run on SDS-PAGE (10%) and transferred to nitrocellulose membranes and probed with HRP-Streptavidin.
[0687] FBW7 selectivity assays were performed in the same manner except using FBW7/Skp1 as E3 component and cyclin E as the substrate. Blots were probed using anti-cyclin E antibody.
EXAMPLES
[0688] The following Examples illustrate the experiments performed by the inventors to arrive at the preset invention. It will be appreciated that modification of detail may be made without departing from the scope of the invention.
Example 1 Construction of Binding Peptides and Analysis Using Fluorescence Polarisation (FP) Assay
[0689] A consensus binding motif is known to be present in IkBa, Vpu and -catenin, all of which bind TrCP (J. Pons et al., Biochemistry, 2008, 47 (1), 14-29). The consensus motif has the sequence DpSGXXpS (SEQ ID NO:69), wherein the two serine residues are phosphorylated.
[0690] A selection of compounds were prepared using the synthesis procedure described above, and shown in
[0691] Binding of the compounds to TrCP was assessed using a fluorescence polarisation (FP) binding assay. The FP assay is an in vitro binding assay using a fluorescein-tagged IkB peptide at 10 nM to mimic substrate binding to TrCP, and was performed as described above.
[0692] Dose response curves for a number of representative peptides are shown in
TABLE-US-00002 TABLE1 DpSGXXpSsequencemodifiedto determinealternatebindingsequences FPAssay Entry Sequence IC.sub.50/M 1 D-pS-GIHS-NH.sub.2 >100 2 GD-pS-GIHS-NH.sub.2 >100 3 AD-pS-GIHS-NH.sub.2 >100 4 VD-pS-GIHS-NH.sub.2 >100 5 DAGIHS-NH.sub.2 >100 6 GDAGIHS-NH.sub.2 >100 7 ADAGIHS-NH.sub.2 >100 8 VDAGIHS-NH.sub.2 >100 9 DDASGIHS-NH.sub.2 >100 10 LDASGIHS-NH.sub.2 >100 11 LD-pS-SGHIS-NH.sub.2 >100 12 DAGIFE-NH.sub.2 >100 13 EAGIFE-NH.sub.2 >100 14 dEGIFE-NH.sub.2 >100 15 dAGIFE-NH.sub.2 >100 16 dAGIFR-NH.sub.2 >100 17 dNGIFR-NH.sub.2 >100 18 E-pS-GIFE-NH.sub.2 34.5 19 D-pS-GIFE-NH.sub.2 1.24 20 DAGNFE-NH.sub.2 >100 21 DEGFFE-NH.sub.2 43.6 22 DAGFFE-NH.sub.2 >100 23 dEGIFD-NH.sub.2 >100 24 dAGIFD-NH.sub.2 >100 25 DAGIFH-NH.sub.2 >100 26 D-pS-GIFH-NH.sub.2 >100 27 D-pS-GNFE-NH.sub.2 >100
[0693] The peptide DEGFFE-NH.sub.2, having an IC.sub.50 of 43.6 M, was selected as a suitable non-phosphorylated candidate for further progression.
[0694] Starting from DEGFFE-NH.sub.2, an array of compounds was prepared, as shown in Table 2, in which the N-terminal amino acid (D) was replaced with various capping groups. All of these compounds have an aminde at the C-terminus, as do those shown in Tables 5 and 6.
TABLE-US-00003 TABLE 2 Optimisation of DEGFFE by replacing N-terminal Asp with capping group (SEQ ID P1 P2 P3 P4 P5 P6 NO:) 1 D E G I F E (70) 2 D E G Y F E 3 D A G Y F E 4 Ac E G I F E (71) 5 MeOCO E G I F E 6 EtOCO E G I F E 7 BnOCO E G I F E 8 4-(MeO)PhOCO E G I F E 9 4-(NO.sub.2)PhOCO E G I F E 10 Piv E G I F E 11 Bz E G I F E 12 4-(MeO)PhCO E G I F E 13 4-(NO.sub.2)PhCO E G I F E 14 Palm E G I F E 15 Stear E G I F E 16 TPCC E G I F E 17 Phth E G I F E 18 Suc E G I F E 19 PhNHCO E G I F E 20 BnNHCO E G I F E 21 4-(NO.sub.2)PhNHCO E G I F E 22 Ms E G I F E 23 PhSO.sub.2 E G I F E 24 Ts E G I F E 25 Ns E G I F E 26 D E G F F E 27 Ac E G F F E (72) 28 MeOCO E G F F E 29 EtOCO E G F F E 30 BnOCO E G F F E 31 4-(MeO)PhOCO E G F F E 32 4-(NO.sub.2)PhOCO E G F F E 33 Piv E G F F E 34 Bz E G F F E 35 4-(MeO)PhCO E G F F E 36 4-(NO.sub.2)PhCO E G F F E 37 Palm E G F F E 38 Stear E G F F E 39 TPCC E G F F E 40 Phth E G F F E 41 Suc E G F F E 42 PhNHCO E G F F E 43 BnNHCO E G F F E 44 4-(NO.sub.2)PhNHCO E G F F E 45 Ms E G F F E 46 PhSO.sub.2 E G F F E 47 Ts E G F F E 48 Ns E G F F E
[0695] Four sequences were selected for re-synthesis and testing in the FP assay, which was performed as described above along with 4 negative controls. The results of the FP assay are shown in Table 3.
TABLE-US-00004 TABLE3 FPassayresultsofselected cappedsequencesandnegativecontrols FPAssay Entry Sequence(SEQIDNO:) IC.sub.50/M 1 DEGIFE-NH.sub.2(13) >100 2 Phth-EGIFE-NH.sub.2(71) >100 3 Suc-AGIFE-NH.sub.2(73) >100 4 Phth-AGIFE-NH.sub.2(73) >100 5 Suc-EGIFE-NH.sub.2(71) >100 6 Phth-EGFFE-NH.sub.2(72) 10.9 7 Suc-EGFFE-NH.sub.2(57) 3.18 8 Phth-AGFFE-NH.sub.2(74) >100 9 Suc-AGFFE-NH.sub.2(74) >100
[0696] The FP assay identified Suc-EGFFE-NH.sub.2 as a low M inhibitor for further optimisation.
[0697] Further peptides were synthesised as described above by replacing residues in the Suc-EGFFE-NH.sub.2 sequence with alternative acidic capping groups and non-natural amino acids. The sequences and abbreviations of the acidic capping groups and non-natural amino acids are shown in
TABLE-US-00005 TABLE 4 Key to capping groups Succinic anhydride Suc PEG 2035 Peg35 PEG2030 Peg30 Trans aconitic acid Taa Cis aconitic acid Caa Fumaric acid Fum Terephthalic acid TA Isophthalic acid Ia 1,4 cyclohexanedicarboxylic acid 1,4-Chda trans-1,2 cyclohexanedicarboxylic acid 1,2-Chda Glutaric anhydride Ga
TABLE-US-00006 TABLE 5 Sequence of -TrCP binding peptides (5-Mers) synthesised P1 P2 P3 P4 P5 P6 (SEQ ID NO:) 1 Peg35 E G F F E (75) 2 Peg30 E G F F E 3 Taa E G F F E 4 Caa E G F F E 5 Fum E G F F E 6 TA E G F F E 7 Ia E G F F E 8 1,4-Chda E G F F E 9 1,2-Chda E G F F E 10 Ga E G F F E 11 Suc E G Y F E 12 Peg35 E G Y F E 13 Peg30 E G Y F E 14 Taa E G Y F E 15 Caa E G Y F E 16 Fum E G Y F E 17 TA E G Y F E 18 Ia E G Y F E 19 1,4-Chda E G Y F E 20 1,2-Chda E G Y F E 21 Ga E G Y F E 22 Suc E A F F E (76) 23 Peg35 E A F F E 24 Peg30 E A F F E 25 Taa E A F F E 26 Caa E A F F E 27 Fum E A F F E 28 TA E A F F E 29 Ia E A F F E 30 1,4-Chda E A F F E 31 1,2-Chda E A F F E 32 Ga E A F F E 33 Suc E A F F E 34 Peg35 E A F F E 35 Peg30 E A F F E 36 Taa E A F F E 37 Caa E A F F E 38 Fum E A F F E 39 TA E A F F E 40 Ia E A F F E 41 1,4-Chda E A F F E 42 1,2-Chda E A F F E 43 Ga E A F F E 44 Suc E A F F E (77) 45 Peg35 E A F F E 46 Peg30 E A F F E 47 Taa E A F F E 48 Caa E A F F E 49 Fum E A F F E 50 TA E A F F E 51 Ia E A F F E 52 1,4-Chda E A F F E 53 1,2-Chda E A F F E 54 Ga E A F F E 55 Suc E P F F E (78) 56 Peg35 E P F F E 57 Peg30 E P F F E 58 Taa E P F F E 59 Caa E P F F E 60 Fum E P F F E 61 TA E P F F E 62 Ia E P F F E 63 1,4-Chda E P F F E 64 1,2-Chda E P F F E 65 Ga E P F F E 66 Suc E p F F E 67 Peg35 E p F F E 68 Peg30 E p F F E 69 Taa E p F F E 70 Caa E p F F E 71 Fum E p F F E 72 TA E p F F E 73 Ia E p F F E 74 1,4-Chda E p F F E 75 1,2-Chda E p F F E 76 Ga E p F F E 77 Suc E Sar F F E (79) 78 Peg35 E Sar F F E 79 Peg30 E Sar F F E 80 Taa E Sar F F E 81 Caa E Sar F F E 82 Fum E Sar F F E 83 TA E Sar F F E 84 Ia E Sar F F E 85 1,4-Chda E Sar F F E 86 1,2-Chda E Sar F F E 87 Ga E Sar F F E 88 Suc E -H-ala F F E 89 Peg35 E -H-ala F F E 90 Peg30 E -H-ala F F E 91 Taa E -H-ala F F E 92 Caa E -H-ala F F E 93 Fum E -H-ala F F E 94 TA E -H-ala F F E 95 Ia E -H-ala F F E 96 1,4-Chda E -H-ala F F E 97 1,2-Chda E -H-ala F F E 98 Ga E -H-ala F F E 99 Suc E G F F E (80) 100 Suc Q G F F E 101 Suc N G F F E 102 Suc (Me)E G F F E 103 Suc E G F F E 104 Suc (Me)D G F F E 105 Suc D G F F E 106 Suc E(Me) G F F E 107 Suc D G F F E 108 Suc Gla G F F E 109 Suc E G Y F E 110 Suc Q G Y F E 111 Suc N G Y F E 112 Suc (Me)E G Y F E 113 Suc E G Y F E 114 Suc (Me)D G Y F E 115 Suc D G Y F E 116 Suc E(Me) G Y F E 117 Suc D G Y F E 118 Suc Gla G Y F E 119 Suc E G W F E 120 Suc Q G W F E 121 Suc N G W F E 122 Suc (Me)E G W F E 123 Suc E G W F E 124 Suc (Me)D G W F E 125 Suc D G W F E 126 Suc E(Me) G W F E 127 Suc D G W F E 128 Suc Gla G W F E 129 Suc E G F(3Br) F E (81) 130 Suc Q G F(3Br) F E 131 Suc N G F(3Br) F E 132 Suc (Me)E G F(3Br) F E 133 Suc E G F(3Br) F E 134 Suc (Me)D G F(3Br) F E 135 Suc D G F(3Br) F E 136 Suc E(Me) G F(3Br) F E 137 Suc D G F(3Br) F E 138 Suc Gla G F(3Br) F E 139 Suc E G F(4NO.sub.2) F E 140 Suc Q G F(4NO.sub.2) F E 141 Suc N G F(4NO.sub.2) F E 142 Suc (Me)E G F(4NO.sub.2) F E 143 Suc E G F(4NO.sub.2) F E 144 Suc (Me)D G F(4NO.sub.2) F E 145 Suc D G F(4NO.sub.2) F E 146 Suc E(Me) G F(4NO.sub.2) F E 147 Suc D G F(4NO.sub.2) F E 148 Suc Gla G F(4NO.sub.2) F E 149 Suc E G 3Pal F E (82) 150 Suc Q G 3Pal F E 151 Suc