Post on 20-Apr-2020
Hit Identification and Lead Confirmation in the Discovery of Drugs Targeting
Bromodomain Proteins
Liu Liu, Shaomeng Wang
Department of Internal Medicine and Comprehensive Cancer CenterUniversity of Michigan, Ann Arbor, MI 48109
Bromodomain (BRD) proteins
Family of Chromatin modification “Reader” proteins Recognizing acetylated histone lysine and recruiting
“Effectors” (Chromatin modifiers and remodeling enzymes)
Critical to maintain proper epigenetic regulationDysfunction linked to a large number of human diseasesTotal 46 human proteins containing 61 BRDs identified
8
9
36
21
4
1
3
1
4
12 2
TRIM24
TRIM28
p300
MLL1BRG1BRM
BRDT
BRD2
BRD3
BRD4
TRIM33
CBP
PB1ATAD2
Number of diseases indications
BRDs involved in various diseases
BET
BET
BET: bromodomain and extra-terminal
BET Bromodomain (BRD) proteins
BRD4 is an activator of c-Myc, a master regulator of diverse cellular functions and a long-time compelling therapeutic target
BRD3 and 4 fuse with NUT in NUT midline carcinomas BET Bromodomain proteins cooperate with MLL (MLL1)
fusion proteins in gene activation in acute leukemia (BRD4) Beyond oncogenesis (BRD3-4), these proteins have potential
roles in other therapeutic areas, including viral infections (BRD2/4), cholesterol regulation (BRD2), inflammation(BRD2/4) and spermatogenesis (BRDT), among others.
Small molecule inhibitors of BET BRDs also selectively suppresses other genes important in cancers, such as Bcl-2and some NF-κB-dependent genes.
Therapeutic potentials of BET-BRD inhibitors in cancers
BRD inhibitors have been shown to be effective in the following tumor types
– NUT midline carcinomas with BRD-NUT fusion protein– Acute leukemia with MLL1 fusion protein– Multiple myeloma (Delmore, et al, Cell, 2001, 146 (6)
904-917)– Lung cancer (Lockwood, et al, PNAS, 2012, 109 (47),
19408–19413)– Breast, prostate and head-neck cancers (our own data)
BET-BRD inhibitors available
MWAffinities to BET-BRDs
BRD2BD1
BRD2BD2
BRD3BD1
BRD3BD2
BRD4BD1
BRD4BD2
(+)JQ-1 457 128(1) 59(1) 82(1) 49(1) 90(1)
IBET762 424 81(1, 2) 70(1, 2) 53(1, 2)
IBET151 415 20(2, 3) 100(2, 3)
Bromosporine 404 410(4) 290(4)
PFI-1 347 123(5) 98(4) 136(1) 303(1)
PFI-1(1) 108 144 80.0 76.3 47.4 195Abbot CMPD 4 409 1.4(6) 2.0(6)
(1) ITC(2) BRD proteins with both BD1 and BD2(3) SPR(4) ALPHA screen IC50s(5) Octet RED96(6) TR-FRET (Ki)
Develop new classes of BET-BRD inhibitors with improved affinities, cellular activities, in vivo efficacy and drug properties
Identify probe molecules for other BRDs with promising therapeutic potentials, which can be used as scaffolds for developing tool molecules and clinical candidates
Rationally design and synthesize a small probe compound library (MW between 133-300) based on existing inhibitors and crystal structures (29 high resolution structures available)
Screen this library to identify new scaffolds for inhibitors targeting both BET and other BRD subfamilies
Obtain high-resolution co-crystal structures of our lead compounds with BRD proteins followed by optimization based on SAR
Determine affinities to all BETs and selectivities to other BRDs Evaluate cellular activities, Mechanism of Action, and in vivo
efficacy
New BRD inhibitors discovery and optimization
Screening small probe molecules against BRD proteins
Natural ligands (e.g. the acetylated histone peptides) of most BRDs are largely unknown or too weak to be utilized
http://www.discoverx.com/services/drug-discovery-development-services/bromoscan-epigenetic-profiling
Bromoscan from DiscoveRx
Label free technologies: ThermoFluor, NMR: semi-quantitative, requiring relatively high
