28.09.2017

Takeda and SGC Announce a Collaboration Agreement Using Patient Tissue-Based Assays for Clinical Target Validation in Irritable Bowel Disease

by: SGC

Osaka, Japan, San Diego, Calif., USA, and Stockholm, Sweden, September 28, 2017— Takeda Pharmaceutical Company Limited (“Takeda”) (TSE: 4502), Karolinska Institutet (“KI”) and The Structural Genomics Consortium (“SGC”) today announced a combined pre-competitive and proprietary collaboration to discover and validate new potential intervention points for the treatment of Inflammatory Bowel Disease (IBD).

16.06.2017

SGC releases TEPs to support research into new drug discovery targets

by: SGC

June 16th, 2017- SGC’s Target Enabling Package (TEP) Evaluation Group has approved five new TEPs for protein targets related to cancer, metabolic diseases and neuropsychiatric disorders.

TEPs enable scientists to research understudied proteins that have been genetically linked to human disease. The SGC generates and disseminates structural data, assays and other key research information and tools on these proteins to the scientific community.

The five TEPs recently released focus on the following genes:

12.06.2017

New support for Structural Genomics Consortium and open science

by: SGC

Left to right: Dr. Martin Osmond, CEO and Scientific Director of the CHEO Research Institute; Marc LePage, President and CEO, Genome Canada; Jennifer Chan, VP, Policy and External Affairs, Merck Canada; Hon. Reza Moridi, Ontario Minister of Research, Innovation and Science; Dr. Cheryl Arrowsmith, Chief Scientist, Structural Genomics Consortium; Hon. Kirsty Duncan, federal Minister of Science; David McGuinty, MP, Ottawa South (Photo courtesy of Genome Canada).

$33 million will drive scientific discoveries into potentially life-saving cures for patients

GSK4027 for PCAF and GCN5 Bromodomains

This probe is available from Sigma and Cayman Chemical. 

Its negative control (GSK4028) is available for purchase from Sigma.

  • overview
  • properties
  • references
overview
Probe Negative control

 

GSK4027

 

GSK4028

p300/CREB binding protein associated factor (PCAF/KAT2B) and general control non-derepressible 5 (GCN5/KAT2A) are multidomain proteins that have been implicated in retroviral infection, inflammation pathways and cancer development. However, outside of viral replication, little is known about the dependence of these effects on the C-terminal bromodomain.  GSK4027 is as a chemical probe for the PCAF/GCN5 bromodomain and  GSK4028 is the enantiomeric negative control. The probe was optimized from a weakly potent, non-selective pyridazinone hit to deliver high potency for the PCAF/GCN5 bromodomain, high solubility, cellular target engagement and ≥18000-fold selectivity over the BET family, together with ≥70 fold selectivity over the wider bromodomain families.

Potency

PCAF IC50 40 nM in a TR-FRET binding competition assay using truncated PCAF bromodomain and a fluorescently tagged bromodomain ligand.

PCAF Ki 1.4 nM in a BROMOscan assay run at DiscoverX.

GCN5 Ki 1.4 nM in a BROMOscan assay run at DiscoverX.

3D structure

Co-crystal structure with GCN5: PDB ID 5MLJ

Selectivity Bromodomains

>70 fold selectivity over other bromodomain targets including BRPF3 (100 nM), BRD1 (110 nM), FALZ (130 nM) and BRPF1 (140 nM) In BROMOscan assay run at DiscoverX.

Non-family targets

GSK internal enhanced, cross-screening panel (eXP); a full curve against 53 biochemical and phenotypic assays did not reveal any off-target binding <3 µM.

Cellular Potency 

IC50 60 nM in Promega NanoBRET assay, measuring displacement of NanoLuc-tagged full length PCAF from Halo-tagged histone H3.3 in HEK293 cells.

Cytoxicity assay: 

Cellular health assay looking at mitochondrial integrity, nuclear size and membrane permeability found no changes up to 200 µM.

properties
Probe Negative control

 

GSK4027

 

GSK4028

Physical and chemical properties for GSK4027
Molecular weight376.1
Molecular formulaC17H21BrN4O
IUPAC name4-bromo-2-methyl-5-(1-methyl-5-phenyl-piperidin-3-ylamino)-2,3-dihydro-pyridazin-3-one
MollogP2.226
PSA41.29
No. of chiral centres2
No. of rotatable bonds3
No. of hydrogen bond acceptors4
No. of hydrogen bond donors1



 