N G 3Pal F E 152 Suc (Me)E G 3Pal F E 153 Suc E G 3Pal F E 154 Suc (Me)D G 3Pal F E 155 Suc D G 3Pal F E 156 Suc E(Me) G 3Pal F E 157 Suc D G 3Pal F E 158 Suc Gla G 3Pal F E 159 Suc E G Y(Me) F E 160 Suc Q G Y(Me) F E 161 Suc N G Y(Me) F E 162 Suc (Me)E G Y(Me) F E 163 Suc E G Y(Me) F E 164 Suc (Me)D G Y(Me) F E 165 Suc D G Y(Me) F E 166 Suc E(Me) G Y(Me) F E 167 Suc D G Y(Me) F E 168 Suc Gla G Y(Me) F E 169 Suc E G Pip F E 170 Suc Q G Pip F E 171 Suc N G Pip F E 172 Suc (Me)E G Pip F E 173 Suc E G Pip F E 174 Suc (Me)D G Pip F E 175 Suc D G Pip F E 176 Suc E(Me) G Pip F E 177 Suc D G Pip F E 178 Suc Gla G Pip F E 179 Suc E G Tic F E 180 Suc Q G Tic F E 181 Suc N G Tic F E 182 Suc (Me)E G Tic F E 183 Suc E G Tic F E 184 Suc (Me)D G Tic F E 185 Suc D G Tic F E 186 Suc E(Me) G Tic F E 187 Suc D G Tic F E 188 Suc Gla G Tic F E 189 Suc E G 1Nal F E 190 Suc Q G 1Nal F E 191 Suc N G 1Nal F E 192 Suc (Me)E G 1Nal F E 193 Suc E G 1Nal F E 194 Suc (Me)D G 1Nal F E 195 Suc D G 1Nal F E 196 Suc E(Me) G 1Nal F E 197 Suc D G 1Nal F E 198 Suc Gla G 1Nal F E 199 Suc E G F(2F) F E (83) 200 Suc Q G F(2F) F E 201 Suc N G F(2F) F E 202 Suc (Me)E G F(2F) F E 203 Suc E G F(2F) F E 204 Suc (Me)D G F(2F) F E 205 Suc D G F(2F) F E 206 Suc E(Me) G F(2F) F E 207 Suc D G F(2F) F E 208 Suc Gla G F(2F) F E 209 Suc E G F(3F) F E 210 Suc Q G F(3F) F E 211 Suc N G F(3F) F E 212 Suc (Me)E G F(3F) F E 213 Suc E G F(3F) F E 214 Suc (Me)D G F(3F) F E 215 Suc D G F(3F) F E 216 Suc E(Me) G F(3F) F E 217 Suc D G F(3F) F E 218 Suc Gla G F(3F) F E 219 Suc E G F(4F) F E 220 Suc Q G F(4F) F E 221 Suc N G F(4F) F E 222 Suc (Me)E G F(4F) F E 223 Suc E G F(4F) F E 224 Suc (Me)D G F(4F) F E 225 Suc D G F(4F) F E 226 Suc E(Me) G F(4F) F E 227 Suc D G F(4F) F E 228 Suc Gla G F(4F) F E 229 Suc E G F F E (84) 230 Suc E G F F Q 231 Suc E G F F N 232 Suc E G F F (Me)E 233 Suc E G F F E 234 Suc E G F F (Me)D 235 Suc E G F F D 236 Suc E G F F E(Me) 237 Suc E G F F D 238 Suc E G F F Gla 239 Suc E G F Y E 240 Suc E G F Y Q 241 Suc E G F Y N 242 Suc E G F Y (Me)E 243 Suc E G F Y E 244 Suc E G F Y (Me)D 245 Suc E G F Y D 246 Suc E G F Y E(Me) 247 Suc E G F Y D 248 Suc E G F Y Gla 249 Suc E G F W E 250 Suc E G F W Q 251 Suc E G F W N 252 Suc E G F W (Me)E 253 Suc E G F W E 254 Suc E G F W (Me)D 255 Suc E G F W D 256 Suc E G F W E(Me) 257 Suc E G F W D 258 Suc E G F W Gla 259 Suc E G F F(3Br) E (85) 260 Suc E G F F(3Br) Q 261 Suc E G F F(3Br) N 262 Suc E G F F(3Br) (Me)E 263 Suc E G F F(3Br) E 264 Suc E G F F(3Br) (Me)D 265 Suc E G F F(3Br) D 266 Suc E G F F(3Br) E(Me) 267 Suc E G F F(3Br) D 268 Suc E G F F(3Br) Gla 269 Suc E G F F(4NO.sub.2) E 270 Suc E G F F(4NO.sub.2) Q 271 Suc E G F F(4NO.sub.2) N 272 Suc E G F F(4NO.sub.2) (Me)E 273 Suc E G F F(4NO.sub.2) r3E 274 Suc E G F F(4NO.sub.2) (Me)D 275 Suc E G F F(4NO.sub.2) D 276 Suc E G F F(4NO.sub.2) E(Me) 277 Suc E G F F(4NO.sub.2) D 278 Suc E G F F(4NO.sub.2) Gla 279 Suc E G F 3Pal E (86) 280 Suc E G F 3Pal Q 281 Suc E G F 3Pal N 282 Suc E G F 3Pal (Me)E 283 Suc E G F 3Pal E 284 Suc E G F 3Pal (Me)D 285 Suc E G F 3Pal D 286 Suc E G F 3Pal E(Me) 287 Suc E G F 3Pal D 288 Suc E G F 3Pal Gla 289 Suc E G F Y(Me) E 290 Suc E G F Y(Me) Q 291 Suc E G F Y(Me) N 292 Suc E G F Y(Me) (Me)E 293 Suc E G F Y(Me) E 294 Suc E G F Y(Me) (Me)D 295 Suc E G F Y(Me) D 296 Suc E G F Y(Me) E(Me) 297 Suc E G F Y(Me) D 298 Suc E G F Y(Me) Gla 299 Suc E G F Pip E 300 Suc E G F Pip Q 301 Suc E G F Pip N 302 Suc E G F Pip (Me)E 303 Suc E G F Pip E 304 Suc E G F Pip (Me)D 305 Suc E G F Pip D 306 Suc E G F Pip E(Me) 307 Suc E G F Pip D 308 Suc E G F Pip Gla 309 Suc E G F Tic E 310 Suc E G F Tic Q 311 Suc E G F Tic N 312 Suc E G F Tic (Me)E 313 Suc E G F Tic E 314 Suc E G F Tic (Me)D 315 Suc E G F Tic D 316 Suc E G F Tic E(Me) 317 Suc E G F Tic D 318 Suc E G F Tic Gla 319 Suc E G F 1Nal E 320 Suc E G F 1Nal Q 321 Suc E G F 1Nal N 322 Suc E G F 1Nal (Me)E 323 Suc E G F 1Nal E 324 Suc E G F 1Nal (Me)D 325 Suc E G F 1Nal D 326 Suc E G F 1Nal E(Me) 327 Suc E G F 1Nal D 328 Suc E G F 1Nal Gla 329 Suc E G F F(2F) E (87) 330 Suc E G F F(2F) Q 331 Suc E G F F(2F) N 332 Suc E G F F(2F) (Me)E 333 Suc E G F F(2F) E 334 Suc E G F F(2F) (Me)D 335 Suc E G F F(2F) D 336 Suc E G F F(2F) E(Me) 337 Suc E G F F(2F) D 338 Suc E G F F(2F) Gla 339 Suc E G F F(3F) E 340 Suc E G F F(3F) Q 341 Suc E G F F(3F) N 342 Suc E G F F(3F) (Me)E 343 Suc E G F F(3F) E 344 Suc E G F F(3F) (Me)D 345 Suc E G F F(3F) D 346 Suc E G F F(3F) E(Me) 347 Suc E G F F(3F) D 348 Suc E G F F(3F) Gla 349 Suc E G F F(4F) E 350 Suc E G F F(4F) Q 351 Suc E G F F(4F) N 352 Suc E G F F(4F) (Me)E 353 Suc E G F F(4F) E 354 Suc E G F F(4F) (Me)D 355 Suc E G F F(4F) D 356 Suc E G F F(4F) E(Me) 357 Suc E G F F(4F) D 358 Suc E G F F(4F) Gla 359 Suc E G F F E (88) 360 Suc E G Y F E 361 Suc E G W F E 362 Suc E G F(3Br) F E 363 Suc E G F(4NO.sub.2) F E 364 Suc E G 3Pal F E 365 Suc E G Y(Me) F E 366 Suc E G Pip F E 367 Suc E G Tic F E 368 Suc E G 1Nal F E 369 Suc E G F(2F) F E 370 Suc E G F(3F) F E 371 Suc E G F(4F) F E 372 Suc E G F Y E 373 Suc E G Y Y E 374 Suc E G W Y E 375 Suc E G F(3Br) Y E 376 Suc E G F(4NO.sub.2) Y E 377 Suc E G 3Pal Y E 378 Suc E G Y(Me) Y E 379 Suc E G Pip Y E 380 Suc E G Tic Y E 381 Suc E G 1Nal Y E 382 Suc E G F(2F) Y E 383 Suc E G F(3F) Y E 384 Suc E G F(4F) Y E 385 Suc E G F W E 386 Suc E G Y W E 387 Suc E G W W E 388 Suc E G F(3Br) W E 389 Suc E G F(4NO.sub.2) W E 390 Suc E G 3Pal W E 391 Suc E G Y(Me) W E 392 Suc E G Pip W E 393 Suc E G Tic W E 394 Suc E G 1Nal W E 395 Suc E G F(2F) W E 396 Suc E G F(3F) W E 397 Suc E G F(4F) W E 398 Suc E G F F(3Br) E (89) 399 Suc E G Y F(3Br) E 400 Suc E G W F(3Br) E 401 Suc E G F(3Br) F(3Br) E 402 Suc E G F(4NO.sub.2) F(3Br) E 403 Suc E G 3Pal F(3Br) E 404 Suc E G Y(Me) F(3Br) E 405 Suc E G Pip F(3Br) E 406 Suc E G Tic F(3Br) E 407 Suc E G 1Nal F(3Br) E 408 Suc E G F(2F) F(3Br) E 409 Suc E G F(3F) F(3Br) E 410 Suc E G F(4F) F(3Br) E 411 Suc E G F F(4NO.sub.2) E 412 Suc E G Y F(4NO.sub.2) E 413 Suc E G W F(4NO.sub.2) E 414 Suc E G F(3Br) F(4NO.sub.2) E 415 Suc E G F(4NO.sub.2) F(4NO.sub.2) E 416 Suc E G 3Pal F(4NO.sub.2) E 417 Suc E G Y(Me) F(4NO.sub.2) E 418 Suc E G Pip F(4NO.sub.2) E 419 Suc E G Tic F(4NO.sub.2) E 420 Suc E G 1Nal F(4NO.sub.2) E 421 Suc E G F(2F) F(4NO.sub.2) E 422 Suc E G F(3F) F(4NO.sub.2) E 423 Suc E G F(4F) F(4NO.sub.2) E 424 Suc E G F 3Pal E 425 Suc E G Y 3Pal E 426 Suc E G W 3Pal E 427 Suc E G F(3Br) 3Pal E 428 Suc E G F(4NO.sub.2) 3Pal E 429 Suc E G 3Pal 3Pal E 430 Suc E G Y(Me) 3Pal E 431 Suc E G Pip 3Pal E 432 Suc E G Tic 3Pal E 433 Suc E G 1Nal 3Pal E 434 Suc E G F(2F) 3Pal E 435 Suc E G F(3F) 3Pal E 436 Suc E G F(4F) 3Pal E 437 Suc E G F Y(Me) E 438 Suc E G Y Y(Me) E 439 Suc E G W Y(Me) E 440 Suc E G F(3Br) Y(Me) E 441 Suc E G F(4NO.sub.2) Y(Me) E 442 Suc E G 3Pal Y(Me) E 443 Suc E G Y(Me) Y(Me) E 444 Suc E G Pip Y(Me) E 445 Suc E G Tic Y(Me) E 446 Suc E G 1Nal Y(Me) E 447 Suc E G F(2F) Y(Me) E 448 Suc E G F(3F) Y(Me) E 449 Suc E G F(4F) Y(Me) E 450 Suc E G F Pip E 451 Suc E G Y Pip E 452 Suc E G W Pip E 453 Suc E G F(3Br) Pip E 454 Suc E G F(4NO.sub.2) Pip E 455 Suc E G 3Pal Pip E 456 Suc E G Y(Me) Pip E 457 Suc E G Pip Pip E 458 Suc E G Tic Pip E 459 Suc E G 1Nal Pip E 460 Suc E G F(2F) Pip E 461 Suc E G F(3F) Pip E 462 Suc E G F(4F) Pip E 463 Suc E G F Tic E 464 Suc E G Y Tic E 465 Suc E G W Tic E 466 Suc E G F(3Br) Tic E 467 Suc E G F(4NO.sub.2) Tic E 468 Suc E G 3Pal Tic E 469 Suc E G Y(Me) Tic E 470 Suc E G Pip Tic E 471 Suc E G Tic Tic E 472 Suc E G 1Nal Tic E 473 Suc E G F(2F) Tic E 474 Suc E G F(3F) Tic E 475 Suc E G F(4F) Tic E 476 Suc E G F 1Nal E 477 Suc E G Y 1Nal E 478 Suc E G W 1Nal E 479 Suc E G F(3Br) 1Nal E 480 Suc E G F(4NO.sub.2) 1Nal E 481 Suc E G 3Pal 1Nal E 482 Suc E G Y(Me) 1Nal E 483 Suc E G Pip 1Nal E 484 Suc E G Tic 1Nal E 485 Suc E G 1Nal 1Nal E 486 Suc E G F(2F) 1Nal E 487 Suc E G F(3F) 1Nal E 488 Suc E G F(4F) 1Nal E 489 Suc E G F F(2F) E 490 Suc E G Y F(2F) E 491 Suc E G W F(2F) E 492 Suc E G F(3Br) F(2F) E 493 Suc E G F(4NO.sub.2) F(2F) E 494 Suc E G 3Pal F(2F) E 495 Suc E G Y(Me) F(2F) E 496 Suc E G Pip F(2F) E 497 Suc E G Tic F(2F) E 498 