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Current Literature!!Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation!
!ACS Med. Chem. Lett. 2017, 139, 6046. !
Evan Carder!Wipf Group Current Literature!
November 18, 2017
11/18/17 1EvanCarder@WipfGroup
NNS
O
O O HN O
NHO
OO NHFF
Cl
O
NN
NHNO
NH
Proteasomal degradation
BCR-ABL BCR-ABL
UbUb
Ub
Ub
BCR-ABL
E3 Ligase
E2
Ub
Ub
11/18/17! 2EvanCarder@WipfGroup
Normal !chromosomal 9!
Normal !chromosomal 22!
break! translocation!
Transformed!chromosomal 9!
Transformed!chromosomal 22!
ABL!
BCR!
JCI 2007, 117, 2036.!Nat. Rev. Cancer 2007, 7, 345. !
BCR-ABL!Fusion gene!
11/18/17! 3EvanCarder@WipfGroup
Transformed!chromosomal 22!
BCR-ABL!Fusion gene!
Cl
NH
O
N
S HN
N N
NN OH
Dasatinib
N
N
NH
N
NH
O
NN
Imatinib
N
CF3
HN
O
NNH
N
N
NNilotinib
ABL Tyrosine Inhibitors!
Constitutively-active !ABL tyrosine kinase!
Nat. Rev. Cancer 2007, 7, 345. !
Pharmacology Models: Inhibition vs Degradation!
11/18/17! 4EvanCarder@WipfGroup
Nat. Drug. Disc. 2017, 16, 101.!Cell Chemical Biology 2017, 24, 1181. !
§ Occupancy-driven pharmacology!
Stoichiometric activity!Mostly target active-site binding!Site occupancy to block function !Specificity defined by binding !
§ Event-driven pharmacology!
Sub-stoichiometric activity!Target active and allosteric sites!Additional layer of specificity !Restoration of function requires re-synthesis! !
Proteindegradation
Proteininhibition
no functional protein
Abnormal protein function
Event-drivenpharmacology
Occupancy-drivenpharmacology
Hea
lthy
Dise
ase
Protein Degradation - Ubiquitin-Proteasome System (UPS)!
11/18/17! 5EvanCarder@WipfGroup
Nat. Rev. Mol. Cell Biol. 2008, 9, 679. !
ProteinSubstrate
Proteasomal degradation
E3 Ligase
Ub E2Ub
ProteinSubstrate
UbUb
UbUb
Ub
ProteinSubstrate E3 LigaseE3 Ligase
Tagged for !protein degradation!
Proteolysis Targeting Chimeras - PROTACs!
11/18/17! 6EvanCarder@WipfGroup
Natl. Acad, Sci. USA 2001, 98, 8554.!
POI E3 Ligase
POI Ligand
E3 LigaseLigand
PROTAC
Small-molecule-mediated Protein degradation:!
Proteasomal degradation
PROTAC
POI POI E3 Ligase
E2Ub
Ub
POI
UbUb
Ub
Ub
Targeting Protien of Interest!
11/18/17! 7EvanCarder@WipfGroup
ACS Cent. Sci. 2017, 3, 925.!Med. Princ. Pract. 2013, 22, 418. !Rev. Pharmacol. Toxicol. 2014, 54, 165.!
§ Orthosteric/Active Site Modulators!
Structurally similar to endogenous ligand!Employ rational drug design!Varying degrees of specificity!Highly susceptible to drug resistance !
!§ Nonothosteric/Allosteric Modulators!
Lack an endogenous ligand!Unique chemical structures!Elevated target selectivity!Reduced off-targets !Spatio-temporal specificity!Reduced susceptibility to drug resistance !
Nonorthosteric site
Orthosteric site
POI
Hypothesis – Application of an allosteric BCR-ABL PROTAC induces BCR-ABL protein degradation !
11/18/17! 8EvanCarder@WipfGroup
PROTAC AllostericABL Ligand
E3 LigaseLigand
ABL
E3 Ligase
E2
Ub
Ub
BCR-ABL protein degradation!Development of an allosteric BCR-ABL PROTAC!
Phase I! Phase II!
ABL Allosteric-site Inhibitors!
11/18/17! 9EvanCarder@WipfGroup
Nature 2010, 463, 28.!Nature 2017, 543, 733. !
PDB: 5MO4
ABL
OFF
ClHN N N
HN N
OH
ABL001
OFF
FHN
N N O
NH2
GNF-2
ABL !Kinase
ATP-binding !site
Myristate!binding site
Target Myristate-binding site
E3-Ubiquitin Ligands!
11/18/17! 10EvanCarder@WipfGroup
Nat. Drug. Disc. 2017, 16, 101.!
N
NO
ONHN
O
Nutlin-3aMDM2 Ligand
O
Cl
Cl
HO
ONH
O
OH
NH2
BestatincIAP Ligand
N
NH2
O
ONH
O
O
PomalidomideCRBN Ligand
N
OHO
HOO
HN
SN
VHL Ligand
IAPs!!MDM2!!Von Hippel-Lindau!!Cereblon!
!!
E3-Ubiquitin Ligase!E3 Ligase
OO
SN
N
ONH
OHN
4cIAP Ligand
Design of PROTAC!
11/18/17! 11EvanCarder@WipfGroup
PROTAC AllostericABL Ligand
E3 LigaseLigand
Linker
OOO
Ethylene glycol linkerOFF
ClHN N N
HN N
OH
ABL001 OO
SN
N
ONH
OHN
4cIAP Ligand
Structural Analysis of ABL001 bound ABL Kinase!
11/18/17! 12EvanCarder@WipfGroup
Nature 2017, 543, 733. !
