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Discovery of Potent 2-Indole-acylsulfonamide Mcl-1 Inhibitors Using Structure Guided Fragment-based
Methods
Subrata ShawPostdoctoral Fellow
Advisor: Prof. Stephen W. Fesik
AACR Annual Meeting, New Orleans 19th April 2016
• A member of Bcl-2 family of anti-apoptotic proteins• Amplification observed in various human cancers• Overexpression emerged as a resistance
mechanism• Causes resistance to chemotherapy and radiation• Knockdown sensitizes cells to apoptosis
Mcl-1 in Cancer
Mcl-1 up-regulated in
Lung cancer
Liver cancer
Prostate cancer
Oral Cancer
Breast Cancer
Pancreatic cancer
Ovarian cancer Cervical cancer
Melanoma
Leukemia M. Myeloma
Neuroblastoma
Goal: To discover a potent, selective, orally bioavailable Mcl-1 inhibitor for the treatment of human cancers.
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Discovery of Early Lead by Fragment-Merging
Structure-guided merging
22 (0.46)
HN
O
Cl
O
ClHO
Ki 55 nM (Mcl1)2190 nM (Bcl-xL)928 nM (Bcl-2)
InitialOptimization
Friberg et al., J. Med. Chem. 2013, 56, 15
S
HO
O
Cl
O
Cl
OOH
S
HO
O
O
Cl
• No cellular efficacy• Need to improve binding affinity
Ki 131 μM
Ki 60 μM
Ki 0.32 μM
A227 R263
F270
3
‘Lead’
How to Improve Binding Affinity?
P2
P4
ABT-737
P3
Binding interface expansion strategy
4
‘Lead’/Mcl-1 ‘Lead’/Mcl-1/BH3-peptide ‘Lead’/Mcl-1/ABT-737
HN
O
OH
O
Cl
Cl Chemical)Linker)
Spacing)Unit)
P4)Site)Binder)
HN
O
Cl
Cl
Pelz et al., J. Med. Chem. 2016, 59, 2054
Acylsulfonamide as the Chemical Linker Unit
X Acid Ki (nM)*
AcylsulfonamideKi (nM)*
H 300 655
Cl 55 209
HN
O
HN
O
Cl
SO
Me
OXH
N
O
X OH
O
Cl
R263
P2 P4
6
*FPA-assay using FITC-BAK
Pelz et al., J. Med. Chem. 2016, 59, 2054
NMR Based 2nd Site Fragment Screening
Fragment Hits
HN
O OH HN
NH
ON
N
S NH2
NO
HO PhN
O
HO
N
NHO
Ph PhHN N
F3CF2C OH
• Cloned, expressed, and purified 15N-labeled Mcl-1• P2 site blocked using lead compound• Screened 14,000 fragments• HSQC of lead/Mcl-1 perturbed upon fragment binding • Identified 7 hits
NMR Screen
5
Kd 1.5 mM
X-ray Ternary Co-crystal Structure
Fragment Hits
P2
P4
3.6 Å
P2 P4
4.8 Å
NH
OOH
HN N
F3CF2C
HO
7
• X-ray suggests flexible spacer length 3 - 4 atoms
X Ki (nM)
H Me 655
H 322
H 430
H 118
H 1098
H 1015
H 251
Spacer Design & Optimization
O Ph
NH
O
Ph
NH
O
NH
O
HN
NH
SOO
Spacer R
HN
O
HN
O
Cl
SO
O
Spacer RX
8
Binding Conformation of the Linked Acylsulfonamide
• Linked acylsulfonamide maintains similar binding pose as methyl acylsulfonamide
• Cyclohexyl residue occupies P4 site• Validates linking strategy
Ki 374 nM
P4
HN
O
Cl HN
O
SO
O
HNO
at P4 ?