Suc E G 1Nal F(2F) E 499 Suc E G F(2F) F(2F) E 500 Suc E G F(3F) F(2F) E 501 Suc E G F(4F) F(2F) E 502 Suc E G F F(3F) E 503 Suc E G Y F(3F) E 504 Suc E G W F(3F) E 505 Suc E G F(3Br) F(3F) E 506 Suc E G F(4NO.sub.2) F(3F) E 507 Suc E G 3Pal F(3F) E 508 Suc E G Y(Me) F(3F) E 509 Suc E G Pip F(3F) E 510 Suc E G Tic F(3F) E 511 Suc E G 1Nal F(3F) E 512 Suc E G F(2F) F(3F) E 513 Suc E G F(3F) F(3F) E 514 Suc E G F(4F) F(3F) E 515 Suc E G F F(4F) E 516 Suc E G Y F(4F) E 517 Suc E G W F(4F) E 518 Suc E G F(3Br) F(4F) E 519 Suc E G F(4NO.sub.2) F(4F) E 520 Suc E G 3Pal F(4F) E 521 Suc E G Y(Me) F(4F) E 522 Suc E G Pip F(4F) E 523 Suc E G Tic F(4F) E 524 Suc E G 1Nal F (4F) E 525 Suc E G F (2F) F (4F) E 526 Suc E G F (3F) F (4F) E 527 Suc E G F (4F) F (4F) E 528 Suc E G F F E (90) 529 Suc Q G F F E 530 Suc N G F F E 531 Suc (Me)E G F F E 532 Suc E G F F E 533 Suc (Me)D G F F E 534 Suc D G F F E 535 Suc E(Me) G F F E 536 Suc D G F F E 537 Suc Gla G F F E 538 Suc Q G F F Q 539 Suc N G F F Q 540 Suc (Me)E G F F Q 541 Suc E G F F Q 542 Suc (Me)D G F F Q 543 Suc D G F F Q 544 Suc E(Me) G F F Q 545 Suc D G F F Q 546 Suc Gla G F F Q 547 Suc E G F F Q 548 Suc Q G F F N 549 Suc N G F F N 550 Suc (Me)E G F F N 551 Suc E G F F N 552 Suc (Me)D G F F N 553 Suc D G F F N 554 Suc E(Me) G F F N 555 Suc D G F F N 556 Suc Gla G F F N 557 Suc E G F F N 558 Suc Q G F F (Me)E 559 Suc N G F F (Me)E 560 Suc (Me)E G F F (Me)E 561 Suc E G F F (Me)E 562 Suc (Me)D G F F (Me)E 563 Suc D G F F (Me)E 564 Suc E(Me) G F F (Me)E 565 Suc D G F F (Me)E 566 Suc Gla G F F (Me)E 567 Suc E G F F (Me)E 568 Suc Q G F F E 569 Suc N G F F E 570 Suc (Me)E G F F E 571 Suc E G F F E 572 Suc (Me)D G F F E 573 Suc D G F F E 574 Suc E(Me) G F F E 575 Suc D G F F E 576 Suc Gla G F F E 577 Suc E G F F E 578 Suc Q G F F (Me)D 579 Suc N G F F (Me)D 580 Suc (Me)E G F F (Me)D 581 Suc E G F F (Me)D 582 Suc (Me)D G F F (Me)D 583 Suc D G F F (Me)D 584 Suc E(Me) G F F (Me)D 585 Suc D G F F (Me)D 586 Suc Gla G F F (Me)D 587 Suc E G F F (Me)D 588 Suc Q G F F D 589 Suc N G F F D 590 Suc (Me)E G F F D 591 Suc E G F F D 592 Suc (Me)D G F F D 593 Suc D G F F D 594 Suc E(Me) G F F D 595 Suc D G F F D 596 Suc Gla G F F D 597 Suc E G F F D 598 Suc Q G F F E(Me) 599 Suc N G F F E(Me) 600 Suc (Me)E G F F E(Me) 601 Suc E G F F E(Me) 602 Suc (Me)D G F F E(Me) 603 Suc D G F F E(Me) 604 Suc E(Me) G F F E(Me) 605 Suc D G F F E(Me) 606 Suc Gla G F F E(Me) 607 Suc E G F F E(Me) 608 Suc Q G F F D 609 Suc N G F F D 610 Suc (Me)E G F F D 611 Suc E G F F D 612 Suc (Me)D G F F D 613 Suc D G F F D 614 Suc E(Me) G F F D 615 Suc D G F F D 616 Suc Gla G F F D 617 Suc E G F F D 618 Suc Q G F F Gla 619 Suc N G F F Gla 620 Suc (Me)E G F F Gla 621 Suc E G F F Gla 622 Suc (Me)D G F F Gla 623 Suc D G F F Gla 624 Suc E(Me) G F F Gla 625 Suc D G F F Gla 626 Suc Gla G F F Gla 627 Suc E G F F Gla (91) 628 Suc E G F F E 629 Suc E G F F F 630 Suc E G F F Y 631 Suc E G F F W 632 Suc E G F F F(3Br) 633 Suc E G F F F(4NO.sub.2) 634 Suc E G F F 3Pal 635 Suc E G F F Y(Me) 636 Suc E G F F Pip 637 Suc E G F F Tic 638 Suc E G F F 1Nal 639 Suc E G F F F(2F) 640 Suc E G F F F(3F) 641 Suc E G F F F(4F) 642 Suc E G Y F E 643 Suc E G Y F F 644 Suc E G Y F Y 645 Suc E G Y F W 646 Suc E G Y F F(3Br) 647 Suc E G Y F F(4NO.sub.2) 648 Suc E G Y F 3Pal 649 Suc E G Y F Y(Me) 650 Suc E G Y F Pip 651 Suc E G Y F Tic 652 Suc E G Y F 1Nal 653 Suc E G Y F F(2F) 654 Suc E G Y F F(3F) 655 Suc E G Y F F(4F) 656 Suc E G F F E (92) 657 Suc E A F F E 658 Suc E a F F E 659 Suc E A F F E 660 Suc E P F F E 661 Suc E p F F E 662 Suc E Sar F F E 663 Suc E -H-ala F F E 664 Suc E G Y F E 665 Suc E A Y F E 666 Suc E a Y F E 667 Suc E A Y F E 668 Suc E P Y F E 669 Suc E p Y F E 670 Suc E Sar Y F E 671 Suc E -H-ala Y F E 672 Suc E G F F E (93) 673 Suc E G F F E(Me) 674 Suc E G Y F E(Me) 675 Suc E G W F E(Me) 676 Suc E G 1Nal F E(Me) 677 Suc E G Tic F E(Me) 678 Suc E G Pip F E(Me) 679 Suc E G F(3Br) F E(Me) 680 Suc E G 3Pal F E(Me) 681 Suc E G F Y E(Me) 682 Suc E G Y Y E(Me) 683 Suc E G W Y E(Me) 684 Suc E G 1Nal Y E(Me) 685 Suc E G Tic Y E(Me) 686 Suc E G Pip Y E(Me) 687 Suc E G F(3Br) Y E(Me) 688 Suc E G 3Pal Y E(Me) 689 Suc E G F W E(Me) 690 Suc E G Y W E(Me) 691 Suc E G W W E(Me) 692 Suc E G 1Nal W E(Me) 693 Suc E G Tic W E(Me) 694 Suc E G Pip W E(Me) 695 Suc E G F(3Br) W E(Me) 696 Suc E G 3Pal W E(Me) 697 Suc E G F 1Nal E(Me) 698 Suc E G Y 1Nal E(Me) 699 Suc E G W 1Nal E(Me) 700 Suc E G 1Nal 1Nal E(Me) 701 Suc E G Tic 1Nal E(Me) 702 Suc E G Pip 1Nal E(Me) 703 Suc E G F(3Br) 1Nal E(Me) 704 Suc E G 3Pal 1Nal E(Me) 705 Suc E G F Tic E(Me) 706 Suc E G Y Tic E(Me) 707 Suc E G W Tic E(Me) 708 Suc E G 1Nal Tic E(Me) 709 Suc E G Tic Tic E(Me) 710 Suc E G Pip Tic E(Me) 711 Suc E G F(3Br) Tic E(Me) 712 Suc E G 3Pal Tic E(Me) 713 Suc E G F Pip E(Me) 714 Suc E G Y Pip E(Me) 715 Suc E G W Pip E(Me) 716 Suc E G 1Nal Pip E(Me) 717 Suc E G Tic Pip E(Me) 718 Suc E G Pip Pip E(Me) 719 Suc E G F(3Br) Pip E(Me) 720 Suc E G 3Pal Pip E(Me) 721 Suc E G F F(3Br) E(Me) 722 Suc E G Y F(3Br) E(Me) 723 Suc E G W F(3Br) E(Me) 724 Suc E G 1Nal F(3Br) E(Me) 725 Suc E G Tic F(3Br) E(Me) 726 Suc E G Pip F(3Br) E(Me) 727 Suc E G F(3Br) F(3Br) E(Me) 728 Suc E G 3Pal F(3Br) E(Me) 729 Suc E G F 3Pal E(Me) 730 Suc E G Y 3Pal E(Me) 731 Suc E G W 3Pal E(Me) 732 Suc E G 1Nal 3Pal E(Me) 733 Suc E G Tic 3Pal E(Me) 734 Suc E G Pip 3Pal E(Me) 735 Suc E G F(3Br) 3Pal E(Me) 736 Suc E G 3Pal 3Pal E(Me) 737 Suc E Ahx F F E (94) 738 Suc Ahx G F F E 739 Suc E Ahx F F E 740 Suc E G Ahx F E 741 Suc E G F Ahx E
TABLE-US-00007 TABLE 6 Sequence of (TrCP binding peptides (4-mers) synthesised (SEQ ID P1 P2 P3 P4 P5 NO:) 1 Suc E G F F (95) 2 Suc E G Y F 3 Suc E G W F 4 Suc E G F(3Br) F 5 Suc E G F(4NO.sub.2) F 6 Suc E G 3Pal F 7 Suc E G Y(Me) F 8 Suc E G Pip F 9 Suc E G Tic F 10 Suc E G 1Nal F 11 Suc E G F(2F) F 12 Suc E G F(3F) F 13 Suc E G F(4F) F 14 Suc E G F Y 15 Suc E G Y Y 16 Suc E G W Y 17 Suc E G F(3Br) Y 18 Suc E G F(4NO.sub.2) Y 19 Suc E G 3Pal Y 20 Suc E G Y(Me) Y 21 Suc E G Pip Y 22 Suc E G Tic Y 23 Suc E G 1Nal Y 24 Suc E G F(2F) Y 25 Suc E G F(3F) Y 26 Suc E G F(4F) Y 27 Suc E G F W 28 Suc E G Y W 29 Suc E G W W 30 Suc E G F(3Br) W 31 Suc E G F(4NO.sub.2) W 32 Suc E G 3Pal W 33 Suc E G Y(Me) W 34 Suc E G Pip W 35 Suc E G Tic W 36 Suc E G 1Nal W 37 Suc E G F(2F) W 38 Suc E G F(3F) W 39 Suc E G F(4F) W 40 Suc E G F F(3Br) 41 Suc E G Y F(3Br) 42 Suc E G W F(3Br) 43 Suc E G F(3Br) F(3Br) 44 Suc E G F(4NO.sub.2) F(3Br) 45 Suc E G 3Pal F(3Br) 46 Suc E G Y(Me) F(3Br) 47 Suc E G Pip F(3Br) 48 Suc E G Tic F(3Br) 49 Suc E G 1Nal F(3Br) 50 Suc E G F(2F) F(3Br) 51 Suc E G F(3F) F(3Br) 52 Suc E G F(4F) F(3Br) 53 Suc E G F F(4NO.sub.2) 54 Suc E G Y F(4NO.sub.2) 55 Suc E G W F(4NO.sub.2) 56 Suc E G F(3Br) F(4NO.sub.2) 57 Suc E G F(4NO.sub.2) F(4NO.sub.2) 58 Suc E G 3Pal F(4NO.sub.2) 59 Suc E G Y(Me) F(4NO.sub.2) 60 Suc E G Pip F(4NO.sub.2) 61 Suc E G Tic F(4NO.sub.2) 62 Suc E G 1Nal F(4NO.sub.2) 63 Suc E G F(2F) F(4NO.sub.2) 64 Suc E G F(3F) F(4NO.sub.2) 65 Suc E G F(4F) F(4NO.sub.2) 66 Suc E G F 3Pal 67 Suc E G Y 3Pal 68 Suc E G W 3Pal 69 Suc E G F(3Br) 3Pal 70 Suc E G F(4NO.sub.2) 3Pal 71 Suc E G 3Pal 3Pal 72 Suc E G Y(Me) 3Pal 73 Suc E G Pip 3Pal 74 Suc E G Tic 3Pal 75 Suc E G 1Nal 3Pal 76 Suc E G F(2F) 3Pal 77 Suc E G F(3F) 3Pal 78 Suc E G F(4F) 3Pal 79 Suc E G F Y(Me) 80 Suc E G Y Y(Me) 81 Suc E G W Y(Me) 82 Suc E G F(3Br) Y(Me) 83 Suc E G F(4NO.sub.2) Y(Me) 84 Suc E G 3Pal Y(Me) 85 Suc E G Y(Me) Y(Me) 86 Suc E G Pip Y(Me) 87 Suc E G Tic Y(Me) 88 Suc E G 1Nal Y(Me) 89 Suc E G F(2F) Y(Me) 90 Suc E G F(3F) Y(Me) 91 Suc E G F(4F) Y(Me) 92 Suc E G F Pip 93 Suc E G Y Pip 94 Suc E G W Pip 95 Suc E G F(3Br) Pip 96 Suc E G F(4NO.sub.2) Pip 97 Suc E G 3Pal Pip 98 Suc E G Y(Me) Pip 99 Suc E G Pip Pip 100 Suc E G Tic Pip 101 Suc E G 1Nal Pip 102 Suc E G F(2F) Pip 103 Suc E G F(3F) Pip 104 Suc E G F(4F) Pip 105 Suc E G F Tic 106 Suc E G Y Tic 107 Suc E G W Tic 108 Suc E G F(3Br) Tic 109 Suc E G F(4NO.sub.2) Tic 110 Suc E G 3Pal Tic 111 Suc E G Y(Me) Tic 112 Suc E G Pip Tic 113 Suc E G Tic Tic 114 Suc E G 1Nal Tic 115 Suc E G F(2F) Tic 116 Suc E G F(3F) Tic 117 Suc E G F(4F) Tic 