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.

2

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

14

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

17

*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

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

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%

19

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

Funding

•  Vanderbilt University•  Pioneer Award•  NCI Experimental Therapeutics (NExT) Program•  Career Development Award/Lung SPORE•  Breast Cancer SPORE

21

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

16