Physical and chemical properties for GSK4028 (Negative Control)
Molecular weight376.1
Molecular formulaC17H21BrN4O
IUPAC name4-bromo-2-methyl-5-(1-methyl-5-phenyl-piperidin-3-ylamino)-2,3-dihydro-pyridazin-3-one
MollogP2.226
PSA41.29
No. of chiral centres2
No. of rotatable bonds3
No. of hydrogen bond acceptors4
No. of hydrogen bond donors1
  • SMILES:
  • GSK4027: O=C1C(Br)=C(N[C@H](C2)CN(C)C[C@H]2C3=CC=CC=C3)C=NN1C
  • GSK4028: O=C1C(Br)=C(N[C@@H](C2)CN(C)C[C@@H]2C3=CC=CC=C3)C=NN1C
  • InChI:
  • GSK4027: InChI=1S/C17H21BrN4O/c1-21-10-13(12-6-4-3-5-7-12)8-14(11-21)20-15-9-19-22(2)17(23)16(15)18/h3-7,9,13-14,20H,8,10-11H2,1-2H3/t13-,14+/m0/s1
  • GSK4028: InChI=1S/C17H21BrN4O/c1-21-10-13(12-6-4-3-5-7-12)8-14(11-21)20-15-9-19-22(2)17(23)16(15)18/h3-7,9,13-14,20H,8,10-11H2,1-2H3/t13-,14+/m1/s1
  • InChIKey:
  • GSK4027: VZAFGXCWAWRULT-UONOGXRCSA-N
  • GSK4028: VZAFGXCWAWRULT-KGLIPLIRSA-N
selectivity profile
in vitro potency
cell based assay data
references

Reference

Humphreys, P. G.; Bamborough, P.; Chung, C. W.; Craggs, P. D.; Gordon, L.; Grandi, P.; Hayhow, T. G.; Hussain, J.; Jones, K. L.; Lindon, M.; Michon, A. M.; Renaux, J. F.; Suckling, C. J.; Tough, D. F.; Prinjha, R. K. Discovery of a potent, cell penetrant, and selective p300/CBP-associated factor (PCAF)/general control nonderepressible 5 (GCN5) bromodomain chemical probe. J. Med. Chem. 2017, 60 (2), 695-709.

pk properties
co-crystal structures
synthetic schemes
materials and methods

BAY-6035 A potent, peptide-competitive chemical probe for SMYD3

This probe is available from Sigma, Tocris and Cayman Chemical.

  • overview
  • properties
overview
Probe Negative control

 

BAY-6035

 

BAY-444

A collaboration between Bayer AG and the SGC has resulted in the discovery of BAY-6035, a potent, peptide-competitive chemical probe for SMYD3. BAY-6035 has a unique chemotype relative to the published SMYD3 inhibitor, EPZ031686. BAY-6035 inhibits in vitro methylation of MEKK2 peptide with IC50 = 88 nM and has more than 100-fold selectivity over other histone methyltransferases. BAY-6035 inhibits the methylation of MEKK2 in cells with IC50 = 70 nM. A control compound, BAY-444, has also been developed. BAY-444 inhibits the in vitro methylation of MEKK2 with IC50 = 23 micromolar.

https://doi.org/10.1177/24725552211019409

properties
Probe Negative control

 

BAY-6035

 

BAY-444

Physical and chemical properties for BAY-6035
Molecular weight396.2
Molecular formulaC22H28N4O3
IUPAC name6-((3-aza-bicyclo[3.1.0]hexan-3-yl)-formyl)-10-((2-cyclopropyl-ethylamino)-formyl)-5-methyl-2,6-diaza-bicyclo[5.4.0]undeca-1(7),8,10-trien-3-one
MollogP2.011
PSA66.79
No. of chiral centres3
No. of rotatable bonds7
No. of hydrogen bond acceptors6
No. of hydrogen bond donors2
Physical and chemical properties for BAY-444 (Negative Control)
Molecular weight410.2
Molecular formulaC23H30N4O3
IUPAC name6-((3-aza-bicyclo[3.1.0]hexan-3-yl)-formyl)-10-(((2-cyclopropyl-ethyl)-methyl-amino)-formyl)-5-methyl-2,6-diaza-bicyclo[5.4.0]undeca-1(7),8,10-trien-3-one
MollogP2.208
PSA58.66
No. of chiral centres3
No. of rotatable bonds7
No. of hydrogen bond acceptors6
No. of hydrogen bond donors1
  • SMILES:
  • BAY-6035: C[C@H]1CC(NC2=C(N1C(N3CC4CC4C3)=O)C=CC(C(NCCC5CC5)=O)=C2)=O
  • BAY-444: C[C@@H]1CC(NC2=C(N1C(N3CC4CC4C3)=O)C=CC(C(N(CCC5CC5)C)=O)=C2)=O
  • InChI:
  • BAY-6035: InChI=1S/C22H28N4O3/c1-13-8-20(27)24-18-10-15(21(28)23-7-6-14-2-3-14)4-5-19(18)26(13)22(29)25-11-16-9-17(16)12-25/h4-5,10,13-14,16-17H,2-3,6-9,11-12H2,1H3,(H,23,28)(H,24,27)/t13-,16?,17?/m0/s1
  • BAY-444: InChI=1S/C23H30N4O3/c1-14-9-21(28)24-19-11-16(22(29)25(2)8-7-15-3-4-15)5-6-20(19)27(14)23(30)26-12-17-10-18(17)13-26/h5-6,11,14-15,17-18H,3-4,7-10,12-13H2,1-2H3,(H,24,28)/t14-,17?,18?/m1/s1)
  • InChIKey:
  • BAY-6035: CKFRXCBNKKOFGO-IGEOTXOUSA-N
  • BAY-444: FNTARNKIMWWTFZ-RWBZWWBESA-N
selectivity profile
in vitro potency
cell based assay data
references
pk properties
co-crystal structures
synthetic schemes
materials and methods
25.04.2017

