UNC1999 A chemical probe for EZH2/1

The probe UNC1999 is available from Cayman Chemical, Sigma and Tocris.

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
overview
Probe Negative control

 

UNC1999

 

UNC2400

Biotinylated UNC1999

UNC2399

The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. A collaboration between the SGC and the Center for Integrative Chemical Biology and Drug Discovery (CICBDD) at the University of North Carolina at Chapel Hill has resulted in the discovery of UNC1999. UNC1999 inhibits EZH2 with an IC50 of 2nM and is over 1000-fold selective for other HMTs except EZH1 (22-fold selectivity). UNC1999 inhibits H3K27 methylation in MCF10A cells with an IC50 of 124nM as measured by immunofluorescence. A dimethylated version, UNC2400, was also used as a negative control compound in key experiments.

properties
UNC1999

N-[(6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl]-1-(propan-2-yl)-6-{6-[4-(propan-2-yl)piperazin-1-yl]pyridin-3-yl}-1H-indazole-4-carboxamide

Click here to download the SD file

Physical and chemical properties
Molecular weight569.74
Molecular formulaC33H43N7O2
IUPAC nameN-[(6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl]-1-(propan-2-yl)-6-{6-[4-(propan-2-yl)piperazin-1-yl]pyridin-3-yl}-1H-indazole-4-carboxamide
logP4.01
PSA95.39
  • SMILES
  • CCCC(C=C(N1)C)=C(CNC(C2=CC(C3=CC=C(N4CCN(C(C)C)CC4)N=C3)=CC5=C2C=NN5C(C)C)=O)C1=O
  • InChi
  • InChI=1S/C33H43N7O2/c1-7-8-24-15-23(6)37-33(42)28(24)19-35-32(41)27-16-26(17-30-29(27)20-36-40(30)22(4)5)25-9-10-31(34-18-25)39-13-11-38(12-14-39)21(2)3/h9-10,15-18,20-22H,7-8,11-14,19H2,1-6H3,(H,35,41)(H,37,42)
  • InChiKey
  • InChIKey=DPJNKUOXBZSZAI-UHFFFAOYSA-N
selectivity profile

UNC1999 is selective for EZH2 over 15 other methyltransferases and proteins in other target classes

  • Less than 20% inhibition on 50 kinase targets @ 10 µM.
  • Less than 50% inhibition on 40 7TM targets @ 10 µM.
  • Greater than 50% inhibition of 4 7TM targets @ 10 µM.
    • H3 (Ki 300 nM), NET (Ki 1500nM), Sigma 1(Ki 4700 nM), Sigma 2 (Ki 65 nM)
    • No functional activity on H3, functional assay not available for Sigma 2
in vitro potency
cell based assay data

UNC1999 is Cellularly Active

A. Treatment of MCF10A cells for 3 days with UNC1999 shows a dose-related decrease in H3K27me3 which is unrelated to cell viability. 
B. UNC2400 did not show any dose-related decrease in H3K27me3.

UNC1999 Kills DB cells with Y641N mutation

DB cells harbor the EZH2 Y641N mutation

C. 8-day treatment of UNC1999 but not UNC2400 kills DB cells.
D. Western blotting of EZH2, total H3 and H3K27me3 following 3-day treatment of UNC1999 shows no change in H3 or EZH2, but significant change in H3K27me3

references

An Orally Bioavailable Chemical Probe of the Lysine Methyltransferases EZH2 and EZH1
 

Kyle D. Konze, Anqi Ma, Fengling Li, Dalia Barsyte-Lovejoy, Trevor Parton, Christopher J.MacNevin, Feng Liu, Cen Gao, Xi-Ping Huang, Ekaterina Kuznetsova, Marie Rougie, Alice Jiang, Samantha G. Pattenden, Jacqueline L. Norris, Lindsey I. James, Bryan L Roth, Peter J. Brown, Stephen V. Frye, Cheryl H. Arrowsmith, Klaus M. Hahn, Gang Greg Wang, Masoud Vedadi, and Jian Jin.