118 Suc E G F 1Nal 119 Suc E G Y 1Nal 120 Suc E G W 1Nal 121 Suc E G F(3Br) 1Nal 122 Suc E G F(4NO.sub.2) 1Nal 123 Suc E G 3Pal 1Nal 124 Suc E G Y(Me) 1Nal 125 Suc E G Pip 1Nal 126 Suc E G Tic 1Nal 127 Suc E G 1Nal 1Nal 128 Suc E G F(2F) 1Nal 129 Suc E G F(3F) 1Nal 130 Suc E G F(4F) 1Nal 131 Suc E G F F(2F) 132 Suc E G Y F(2F) 133 Suc E G W F(2F) 134 Suc E G F(3Br) F(2F) 135 Suc E G F(4NO.sub.2) F(2F) 136 Suc E G 3Pal F(2F) 137 Suc E G Y(Me) F(2F) 138 Suc E G Pip F(2F) 139 Suc E G Tic F(2F) 140 Suc E G 1Nal F(2F) 141 Suc E G F(2F) F(2F) 142 Suc E G F(3F) F(2F) 143 Suc E G F(4F) F(2F) 144 Suc E G F F(3F) 145 Suc E G Y F(3F) 146 Suc E G W F(3F) 147 Suc E G F(3Br) F(3F) 148 Suc E G F(4NO.sub.2) F(3F) 149 Suc E G 3Pal F(3F) 150 Suc E G Y(Me) F(3F) 151 Suc E G Pip F(3F) 152 Suc E G Tic F(3F) 153 Suc E G 1Nal F(3F) 154 Suc E G F(2F) F(3F) 155 Suc E G F(3F) F(3F) 156 Suc E G F(4F) F(3F) 157 Suc E G F F(4F) 158 Suc E G Y F(4F) 159 Suc E G W F(4F) 160 Suc E G F(3Br) F(4F) 161 Suc E G F(4NO.sub.2) F(4F) 162 Suc E G 3Pal F(4F) 163 Suc E G Y(Me) F(4F) 164 Suc E G Pip F(4F) 165 Suc E G Tic F(4F) 166 Suc E G 1Nal F(4F) 167 Suc E G F(2F) F(4F) 168 Suc E G F(3F) F(4F) 169 Suc E G F(4F) F(4F) 170 Suc E(Me) G F F (96) 171 Suc E(Me) G Y F 172 Suc E(Me) G W F 173 Suc E(Me) G F(3Br) F 174 Suc E(Me) G F(4NO.sub.2) F 175 Suc E(Me) G 3Pal F 176 Suc E(Me) G Y(Me) F 177 Suc E(Me) G Pip F 178 Suc E(Me) G Tic F 179 Suc E(Me) G 1Nal F 180 Suc E(Me) G F(2F) F 181 Suc E(Me) G F(3F) F 182 Suc E(Me) G F(4F) F 183 Suc E(Me) G F Y 184 Suc E(Me) G Y Y 185 Suc E(Me) G W Y 186 Suc E(Me) G F(3Br) Y 187 Suc E(Me) G F(4NO.sub.2) Y 188 Suc E(Me) G 3Pal Y 189 Suc E(Me) G Y(Me) Y 190 Suc E(Me) G Pip Y 191 Suc E(Me) G Tic Y 192 Suc E(Me) G 1Nal Y 193 Suc E(Me) G F(2F) Y 194 Suc E(Me) G F(3F) Y 195 Suc E(Me) G F(4F) Y 196 Suc E(Me) G F W 197 Suc E(Me) G Y W 198 Suc E(Me) G W W 199 Suc E(Me) G F(3Br) W 200 Suc E(Me) G F(4NO.sub.2) W 201 Suc E(Me) G 3Pal W 202 Suc E(Me) G Y(Me) W 203 Suc E(Me) G Pip W 204 Suc E(Me) G Tic W 205 Suc E(Me) G 1Nal W 206 Suc E(Me) G F(2F) W 207 Suc E(Me) G F(3F) W 208 Suc E(Me) G F(4F) W 209 Suc E(Me) G F F(3Br) 210 Suc E(Me) G Y F(3Br) 211 Suc E(Me) G W F(3Br) 212 Suc E(Me) G F(3Br) F(3Br) 213 Suc E(Me) G F(4NO.sub.2) F(3Br) 214 Suc E(Me) G 3Pal F(3Br) 215 Suc E(Me) G Y(Me) F(3Br) 216 Suc E(Me) G Pip F(3Br) 217 Suc E(Me) G Tic F(3Br) 218 Suc E(Me) G 1Nal F(3Br) 219 Suc E(Me) G F(2F) F(3Br) 220 Suc E(Me) G F(3F) F(3Br) 221 Suc E(Me) G F(4F) F(3Br) 222 Suc E(Me) G F F(4NO.sub.2) 223 Suc E(Me) G Y F(4NO.sub.2) 224 Suc E(Me) G W F(4NO.sub.2) 225 Suc E(Me) G F(3Br) F(4NO.sub.2) 226 Suc E(Me) G F(4NO.sub.2) F(4NO.sub.2) 227 Suc E(Me) G 3Pal F(4NO.sub.2) 228 Suc E(Me) G Y(Me) F(4NO.sub.2) 229 Suc E(Me) G Pip F(4NO.sub.2) 230 Suc E(Me) G Tic F(4NO.sub.2) 231 Suc E(Me) G 1Nal F(4NO.sub.2) 232 Suc E(Me) G F(2F) F(4NO.sub.2) 233 Suc E(Me) G F(3F) F(4NO.sub.2) 234 Suc E(Me) G F(4F) F(4NO.sub.2) 235 Suc E(Me) G F 3Pal 236 Suc E(Me) G Y 3Pal 237 Suc E(Me) G W 3Pal 238 Suc E(Me) G F(3Br) 3Pal 239 Suc E(Me) G F(4NO.sub.2) 3Pal 240 Suc E(Me) G 3Pal 3Pal 241 Suc E(Me) G Y(Me) 3Pal 242 Suc E(Me) G Pip 3Pal 243 Suc E(Me) G Tic 3Pal 244 Suc E(Me) G 1Nal 3Pal 245 Suc E(Me) G F(2F) 3Pal 246 Suc E(Me) G F(3F) 3Pal 247 Suc E(Me) G F(4F) 3Pal 248 Suc E(Me) G F Y(Me) 249 Suc E(Me) G Y Y(Me) 250 Suc E(Me) G W Y(Me) 251 Suc E(Me) G F(3Br) Y(Me) 252 Suc E(Me) G F(4NO.sub.2) Y(Me) 253 Suc E(Me) G 3Pal Y(Me) 254 Suc E(Me) G Y(Me) Y(Me) 255 Suc E(Me) G Pip Y(Me) 256 Suc E(Me) G Tic Y(Me) 257 Suc E(Me) G 1Nal Y(Me) 258 Suc E(Me) G F(2F) Y(Me) 259 Suc E(Me) G F(3F) Y(Me) 260 Suc E(Me) G F(4F) Y(Me) 261 Suc E(Me) G F Pip 262 Suc E(Me) G Y Pip 263 Suc E(Me) G W Pip 264 Suc E(Me) G F(3Br) Pip 265 Suc E(Me) G F(4NO.sub.2) Pip 266 Suc E(Me) G 3Pal Pip 267 Suc E(Me) G Y(Me) Pip 268 Suc E(Me) G Pip Pip 269 Suc E(Me) G Tic Pip 270 Suc E(Me) G 1Nal Pip 271 Suc E(Me) G F(2F) Pip 272 Suc E(Me) G F(3F) Pip 273 Suc E(Me) G F(4F) Pip 274 Suc E(Me) G F Tic 275 Suc E(Me) G Y Tic 276 Suc E(Me) G W Tic 277 Suc E(Me) G F(3Br) Tic 278 Suc E(Me) G F(4NO.sub.2) Tic 279 Suc E(Me) G 3Pal Tic 280 Suc E(Me) G Y(Me) Tic 281 Suc E(Me) G Pip Tic 282 Suc E(Me) G Tic Tic 283 Suc E(Me) G 1Nal Tic 284 Suc E(Me) G F(2F) Tic 285 Suc E(Me) G F(3F) Tic 286 Suc E(Me) G F(4F) Tic 287 Suc E(Me) G F 1Nal 288 Suc E(Me) G Y 1Nal 289 Suc E(Me) G W 1Nal 290 Suc E(Me) G F(3Br) 1Nal 291 Suc E(Me) G F(4NO.sub.2) 1Nal 292 Suc E(Me) G 3Pal 1Nal 293 Suc E(Me) G Y(Me) 1Nal 294 Suc E(Me) G Pip 1Nal 295 Suc E(Me) G Tic 1Nal 296 Suc E(Me) G 1Nal 1Nal 297 Suc E(Me) G F(2F) 1Nal 298 Suc E(Me) G F(3F) 1Nal 299 Suc E(Me) G F(4F) 1Nal 300 Suc E(Me) G F F(2F) 301 Suc E(Me) G Y F(2F) 302 Suc E(Me) G W F(2F) 303 Suc E(Me) G F(3Br) F(2F) 304 Suc E(Me) G F(4NO.sub.2) F(2F) 305 Suc E(Me) G 3Pal F(2F) 306 Suc E(Me) G Y(Me) F(2F) 307 Suc E(Me) G Pip F(2F) 308 Suc E(Me) G Tic F(2F) 309 Suc E(Me) G 1Nal F(2F) 310 Suc E(Me) G F(2F) F(2F) 311 Suc E(Me) G F(3F) F(2F) 312 Suc E(Me) G F(4F) F(2F) 313 Suc E(Me) G F F(3F) 314 Suc E(Me) G Y F(3F) 315 Suc E(Me) G W F(3F) 316 Suc E(Me) G F(3Br) F(3F) 317 Suc E(Me) G F(4NO.sub.2) F(3F) 318 Suc E(Me) G 3Pal F(3F) 319 Suc E(Me) G Y(Me) F(3F) 320 Suc E(Me) G Pip F(3F) 321 Suc E(Me) G Tic F(3F) 322 Suc E(Me) G 1Nal F(3F) 323 Suc E(Me) G F(2F) F(3F) 324 Suc E(Me) G F(3F) F(3F) 325 Suc E(Me) G F(4F) F(3F) 326 Suc E(Me) G F F(4F) 327 Suc E(Me) G Y F(4F) 328 Suc E(Me) G W F(4F) 329 Suc E(Me) G F(3Br) F(4F) 330 Suc E(Me) G F(4NO.sub.2) F(4F) 331 Suc E(Me) G 3Pal F(4F) 332 Suc E(Me) G Y(Me) F(4F) 333 Suc E(Me) G Pip F(4F) 334 Suc E(Me) G Tic F(4F) 335 Suc E(Me) G 1Nal F(4F) 336 Suc E(Me) G F(2F) F(4F) 337 Suc E(Me) G F(3F) F(4F) 338 Suc E(Me) G F(4F) F(4F) 339 Suc E G F F (97) 340 Suc Q G F F 341 Suc N G F F 342 Suc (MeE G F F 343 Suc E G F F 344 Suc (MeD G F F 345 Suc D G F F 346 Suc E(Me G F F 347 Suc D G F F 348 Suc Gla G F F 349 Suc E G Y F 350 Suc Q G Y F 351 Suc N G Y F 352 Suc (Me)E G Y F 353 Suc E G Y F 354 Suc (Me)D G Y F 355 Suc D G Y F 356 Suc E(Me) G Y F 357 Suc D G Y F 358 Suc Gla G Y F 359 Suc E G W F 360 Suc Q G W F 361 Suc N G W F 362 Suc (Me)E G W F 363 Suc E G W F 364 Suc (Me)D G W F 365 Suc D G W F 366 Suc E(Me) G W F 367 Suc D G W F 368 Suc Gla G W F 369 Suc E G F(3Br) F 370 Suc Q G F(3Br) F 371 Suc N G F(3Br) F 372 Suc (Me)E G F(3Br) F 373 Suc E G F(3Br) F 374 Suc (Me)D G F(3Br) F 375 Suc D G F(3Br) F 376 Suc E(Me) G F(3Br) F 377 Suc D G F(3Br) F 378 Suc Gla G F(3Br) F 379 Suc E G F(4NO.sub.2) F 380 Suc Q G F(4NO.sub.2) F 381 Suc N G F(4NO.sub.2) F 382 Suc (Me)E G F(4NO.sub.2) F 383 Suc E G F(4NO.sub.2) F 384 Suc (Me)D G F(4NO.sub.2) F 385 Suc D G F(4NO.sub.2) F 386 Suc E(Me) G F(4NO.sub.2) F 387 Suc D G F(4NO.sub.2) F 388 Suc Gla G F(4NO.sub.2) F 389 Suc E G 3Pal F 390 Suc Q G 3Pal F 391 Suc N G 3Pal F 392 Suc (Me)E G 3Pal F 393 Suc E G 3Pal F 394 Suc (Me)D G 3Pal F 395 Suc D G 3Pal F 396 Suc E(Me) G 3Pal F 397 Suc D G 3Pal F 398 Suc Gla G 3Pal F 399 Suc E G Y(Me) F 400 Suc Q G Y(Me) F 401 Suc N G Y(Me) F 402 Suc (Me)E G Y(Me) F 403 Suc E G Y(Me) F 404 Suc (Me)D G Y(Me) F 405 Suc D G Y(Me) F 406 Suc E(Me) G Y(Me) F 407 Suc D G Y(Me) F 408 Suc Gla G Y(Me) F 409 Suc E G Pip F 410 Suc Q G Pip F 411 Suc N G Pip F 412 Suc (Me)E G Pip F 413 Suc E G Pip F 414 Suc (Me)D G Pip F 415 Suc D G Pip F 416 Suc E(Me) G Pip F 417 Suc D G Pip F 418 Suc Gla G Pip F 419 Suc E G Tic F 420 Suc Q G Tic F 421 Suc N G Tic F 422 Suc (Me)E G Tic F 423 Suc E G Tic F 424 Suc (Me)D G Tic F 425 Suc D G Tic F 426 Suc E(Me) G Tic F 427 Suc D G Tic F 428 Suc Gla G Tic F 429 Suc E G 1Nal F 430 Suc Q G 1Nal F 431 Suc N G 1Nal F 432 Suc (Me)E G 1Nal F 433 Suc E G 1Nal F 434 Suc (Me)D G 1Nal F 435 Suc D G 1Nal F 436 Suc E(Me) G 1Nal F 437 Suc D G 1Nal F 438 Suc Gla G 1Nal F 439 Suc E G F(2F) F 440 Suc Q G F(2F) F 441 Suc N G F(2F) F 442 Suc (Me)E G F(2F) F 443 Suc E G F(2F) F 444 Suc (Me)D G F(2F) F 445 Suc D G F(2F) F 446 Suc E(Me) G F(2F) F 447 Suc D G F(2F) F 448 Suc Gla G F(2F) F 449 Suc E G F(3F) F 450 Suc Q G F(3F) F 451 Suc N G F(3F) F 452 Suc (Me)E G F(3F) F 453 Suc E G F(3F) F 454 Suc (Me)D G F(3F) F 455 Suc D G F(3F) F 456 Suc E(Me) G F(3F) F 457 Suc D G F(3F) F 