Game-changing PanDDA method unveils previously hidden 3D structure data

by: SGC

Scientists have utilised Diamond Light Source to develop a new method to extract previously hidden information from the X-ray diffraction data that are measured when resolving the three-dimensional (3D) atomic structures of proteins and other biological molecules.

FM-381 A Chemical Probe For JAK3

This probe is available from Cayman Chemical and Sigma.

Its negative control (FM-479) is available for purchase from Sigma.

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
  • co-crystal structures
overview
Probe Negative control

 

FM-381

 

FM-479

Biology of the JAK3 kinase

Janus kinases (JAKs) belong to the family of cytoplasmic tyrosine kinases. In human, the JAK family itself consist of 4 members (JAK1, JAK2, JAK3 and TYK). JAK family members are multi-domain proteins of about 130 kDa, which are highly homologous with respect to their domain structure and residue conservation within their structured domains.


All JAK family members possess a catalytic kinase domain (KD) located at the C-terminus, an adjacent pseudokinase domain (PKD) flanked by a Src homology 2 (SH2) domain.  The N-terminal FERM (four-point-one, ezrin, radixin and moesin homology) domain (B41) serves to mediate the interaction between the JAK and the cytokine receptor [1]. 

JAK kinases are effectors of the JAK-STAT signaling pathway, which is triggered by ligand binding to a cognate receptor resulting in activation of JAK by phosphorylation of key tyrosine residues within the catalytic domain. After activation, tyrosine resides in the receptor intracellular region are also phosphorylated which triggers recruitment and phosphorylation of the principal downstream effectors, the STATs. Phosphorylated STATs dimerize and translocate to the nucleus where they initiate transcription of specific responsive genes [1, 2, 3].
Contrary to the other JAK family members, JAK3 expression is restricted to the hematopoietic system, where it plays a specific role in the development of immune-competent cells [4]. This key function of JAK3 has been confirmed by loss-of-function mutations of JAK3, which cause severe combined immunodeficiency syndrome (SCID) [5].  Aberrant activation in kinase domain has been described in lymphoproliferative disorders (T-ALL; T-PLL). JAK3 is required for cytokine signalling downstream of receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 and it acts via activation of the gamma chain receptors (γc). JAK1 and JAK3 co-localise on cytokine receptor dimers suggesting that dual JAK1 and JAK3 inhibition may be required for efficient suppression of cytokine signaling [6].  In order to study JAK3 specific functions we developed in collaboration with the laboratory of Stefan Laufer a JAK3 specific reversible covalent inhibitor.
 

We recommend using FM-381 at 100-300 nM for specific JAK3 inhibition. The inactive control FM-479 has no activity on JAK3 or other kinases when used at similar concentration.
As a non-covalent control inhibitor we recommend NIBR3049 (Novartis) at 1 µM (this potent reversible compound is selective for JAK3 within the JAK family, but shows also potent inhibition of other kinases such as GSK3, PKCα, PCKθ).

FM-381: A CHEMICAL PROBE FOR JAK3

An additional non-covalent control compound (NIBR3049) has been donated by Novartis [7]. This inhibitor is highly selective for JAK3 within the JAK kinase family but inhibits also other kinases with considerable activity including GSK3, PKCα, PKCθ. NIBR3049 is therefore not suitable as an independent JAK3 specific probe. 

Co-crystal structure

Details of the co-crystal structure of FM-381 with the kinase domain of JAK3 kinase in its reversible binding mode, click on the 'Co-Crystal structures' tab above for more details.