ACS Chem. Biol., 2013, 8 (6), pp 1324–1334 

DOI: 10.1021/cb400133j • Publication Date (Web): 08 Apr 2013

pk properties
co-crystal structures
synthetic schemes
materials and methods
02.04.2013

Novel structure of a human membrane enzyme sheds light on molecular mechanisms of rare ageing disorders and metabolic syndromes

by: SGC

Laminopathies are a group of rare genetic disorders caused by mutations in genes encoding proteins involved in the building and maintenance of the nuclei of cells. Severe laminopathies such as the premature ageing syndrome pregeria leads to death in the teens from heart disease and other symtoms normally found in people in their 80s. The structure of the nucleus is maintained by a class of proteins called lamins. When one of these proteins, prelamin A is not properly processed by an enzyme ZMPSTE24, laminopathies occur.

I-CBP112 A CREBBP/EP300-selective chemical probe

This probe is available from Sigma and Cayman Chemical.

This probe (as hydrochloride) is available from Cayman Chemical.

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

I-CBP112

CREBBP (CBP) and EP300 are general transcriptional co-activators, which are involved in many biological processes like maintenance of genomic stability by affecting DNA replication and DNA repair as well as cell growth, transformation and development. They  also plays and essential role in neuronal plasticity/ memory formation, hematopoiesis and energy homeostasis as demonstrated in a variety of mouse models. By acetylation of non-histone proteins CREBBP can have a positive or negative effect on transcriptional regulation affecting protein- protein interactions, protein-DNA interactions, nuclear retention or protein half-life.

Mutations of CREBBP and EP300 lead to Rubinstein-Taybi syndrome (RTS), characterised by growth retardation, facial abnormalities, organ abnormalities, mental retardation and proneness to tumors. Chromosomal translocations of CREBBP or EP300 with MOZ, MLL have been observed in acute myeloid leukemia. CREBBP has also been associated with Amyotrophic lateral sclerosis (ALS), Lou Gherig’s disease, a neurodegenerative disease with progressive degeneration of motor neurons in the brain and spinal cord, Alzheimer's disease and polyglutamine diseases such as Spinal and Bulbar Muscular Atrophy and Huntington’s disease.

We have developed an inhibitor, I-CBP112, against the CREBBP and EP300 Bromodomains.

In vitro Potency
AssayIC50/Kd
CREBBP (Alphascreen)0.170
CREBBP (BLI)0.170
CREBBP (ITC)0.151
EP300 (ITC)0.625
properties
I-CBP112
1-[7-(3,4-dimethoxyphenyl)-9-{[(3S)-1-methylpiperidin-3-yl]methoxy}-2,3,4,5-tetrahydro-1,4-benzoxazepin-4-yl]propan-1-one
Click here to download SDF file
Physical and chemical properties
Molecular weight468.3
clogP4.5
PSA50.2
Storage2-8°C as powder. NB making aliquots rather than freeze-thawing is recommended
DissolutionSoluble in DMSO at least up to 50 mM
  • SMILES:
  • CN1CCC[C@H](COC2=C3C(CN(CCO3)C(CC)=O)=CC(C4=CC=C(C(OC)=C4)OC)=C2)C1
  • InChI:
  • InChI=1S/C27H36N2O5/c1-5-26(30)29-11-12-33-27-22(17-29)13-21(20-8-9-23(31-3)24(14-20)32-4)15-25(27)34-18-19-7-6-10-28(2)16-19/h8-9,13-15,19H,5-7,10-12,16-18H2,1-4H3/t19-/m0/s1
  • InChIKey:
  • YKNAKDFZAWQEEO-IBGZPJMESA-N
selectivity profile

Selectivity against other Bromodomains

BLI@ 0.2 and 1 µM:

No interaction was detected by BLI for the selectivity panel comprising bromodomains:

ATAD2

BAZ2B

BRD2(2)

BRD4 (1)

PB1(5)

PCAF

PHIP(2)

TIF1α

Against the entire bromodomain family, weak cross-reactivity was only observed for the BET bromodomains. 

in vitro potency
cell based assay data
Probe

I-CBP112

CREBBP (3x bromodomain) (FRAP assay) - Accelerated FRAP recovery at 1 µM.