458 Suc Gla G F(3F) F 459 Suc E G F(4F) F 460 Suc Q G F(4F) F 461 Suc N G F(4F) F 462 Suc (Me)E G F(4F) F 463 Suc E G F(4F) F 464 Suc (Me)D G F(4F) F 465 Suc D G F(4F) F 466 Suc E(Me) G F(4F) F 467 Suc D G F(4F) F 468 Suc Gla G F(4F) F 469 Suc E G F F (98) 470 Suc Q G F F 471 Suc N G F F 472 Suc (Me)E G F F 473 Suc E G F F 474 Suc (Me)D G F F 475 Suc D G F F 476 Suc E(Me) G F F 477 Suc D G F F 478 Suc Gla G F F 479 Suc E G F Y 480 Suc Q G F Y 481 Suc N G F Y 482 Suc (Me)E G F Y 483 Suc E G F Y 484 Suc (Me)D G F Y 485 Suc D G F Y 486 Suc E(Me) G F Y 487 Suc D G F Y 488 Suc Gla G F Y 489 Suc E G F W 490 Suc Q G F W 491 Suc N G F W 492 Suc (Me)E G F W 493 Suc E G F W 494 Suc (Me)D G F W 495 Suc D G F W 496 Suc E(Me) G F W 497 Suc D G F W 498 Suc Gla G F W 499 Suc E G F F(3Br) 500 Suc Q G F F(3Br) 501 Suc N G F F(3Br) 502 Suc (Me)E G F F(3Br) 503 Suc E G F F(3Br) 504 Suc (Me)D G F F(3Br) 505 Suc D G F F(3Br) 506 Suc E(Me) G F F(3Br) 507 Suc D G F F(3Br) 508 Suc Gla G F F(3Br) 509 Suc E G F F(4NO.sub.2) 510 Suc Q G F F(4NO.sub.2) 511 Suc N G F F(4NO.sub.2) 512 Suc (Me)E G F F(4NO.sub.2) 513 Suc E G F F(4NO.sub.2) 514 Suc (Me)D G F F(4NO.sub.2) 515 Suc D G F F(4NO.sub.2) 516 Suc E(Me) G F F(4NO.sub.2) 517 Suc D G F F(4NO.sub.2) 518 Suc Gla G F F(4NO.sub.2) 519 Suc E G F 3Pal 520 Suc Q G F 3Pal 521 Suc N G F 3Pal 522 Suc (Me)E G F 3Pal 523 Suc E G F 3Pal 524 Suc (Me)D G F 3Pal 525 Suc D G F 3Pal 526 Suc E(Me) G F 3Pal 527 Suc D G F 3Pal 528 Suc Gla G F 3Pal 529 Suc E G F Y(Me) 530 Suc Q G F Y(Me) 531 Suc N G F Y(Me) 532 Suc (Me)E G F Y(Me) 533 Suc E G F Y(Me) 534 Suc (Me)D G F Y(Me) 535 Suc D G F Y(Me) 536 Suc E(Me) G F Y(Me) 537 Suc D G F Y(Me) 538 Suc Gla G F Y(Me) 539 Suc E G F Pip 540 Suc Q G F Pip 541 Suc N G F Pip 542 Suc (Me)E G F Pip 543 Suc E G F Pip 544 Suc (Me)D G F Pip 545 Suc D G F Pip 546 Suc E(Me) G F Pip 547 Suc D G F Pip 548 Suc Gla G F Pip 549 Suc E G F Tic 550 Suc Q G F Tic 551 Suc N G F Tic 552 Suc (Me)E G F Tic 553 Suc E G F Tic 554 Suc (Me)D G F Tic 555 Suc D G F Tic 556 Suc E(Me) G F Tic 557 Suc D G F Tic 558 Suc Gla G F Tic 559 Suc E G F 1Nal 560 Suc Q G F 1Nal 561 Suc N G F 1Nal 562 Suc (Me)E G F 1Nal 563 Suc E G F 1Nal 564 Suc (Me)D G F 1Nal 565 Suc D G F 1Nal 566 Suc E(Me) G F 1Nal 567 Suc D G F 1Nal 568 Suc Gla G F 1Nal 569 Suc E G F F(2F) 570 Suc Q G F F(2F) 571 Suc N G F F(2F) 572 Suc (Me)E G F F(2F) 573 Suc E G F F(2F) 574 Suc (Me)D G F F(2F) 575 Suc D G F F(2F) 576 Suc E(Me) G F F(2F) 577 Suc D G F F(2F) 578 Suc Gla G F F(2F) 579 Suc E G F F(3F) 580 Suc Q G F F(3F) 581 Suc N G F F(3F) 582 Suc (Me)E G F F(3F) 583 Suc E G F F(3F) 584 Suc (Me)D G F F(3F) 585 Suc D G F F(3F) 586 Suc E(Me) G F F(3F) 587 Suc D G F F(3F) 588 Suc Gla G F F(3F) 589 Suc E G F F(4F) 590 Suc Q G F F(4F) 591 Suc N G F F(4F) 592 Suc (Me)E G F F(4F) 593 Suc E G F F(4F) 594 Suc (Me)D G F F(4F) 595 Suc D G F F(4F) 596 Suc E(Me) G F F(4F) 597 Suc D G F F(4F) 598 Suc Gla G F F(4F) 599 Suc E G F F (99) 600 Peg35 E G F F 601 Peg30 E G F F 602 Taa E G F F 603 Caa E G F F 604 Fum E G F F 605 TA E G F F 606 Ia E G F F 607 1,4-Chda E G F F 608 1,2-Chda E G F F 609 Ga E G F F 610 Suc E G Y F 611 Peg35 E G Y F 612 Peg30 E G Y F 613 Taa E G Y F 614 Caa E G Y F 615 Fum E G Y F 616 TA E G Y F 617 Ia E G Y F 618 1,4-Chda E G Y F 619 1,2-Chda E G Y F 620 Ga E G Y F 621 Suc E A F F (100) 622 Peg35 E A F F 623 Peg30 E A F F 624 Taa E A F F 625 Caa E A F F 626 Fum E A F F 627 TA E A F F 628 Ia E A F F 629 1,4-Chda E A F F 630 1,2-Chda E A F F 631 Ga E A F F 632 Suc E a F F 633 Peg35 E a F F 634 Peg30 E a F F 635 Taa E a F F 636 Caa E a F F 637 Fum E a F F 638 TA E a F F 639 Ia E a F F 640 1,4-Chda E a F F 641 1,2-Chda E a F F 642 Ga E a F F 643 Suc E r3A F F 644 Peg35 E r3A F F 645 Peg30 E r3A F F 646 Taa E r3A F F 647 Caa E r3A F F 648 Fum E r3A F F 649 TA E r3A F F 650 Ia E r3A F F 651 1,4-Chda E r3A F F 652 1,2-Chda E A F F 653 Ga E A F F 654 Suc E P F F 655 Peg35 E P F F 656 Peg30 E P F F 657 Taa E P F F 658 Caa E P F F 659 Fum E P F F 660 TA E P F F 661 Ia E P F F 662 1,4-Chda E P F F 663 1,2-Chda E P F F 664 Ga E P F F 665 Suc E P F F 666 Peg35 E p F F 667 Peg30 E p F F 668 Taa E p F F 669 Caa E p F F 670 Fum E p F F 671 TA E p F F 672 Ia E p F F 673 1,4-Chda E p F F 674 1,2-Chda E p F F 675 Ga E p F F 676 Suc E Sar F F 677 Peg35 E Sar F F 678 Peg30 E Sar F F 679 Taa E Sar F F 680 Caa E Sar F F 681 Fum E Sar F F 682 TA E Sar F F 683 Ia E Sar F F 684 1,4-Chda E Sar F F 685 1,2-Chda E Sar F F 686 Ga E Sar F F 687 Suc E -H-ala F F 688 Peg35 E -H-ala F F 689 Peg30 E -H-ala F F 690 Taa E -H-ala F F 691 Caa E -H-ala F F 692 Fum E -H-ala F F 693 TA E -H-ala F F 694 Ia E -H-ala F F 695 1,4-Chda E -H-ala F F 696 1,2-Chda E -H-ala F F 697 Ga E -H-ala F F 698 Suc E Ahx F F 699 Peg35 E Ahx F F 700 Peg30 E Ahx F F 701 Taa E Ahx F F 702 Caa E Ahx F F 703 Fum E Ahx F F 704 TA E Ahx F F 705 Ia E Ahx F F 706 1,4-Chda E Ahx F F 707 1,2-Chda E Ahx F F 708 Ga E Ahx F F 709 Suc E F F 710 Peg35 E F F 711 Peg30 E F F 712 Taa E F F 713 Caa E F F 714 Fum E F F 715 TA E F F 716 Ia E F F 717 1,4-Chda E F F 718 1,2-Chda E F F 719 Ga E F F 720 Suc E F F E 721 Suc E Ahx E 722 Suc E Ahx F E (101) 723 Suc E Ahx F Y 724 Suc E Ahx F W 725 Suc E Ahx F F(3Br) 726 Suc E Ahx F F(4NO.sub.2) 727 Suc E Ahx F 3Pal 728 Suc E Ahx F Y(Me) 729 Suc E Ahx F Pip 730 Suc E Ahx F Tic 731 Suc E Ahx F 1Nal 732 Suc E Ahx F F(2F) 733 Suc E Ahx F F(3F) 734 Suc E Ahx F F(4F) 735 Suc E Ahx F F (102) 736 Suc Q Ahx F F 737 Suc N Ahx F F 738 Suc (Me)E Ahx F F 739 Suc E Ahx F F 740 Suc (Me)D Ahx F F 741 Suc D Ahx F F 742 Suc E(Me) Ahx F F 743 Suc D Ahx F F 744 Suc Gla Ahx F F 745 Ga E G F Y(Me) (103) 746 Suc E Ahx Y F (104) 747 Suc E Ahx W F 748 Suc E Ahx F(3Br) F 749 Suc E Ahx F(4NO.sub.2) F 750 Suc E Ahx 3Pal F 751 Suc E Ahx Y(Me) F 752 Suc E Ahx Pip F 753 Suc E Ahx Tic F 754 Suc E Ahx 1Nal F 755 Suc E Ahx F(2F) F 756 Suc E Ahx F(3F) F 757 Suc E Ahx F(4F) F 758 Suc E Ahx F F 759 Suc Ahx G F F (105) 760 Suc E Ahx F F (106) 761 Suc E G Ahx F (107) 762 Suc E G F Ahx (108) 763 Suc E F F E (109) 764 Peg35 E F F E 765 Peg30 E F F E 766 Taa E F F E 767 Caa E F F E 768 Fum E F F E 769 TA E F F E 770 Ia E F F E 771 1,4-Chda E F F E 772 1,2-Chda E F F E 773 Ga E F F E 774 Suc E Y F E 775 Peg35 E Y F E 776 Peg30 E Y F E 777 Taa E Y F E 778 Caa E Y F E 779 Fum E Y F E 780 TA E Y F E 781 Ia E Y F E 782 1,4-Chda E Y F E 783 1,2-Chda E Y F E 784 Ga E Y F E 785 Peg35 E F Y(Me) E 786 Peg30 E F Y(Me) E 787 Taa E F Y(Me) E 788 Caa E F Y(Me) E 789 Fum E F Y(Me) E 790 TA E F Y(Me) E 791 Ia E F Y(Me) E 792 1,4-Chda E F Y(Me) E 793 1,2-Chda E F Y(Me) E 794 Ga E F Y(Me) E Ahx = aminohexanoic acid
[0698] Selected peptides from the arrays shown in Tables 5 and 6 were re-synthesised and analysed using the FP assay described above. The results of this assay are shown in Table 7.
TABLE-US-00008 TABLE7 FPassayresultsofselectedpeptides FPAssay Entry Sequence IC.sub.50/M 1 1,4-Chda-EGFFE-NH.sub.2 >100 2 EAFFE-NH.sub.2 28 3 Ga-EGFFE-NH.sub.2 65 4 Suc-EG-1Nal-F(4NO.sub.2)-E-NH.sub.2 68 5 Suc-EG-1Nal-Y(Me)-E-NH.sub.2 5.4 6 Suc-EG-F(2F)-F(3F)-NH.sub.2 >100 7 Suc-EG-F(2F)-F(4NO.sub.2)-E-NH.sub.2 4.9 8 Suc-EG-F(2F)-Y(4Me)-E-NH.sub.2 3.2 9 Suc-EG-F(3F)-3Pal-NH.sub.2 70 10 Suc-EG-F(3F)-F(3F)-NH.sub.2 44 11 Suc-EG-F(3F)-F(4NO2)-E-NH.sub.2 0.52 12 Suc-EG-F(3F)-F(4NO2)-NH.sub.2 >100 13 Suc-EG-F(4Br)-F(3F)-NH.sub.2 81 14 Suc-EG-F(4Br)-F-NH.sub.2 38 15 Suc-EG-F(4Br)-F-NH.sub.2 39 16 Suc-EG-F(4F)-F(4NO.sub.2)-E-NH.sub.2 1.2 17 Suc-EG-F(4NO.sub.2)-1Nal-NH.sub.2 14 18 Suc-EG-F(4NO.sub.2)-F(4NO2)-NH.sub.2 78 19 Suc-EGF-F(3F)-NH.sub.2 >100 20 Suc-EGF-F(4F)-NH.sub.2 >100 21 Suc-EGF-F(4NO.sub.2)-NH.sub.2 >100 22 Suc-EGFF-NH.sub.2 46 23 Suc-EGF-Y(Me)-E(OMe)-NH.sub.2 71 24 Suc-EGY-F(3F)-NH.sub.2 91 25 Suc-EGY-F(4F)-NH.sub.2 71 26 Suc-EGY-F(4NO.sub.2)-NH.sub.2 91 27 Suc-EGYFE-NH.sub.2 1 28 Suc-EGYF-NH.sub.2 31 29 Suc-QGYF-NH.sub.2 49 30 Suc-E-GYFE-NH.sub.2 30
[0699] X-EGXXE-NH.sub.2 was identified as a useful consensus binding motif and further peptides were designed and synthesised to increase potency. These peptides were tested in the FP assay described above and the results are shown in Table 8.