Potency Against Target Family

FM-381 is a potent covalent reversible inhibitor of JAK3 targeting the unique Cys909 at the gatekeeper (GK) position +7 in JAK3. FM-381 exhibited apparent JAK3 IC50 values of 0.154 nM, with 410, 2700 and 3600-fold selectivity over JAK1, JAK2 and TYK2, respectively.

aIC50 values were calculated from the results of a radiometric assay. Data were obtained as 5-dose singlicate IC50 with 10-fold serial dilution starting at 1 µM. [ATP] = 10 µM

FM-381 shows good selectivity over other kinases with a Cys in a similar position (GK+7)


 

Selectivity within the kinase family

Selectivity of FM-381 was assessed in an activity assay against a panel of 410 kinases (ProQinase) at 100 nM and the compound was found to be exquisitely selective (800 fold selective over nearest kinase). Some weak interactions were observed in vitro at higher compound concentration (500 nM)

Selectivity profile of FM-381 assessed against 410 protein kinases (ProQinase). No significant activity was detected at 100 nM concentration (left panel). At 500 nM inhibitor concentration weak activity was detected (between 50 and 90% inhibition, as indicated by spheres).

Selectivity Beyond Target Famil

FM-381 was found to be inactive in a selectivity panel of frequently hit BRDs (BRD4, BRPF, CECR, FALZ, TAF1, BRD9). Strongest hits was 500 nM for TAF1@2

Cellular Activity

FM-381 shows an apparent EC50 of 100 nM in a dose dependent BRET assay and blocks IL2 stimulated (JAK3/JAK1 dependent) STAT5 phosphorylation at 100 nM, but not JAK3 independent IL6  (JAK1/2/TYK dependent) stimulated STAT3 signalling in Human CD4+ T cells up to 1 µM.

properties
FM-381

2-cyano-3-(5-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N,N-dimethylacrylamide

Smiles: CN(C(/C(C#N)=C/C(O1)=CC=C1C2=NC3=CN=C(NC=C4)C4=C3N2C5CCCCC5)=O)C

For SDF click here

Physical and chemical properties
Molecular weight428.20
Molecular formulaC24H24N6O2
IUPAC name2-cyano-3-(5-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N,N-dimethylacrylamide
clogP4.2
PSA71.4
InChiInChI=1S/C24H24N6O2/c1-29(2)24(31)15(13-25)12-17-8-9-20(32-17)23-28-19-14-27-22-18(10-11-26-22)21(19)30(23)16-6-4-3-5-7-16/h8-12,14,16H,3-7H2,1-2H3,(H,26,27)/b15-12+
InChiKeyGJMZWYLOARVASY-NTCAYCPXSA-N
Storage-20 as DMSO stock
DissolutionSoluble in DMSO at least up to 50mM
selectivity profile

Potency against Target

FM-381 is a potent reversible covalent inhibitor of JAK3 with an IC50 of 0.154 nM as shown in enzyme kinetic assays (ProQinase).

  IC50 [nM]a
Compd. JAK1JAK2JAK3TYK2
FM-381 523460.127459

aIC50 values were calculated from the results of radiometric assay. Data were obtained as 5-dose singlicate IC50 with 10-fold serial dilution starting at 1 µM. [ATP] = 10 µM

JAK3 Selectivity
JAK1/JAK3JAK2/JAK3TYK2/JAK3
41027243614

Materials and Methods


Inhibition of JAK3 kinase activity was measured using a FlashPlate™-based radiometric assay (Kinase 410-Profiler, ProQinase). Data were obtained as 5-dose singlicate IC50 with 10-fold serial dilution starting at 1 µM. [ATP] = 10 µM.

Selectivity within Target Family

ProQinase kinase assay panel

FM-381 is highly selective against 410 kinases tested at 10 µM with a selectivity score of 0.002.

 % Inhibition
  100 nM 500 nM
 LRRK2            25          66  
 MAP3K11            26          51  
 SNARK            28         61  
 RPS6KA6            30         60  
 PRK2            30          52  
 BLK            33         43  
 PKC-beta2            39         69  
 RPS6KA2            45          74  
 JAK3              92            97 

Selectivity within Target Family

FM-381 shows no cross-reactivity in a selectivity panel of frequently hit BRDs by HTRF assay (BRD4, BRPF, CECR, FALZ, TAF1, BRD9). Strongest hit was 500 nM for TAF1@2.
 
Control compounds:
Negative control FM-479 showed no significant activity against kinome screened at 1 µM (> 8000 fold IC50).  Highest activity of 82% was reported to binding to the lipid kinase PIK3C2G in DiscoverX Kinomescan). No activity was found in a selectivity panel of frequently hit BRDs by HTRF assay (BRD4, BRPF, CECR, FALZ, TAF1, BRD9) with the exception of TAF1@2 (170 nM) which results in a 1000 fold window to FM-381 on JAK3)
Positive control NIBR3049 is highly selective within the JAK/TYK family.
 