No significant cytotoxicity up to 50 μM in U2OS cells.

references

Generation of a selective small molecule inhibitor of the CBP/p300 bromodomain for leukemia therapy.
Picaud S, Fedorov O, Thanasopoulou A, Leonards K, Jones K, Meier J, Olzscha H, Monteiro O, Martin S, Philpott M, Tumber A, Filippakopoulos P, Yapp C, Wells C, Hing Che K, Bannister A, Robson S, Kumar U, Parr N, Lee K, Lugo D, Jeffrey P, Taylor S, Vecellio ML, Bountra C, Brennan P, O'Mahony A, Velichko S, Muller S, Hay D, Daniels DL, Urh M, La Thangue NB, Kouzarides T, Prinjha R, Schwaller J, Knapp S.
Cancer Res. 2015;75(23):5106-19.

pk properties
co-crystal structures

I-CBP112 has been co-crystallised with CREBBP and is deposited in the PDB - 4NR6

synthetic schemes
materials and methods

SGC-CBP30 A CREBBP/EP300-selective chemical probe

This probe is available from Tocris, Sigma and Cayman Chemical

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

 

SGC-CBP30

 

BDOIA513 

In vitro Potency
AssayIC50/Kd µM
CREBBP (Alphascreen)0.069
CREBBP (BLI)0.041
CREBBP (ITC)0.021
EP300 (ITC)0.038

CREBBP (CBP) and EP300 are general transcriptional co-activators, which are involved in many biological processes like maintenance of genomic stability by affecting DNA replication and DNA repair as well as cell growth, transformation and development. They also play and essential role in neuronal plasticity/ memory formation hematopoiesis and energy homeostasis as demonstrated in a variety of mouse models. They possess both acetyl-transferase enzymatic and bromodomain containing regions. Through acetylation of non-histone proteins CREBBP can have a positive or negative effect on transcriptional regulation by affecting protein- protein interactions, protein-DNA interactions, nuclear retention or protein half-life.

Mutations of CREBBP and EP300 lead to Rubinstein-Taybi syndrome (RTS), characterised by growth retardation, facial abnormalities, organ abnormalities, mental retardation and altered tumor susceptibility. Chromosomal translocations of CREBBP or EP300 with MOZ, MLL have been observed in acute myeloid leukemia.

CREBBP has also been associated with Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig’s disease, a neurodegenerative disease with progressive degeneration of motor neurons in the brain and spinal cord, Alzheimer's disease and poly glutamine repeat diseases such as Spinal and Bulbar Muscular Atrophy and Huntington’s disease.

We have developed an inhibitor, SGC-CBP30* against the CREBBP and EP300 Bromodomains.

* This is officially pronounced SGC-CBP-three-oh

SGC-CBP30N (BDOIA513) is the negative control for SGC-CBP30. This compound has moderate activity for CREBBP (Thermal shift 2°C compared to 9.7°C for SGC-CBP30). This compound is inactive against BRD4 (Thermal shift 0.4°C compared to 1.8°C for SGC-CBP30)

properties
SGC-CBP30
8-(3-chloro-4-methoxy-phenethyl)-4-(3,5-dimethyl-isoxazol-4-yl)-9-(2-(morpholin-4-yl)-propyl)-7,9-diaza-bicyclo[4.3.0]nona-1(6),2,4,7-tetraene
Click here to download SDF file
Physical and chemical properties
Molecular weight508.2
Molecular formulaC28H33ClN4O3
IUPAC name8-(3-chloro-4-methoxy-phenethyl)-4-(3,5-dimethyl-isoxazol-4-yl)-9-(2-(morpholin-4-yl)-propyl)-7,9-diaza-bicyclo[4.3.0]nona-1(6),2,4,7-tetraene
logP4.89
PSA51.8
Storage2-8°C as powder. NB making aliquots rather than freeze-thawing is recommended
DissolutionSoluble in DMSO at least up to 50mM
  • SMILES:
  • CC1=NOC(C)=C1C2=CC(N=C(N3C[C@@H](N4CCOCC4)C)CCC5=CC(Cl)=C(OC)C=C5)=C3C=C2
  • InChI:
  • InChI=1S/C28H33ClN4O3/c1-18(32-11-13-35-14-12-32)17-33-25-8-7-22(28-19(2)31-36-20(28)3)16-24(25)30-27(33)10-6-21-5-9-26(34-4)23(29)15-21/h5,7-9,15-16,18H,6,10-14,17H2,1-4H3/t18-/m0/s1
  • InChIKey:
  • InChIKey=GEPYBHCJBORHCE-SFHVURJKSA-N

Measured Properties


(click for a larger version)

selectivity profile

Differential Scanning Fluorimetry (DSF)

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 µM in 20 µl volume. BDOIA518 was added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence during the protein denaturation process was fit to the Boltzmann equation.