TABLE-US-00009 TABLE8 Furthermodificationof peptidesequenceX-EGXXE-NH.sub.2 FPAssay Entry Sequence(SEQIDNO:) IC.sub.50/M 1 (MeO)Suc-EG-F(3F)-1Nal-E-NH.sub.2(110) 1.6 2 2-NaphthylSO.sub.2-dEG-F(3F)-WE-NH.sub.2 0.044 3 3,4-(MeO)2PhSO.sub.2-dEG-F(3F)-WE-NH.sub.2 0.042 4 4-(BuO)PhSO.sub.2-dEG-F(3F)-WE-NH.sub.2 0.056 5 4-(MeO)PhSO.sub.2-dEG-F(3F)-WE-NH.sub.2 0.017 6 4-(MeO)PhSO.sub.2-DEG-F(3F)-WE-NH.sub.2(111) 0.027 7 4-(PhO)PhSO.sub.2-dEG-F(3F)-WE-NH.sub.2 0.063 8 Ac-dEG-F(3F)-1Nal-E-NH.sub.2 0.112 9 Ac-dEG-F(3F)-WE-NH.sub.2 0.102 10 AG-F(3F)-F(4NO.sub.2)-E-NH.sub.2(112) >100 11 AGYFE-NH.sub.2(112) >100 12 Bz-dEG-F(3F)-WE-NH.sub.2 0.173 13 EA-F(3F)-F(4NO.sub.2)-E-NH.sub.2(113) >100 14 EAFFE-NH.sub.2(113) >100 15 EAYFE-NH.sub.2(113) >100 16 EG-F(3F)-1Nal-E-NH.sub.2(113) >100 17 EG-F(4NO.sub.2)-1Nal-E-NH.sub.2(113) >100 18 EGY-1Nal-E-NH.sub.2(113) >100 19 EGY-F(4NO.sub.2)-E-NH.sub.2(113) >100 20 EtCO-dEG-F(3F)-WE-NH.sub.2 0.145 21 EtOCO-dEG-F(3F)-WE-NH.sub.2 0.045 22 Fum-EG-F(3F)-F(4NO.sub.2)-E-NH.sub.2(114) 60.4 23 Mal-EG-F(3F)-F(4NO.sub.2)-E-NH.sub.2(114) 2.17 24 MeoCO-dEG-F(3F)-WE-NH.sub.2 0.076 25 QG-F(3F)-F(4NO.sub.2)-E-NH.sub.2(115) >100 26 QGFFE-NH.sub.2(115) >100 27 QGYFE-NH.sub.2(115) >100 28 Suc-AG-F(3F)-F(4NO2)-E-NH.sub.2(116) 8.01 29 Suc-AGYFE-NH.sub.2(116) 22.2 30 Suc-EA-F(3F)-F(4NO.sub.2)-E-NH.sub.2(117) 27.5 31 Suc-EAFFE-NH.sub.2(117) 59.8 32 Suc-EAYFE-NH.sub.2(117) 82.1 33 Suc-EG-3Pal-1Nal-E-NH.sub.2(117) 3.63 34 Suc-EG-F(3F)-1Nal-E-NH.sub.2(117) 0.157 35 Suc-EG-F(3F)-1Nal-Q-NH.sub.2(117) 7.22 36 Suc-EG-F(3F)-HE-NH.sub.2(117) 0.521 37 Suc-EG-F(3F)-WE-NH.sub.2(117) 0.095 38 Suc-EG-F(3F)-Y(Me)-E-NH.sub.2(117) 1.4 39 Suc-EG-F(4NO.sub.2)-1Nal-E-NH.sub.2(117) 1.31 40 Suc-EGI-1Nal-E-NH.sub.2(117) 15.09 41 Suc-EGY-1Na1-E-NH.sub.2(117) 1.15 42 Suc-EGY-F(4NO.sub.2)-E-NH.sub.2(117) 2.07 43 Suc-QG-F(3F)-F(4NO.sub.2)-E-NH.sub.2(115) 2.56 44 Suc-QGFFE-NH.sub.2(115) 10 45 Suc-QGYFE-NH.sub.2(115) 10.7 46 Ts-dDG-F(3F)-WD-NH.sub.2 0.081 47 Ts-dDG-F(3F)-WE-NH.sub.2 0.025 48 Ts-dEGF(3Cl)WE(Me)-NH.sub.2 0.017 49 Ts-dEGF(3Cl)WE-NH.sub.2 0.01 50 Ts-dEG-F(3F)-WD-NH.sub.2 0.034 51 Ts-dEGF(3F)W-E(Me)-NH.sub.2 0.022 52 Ts-dEG-F(3F)-WE-NH.sub.2 0.029 53 Ts-DEG-F(3F)-WE-NH.sub.2 0.018
Example 2in Silico Biotin Capture Assay of Lead Test Compounds
[0700] A non-fluorescent based assay was used to validate potential binding peptides. The phosphopeptide substrate KKERLLDDRHDpSGLDpSMKDEE was biotinylated by coupling a biotinamido-hexanoic acid succinimide ester to a lysine in the peptide. 0.5 g of each protein was mixed with 50 picomoles of biotinylated peptide in a total volume of 50 l. The buffer conditions were 50 mM Hepes 7.5 and 100 mM NaCl. Compounds were then added to a final concentration of 60 M. The reaction was allowed to incubate for 1 hour at room temperature and then 5 l of streptavidin-agarose beads was added and incubated for 1 hour. Beads were washed twice with buffer and run on an SDS-PAGE gel. Proteins were visualized by anti-His antibody. The results of this assay are shown in
Example 3Compound Hits from In-Vitro Assays Tested with SPR
[0701] Binding of SucEGF(4NO.sub.2)1NalENH.sub.2 to TrCP1 was tested using the Surface Plasmon Resonance (SPR) method described above.
Example 4Measurement of Inhibition of the E3 Ligase Cascades TrCP(IB) and TrCP(-Catenin)
[0702] Candidate compounds were tested in duplicate at both 10 and 100 M against the E3 ligase cascades TrCP(IB) and TrCP(-catenin). The E3 assays were carried out according to the component concentrations detailed in Table 9. In each case the E2 was HA,6His-UbcH3-(hu,FL), the E1 was 6His-UBE1-(hu,FL) and the ubiquitin (Sigma U6253) was biotinylated at a 5:1 ratio. The E3 tetramer constructs and substrate pairings are shown in Table 10. Substrate phosphorylation was performed in the absence of compound; consequently any observed signal modulation should not reflect inhibition of the up-stream kinase reaction. All other steps (E1, E2 and substrate ubiquitination) were carried out in the presence of compound. The stopped reaction mix (10 l) was added to an ECL plate loaded with anti c-Myc ( 1/500 Dilution, Millipore 05-724) and blocked with 5% BSA. Binding was allowed to proceed for 1 hour at RT before a wash step (3 40 l PBST washes). Detection was achieved by binding SA-Ru TAG at 1 g/ml (1 hour @ RT, 3 40 l PBST washes) before reading on an MSD Sector Imager 6000.
TABLE-US-00010 TABLE 9 Assay concentrations TrCP(IB) TrCP(-catenin) Ub 2 M 2 M ATP 10 M 10 M E2 250 nM 250 nM E1 2.5 nM 2.5 nM E3 Tetramer 0.3 g/well 0.3 g/well Sub 200 nM 200 nM
TABLE-US-00011 TABLE 10 E3 Ligase and substrate pairings E3 Tetramer Substrate GST-TrCP1-(iso1,hu,53-end,E353D)/GST-Skp1- cMyc, 6His-IB- (hu,fl)/6His-Cul1-(hu,fl)/UT-Rbx1-(hu,fl) (hu,FL) GST-TrCP1-(iso1,hu,53-end,E353D)/GST-Skp1- cMyc,6His-- (hu,fl)/6His-Cul1-(hu,fl)/UT-Rbx1-(hu,fl) catenin-(hu,FL)
[0703] Assay results are shown in
Example 5Selectivity Analysis of Top CompoundsFP Against Fbw 7
[0704] Candidate compounds were tested for inhibition of Fbw7 and Skp2 as described above. As shown in
Example 6Collation of Data Collected on Candidate Compounds
[0705] Table 11 (below) shows the collation of all data points collected for the most promising compounds.
TABLE-US-00012 TABLE11 Summaryofpeptidedata Biotin Pulldown %Inhibition Assay UbAssay Skp2@ Fbw7@ FP SPR 10M/ 10M/ 10M 10M Entry Sequence IC.sub.50/M Kd/M 100M 100M (100M) (100M) 1 DpSGIFE-NH.sub.2 1.24 0.145 1.477 0.286 reduces/ reduces/ 8(13) 0(0) inhibits inhibits 2 Suc-EGFFE-NH.sub.2 3.18 0.357 29.456 4.613 noeffect/ reduces/ 14(0) 0(0) reduces inhibits 3 Suc-EGF(2F)F(4NO.sub.2)E-NH.sub.2 4.91 0.702 8.000 0.260 noeffect/ noeffect/ 8(11) 0(0) reduces inhibits 4 Suc-EGF(3F)F(4NO.sub.2)E-NH.sub.2 0.522 0.049 4.530 0.681 reduces/ inhibits/ 0(3) 0(2) inhibits inhibits 5 Suc-EGF(4F)F(4NO2)E-NH.sub.2 1.21 0.093 0.507 0.267 noeffect/ reduces/ 0(0) 0(0) inhibits inhibits 6 Suc-EGF(2F)Y(Me)E-NH.sub.2 3.28 0.500 4.400 0.820 noeffect/ noeffect/ 0(4) 0(4) reduces inhibits 7 Suc-EGYFE-NH.sub.2 1.04 0.101 0.537 0.117 noeffect/ reduces/ 11(28) 0(0) inhibits inhibits 8 Mal-EGF(3F)F(4NO.sub.2)E-NH.sub.2 2.17 0.425 1.463 0.035 noeffect/ noeffect/ 0(14) 0(18) inhibits inhibits 9 Suc-EGY1NalE-NH.sub.2 1.15 0.222 1.490 0.625 noeffect/ reduces/ 0(6) 1(5) inhibits inhibits 10 SucEGF(3F)1NalE-NH.sub.2 0.157 0.037 0.320 0.240 inhibits/ inhibits/ 0(12) 3(0) inhibits inhibits 11 Suc-EGF(4NO2)1NalE-NH.sub.2 1.31 0.198 1.965 0.635 reduces/ reduces/ 12(6) 14(28) inhibits inhibits 12 Suc-QGF(3F)F(4NO.sub.2)E-NH.sub.2 2.56 0.374 5.491 2.271 noeffect/ noeffect/ 0(0) 0(0) reduces inhibits 13 Suc-EGYF(4NO.sub.2)E-NH.sub.2 2.07 0.346 17.713 5.196 noeffect/ noeffect/ 11(31) 0(0) inhibits inhibits 14 Suc-EGF(3F)WE-NH.sub.2 0.095 0.009 0.455 0.030 reduces/ reduces/ 5(6) 0(0) inhibits inhibits 15 Suc-EGF(3F)HE-NH.sub.2 0.521 0.059 0.400 0.021 reduces/ reduces/ 3(18) 12(83) inhibits inhibits 16 Ac-dEGF(3F)1NalE-NH.sub.2 0.112 0.017 0.590 0.282 reduces/ reduces/ 0(7) 3(8) inhibits inhibits 17 Ac-dEGF(3F)WE-NH.sub.2 0.102 0.017 0.250 0.059 reduces/ reduces/ 10(27) 3(0) inhibits inhibits 18 Bz-dEGF(3F)WE-NH.sub.2 0.173 0.072 0.4240 0.067 reduces/ reduces/ 0(0) 0(5) inhibits inhibits 19 Et(CO)-dEGF(3F)WE-NH.sub.2 0.145 0.026 0.150 0.065 reduces/ reduces/ 1(10) 0(10) inhibits inhibits 20 MeO(CO)-dEGF(3F)WE-NH.sub.2 0.076 0.014 0.053 0.015 reduces/ inhibits/ 0(16) 6(8) inhibits inhibits 21 Ts-dEGF(3F)WE-NH.sub.2 0.029 0.005 0.190 0.042 reduces/ inhibits/ 19(25) 9(0) inhibits inhibits 22 4-(MeO)-PhSO.sub.2- 0.017 0.002 0.048 0.003 s.reduces/ inhibits/ 7(12) 0(18) dEGF(3F)WE-NH.sub.2 inhibits inhibits 23 EtO(CO)-dEGF(3F)WE-NH.sub.2 0.045 0.005 0.034 0.017 reduces/ inhibits/ 4(4) 0(12) inhibits inhibits 24 Ts-DEGF(3F)WE-NH.sub.2 0.018 0.002 0.019 0.013 s.reduces/ inhibits/ 0(7) 0(8) inhibits inhibits 25 Ts-dDGF(3F)WE-NH.sub.2 0.025 0.004 0.292 0.106 s.reduces/ inhibits/ 0(22) 1(8) inhibits inhibits 26 4-(MeO)-PhSO.sub.2- 0.027 0.002 0.495 0.163 s.reduces/ inhibits/ 15(3) 0(8) DEGF(3F)WE-NH.sub.2 inhibits inhibits 27 Ts-dEGF(3F)WD-NH.sub.2 0.034 0.005 0.680 0.152 s.reduces/ inhibits/ 4(6) 3(8) inhibits inhibits 28 Ts-dDGF(3F)WD-NH.sub.2 0.081 0.020 0.637 0.114 reduces/ inhibits/ 0(14) 4(10) inhibits inhibits 29 3,4-(MeO).sub.2-PhSO.sub.2- 0.042 0.001 0.603 0.144 reduces/ reduces/ 19(19) 2(0) dEGF(3F)WE-NH.sub.2 inhibits inhibits 30 4-(BuO)-PhSO.sub.2- 0.056 0.008 0.767 0.009 reduces/ reduces/ 0(13) 7(12) dEGF(3F)WE-NH.sub.2 inhibits inhibits 31 2-NaphthylSO.sub.2- 0.044 0.005 0.262 0.021 reduces/ reduces/ 0(17) 10(11) dEGF(3F)WE-NH.sub.2 inhibits inhibits 32 Ts-dEGF(3F)WE(Me)-NH.sub.2 0.022 0.003 0.170 0.092 s.reduces/ inhibits/ 0(0) 4(10) inhibits inhibits 33 Ts-dEGF(3Cl)WE(Me)-NH.sub.2 0.017 0.003 0.047 0.025 s.reduces/ inhibits/ 0(10) 7(11) inhibits inhibits 34 Ts-dEGF(3Cl)WE-NH.sub.2 0.01 0.002 0.154 0.057 s.reduces/ inhibits/ 0(0) 8(6) inhibits inhibits
Example 7Cell-Based Activity of Peptidomimetics
[0706] The activity of 4-(MeO)-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 in a cell was investigated as an example of activity seen with this family of compounds.