AssayNIBR3049
JAK1 IC50 (nM) 
JAK2 IC50 (nM) 
JAK3 IC50 (nM) 
TYK2 IC50 (nM)		1017 (> 100-fold)
2550 (> 300-fold)
8
8055 (> 1000-fold)

NIBR3049 does show shows potent inhibition of GSK3, PKCa, PKCq, typically seen for maleimide scaffolds. Details have been reported in [7]. The compound is poorly soluble (, 0.004 g/L at pH 6.8 and only moderately cell permeable (see below).

aterials and Methods


ProQinase Selectivity panel:
FM-381 was profiled by using a Kinase 410- Profiler, a FlashPlate™-based radiometric assay (ProQinase https://www.proqinase.com). For lipid kinases and ADP-Glo™ assay technology (Promega) was used. Data were obtained as 5-dose singlicate IC50 with 10-fold serial dilution starting at 1 µM. [ATP] = 10 µM.
 
Binding Assay
Binding assays were performed at DiscoverX coporation in the KINOMEscan assay. Compound binding constants (Kd values) are calculated from duplicate 11-point dose-response curves using Hill equation. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

 

in vitro potency
cell based assay data

The cellular efficacy of FM-381 on JAK3 was assed using a Promega NanoBRET assay demonstrating on-target effect at an apparent EC50 of 100 nM in HeLa cells. The negative control compound FM-479 was negative in this assay and the positive control compound NIBR3049 showed an apparent EC50 of 1 µM. Other Cys containing kinases tested in NanoBRET assay (TEC, BTK) were negative. Only BLK showed some inhibition at micromolar concentrations. Washout experiments demonstrated that FM-381 has a residence time of ~60 min on full length JAK3 in BRET assay [4].


Dose dependent BRET experiment using FM-381 and the 2 control compounds showing displacement of the fluorescent tracer in C-terminally NanoLuc tagged JAK3 in HEK293 cells.

 

Materials and Methods

NanoBRET assay
Dose-response experiments were conducted in HeLa cells expressing NanoLuc fused to the C-terminus of full-length JAK3 using Promega tracer 5 at 2 µM in HEK293 cells in a 96 well format. A detailed description is provided in the supplemental of [4].  
 

references

 
1.  Babon JJ, Lucet IS, Murphy JM, Nicola NA, Varghese LN. The molecular regulation of Janus kinase (JAK) activation. Biochem J 2014;462(1):1-13.
2.  Gurzov EN, Stanley WJ, Pappas EG, Thomas HE, Gough DJ. The JAK/STAT pathway in obesity and diabetes. FEBS J 2016;283(16):3002-3015.
3. Villarino AV, Kanno Y, Ferdinand JR, O'Shea JJ. Mechanisms of Jak/STAT signaling in immunity and disease. J Immunol 2015;194(1):21-27.
4. Forster M, Chaikuad A, Bauer SM, Holstein J, Robers MB, Corona CR, Gehringer M, Pfaffenrot E, Ghoreschi K, Knapp S et al. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket. Cell Chem Biol 2016;23(11):1335-1340.
5. Pesu M, Candotti F, Husa M, Hofmann SR, Notarangelo LD, O'Shea JJ. Jak3, severe combined immunodeficiency, and a new class of immunosuppressive drugs. Immunol Rev 2005;203:127-142.
6. Haan C, Rolvering C, Raulf F, Kapp M, Druckes P, Thoma G, Behrmann I, Zerwes HG. Jak1 has a dominant role over Jak3 in signal transduction through gammac-containing cytokine receptors. Chem Biol 2011;18(3):314-323.
7. Thoma G, Nuninger F, Falchetto R, Hermes E, Tavares GA, Vangrevelinghe E, Zerwes HG. Identification of a potent Janus kinase 3 inhibitor with high selectivity within the Janus kinase family. J Med Chem 2011;54(1):284-288.
 

pk properties
co-crystal structures

The co-crystal structures of FM-381 with the catalytic domain of JAK3 has been refined at a resolution of 1.55 Å resolution. Shown are the details of interaction of FM-381 in its non-covalent binding mode. The pyrrolopyridine hinge binding motive forms the canonical hydrogen bonds with the hinge backbone. The nitrile attached to the reactive double bond induces a pocket by re-orienting two conserved arginine residues (R953 and R911). In this structure (pdb-code: 5LWM) the covalent bond with C908 is not formed. However, the covalent as well as the non-covalent binding mode was evident in a close derivative of (FM-409; pdb-code: 5LWN). The reversible covalent interaction of FM-381 with the C908 resulted in a prolonged target residence time in cells (T1/2 ~60 min).

synthetic schemes
materials and methods
12.01.2017

SGC Scientists Report the First Chemical Probe for PCAF Bromodomain

by: SGC

Oxford, UK, January 12, 2017, Scientists from the Structural Genomics Consortium (SGC) and Oxford University report the first potent and selective chemical probe for the bromodomain of PCAF in an article published in Angewandte Chemie International Edition.