concentrations for both compounds and proteins ITC: too low throughput, large amount of proteins Optical: SPR, BLI (Octet RED96)
Higher throughput, faster workflow Reads 8 channels simultaneously so wide concentration ranges can be
run in parallel Multiple assay conditions can be used simultaneously Analyzes more samples in a shorter timeframe than Biacore Assays can be developed in hours and days
The Octet is fluidics-free so there is no leaking, or clogging to deal with The Octet can analyze samples that are not feasible on a SPR: phage
samples, cell lysates, culture media, 100% serum, etc. Octet assays are not affected by changes in the refractive index of the buffer
used leading to significantly higher tolerance to DMSO The Octet replaces ELISA assays with a single-step, no wash assay The Octet uses a non-destructive, dip-and-read measurement method that
allows for full protein/sample recovery after analysis Operational advantages (lower maintenance and cost)
The advantages of Octet RED96 system over SPR
systems
Influence of BSA and Tween-20
BRD4 BD2/PFI-1 Super Streptavidin (SSA) sensors Inactive blocked Streptavidin and buffer as double references
0.1% BSA 0.01% Tween-20
DMSO influence the interaction between BET-BRDs and inhibitors
DMSO controlled lower than 0.5%
O N
N
Cl
NN
NHO
NH
OO
SO3H
NH2
NH2
SO3HO
O
FAM-762
KD values of reference compounds to BRD4 BD1-2 determined by BLI
KD = 418nM
KD = 42nM
CMPDs IBET-762 PFI-1
MW 424 374
BRD4BD1
Octet RED96 89 131
Published 53* 136
BRD4BD2
Octet RED96 42 418
Published 53* 303* BRD4 BD1-2, both reported data and our own data suggested no significant difference between BD1-2 and BD1 and BD2.
Super Streptavidin (SSA) sensors Inactive blocked Streptavidin (SAB4) and buffer as double references 0.1% BSA, 0.01% Tween-20, 0.5% DMSO
Screening to BRD4BD2
250µM, 0.5% DMSO, finished in a few hours Positive hits: > average of buffer + 3 SD Hit 1, 2, 3 are derivatives from the same scaffold.
Hit 1 Hit 2
Hit 3
Hit confirmation by dose dependence titration
CMPD MW KD (µM) kon (1/Ms) kdis (1/s)
Hit 1 133 89.2 2.21E+02 1.97E-02
Hit 2 210 42.5 5.57E+02 2.37E-02
Hit 3 211 14.9 1.52E+03 2.27E-02
Hit 1 (89µM)
Hit 1
Hit 3 (15µM)
Hit 3
N433
L387I385
W374
P375 F376
BRD4-BD2
N433
L387I385
W374
P375F376
BRD4-BD2
BRD4-BD1 BRD4-BD1
N140
L94L92
W81
P82F83 N140
L94L92
W81
P82F83
Docking of hit compounds to BRD4 BD1 and BD2
Co-crystal structures of BRD4 BD2 complexed with lead compound
Co-Crystal structure of Lead 1complexed with BRD4 BD2 (1.4 Å)
Modifications of hit 3 leading to the discovery of lead compound 1 with sub-µM affinities to BETs
KD values of optimized BRD inhibitors to BRD2-4 BD1 and BD2
Inhibitors MW
KD SD (nM)*
BRD2 BRD3 BRD4
BD1 BD2 BD1 BD2 BD1 BD2
IBET-762 424 12911 538 8517 528 8932 426
CMPD 1 415 342 246 189 185 406 287
CMPD 2 413 264 204 183 214 299 132
CMPD 3 450 412 369 218 4218 6018 3611
CMPD 4 423 348 114 177 111 2715 104
* SD obtained from three independent experiments
More than 50 compounds of this class were synthesized with significantly improved affinities.
Ki values to BRD2/4 BD2 determined by competitive FP assay
IDBRD2 BD2 BRD4 BD2
IC50 (nM) Ki (nM) IC50 (nM) Ki (nM)
IBET151 29839 6614 451111 8820
IBET762 14624 2611 14922 218
CMPD 1 28234 386 15915 N/A
CMPD 2 21232 269 25630 327
CMPD 3 33579 6620 379123 6925
CMPD 4 9831 N/A 13130 N/A
O N
N
Cl
NN
NHO
NH
OO
SO3H
NH2
NH2
SO3HO
O
FAM-762
FAM labeled IBET762 was used as tracer.
Only assays for BD2 proteins are available.
Assays for BD1 are not available due to too low dynamic range.
Ki of CMPD 4 can’t be accurately determined since they are lower than assay limits.