[0707] In-Cell Western Assay for PDCD4 Accumulation.
[0708] PDCD4 is a substrate of TrCP and so an inhibitor of TrCP should result in the stabilisation and accumulation of PDCD4 in cells. To measure this an in-cell western assay was used and the peptidomimetics were delivered to the cells by nucleofection.
[0709] Nucleofection
[0710] MCF7 cells were grown in 10 cm dishes in 10 ml DMEM+10% FBS and 1% Pen/Strep at 37 C./5% CO.sub.2. On the day of nucleofection, the dish was washed with 90% confluent MCF7 cells with 5 ml PBS, then 3 ml of Trypsin/EDTA was added and incubated at 37 C./5% CO.sub.2 for approximately 5 mins until the cells detached from the plastic. 7 ml of normal growth media was added and the number of cells present was counted using a haemocytometer.
[0711] The cells were centrifuged cells at 90 g for 10 mins at RT and the supernatant was removed. 100 l of Nucleofection Buffer V+ Supplement was added (Lonza Biologics Cat no VCA-1003) per 810.sup.5 cells, and the cells were added to peptidomimetics dissolved in <3% DMSO.
[0712] The cell/peptidomimetic mix was added to cuvettes supplied for the nucleofector and the sample was nucleofected using the recommended MCF7 programme for high cell viability (i.e. E-014). 500 l of TPA (12-O-tetradecanoylphorbol-13-acetate)-supplemented growth media (i.e. DMEM/10% FBS/1% Pen-Strep/10 nM TPA) was added to the cuvette and 100 l of this solution was transferred to a well of a 96 well plate and incubated at 37 C./5% CO.sub.2 for 8 hours.
[0713] In Cell Western
[0714] The celled were fixed by removing the growth media, adding 3.7% Formaldehyde in PBS and incubating at RT for 20 mins. The plate was washed three times in PBS. And the cells were permeabilised by washing 5 for 5 mins each in PBS+0.1% Triton X-100.
[0715] The cells were blocked by adding 3% BSA in PBS-Tween to the cells and incubating at RT for 1.5 hours. An anti-PDCD4 antibody (Abcam cat no: ab80590) was added at a concentration of 1:1000 in 3% BSA in PBS-T (50 ul/well) and incubated at RT for 2.5 hours. The cells were washed 5 for 5 mins each with PBS-T before the secondary antibody (LiCor Biosciences Donkey Anti-Rabbit IRDye 800CW cat no 926-32213) was added at a concentration of 1:1000, along with the DNA stain DRAQ5 at a concentration of 1:10000, in 3% BSA in PBS-T (50 ul/well) and incubated at RT for 1 hour, protected from light.
[0716] The cells were washed cells 5 for 5 mins each with PBS-T and all the liquid was removed from the wells before the plate was read on the LiCor Biosciences Odyssey at 700 nm and 800 nm. The reading was normalised in the 800 nm channel to that in the 700 nm channel. The data are shown in
[0717] Fluorescence Reporter Assay for PDCD4 Accumulation
[0718] This assay uses two stable cell lines expressing PDCD4 fused to a GFP tag. One cell line (MCF7:GFP-PDCD4.sup.WT) shows an increase in nuclear fluorescence when TrCP is inhibited due to the accumulation of GFP-PDCD4 in the nucleus. The other cell line (MCF7:GFP-PDCD4.sup.S71A/S76A or or MCF7:GFP-PDCD4.sup.Mut) does not show an increase in nuclear fluorescence when TrCP is inhibited because of a mutation of two serine residues in the phosphodegron of PDCD4 that are required for TrCP recognition. This allows false positives to be identified, where accumulation of PDCD4 is not due to stabilisation by inhibiting TrCP.
[0719] Nucleofection
[0720] The two stable cell lines MCF7:GFP-PDCD4.sup.WT and MCF7:GFP-PDCD4.sup.S71A/S76Awere grown in 10 ml DMEM+10% FBS and 1% Pen/Strep supplemented with 2 mg/ml Geneticin at 37 C./5% CO.sub.2. These cell lines were nucleofected as described above except for the additional supplementation of all media with 2 mg/ml Geneticin.
[0721] Fluorescent Reporter Assay
[0722] The cells were fixed by removing the growth media and adding 3.7% Formaldehyde in PBS to the cells. The cells were then incubated at RT for 20 mins and the plates washed 3 in PBS.
[0723] DRAQ5 DNA stain was added to the cells in PBS at a concentration of 1:10000 and incubated at RT for 1 hour protecting from light. The plate was washed 3 in PBS and read on the Perkin Elmer OPERA platform using the nuclei counting algorithm F in both the 488 nm channel (GFP) and 640 nm channel (DRAQ5). The number of GFP-positive nuclei was expressed as a percentage of total number of nuclei, and the data are shown in
Example 8Cell Based Activity of Cell Permeable Compounds
[0724] The compounds shown in
[0725]
[0726] Testing of UBP037 and UBP038 by in cell western provided inconclusive results. The fluorescence readings were consistently under those of the DMSO negative control. This suggested that the compounds were interfering in some way with the fluorescence readout assay. When these compounds were tested by traditional western blot however, they exhibited cellular activity. The results for PDCD4 accumulation in MCF7 cells are shown in
[0727] -Catenin in Cell Western
[0728] Plating of the MCF7 cells onto 96 well platesthis step is carried out 1 day before the treatment of the cells to allow the cells to adhere well to the tissue culture plastic. MCF7 cells to be used for seeding should be less than 100% confluent in a 10 cm dish. Add 3 ml of RT trypsin/EDTA to the cells. Incubate at 37 C./5% CO.sub.2 for a few minutes until the cells easily come away from the plastic by gentle swirling. Add 7 ml of media to the cells. Wash the bottom of the plate gently with the 10 ml to ensure all cells are captured and dispense into a 15 ml falcon. Add 1001l of the cells to 1001l of Trypan blue and add to haemocytometer to count the number of cells present. Make up approx 10 ml of cells in media at 210.sup.4 cells/1001l (well) with fresh media. Add this correct seeding density to a 10 cm dish. Dispense 1001l of cells/well into a 96 well plate without using the outside wells. Incubate at 37 C./5% CO.sub.2 overnight
[0729] Treatment of MCF7 cells with compound of interest (COI) and controlsPut OptiMEM into 37 C. water bath to warm. Add 4 ul of 50 mM compound X for testing to eppendorfthis is tube 1. Add 2 al DMSO to 6 tubes marked tube 2-7. Take 21 al of 50 mM (COI) and add to 2 al of DMSO in tube 2 and pipette up and down to mix. Take 21 al from tube 2 and add to 2 l DMSO in next tube and mix. Repeat until 2 l in all 7 tubes and have 1:2 serial dilutions from tube 1 to 7 (final conc.: 250 PM to 2 M). Add 3.5 l DMSO in tube marked DMSO (final percentage 0.5% as for all compounds). Add 0.7 l of 10 mM MG132 and 2.8pal DMSO in tube marked MG132 (final conc.: 10 M). Add 0.7pal of 10 mM MLN4924 and 2.8 M DMSO in tube marked MLN4924 (final conc. 10 uM). Add 4001l of pre-warmed OptiMEM to each of tubes 1-7 with serial dilution of compound X. Add 700 ul of pre-warmed OptiMEM to tubes marked DMSO, MG132 and MLN4924. Take plate with seeded MCF7 cells and remove the media from the first column of wells. Add 100 l of DMSO to each of six wells in first column of wells. Remove media from the second column of wells and add 100 l of MG132 to each of six wells in second column of wells. Remove media from the third column of wells and add 100 l of MLN4924 to each of six wells in third column of wells. Remove media from the remaining columns of wells in small batches and add 100 l of each dilution of compound X to three wells in fourth to tenth column of wells. Incubate at 37 C./5% CO.sub.2 for 8 hrs. Add 1 ml of 37% formaldehyde to 9 ml PBS. Remove the media from the plate and add 100 l of 3.7% formaldehyde to each well. Incubate at RT for 20 mins. Remove formaldehyde and add 100 l of PBS to each well. Remove PBS and add another 100 l PBS to each well. Store at 4 C. overnight.
[0730] Immunostaining of -catenin using In Cell Western (ICW) protocolAdd 100 l of PBS+0.1% triton to each well and incubate at RT with gentle mixing for 5 mins. Replace with fresh PBS+0.1% triton and repeat 4 times. Noteall washing steps must be carried out gently to avoid dislodging cells. Add 100 l of 3% BSA in PBS-Tween to each well and incubate at RT with gentle mixing for 1 hour. Add 6.51 al of -catenin antibody to 6.5 ml of 3% BSA in PBS-Tween. Add 100 l of primary antibody solution to all bar one of the DMSO-treated wells. This will act as a negative control. It is also possible to not add primary antibody to one well of each treated column of wells in order to have a no primary control for all positive controls and each concentration of COI. Incubate at RT with gentle mixing for 2.5 hours or overnight at 4 C. Add 100 l of PBS-Tween to each well and incubate at RT with gentle mixing for 5 mins. Replace with fresh PBS-Tween and repeat 4 times. Spin down the vial of anti-rabbit IR800 (LI-COR Biosciences: cat no 926-32213 Donkey Anti-Rabbit IRDye 800CW) at top speed for a few seconds and add 6.5 l of this to 6.5 ml of 3% BSA in PBS-Tween. Add 50 l of this secondary antibody solution to one well as a control for DRAQ5. Add 0.65pal of DRAQ5 to the secondary antibody solution and add 100 l of this to all other wells. Incubate at RT with gentle mixing for 1 hr with the plates protected from light with tin foil. Add 100 l of PBS-Tween to each well and incubate at RT with gentle mixing for 5 mins. Replace with fresh PBS-Tween and repeat 4 times
[0731] Traditional Western Blot [0732] Seed MCF7 or LNCaP cells in 12 well plates at 100,000cells/well in complete medium (RPMI 1640, 10% fetal bovine serum, 100 units/mL penicillin, 100 ug/mL streptomycin, and 2 mmol/L glutamine, all from GIBCO) and incubate at 37 C./5% CO.sub.2 overnight. [0733] 24 hrs later change media to complete media+compound of interest+10 nM TPA and incubate for 8 hrs [0734] After incubation, wash cells three times in cold PBS and lyse with 3T3 lysis buffer containing protease inhibitors (Roche). [0735] Protein quantification was determined by the Bradford assay (BIORAD). [0736] Run 5 ug of protein samples on 4-12% NuPAGE Bis-Tris gels (Invitrogen) and transfer onto nitrocellulose membrane (Whatman). [0737] Block membranes in 50% PBS, 50% Odyssey block (LI-COR) for 1 hour. [0738] Incubate blots with primary antibodies diluted in 49% PBST, 49% Odyssey block and 0.5% 10% Tween-20 (Sigma) overnight at the following concentrations: [0739] Anti-PDCD4 (Abcam) at 1/10000 [0740] Anti-Tubulin (Abcam) at 1/20000 [0741] Anti-Actin (Abcam) at 1/5000. [0742] Wash blots 3 times for 5 min in PBST. [0743] Incubate with secondary antibodies on blots for 1 hour, protected from light. [0744] Antibodies were diluted in the same solution as primary antibodies [0745] 1/20000 goat anti-mouse alexa 680 (LI-COR), [0746] 1/10000 donkey anti-rabbit alexa 800 (LI-COR). [0747] Wash blots 3 times for 5 min in PBST. [0748] Observe protein bands using LI-COR Odyssey reader and quantitate strength of bands with Odyssey software.
Example 9Cell Based Activity of Cell Permeable Compounds on the xCELLigence Platform
[0749] The effects of the compounds shown in
[0750] Different doses of the compounds were tested on MCF7 cells to see if there would be an effect on their cell viability.
[0751] In order to prove that this activity was specific to the active compounds of the invention, a control compound was developed that is identical to the active compound except for the amino acid sequence that confers the specificity of the active compound. The control compound was compared to its partner active compound (UBP037).
[0752] xCELLigence Method [0753] Before starting the experiment add 50 L of medium (RPMI 1640, 10% fetal bovine serum, 100 units/mL penicillin, 100 ug/mL streptomycin, and 2 mmol/L glutamine, all from GIBCO) to each well of 96 well E-plate (Roche) and place the plate in the xCELLigence platform to measure the background. [0754] LNCaP/PNT-1 cells are seeded (LNCaPs 80000 cells/well; PNT1 3000 cells/well) in the plate in complete medium and equilibrated for 60 mins at RT before returning to the xCELLigence platform and incubating at 37 C./5% CO.sub.2. [0755] 24 hrs after seeding, remove 100 L of medium was removed from each well and cells were treated with compounds or DMSO diluted in 100 L of OptiMEM (GIBCO). [0756] Replace the plate in the xCELLigence platform [0757] Cells growth can then be monitored in real-time by the cell index profile on the xCELLigence readout screen [0758] Cell growth can be expressed as normalized cell index and doubling time/slope calculated using the RTCA software.
Example 10Activity in Cancer Cells Lines Compared to Non-Cancer Cell Lines
[0759] The susceptibility of cancer cell lines versus non-cancer cell lines following treatment with the compounds was also investigated.
[0760] Analysis
[0761] 1. Demonstration of Cell Based Activity by Active Compounds in Comparison to an Inactive Compound of a Similar Physicochemical Nature
[0762] This has been demonstrated by the xCELLigence assay that compared UBP037 with the control compound (Ts-EdFEGW-Ahx-K(Stearic)-NH.sub.2, a scrambled version of a compound of the present invention (see
[0763] 2. Demonstration of Target-Specific Activity in a Cell Based Assay Format
[0764] This is illustrated for compound UBP036 when examined in the GFP reporter (
[0765] In addition, the use of two TrCP substrates; PDCD4 and 1-catenin, in the in cell western assay would also suggest that any effect seen is due to TrCP inhibition.