GSK6853 A chemical probe for BRPF1

GSK6853 is available for purchase from Cayman Chemical, Sigma and Tocris.

Its negative control (GSK9311) is available for purchase from Sigma and Tocris (hydrochloride).

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
  • co-crystal structures
  • materials and methods
overview
Probe Negative control

 

GSK6853

 

GSK9311

A chemical probe for the bromodomains of the BRPF (BRomodomain and PHD Finger containing) family of proteins (BRPF1/2/3) has been discovered by the SGC.

BRPF1, BRPF2 (BRD1) and BRPF3 are scaffolding proteins, assembling HAT complexes of the MOZ/MORF family (MOZ, Ybf2/Sas3, Sas2, and Tip60) (1). These MYST complexes have a tetrameric core containing BRPF, the tumour suppressor ING and Eaf6/EPC (enhancer of polycomb)-related scaffold subunits. MYST complexes play crucial roles in DNA repair, recombination, and replication as well as in transcription activation (2,3). MOZ is frequently translocated in AML (acute myeloid leukemia) and is required for HSC proliferation (4).

Two BRPF1 isoforms (isoform A and B) can be generated by alternative splicing. In contrast to BRPF1B, the isoform A harbours a residue insertion in the ZA-loop that prevents binding to acetylated histone peptides (5).

Phylogenetic tree of bromodomains and detailed view at the BRPF family.

GSK6853 has excellent BRPF1 potency with an IC50 of 8 nM in a TR-FRET assay and a KD of 0.3 nM as determined by Bromoscan. In a panel of 48 bromodomains GSK6853 shows a greater than 1600-fold selectivity over all other bromodomains. In addition, GSK6853 exhibits potent binding to full-length endogenous BRPF1 (pIC50 of 8.6 nM) in a chemoproteomic competition binding assay.

A NanoBRETTM cellular target engagement assay evaluating the interaction of BRPF1B with histones suggested IC50 of 20 nM for GSK6853.

Dosage

To minimize the chance of off-target effects we recommend that a concentration of no higher than 1 μM should be used in cell-based assays.

Cellular Activity

In a NanoBRETTM cellular target engagement assay using isolated BRPF1B BRD with NLS and Halo-tagged histone H3.3 BRPF1 showed a dose-dependent displacement from histone H3.3, with cellular IC50 of 20 nM.

Chemoproteomic competition binding assay in HUT-78 cell lysate shows binding to endogenous BRPF-1 with selectivity over BRD3.

Properties

Chrom logD pH 7.42.0
CLND solubility (μg/mL)140
iv CLb (mL/min/kg)/t1/2 (h)107/1.7
F% ip/po (3 mg/kg)85/22
properties
Probe Negative control

 

GSK6853

 

GSK9311

Physical and chemical properties for GSK-6853
Molecular weight409.2
Molecular formulaC22H27N5O3
IUPAC name3-((2-methoxy-phenyl)-formylamino)-7,9-dimethyl-4-(2-methyl-piperazin-1-yl)-7,9-diaza-bicyclo[4.3.0]nona-1,3,5-trien-8-one
MollogP1.779
PSA62.28
No. of chiral centres1
No. of rotatable bonds5
No. of hydrogen bond acceptors6
No. of hydrogen bond donors2
Physical and chemical properties for GSK-9311 (Negative Control)
Molecular weight437.2
Molecular formulaC24H31N5O3
IUPAC name3-(ethyl-((2-methoxy-phenyl)-formyl)-amino)-7,9-dimethyl-4-(2-methyl-piperazin-1-yl)-7,9-diaza-bicyclo[4.3.0]nona-1,3,5-trien-8-one
MollogP2.369
PSA54.93
No. of chiral centres1
No. of rotatable bonds6
No. of hydrogen bond acceptors6
No. of hydrogen bond donors1
  • SMILES:
  • GSK-6853: C[C@@H]1CNCCN1C2=CC3=C(N(C(N3C)=O)C)C=C2NC(C4=C(OC)C=CC=C4)=O
  • GSK-9311: CCN(C1=CC2=C(N(C(N2C)=O)C)C=C1N3CCNC[C@H]3C)C(C4=C(OC)C=CC=C4)=O
  • InChI:
  • >GSK-9311: InChI=1S/C24H31N5O3/c1-6-28(23(30)17-9-7-8-10-22(17)32-5)20-13-18-19(27(4)24(31)26(18)3)14-21(20)29-12-11-25-15-16(29)2/h7-10,13-14,16,25H,6,11-12,15H2,1-5H3/t16-/m1/s1
  • InChIKey:
  • GSK-6853: FQWDVNSBYDXPIO-CQSZACIVSA-N
  • GSK-9311: WFXIHQFRQPGCCR-MRXNPFEDSA-N
selectivity profile

Temperature Shift

The temperature shifts mapped onto the phylogenetic tree using red circles corresponding to ΔTm as indicated in the figure.