Activity against BRD4(1) by ITC 0.86 mM: 40 fold selective for CBP. (click for larger version)

SGC-CBP30 was profiled against the CEREP panel of Receptors, Ion Channels and Enzymes with the following results

 
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(click for larger version)

in vitro potency
cell based assay data
Activity In cells
CREBBP (3x bromodomain) (FRAP assay)Accelerated FRAP recovery at 1 µM

Moderate cytotoxicity in U2OS cells and HeLa cells.

references

Discovery and Optimization of Small-Molecule Ligands for the CBP/p300 Bromodomains

Duncan A. Hay, Oleg Fedorov, Sarah Martin, Dean C. Singleton, Cynthia Tallant,Christopher Wells, Sarah Picaud, Martin Philpott, Octovia P. Monteiro, Catherine M. Rogers, Stuart J. Conway, Timothy P. C. Rooney, Anthony Tumber, Clarence Yapp,Panagis Filippakopoulos, Mark E. Bunnage,Susanne Müller, Stefan Knapp, Christopher J. Schofield, and Paul E. Brennan

J. Am. Chem. Soc., 2014, doi: 10.1021/

 

CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses.

Hammitzsch A, Tallant C, Fedorov O, O'Mahony A, Brennan PE, Hay DA, Martinez FO, Al-Mossawi MH, de Wit J, Vecellio M, Wells C, Wordsworth P, Müller S, Knapp S, Bowness P.

Proc Natl Acad Sci U S A. 2015, 112(34), 10768-73. doi: 10.1073/pnas.1501956112

pk properties
co-crystal structures

SGC-CBP30 has been co-crystallised with CREBBP and is deposited in the PDB - 4NR7

synthetic schemes
materials and methods

Bromosporine (BSP)

This compound is available from Tocris, Sigma and Cayman Chemical

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
  • synthetic schemes
overview
Probe

Bromosporine (BSP)

Bromodomains (BRDs) are protein interaction modules that read epigenetic marks recognizing ε-N-lysine acetylation motifs. The conserved BRD fold contains a deep, largely hydrophobic acetyllysine binding site, an attractive pocket for the development of small, pharmaceutically active molecules. BRDs have an important role in the targeting of chromatin-modifying enzymes to specific sites, including methyltransferases, HATs and transcription factors and regulate diverse biological processes from cell proliferation and differentiation to energy homeostasis and neurological processes.


Co-crystal structure of Bromosporine with BRD4(1)

Proteins that contain BRDs have been implicated in the development of a large variety of diseases, including various cancers, inflammatory diseases and neurological diseases and the therapeutic potential of bromodomain inhibition has been shown in several of these diseases, such as HIV, cancer and inflammation. Bromosporine is a broad spectrum inhibitor for bromodomains and as such will be very useful in elucidating further biological roles of reader domains as well as a tool for the validation of functional assays.
properties

ethyl N-[6-(3-methanesulfonamido-4-methylphenyl)-3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl]carbamate
Click here to download SDF file

Physical and chemical properties
Molecular weight404.44
Molecular formulaC17H20N6O4S
IUPAC Nameethyl N-[6-(3-methanesulfonamido-4-methylphenyl)-3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl]carbamate
clogP0.78
PSA105.64
Storage2-8°C as powder. NB making aliquots rather than freeze-thawing is recommended
DissolutionSoluble in DMSO at least up to 50mM
SMILES:

CC1=NN=C2N1N=C(C3=CC=C(C)C(NS(=O)(C)=O)=C3)C=C2NC(OCC)=O

InChI:

InChI=1S/C17H20N6O4S/c1-5-27-17(24)18-15-9-14(21-23-11(3)19-20-16(15)23)12-7-6-10(2)13(8-12)22-28(4,25)26/h6-9,22H,5H2,1-4H3,(H,18,24)

InChIKey:

UYBRROMMFMPJAN-UHFFFAOYSA-N

selectivity profile

Differential Scanning Fluorimetry (DSF)

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 µM in 20 µl volume. Bromosporine was added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence during the protein denaturation process was approximated by the equation (displayed below) where ΔuG(T) is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant and yF and yU are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein respectively. The baselines of the denatured and native states were approximated by a linear fit. The observed temperature shifts, ΔTmobs, were recorded as the difference between the transition midpoints of sample and reference wells containing protein without ligand in the same plate and determined by non-linear least squares fit.

