1. Discovery of Potent 2-Indole-acylsulfonamide Mcl-1
Inhibitors Using Structure Guided Fragment-based
Methods
Subrata Shaw
Postdoctoral Fellow
Advisor: Prof. Stephen W. Fesik
AACR Annual Meeting, New Orleans
19th April 2016
2. • 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.
2
3. Discovery of Early Lead by Fragment-Merging
Structure-
guided merging
22 (0.46)
H
N
O
Cl
O
ClHO
Ki 55 nM (Mcl1)
2190 nM (Bcl-xL)
928 nM (Bcl-2)
Initial
Optimization
Friberg et al., J. Med. Chem. 2013, 56, 15
S
HO
O
Cl
O
Cl
O
OH
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’
4. 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
H
N
O
OH
O
Cl
Cl Chemical)
Linker)
Spacing)
Unit)
P4)Site)
Binder)
H
N
O
Cl
Cl
Pelz et al., J. Med. Chem. 2016, 59, 2054
5. Acylsulfonamide as the Chemical Linker Unit
X
Acid
Ki (nM)*
Acylsulfonamide
Ki (nM)*
H 300 655
Cl 55 209
H
N
O
HN
O
Cl
S
O
Me
O
X
H
N
O
X OH
O
Cl
R263
P2 P4
6
*FPA-assay using FITC-BAK
Pelz et al., J. Med. Chem. 2016, 59, 2054
6. NMR Based 2nd Site Fragment Screening
Fragment Hits
H
N
O OH H
N
NH
O
N
N
S
NH2
N
O
HO Ph
N
O
HO
N
N
HO
Ph Ph
HN 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
7. X-ray Ternary Co-crystal Structure
Fragment Hits
P2
P4
3.6 Å
P2
P4
4.8 Å
NH
O
OH
HN N
F3CF2C
HO
7
• X-ray suggests flexible spacer length 3 - 4 atoms
8. X Ki (nM)
H Me 655
H 322
H 430
H 118
H 1098
H 1015
H 251
Spacer Design & Optimization
O
Ph
N
H
O
Ph
N
H
O
N
H
O
H
N
N
H
S
OO
Spacer R
H
N
O
HN
O
Cl
S
O
O
Spacer R
X
8
9. 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
H
N
O
Cl HN
O
S
O
O
HN
O
at P4 ?
9
10. R Ki (nM)
199
311
193
37
Fragments Containing 6,5-Fused Heterocycles
R Ki (nM)
269
278
432
160
298
H
N
H
N
N
H
H
N
N
Me
N
Me
O
P4 Site Fragment Hits: indoles
H
N
O
HN
O
S
O
O
HN
O
R
Cl
• Flat SAR with indoles irrespective of attachment point
• Nonplanar geometry at the linking position preferred
10
H
N
O OH H
N
NH
O
118
H
N
H
N
11. R Ki (nM)
148
139
782
90
306
120
Fragments Containing 5-Membered Heterocycles
N
Bn
N
Ph
N
Me
N
H
O
O
P4 Site Fragment Hits: 5-
membered heterocycles
R Ki (nM)
842
860
2553
907
N
N
Bn
N
S
N
O
O
N
H
N
O
HN
O
S
O
O
HN
O
R
Cl
• H-bonding donor not suitable for P4 site
• Residues with additional hetero-atom not tolerated
11
N
O
HO Ph
N
O
HO Bn
N
N
HO
Bn
118
12. 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
H
N
O
X HN
O
S
O
O
HN
O
R
Cl
OHHN N
P2
P4
4.8Å
12
HN N
F3CF2C
OH
13. Fragments Containing Aliphatic Residues
R Ki (nM)
55
16
66
87
CH3 209
CF3 116
26
10
• Aliphatic residues favored at P4 site
H
N
O
Cl HN
O
S
O
O
HN
O
R
Cl
13
14. Aromatic Spacers
X Ar Ki (nM)
H 361
Cl 91
H 335
Cl 116
N
O
H
N
O
HN
O
Cl
S
O
Ar
O
X
Cl Me 209
H
N
O
HN
O
Cl
S
O
O
14
15. 7-Aryl Group Optimization
H
N
O
X HN
O
S
O
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
16. 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
H
N
O
F3C
H
N
O
N
H
N
O
O
H
N
O
N
N
H
N
N
H
N
N
H
• Highly potent Mcl-1 inhibitors
• Still no cell efficacy: highly
protein bound, poor aqueous
solubility and permeability
17
*FITC-SM: Small molecule FITC probe
Spacer R
H
N
O
HN
O
Cl
S
O
O
Spacer R
Cl
NN
17. 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
18
18. 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
H
N
NH
O
O
Cl
O
HO
H
N
HO
O
O
Cl
Inhibitor Design Evolution
Binding interface
Expansion
Ki = 570 µM
Old Lead
19. 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%
19
20. 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
20
NCI
Bill Moore
Craig Thomas
Damien Duveau