BROMO scanTM

Bromodomain selectivity was evaluated in the BROMO scan™ panel (DiscoveRx Corp., Fremont, CA, USA, http://www.discoverx.com). This screen measures competition against reference immobilized ligands for 35 DNA-tagged bromodomains. GSK6853 is more than 1600 selective for BRPF1B over tested bromodomains.

GSK6853 was tested in a panel of 48 unrelated targets only weak off-target activity has been observed compared to the activity on BRPF1B.

No potent off-targets identified

in vitro potency
cell based assay data

In a NanoBRETTM cellular target engagement assay using isolated BRPF1B BRD with NLS and Halo-tagged histone H3.3 BRPF1 showed a dose-dependent displacement from histone H3.3, with cellular IC50 of 20 nM, but no effect of the less active control GSK9311.

Chemoproteomic competition binding assay in HUT-78 cell lysate shows binding to endogenous BRPF-1 with selectivity over BRD3 (8).

references

Work on this probe has been published in 'GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain’.

  1. Laue K., et al., The multidomain protein Brpf1 binds histones and is required for Hox gene expression and segmental identity. Development 2008, 135, 1935–194610
  2. Vezzoli A. et al., Molecular basis of histone H3K36me3 recognition by the PWWP domain of Brpf1. Nat. Struct. Mol. Biol. 2010, 17, 617–61910
  3. Hibiya K. et al., Brpf1, a subunit of the MOZ histone acetyl transferase complex, maintains expression of anterior and posterior Hox genes for proper patterning of craniofacial and caudal skeletons. Dev Biol. 2009, 329, 176–190
  4. Johnstone R. W., et al., Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discovery 2014, 13, 673–691
  5. Meier C. M., et al., Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation. ACS Chem Biol. 2017, 12, 2619–2630
  6. Filippakopoulos P. et al., Histone recognition and largescale structural analysis of the human bromodomain family. Cell. 2012, 149, 214-231.
  7. Philpott M., et al., Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery. Mol Biosyst. 2011, 10, 2899-908.
  8. Bamborough P. et al., GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain. ACS Med Chem Lett. 2016, 7, 552-7. doi: 10.1021/acsmedchemlett.6b00092. eCollection 2016 Jun 9.
pk properties
co-crystal structures

4 chains in asymmetric unit (not yet released on PDB)

X-ray structure of BRPF-1 (cyan) with GSK6853, overlayed with BRPF-2 apo (magenta) (not yet released on PDB)

synthetic schemes
materials and methods

NanoBRET assay

HEK293 cells (8 x 105) were plated in each well of a 6-well plate and co-transfected with Histone H3.3-HaloTag (NM_002107) and NanoLuc-BRPF1 isoform 1 (P55201-1) bromodomain amino acids 625-735 or isoform 2 (P55201-2) bromodomain amino acids 625-741. Isoform 2 has an insertion 660 -> SEVTELD in the bromodomain. Twenty hours post-transfection cells were collected, washed with PBS, and exchanged into media containing phenol red-free DMEM and 4% FBS in the absence (control sample) or the presence (experimental sample) of 100 nM NanoBRET 618 fluorescent ligand (Promega). Cell density was adjusted to 2 x 105 cells/ml and then re-plated in a 96-well assay white plate (Corning Costar #3917). Inhibitors were then added directly to media at final concentrations between 0-33 μM and the plates were incubated for 18hrs at 37 °C in the presence of 5% CO2. NanoBRET furimazine substrate (Promega) was added to both control and experimental samples at a final concentration of 10 μM. Readings were performed within 5 minutes using the CLARIOstar BMG) equipped with 450/80 nm bandpass and 610 nm longpass filters with a 0.5sec reading setting. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/450 nm for experimental samples (i.e. those treated with NanoBRET fluorescent ligand) subtracted by the emission at 610 nm/450 nm for control samples (not treated with NanoBRET fluorescent ligand). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000.

Chemoproteomic assay with dose-dependent competition

Chemical probe was spiked into HUT-78 nuclear and chromatin extracts and incubated for 45 min at 4 °C. Derivatized sepharose beads (35 μL beads per sample) were equilibrated in lysis buffer and incubated with cell extract pre-incubated with compound. Beads were washed with lysis buffer containing 0.2 % NP-40 and eluted with 2x SDS sample buffer supplemented with DTT. Proteins were alkylated with iodoacetamide, separated on 4–12 % NuPAGE (Invitrogen), and stained with colloidal Coomassie.