[0766] Again the active species of all the compounds in
[0767] 3. Demonstration of Activity in Several Cancer Cell Lines
[0768] In the development of the assays for a TrCP inhibitor a number of cell-line biomarker combinations were surveyed to identify the most sensitive assay for TrCP inhibitors. The breast cancer cell-line MCF7 proved extremely responsive to TrCP inhibition and this could be measured by the rapid and robust accumulation of the TrCP substrates PDCD4 and 1-catenin.
[0769] As can been seen from the data presented here all compounds exhibit this activity (to various degrees) in MCF7 cells.
[0770] The next phase of development involved addressing the potential therapeutic benefit of TrCP inhibition in a cell line that could be replicated in an animal model. Here LNCaPs were chosen given previous evidence that TrCP inhibition inhibited cell proliferation both in vitro and in vivo (PLoS One. 2010 Feb. 5; 5(2):e9060.) Again it can be seen from the data that all compounds tested exhibit a reduction in proliferation (to various degrees) in LNCaP cells.
[0771] In addition to the cellular activity seen in each of these cell lines, it is also becoming apparent that the biomarker activity assays in MCF7 cells appear to predict the therapeutic activity seen in LNCaP cells.
[0772] There is also cell viability inhibition in MCF7 cells upon treatment with these compounds (see
[0773] Medical Applications of TrCP Inhibitors
[0774] There are a number of potential indications for TrCP inhibitors including many forms of cancer.
[0775] Two key indications exemplified are prostate cancer and breast cancer.
[0776] Breast Cancer.
[0777] There is clinical evidence that TrCP2 is over expressed in a number of cancers including breast cancer [J Biol Chem. 2002, 277, 36624-30]. This study also demonstrated that the cell lines used to model breast cancer such as MCF7 cells also display this same overexpression when compared to non-cancer cell lines such as MCF10A. This implies that work done on TrCP inhibition in these cancer cell lines could indeed reflect potential outcomes in a clinical setting.
[0778] There have been numerous in vivo studies to demonstrate the importance of TrCP in mammary development. In TrCP1/ mice there is a hypoplastic phenotype observed where cell proliferation is reduced by 50% in the mammary gland with other organs unaffected. Furthermore, when there is exogenous high expression of TrCP1 introduced in the mammary epithelia, approx 40% of mice develop carcinomas. [Mol Cell Biol. 2004, 24, 8184-94.]. The value of this study is two-fold. It demonstrates that despite the widespread expression of TrCP, a systemic reduction in TrCP levels (via the genetic ablation of TrCP1) has a preferential effect on the mammary gland. Also it reveals that overexpression of TrCP1 can in itself result in an increased cancer risk in this tissue. This suggests inhibition of TrCP may be of value in both breast cancers that do not display TrCP overexpression (as inhibition of TrCP in healthy animals appears to preferentially target the mammary gland for reduced cell proliferation) and those that do (due to the potential causative effect of TrCP mis-regulation).
[0779] In addition to the value of inhibiting TrCP alone to affect favourable outcomes in breast cancer, there is also work to suggest that combining TrCP inhibition with some of the current therapies for breast cancer could improve the outcome of these therapies. Inhibition of TrCP by an RNAi approach suppressed growth and survival of human breast cancer cells [Cancer Res. 2005 Mar. 1; 65(5):1904-8]. It is worth noting that these experiments were carried out on both ER-positive and ER-negative breast cancer cell lines with TrCP inhibition having a similar impact on both. In addition, inhibition of TrCP augmented the anti-proliferative effects of anticancer drugs such as doxorubicin, tamoxifen, and paclitaxel on human breast cancer cells. These data suggest that TrCP inhibition could be effective as a front line adjuvant therapy or in combination with an existing breast cancer treatment regime.
[0780] We have shown that the TrCP inhibitors described here inhibit binding of TrCP to IB (a well-known TrCP substrate) in in vitro binding assays and stabilise several TrCP substrates in cell based assays. In addition this inhibitor can reduce the cell viability of a breast cancer cell line in a similar fashion to TrCP RNAi.
[0781] These data and the studies described above show that TrCP is a validated, novel target in breast cancer, and that its inhibition is tractable and of clinical significance.
[0782] Prostate Cancer.
[0783] Here the main evidence for the role of TrCP is from the work of Yinon Ben-Neriah and Eli Pikarsky [PLoS One. 2010 Feb. 5; 5(2):e9060.] Their key finding here was not only that inhibition of TrCP resulted in the loss of cell viability of LNCaP cells in vitrobut also that when this inhibition is transferred from cells to animals through the use of LNCaP xenograftsit results in a loss of growth of prostate tumours and in combination with androgen ablationthe lack of tumour growth entirely.
Example 11Effect of Capping Groups on Peptide Activity
[0784] A number of different C-terminal and N-terminal capping groups were added to peptide d-E-G-F(3F)W-E-NH.sub.2 in order to demonstrate how the inhibitory activity of d-E-G-F(3F)W-E-NH.sub.2 is affected by particular capping groups, which act to increase cell penetration (as illustrated by c Log P, relative to UBP022). K.sub.i and c Log P values for the the capped compounds are shown in Table 12.
TABLE-US-00013 TABLE12 K.sub.iandcLogPforN-andC-terminal cappedpeptided-E-G-F(3F)-W-E-NH.sub.2 Average Code Sequence Ki/nM cLogP Kd/cLogP UBP022 4-(MeO)PhSO.sub.2-dEGF(3F)WE-NH.sub.2 5.775 UBP054 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(t-Bu-Ph))-NH.sub.2 1.938 3.84 0.505 UBP055 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-2-Naph)-NH.sub.2 1.851 3.51 0.527 UBP056 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(2,4,6-(Me)3-Ph))-NH.sub.2 3.387 3.27 1.036 UBP057 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(ME)-Ph))-NH.sub.2 2.884 2.64 1.092 UBP058 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(Br)-Ph))-NH.sub.2 3.3455 3.03 1.104 UBP059 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHSO.sub.2-(4-(Br)-Ph))-NH.sub.2 2.960 2.40 1.2333 UBP060 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(Cl)-Ph))-NH.sub.2 3.641 2.94 1.238 UBP061 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCOPh)-NH.sub.2 3.105 2.33 1.333 UBP062 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(3,5-(Cl).sub.2-Ph))-NH.sub.2 6.229 3.55 1.755 UBP063 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(CH.sub.2).sub.4CH.sub.3)-NH.sub.2 5.374 2.71 1.983 UBP064 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(CF.sub.3)-Ph))-NH.sub.2 6.391 3.09 2.068 UBP065 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-Ph)-NH.sub.2 5.939 2.55 2.329 UBP066 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-(4-(OMe)-Ph))-NH.sub.2 5.1745 2.22 2.331 UBP068 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCONH-Ph)-NH.sub.2 5.608 2.22 2.526 UBP068 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHCO-CH.sub.2CH.sub.2Ch(CH.sub.3).sub.2)-NH.sub.2 5.378 2.12 2.537 UBP069 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHOCO-1-Naph)-NH.sub.2 11.608 3.73 3.112 UBP070 4-(MeO)PhSO.sub.2dEGF(3F)WE-Ahx-K(NHCO-(4-(Cl)-2,6(Fl.sub.2-Ph))-NH.sub.2 11.455 3.06 3.743 UBP071 4-(MeO)PhSO.sub.2dEGF(3F)WE-Ahx-K(NHCO-(4-(Me.sub.2N)-Ph))-NH.sub.2 9.506 2.33 4.080 UBP072 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHSO.sub.2-(4-i-Pr)-Ph))-NH.sub.2 12.941 2.80 4.622 UBP073 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHSO.sub.2Ph)-NH.sub.2 13.005 1.70 7.65 UBP074 4-(MeO)PhSO.sub.2-dEGF(3F)WE-Ahx-K(NHSO.sub.2-(4-(n-Pr)-Ph)-NH.sub.2 28.585 2.84 10.055 UBP040 4-i-Pr-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1.603 1.20 1.336 UBP041 4-Pr-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 1.768 1.24 1.426 UBP042 4-Br-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 2.519 0.8 3.14 UBP043 4-Br-2-CH.sub.3-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 3.961 1.12 3.537 UBP044 2-Naph-SO.sub.2-dEGF(3F)WE-NH.sub.2 5.814 1.29 4.507 UBP045 4-OCF.sub.3-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 4.774 0.94 5.079 UBP046 4-Br-3-CF.sub.3-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 14.217 1.57 9.055 UBP047 4-CF.sub.3-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 9.450 0.94 10.862 UBP048 2,4-(Cl).sub.2-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 27.025 1.33 20.320 UBP049 2,4-(Br).sub.2-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 45.255 1.50 30.17 UBP050 3,5-(CH.sub.3).sub.2-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 24.645 0.74 33.30 UBP051 4-Br-2-OCF.sub.3-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 109.55 1.63 67.209 UBP052 4-I-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 252.9 1.04 243.17 UBP053 4-Cl-PhSO.sub.2-dEGF(3F)WE-NH.sub.2 330.5 0.72 459.0
[0785] As suggested by the data in table 12, modification of the C-terminal capping group has little effect upon activity. Modification of the N-terminal capping group has a more profound effect. The function of the capping groups is to aid cell penetration as demonstrated by the c Log P values. c Log P values are calculated by means well known to the person skilled in the art.
REFERENCES
[0786] Pons et al. (2008) Biochemistry 47, pg. 14-29 [0787] Tapia et al. (2008) J. Pept. Sci. 14, pg. 1309-1314 [0788] Rautio et al. (2008) Nat. Rev. Drug Discov. 7, pg 255-270. using anti-His and anti-TrCP antibodies
Remington's Pharmaceutical Sciences
[0789] Stocks et al. (2007) On Medicinal Chemistry [0790] Werle et al. (1997) British Journal of Cancer [0791] Bungaard et al. Design of Prodrugs [0792] Ornstein et al. (1993) Bioorg. Med. Chem. Lett [0793] Lakshmann et al (2008) Expert Opinion in Therapeutic Targets 12(7):855-870. [0794] Frescas and Pagano (2008) Nature Reviews Cancer Jun; 8(6):438-49 [0795] Nalepa, Rolfe and Harper (2006). Nature Reviews Drug Discovery 5:596-613 [0796] Crosetto, Bienko and Dikic (2006) Molecular Cancer Research 4(12): 899-904
SEQUENCES
[0797]
TABLE-US-00014 (consensussequence) SEQIDNO:1 XXGFXX (preferredpeptide) SEQIDNO:2 dEGF(3F)WE (preferredpeptide) SEQIDNO:3 DEGF(3F)WE (preferredpeptide) SEQIDNO:4 DEGF(3F)WD (preferredpeptide) SEQIDNO:5 dDGF(3F)WD (preferredpeptide) SEQIDNO:6 EGF(3F)WE (preferredpeptide) SEQIDNO:7 dEGF(3F)1NalE (preferredpeptide) SEQIDNO:8 EGF(3F)1NalE (phosphopeptidesubstrate) SEQIDNO:9 KKERLLDDRHDpSGLDpSMKDEE (optimisationstartingsequence) SEQIDNO:10 LDpSGIHS (Vpuphosphodegeneron) SEQIDNO:11 DpSGIHS (bindingpeptide) SEQIDNO:12 DpSGIFE (bindingpeptide) SEQIDNO:13 DEGIFE (bindingpeptide) SEQIDNO:14 ERAEDpSGNEpSEGEIS (bindingpeptide) SEQIDNO:15 ERAEDAGNEpSEGEIS (bindingpeptide) SEQIDNO:16 ERAEDpSGNEpSEGEHS (bindingpeptide) SEQIDNO:17 ERADDpSGNEpSEGEIS (bindingpeptide) SEQIDNO:18 dEGIFE (bindingpeptide) SEQIDNO:19 dEGIFD (bindingpeptide) SEQIDNO:20 dNGIFR (bindingpeptide) SEQIDNO:21 DEGFFE (bindingpeptide) SEQIDNO:22 dNGFFR (bindingpeptide) SEQIDNO:23 EGIFE (bindingpeptide) SEQIDNO:24 EGFFE (bindingpeptide) SEQIDNO:25 DEGYFE (bindingpeptide) SEQIDNO:26 EpSGIFE (bindingpeptide) SEQIDNO:27 DpSGIFH (bindingpeptide) SEQIDNO:28 DpSGNFE (bindingpeptide) SEQIDNO:29 DDpSSGIHS (bindingpeptide) SEQIDNO:30 LDpSSGIHS (bindingpeptide) SEQIDNO:31 GDpSGIHS (bindingpeptide) SEQIDNO:32 ADpSGIHS (bindingpeptide) SEQIDNO:33 VDpSGIHS (controlpeptide) SEQIDNO:34 DAGIFE (controlpeptide) SEQIDNO:35 AGIFE (controlpeptide) SEQIDNO:36 AGFFE (controlpeptide) SEQIDNO:37 dAGIFR (controlpeptide) SEQIDNO:38 dAGIFD (controlpeptide) SEQIDNO:39 dAGIFE (controlpeptide) SEQIDNO:40 DAGFFE (controlpeptide) SEQIDNO:41 DAGYFE (controlpeptide) SEQIDNO:42 EAGIFE (controlpeptide) SEQIDNO:43 DAGIFH (controlpeptide) SEQIDNO:44 DAGNFE (controlpeptide) SEQIDNO:45 DAGIHS (controlpeptide) SEQIDNO:46 DDASGIHS (controlpeptide) SEQIDNO:47 LDASGIHS (controlpeptide) SEQIDNO:48 GDAGIHS (controlpeptide) SEQIDNO:49 ADAGIHS (controlpeptide) SEQIDNO:50 VDAGIHS (bindingpeptide) SEQIDNO:51 EGF(3F)HE-NH2 (bindingpeptide) SEQIDNO:52 dEGF(3F)1Nal-E-NH2 (bindingpeptide) SEQIDNO:53 EGI1NalE-NH2 (bindingpeptide) SEQIDNO:54 EGF(3F)1NalQ-NH2 (bindingpeptide) SEQIDNO:55 EGF(3F)Y(4Me)E-NH2 (bindingpeptide) SEQIDNO:56 dEGF(3F)WD-NH2