Comparison between KD from BLI (Octet RED96) and Ki from FP assays
BRD2 BD2 BRD3 BD2 BRD4 BD2
Ki (nM)(FP)
KD (nM)Octet
RED96
Ki (nM)(FP)
KD (nM)Octet
RED96
Ki (nM)(FP)
KD (nM)Octet
RED96
IBET-762 26±11 538 16±4 528 20±8 426
CMPD 1 38±6 246 21±5 185 8±3 287
CMPD 2 26±9 204 12±3 214 32±7 132
CMPD 3 66±20 369 41±26 4418 69±25 3611
CMPD 4 N/A 114 N/A 111 N/A 104
Compounds with low nM affinities determined by Octet RED96
BRD2 BD2 BRD3 BD2 BRD4 BD2
CMPD 5 12.74.2 13.92.0
CMPD 6 4.10.7 4.50.9 14.41.1
CMPD 7 3.50.7 1.50.3 3.60.1
CMPD 7/BRD3BD2
Very low dissociation rates (koff < 10-3 s-1) Column format required
Binding affinities to other BRD Proteins
KD (nM) SelectivityBRD4
BD2/CREBBPBRD4 BD2 ADAT2A ADAT2B ASH1L CREBBP*
IBET-762 426 > 5000** > 5000 > 5000 > 5000 > 100
CMPD 1 287 > 5000 > 5000 > 5000 4758 170
CMPD 2 132 > 5000 > 5000 > 5000 1510400 117
CMPD 3 4014 > 5000 > 5000 > 5000 39591766 100
CMPD 4 104 > 5000 > 5000 > 5000 47982219 470
*CREBBP has the closest structural and sequence homology to BET BRD proteins** Compounds precipitate when concentration higher than 10 µM with 0.5% DMSO.
Cellular activities of selected BET-BRD inhibitors
ID
Cell growth assay IC50 (nM)
MV4;11 MOLM-13 AML-2 K562
MLL-AF4 MLL-AF9 MLL-AF6 BCR-ABL
IBET151 162112 22852 8131 >2000
IBET762 9345 24158 12754 >2000
CMPD 1 2310 7812 7421 >2000
CMPD 2 209 6614 5030 >2000
CMPD 3 3415 14431 10062 >2000
CMPD 4 10 74 72 460
CellTiter-Glo® Luminescent Cell Viability AssayData are meanSD (n=3-4)
Cellular Mechanism of Action (MOA)
AML-2 AML Cell Line with MLL1 Fusion Protein
µM 0 0.1 0.3 1 0.1 0.3 1 0.1 0.3 1 0.1 0.3 1
I-BET-762 CMPD 1 CMPD 2 CMPD 3
0 0.1 0.3 1 0.1 0.3 1 0.1 0.3 1 0.1 0.3 1
I-BET-762 CMPD 1 CMPD 2 CMPD 3
PARP
Cas-3
c-Myc
Actin
Bcl-2
40 h 60 h
BRD inhibitors induce down-regulation of c-Myc and cleavage of PARP in AML-2 cell line
Similar effects seen in Molm-13 AML cell line with MLL1 Fusion Protein
60 h0 0.1 0.3 1 0.1 0.3 1 0.1 0.3 1 0.1 0.3 1
I-BET-762 CMPD 1 CMPD 2 CMPD 3
PARP
Cas-3
p21
c-Myc
Actin
K562 without MLL1 Fusion Protein
BRD inhibitors do not induce down-regulation of c-Mycand cleavage of PARP in K562 cell line
Cellular Mechanism of Action
Antitumor activity of BRD inhibitors in MV4;11 model
CMPD6 has good microsomal stability (T1/2 >50 min) in human (58 min), mouse (57 min) and rat (>60 min) microsomes.
Summary
Novel chemical scaffolds for BET subfamily BRD protein inhibitors were successfully identified by screening low MW (133-300) probe compound library utilizing label free Octet RED96 assay
New classes of BET subfamily BRD protein inhibitors were developed based on rational design and SAR based optimization
3-10 times more potent than GSK clinical trial IBET-762 in both binding and cell viability assays
Good microsomal stability Comparable tumor growth inhibition than GSK IBET
compounds
Medicinal chemistryDr. Yujun ZhaoXu RanDr. Yongqiang ZhuHacer Karata
Biology and in vivo modelDr. Longchuan BaiDonna McEachern
Computational simulationDr. Chao-Yie Yang
X-ray crystallography core facility of the Life Science Institute at U of MichiganDr. Jennifer MeagherDr. Jeanne Stuckey
Pharmocokinetics (PK) core facility at U. of MichiganDr. Duxin SunDr. Xiaoqin Li
Acknowledgement