Selectivity
BromodomainThermal melt
10 µM
ΔTm
BRD2(1)4.4
BRD2(2)5.9
BRD3(1)5.3
BRD3(2)5.9
BRD4(1)6.9
BRD4(2)6.2
BRDT(1)7.3
BRDT(2)5.2
CECR28.3
PB1(5)0.4
TAF1(1)1.3
TAF1(2)5.2
TAF1L(1)0.9
TAF1L(2)0.9
BAZ2A1.2
TIF1α0.4
ATAD2-0.2
BRD93.9
CREBBP3.4
in vitro potency
cell based assay data
Potency in Cells
BRD4Accelerated FRAP recovery at 1 µM
CREBBPAccelerated FRAP recovery at 1 µM
TIF1αInactive at 10 µM
BAZ2AInactive at 10 µM
SMARCA2Inactive at 10 µM

Bromosporine shows moderate cytotoxicity in HeLa cells at 18 µM

references

 

Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

Sarah Picaud, Katharina Leonards, Jean-Philippe Lambert, Oliver Dovey, Christopher Wells, Oleg Fedorov, Octovia Monteiro, Takao Fujisawa, Chen-Yi Wang, Hannah Lingard, Cynthia Tallant, Nikzad Nikbin, Lucie Guetzoyan, Richard Ingham, Steven V. Ley, Paul Brennan, Susanne Muller, Anastasia Samsonova, Anne-Claude Gingras, Juerg Schwaller, George Vassiliou, Stefan Knapp and Panagis Filippakopoulos. Science Advances  12 Oct 2016: Vol. 2, no. 10, e1600760. DOI: 10.1126/sciadv.1600760

pk properties
co-crystal structures
synthetic schemes

Scheme 1

Scheme 2

materials and methods
16.11.2012

GlaxoSmithKline releases EZH2 inhibitor as part of SGC epigenetics initiative

by: SGC

GlaxoSmithKline has developed a potent, selective, cell-active EZH2 inhibitor, GSK343, and has made this available as a chemical probe as part of the SGC epigenetics initiative.  The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer.  GSK343 inhibits EZH2 with an IC50 of 4nM and is over 1000-fold selective for other HMTs except EZH1 (60-fold selectivity).

19.09.2012

Pfizer and SGC Oxford receive the first Wellcome Trust Pathfinder Award for Rare Diseases

by: SGC

The Pfizer-SGC project aims to tackle a rare, hereditary metabolic disorder called homocystinuria, which leaves patients unable to metabolise the amino acid methionine. A team of researchers at Pfizer Rare Disease unit will work with Dr Wyatt Yue at the Structural Genomics Consortium at the University of Oxford to investigate methods of restoring normal metabolism and preventing the progression of the disease. The team will study the enzyme that is affected by the disease and investigate ways to restore its function.

GSK343 A potent, selective inhibitor of EZH2 with cellular activity

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

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
overview
Probe

GSK343

The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer.  GlaxoSmithKline has developed a potent, selective, cell-active EZH2 inhibitor, GSK343, and has made this available as a chemical probe as part of the SGC epigenetics initiative.  GSK343 inhibits EZH2 with an IC50 of 4nM and is over 1000-fold selective for other HMTs except EZH1 (60-fold selectivity).  GSK343 inhibits H3K27 methylation in HCC1806 cells with an IC50 of <200nM as measured by immunofluorescence.

properties

N-[(6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl]
-6-[2-(4-methylpiperazin-1-yl)pyridin-4-yl]
-1-(propan-2-yl)-1H-indazole-4-carboxamide
Click here to download SDF file