PDB: 5MO4
Positioning of the linker
solvent !exposed region
Surface view of ABL Kinase
ABL
OFF
ClHN N N
HN N
OH
ABL001
OO
SN
N
ONH
OHN
4cIAP Ligand
Structural Analysis of IAP Ligand !
11/18/17! 13EvanCarder@WipfGroup
J. Biological Chemistry 2017, 292, 4556.!J. Med. Chem. Article ASAP!Bioorg. & Medi.Chem. Lett. 2010, 20, 2229.!
Positioning of the linker
PDB: 3GT9
Surface view of IAP
IAP
solvent !exposed region
PROTAC Development!
11/18/17! 14EvanCarder@WipfGroup
PROTAC
NNS
O
O O HN O
NHO
OO NHFF
Cl
O
NN
NHNO
NH
6
ABL Allosteric Ligand cIAP LigandLinker
Synthesis of the ABL Allosteric Ligand!
11/18/17! 15EvanCarder@WipfGroup
Nature 2010, 463, 28.!Nature 2017, 543, 733. !
1. SOCl2, DMF Toluene, 80 oC
2. DIPEA, THF -16 to rt, 77%
N
O
HO Br
Cl
O
NH2FF
Cl+
O
NH
FFCl O
N
Br
Cl
NH HCl
DIPEA, i-PrOH130 oC, 65%
O
NH
FFCl O
N
Br
N
NNB
O
O
O
Pd(PPh3)4, XPhos K3PO4, THF, H2O
100 oC, 92%
O
NH
FFCl O
N N
NN
O
LiOH, H2O
MeOH, rt, 55%
OO
O
O
O
O
O
NH
FFCl O
N N
NN
O
O
OHI
AllostericABL Ligand
Synthesis of the cIAP Ligand!
11/18/17! 16EvanCarder@WipfGroup
J. Biological Chemistry 2017, 292, 4556.!
NBoc
HOO
1. EDC, HOBt NH3 DMF, THF, 0 oC to rt
2. Lawesson’s reagent THF, 60 oC, 82%
NBoc
H2NS
BrO
OEt
O
1. A, EtOH, 60 oC
2. Boc2O, NaHCO3 H2O, THF 0 oC to rt, 89%
A
NBoc
NSEtO
O
1. NaOH, H2O, THF 60 oC, 82%
2. MeO(Me)NH HCl, EDC HOBt, DIPEA, DMF rt, 97%
NBoc
NSN
O
O
THF, -55 to -10 oC, 99%
THPO MgBr NBoc
NS
O
THPO1. HCl, MeOH, rt2. B, EDC, HOBt NaHCO3, THF 0 oC to rt3. K2CO3, MeOH H2O, 0 oC, 99%
NH
HO
O
Boc
B NNS
O
HOO HN Boc
HO
ONBoc
1. HCl, CPME THF, MeOH, rt2. C, EDC, HOBt DIPEA, DMF, rt3. K2CO3, MeOH H2O, 0 oC, 42%
C
NNS
O
HOO HN O
NBoc
E3 LigaseLigand
Synthesis of PROTAC – Compound 6 !
11/18/17! 17EvanCarder@WipfGroup
ACS Med. Chem. Lett. 2017, 8, 1042.!
NNS
O
HOO HN O
NBoc K2CO3, DMF
50 oC, 75%
OTsO O OTs
NNS
O
OO HN O
NBocOOTsO
1. NH3, H2O EtOH, 100 oC
2. I, HATU, DIPEA MeCN, rt, 40%
NNS
O
O O HN O
NBocO
OO NHFF
Cl
O
NN
NNO
O
NH
TFA
rt, 50%
NNS
O
O O HN O
NHO
OO NHFF
Cl
O
NN
NHNO
NH
6
PROTAC
11/18/17! 18EvanCarder@WipfGroup
NNS
O
O O HN O
NHO
OO NHFF
Cl
O
NN
NHNO
NH
6
OO
SN
N
ONH
OHN
4 - LCL161 DerivativeE3 Ligand
OFF
ClNH N N
NH N3 - ABL001 DerivativeABL Ligand
O
O
11/18/17! 19EvanCarder@WipfGroup
Compound 6-induced BCR-ABL Protein Degradation!
Proteasomal degradation
BCR-ABL BCR-ABL
UbUb
Ub
Ub
BCR-ABL
E3 Ligase
E2
Ub
Ub
6
Compound 6 Effect on Cell Proliferation !
11/18/17! 20EvanCarder@WipfGroup
6
Summary!
11/18/17! 21EvanCarder@WipfGroup
§ Designed and developed an allosteric BCR-ABL PROTAC that potently induces BCR-ABL protein degradation in cell assays.!
Half-maximal degradation concentration (DC50): ~30 nM ! Maximum degradation efficacy (Dmax): ~70% between 100-300 nM!!Further developments:!!§ LCL161 derivative 4 induces auto-ubiquitination leading to IAP protein degradation !
§ Evaluation of compound 6 in in-vivo models!!
NNS
O
O O HN O
NHO
OO NHFF
Cl
O
NN
NHNO
NH
6
ABL Allosteric Ligand cIAP LigandLinker
Drugging the “Undruggable”!
11/18/17! 22EvanCarder@WipfGroup
Science 2017, 355, 1158.!
§ The RAS genes constitute the most frequently mutated oncogene family in cancer.!
§ Approximately ~25% of human tumors have a RAS mutation.!
§ Despite more than 30 years of effort to develop a pharmacologic inhibitor of RAS, a clinically effective anti-RAS therapy does not exist.!
§ What approach will prove to be the most effective RAS modulator? Which approach will produce the first clinical anti-RAS agent?!
Perhaps an allosteric RAS PROTAC to! induce RAS protein degradation?!!