9
R Ki (nM)
199
311
193
37
Fragments Containing 6,5-Fused Heterocycles
R Ki (nM)
269
278
432
160
298
HN
HN
NH
HN
NMe
NMe
O
P4 Site Fragment Hits: indoles
HN
O
HN
O
SO
O
HNO
R
Cl
• Flat SAR with indoles irrespective of attachment point• Nonplanar geometry at the linking position preferred
10
HN
O OH HN
NH
O
118
HN
HN
R Ki (nM)
148
139
782
90
306
120
Fragments Containing 5-Membered Heterocycles
NBn
NPh
NMe
NH
O
O
P4 Site Fragment Hits: 5-membered heterocycles
R Ki (nM)
842
860
2553
907
N
NBn
N
S
N
O
ON
HN
O
HN
O
SO
O
HNO
R
Cl
• H-bonding donor not suitable for P4 site• Residues with additional hetero-atom not tolerated
11
NO
HO PhN
O
HO Bn
N
NHO
Bn
118
Fragments Containing Substituted Phenyl
X R Ki (nM)
Cl 308
H 336
H 110
H 117
Cl 18
Cl 79
Cl 119
CF3
CF3
CF3
F
F
F
HN
O
X HN
O
SO
O
HNO
R
Cl
OHHN N
P2
P4
4.8Å
12
HN NF3CF2C OH
Fragments Containing Aliphatic Residues
R Ki (nM)
55
16
66
87
CH3 209
CF3 116
26
10
• Aliphatic residues favored at P4 site
HN
O
Cl HN
O
SO
O
HNO
R
Cl
13
Aromatic Spacers
X Ar Ki (nM)
H 361
Cl 91
H 335
Cl 116
N
O
HN
O
HN
O
Cl
SO
ArO
X
Cl Me 209
HN
O
HN
O
Cl
SO
O
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7-Aryl Group Optimization
HN
O
X HN
O
SO
Ph
O
Cl
Ar
X Ar Ki (nM)
H 19
H 11
H 21
H <10
Cl <10
Cl <10
N
ON
NHN
NN
NHN
• A library of > 50 compounds• 7-substituent improve Ki to 10 fold or better• Binding affinities below detection the limit of FITC-BAK assay• Developed potent small molecule probe
15
Incorporation of Preferred P4 Site Binders
Ki (nM)Bak
Ki (nM)FITC-SM*
<10 5.0
<10 10.0
<10 2.9
<10 5.6
<10 5.6
<10 1.2
<10 1.0
HN
O
F3C
HN
ON
HN
O
O
HN
O
N
NH
N
NH
N
NH
• Highly potent Mcl-1 inhibitors • Still no cell efficacy: highly
protein bound, poor aqueous solubility and permeability
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*FITC-SM: Small molecule FITC probe
Spacer R
HN
O
HN
O
Cl
SO
O
Spacer RCl
NN
Summary
• Fragment-based method and structure-based design is a powerful tool to generate lead
• Binding affinity (Ki) improved > 50,000 – fold from initial fragment hits
• Binding affinity can be further improved efficiently based on structural information
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Current Effort: Tricyclic Indole Amide Series
Ki = 300 µM
Initial Lead Binding Affinity Optimization
Structure guided Tethering
Mcl-1 Ki = 23 nM +1% FBS = 108 nM
Mcl-1 Ki = 1.0 nM +1% FBS = 3.5 nM H929 GI50 = 1.2 µM K562 GI50 = 12 (µM)
Current Leads
Mcl-1 Ki = <0.3 nM +1% FBS = <1.0 nM H929 GI50 = <0.3 µM K562 GI50 = >12 µM
Med. Chem. Optimization
HN
NH
O
O
Cl
O
HO
HN
HO
O
O
Cl
Inhibitor Design Evolution
Binding interface Expansion
Ki = 570 µM
Old Lead
Mcl-1 Inhibitors Exhibit Anti-Cancer Activity
Caspase Activation Apoptosis
Target Engagement Assays
• 79 Cancer cell lines screened using current best Mcl-1 inhibitor
• Cancer lines include: Colon, Breast, Prostate, Lung, Leukemia, Melanoma, Ovarian and Renal Cancer
• Mcl-1 inhibitor exhibits GI50 < 2 μM in 16/79 lines
Kill Multiple Cancer Cells
Program goal: To select a candidate for human cancer pre-clinical trials
Breast'
CNS'
M.'Myeloma'
Leukemia'Melanoma'
Lung'
Renal'
Lung%cancer%
Breast%Cancer% Melanoma%
Leukemia%M.%Myeloma%
Neuroblastoma%%
Renal%Cancer%
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Medicinal Chemistry Taekyu Lee Alex Waterson Chris Tarr Nick Pelz Zhiguo Bian Johannes Belmar Claire Gregg Brian Choudar
Structural Biology Ed Olejniczak Anders Friberg Bin Zhao
Biology Olivia Rossanese Craig Goodwin Carrie Browning Allison Arnold John Sensintaffar Leah Hogdal Demarco Camper Dom Vigil
Acknowledgements
Advisor: Prof. Stephen Fesik
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NCI Bill Moore Craig Thomas Damien Duveau
Funding
• Vanderbilt University• Pioneer Award• NCI Experimental Therapeutics (NExT) Program• Career Development Award/Lung SPORE• Breast Cancer SPORE
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OH
O
O OSCl
O
HN
Cl
O
HN
O
NHN
R263
N260
P2 P4
O
OH
HN
O
Cl HN
O
Cl
NN
ONH(CH2)6NH
SO
OO
O
OH
O
COOH
O
Developing Potent Small Molecule Probe
1IC50 < 15 nM (Bak-probe)[Mcl-1] = 30 nM
DM
SO -11 -10 -9 -8 -7 -6
240
250
260
270
280
290
300
Log Mcl-1 (M)F
luor
esce
nce
Ani
sotro
py
FITC-1 Kd = 0.46 nM
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