Physical and chemical properties
Molecular weight541.3
Molecular formulaC31H39N7O2
IUPAC nameN-[(6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl]
-6-[2-(4-methylpiperazin-1-yl)pyridin-4-yl]
-1-(propan-2-yl)-1H-indazole-4-carboxamide
logP3.23
PSA95.39
  • SMILES:
  • CCCC1=C(CNC(=O)C2=C3C=NN(C(C)C)C3=CC(=C2)C2=CC=NC(=C2)N2CCN(C)CC2)C(=O)NC(C)=C1
  • InChI:
  • InChI=1S/C31H39N7O2/c1-6-7-23-14-21(4)35-31(40)26(23)18-33-30(39)25-15-24(16-28-27(25)19-34-38(28)20(2)3)22-8-9-32-29(17-22)37-12-10-36(5)11-13-37/h8-9,14-17,19-20H,6-7,10-13,18H2,1-5H3,(H,33,39)(H,35,40)
  • InChIKey:
  • ULNXAWLQFZMIHX-UHFFFAOYSA-N
selectivity profile

in vitro potency
cell based assay data

GSK343 inhibits H3K27 methylation in HCC1806 cells with an IC50 of <200nM as measured by immunofluorescence.


Biological characterization of GSK343. (a) H3K27me3 imaging in HCC1806 cells treated with DMSO or GSK343 for 72 hours; (b) Dose-response of GSK343-treated HCC 1806 cells.

references

Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2 

Sharad K. Verma*, Xinrong Tian, Louis V. LaFrance, Céline Duquenne, Dominic P.Suarez, Kenneth A. Newlander, Stuart P. Romeril, Joelle L. Burgess, Seth W. Grant, James A. Brackley, Alan Graves, Daryl A. Scherzer, Art Shu, Christine S. Thompson, Heidi Morgan-Ott, Glenn S. Van Aller, Carl A. Machutta, Elsie Diaz, Yong Jiang, Neil W. Johnson, Steven Knight, Ryan G. Kruger, Michael T. McCabe, Dashyant Dhanak, Peter J. Tummino, Caretha L. Creasy and William H. Miller.  ACS Med. Chem. Lett., 2012, 3 (12), pp 1091–1096 DOI: 10.1021/ml3003346.

pk properties
co-crystal structures
synthetic schemes
materials and methods

UNC0642 A potent, selective inhibitor of G9a/GLP with improved PK properties

This probe is available from Cayman Chemical, Tocris and Sigma

  • overview
  • properties
  • selectivity profile
  • cell based assay data
  • references
overview
Probe

UNC0642

G9a (EHMT2) and GLP (EHMT1) catalyze the mono and dimethylation of lysine 9 of histone 3 (H3K9) and other non-histone substrates such as p53 and WIZ.

Here we present UNC0642, a G9a/GLP chemical probe with improved PK properties relative to UNC0638. UNC0642 exhibits an in vitro IC50 <15 nM with selectivity > 100-fold over 13 other HMTs and selected representatives of kinases, ion channels, 7TMs, and other epigenetic proteins.

In cells, UNC0642 results in a potent reduction of H3K9me2 in MDA MB231 cells with IC50 = 106 nM.

properties

2-(4,4-difluoropiperidin-1-yl)-6-methoxy-N-[1-(propan-2-yl)piperidin-4-yl]-7-[3-(pyrrolidin-1-yl)propoxy]quinazolin-4-amine
Click here to download SDF file

Physical and chemical properties
Molecular weight546.3
Molecular formulaC29H44F2N6O2
IUPAC name2-(4,4-difluoropiperidin-1-yl)-6-methoxy-N-[1-(propan-2-yl)piperidin-4-yl]-7-[3-(pyrrolidin-1-yl)propoxy]quinazolin-4-amine
clogP5.96
PSA54.6 A
  • SMILES:
  • N1C2=C(C=C(C(=C2)OCCCN(CCC2)C2)OC)C(=NC=1N(CCC(C1)(F)F)C1)NC(CCN(C(C)C)C1)C1
  • InChI:
  • InChI=1S/C29H44F2N6O2/c1-21(2)36-14-7-22(8-15-36)32-27-23-19-25(38-3)26(39-18-6-13-35-11-4-5-12-35)20-24(23)33-28(34-27)37-16-9-9(30,31)10-17-37/h19-22H,4-18H2,1-3H3,(H,32,33,34)
  • InChIKey:
  • RNAMYOYQYRYFQY-UHFFFAOYSA-N
selectivity profile

Below we show UNC0642 is equipotent to UNC0638 in the G9a in vitro assay:

 

G9a: Ki = 4 ± 2 (nM)

Competitive with peptide substrate

Non-competitive with SAM cofactor

Similar potency as UNC0638 (Ki = 3 nM)


In addition, UNC0642 has been shown to be inactive versus 50 kinases at 10 uM and has a similar GPCR selectivity as UNC0638.

in vitro potency
cell based assay data

a i.p. administration of a single 5 mg/kg dose in male Swiss Albino mice. 8 time points and 3 animals per time point 

Materials and Methods

MDA-MB231 cells were cultured in RPMI with 10% FBS and MCF7 cells cultured in DMEM with 10% FBS.