GSK8814 A chemical probe for ATAD2 and ATAD2B

This probe and its negative control are available from Sigma.

  • overview
  • properties
  • references
overview
Probe Negative control

 

GSK8814

 

GSK8815

ATAD2 and ATAD2B are chromatin remodelling factors and modulate the expression of multiple tumour cell growth factors. ATAD2 overexpression correlates with poor outcomes in several cancers. The bromodomain module of ATAD2 has been shown to be essential for its association with acetylated chromatin and presumable function.
GSK8814 is a chemical probe for the ATAD2/2B bromodomain, with a binding constant pKd=8.1 as measured by ITC (Bamborough et al, 2016). GSK8814 displaces acetylated H4 peptide from the ATAD2 bromodomain with pIC50 =7.3 and is also active in BROMOscan with pKi=8.9. It is more than 100 fold selective over all other bromodomains in the BROMOscan. Importantly, it is more than 1000 fold selective over BRD4. GSK8814 shows cellular target engagement with an EC50 of 2 µM in a NanoBRET assay evaluating the interaction of the NanoLuc-ATAD2 bromodomain with histone H3.3-HaloTag.
GSK8815 is a companion control compound with strongly reduced potency against ATAD2 (pKd=5.5).p>

properties
Probe Negative control

 

GSK8814

 

GSK8815

Physical and chemical properties for GSK8814
Molecular weight527.3
Molecular formulaC28H35F2N5O3
IUPAC name10-(3-((4,4-difluoro-cyclohexyl)-methoxy)-5-methoxy-piperidin-4-ylamino)-4-methyl-7-(5-methyl-pyridin-3-yl)-2,9-diaza-bicyclo[4.4.0]deca-1(10),4,6,8-tetraen-3-one
MollogP3.212
PSA78.42
No. of chiral centres3
No. of rotatable bonds7
No. of hydrogen bond acceptors7
No. of hydrogen bond donors3
Physical and chemical properties for GSK8815 (Negative Control)
Molecular weight527.3
Molecular formulaC28H35F2N5O3
IUPAC name10-(3-((4,4-difluoro-cyclohexyl)-methoxy)-5-methoxy-piperidin-4-ylamino)-4-methyl-7-(5-methyl-pyridin-3-yl)-2,9-diaza-bicyclo[4.4.0]deca-1(10),4,6,8-tetraen-3-one
MollogP3.212
PSA78.42
No. of chiral centres3
No. of rotatable bonds7
No. of hydrogen bond acceptors7
No. of hydrogen bond donors3
  • SMILES:
  • GSK8814: CC1=CC2=C(NC1=O)C(N[C@@H]3[C@@H](OC)CNC[C@H]3OCC4CCC(F)(F)CC4)=NC=C2C5=CN=CC(C)=C5
  • GSK8815: CC1=CC2=C(C3=CN=CC(C)=C3)C=NC(N[C@H]4[C@H](OC)CNC[C@@H]4OCC5CCC(CC5)(F)F)=C2NC1=O
  • InChI:
  • GSK8814: InChI=1S/C28H35F2N5O3/c1-16-8-19(11-31-10-16)21-12-33-26(24-20(21)9-17(2)27(36)35-24)34-25-22(37-3)13-32-14-23(25)38-15-18-4-6-28(29,30)7-5-18/h8-12,18,22-23,25,32H,4-7,13-15H2,1-3H3,(H,33,34)(H,35,36)/t22-,23+,25+/m0/s1
  • GSK8815: InChI=1S/C28H35F2N5O3/c1-16-8-19(11-31-10-16)21-12-33-26(24-20(21)9-17(2)27(36)35-24)34-25-22(37-3)13-32-14-23(25)38-15-18-4-6-28(29,30)7-5-18/h8-12,18,22-23,25,32H,4-7,13-15H2,1-3H3,(H,33,34)(H,35,36)/t22-,23+,25+/m1/s1
  • InChIKey:
  • GSK8814: YDPMMWAOCCOULO-JBRSBNLGSA-N
  • GSK8815: YDPMMWAOCCOULO-CUYJMHBOSA-N
selectivity profile
in vitro potency
cell based assay data
references

Bamborough P, Chung CW, Demont EH, Furze RC, Bannister AJ, Che KH, Diallo H, Douault C, Grandi P, Kouzarides T, Michon AM, Mitchell DJ, Prinjha RK, Rau C, Robson S, Sheppard RJ, Upton R, Watson RJ. A Chemical Probe for the ATAD2 Bromodomain. Angew Chem Int Ed Engl. 2016, 55(38):11382-6.
 

pk properties
co-crystal structures
synthetic schemes
materials and methods
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