MTT Toxicity Assay

Cells were grown in the presence or absence of inhibitors for stated amount of time. The media was removed and replaced with DMEM 10% FBS without phenol red supplemented with 1mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and incubated for 1-2h. Live cells reduce yellow MTT to purple formazan. The resulting formazan was solubilized in acidified isopropanol and 1% Triton and absorbance measured at 570nm, corrected for 650nm background.

In-Cell Western (ICW)

Cells were grown in 96-well plates in the presence of inhibitors as stated in figures. Media was removed by flicking and 2% formaldehyde in PBS added for 15min. After five washes with 0.1% Triton X100 in PBS, cells were blocked for 1h with 1% BSA in PBS. Three out of four replicates were exposed to primary H3K9m2 antibody, Abcam #1220 at 1/800 dilution in 1% BSA, PBS for 2h. One replicate was reserved for background control. The wells were washed five times with 0.1% Tween 20 in PBS, then secondary IR800 conjugated antibody (LiCor) and DNA-intercalating dye, DRAQ5 (LiCor) added for 1h. After 5 washes with 0.1% Tween 20 in PBS, the plates were read on Odyssey (LiCor) scanner at 800nm (H3K9m2 signal; 764nm excitation) and 700nm (DRAQ5 signal; 683nm excitation). Fluorescence intensity was quantified, normalized to background and DRAQ5 signal expressed as percentage of control.

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references

Discovery of an in Vivo Chemical Probe of the Lysine Methyltransferases G9a and GLP; Feng Liu, Dalia Barsyte-Lovejoy, Fengling Li, Yan Xiong, Victoria Korboukh, Xi-Ping Huang, Abdellah Allali-Hassani, William P. Janzen, Bryan L. Roth, Stephen V. Frye, Cheryl H. Arrowsmith, Peter J. Brown, Masoud Vedadi, and Jian Jin; J. Med. Chem., 2013, 56 (21), pp 8931–8942.

pk properties
co-crystal structures
synthetic schemes
materials and methods

UNC1215 A potent, selective antagonist of L3MBTL3 with cellular activity

This probe is available from Cayman Chemical, Sigma and Tocris (acid).
Its control compound UNC1079 is available from Cayman Chemical

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

UNC1215

 

 

UNC1079

Methyl-lysine (Kme) recognition domains play a central role in epigenetic regulation during cellular differentiation, development, and gene transcription with more than 200 known “reader” domains in the human proteome. In collaboration with the Center for Integrative Chemical Biology and Drug Discovery (CICBDD) at The University of North Carolina at Chapel Hill, we have developed the first chemical probe for a Kme-binding protein.  UNC1215 is a potent and selective chemical probe for the Kme reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds the MBT domains of L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides.  This probe is greater than 50-fold selective versus other members of the human MBT family and also demonstrates selectivity against more than 200 other Kme reader domains examined. UNC1079 is a structurally similar but substantially less potent antagonist and negative control.

properties

N-phenyl-2,5-bis[4-(pyrrolidin-1-yl)piperidine-1-carbonyl]aniline
Click here to download SDF file

Physical and chemical properties
Molecular weight529.3
Molecular formulaC32H43N5O2
IUPAC NameN-phenyl-2,5-bis[4-(pyrrolidin-1-yl)piperidine-1-carbonyl]aniline
logP4.17
PSA59.13
SMILES:

O=C(N1CCC(N2CCCC2)CC1)C3=C(NC4=CC=CC=C4)C=C(C=C3)C(N(CC5)CCC5N6CCCC6)=O

InChI:

InChI=1S/C32H43N5O2/c38-31(36-20-12-27(13-21-36)34-16-4-5-17-34)25-10-11-29(30(24-25)33-26-8-2-1-3-9-26)32(39)37-22-14-28(

InChIKey:

PQOOIERVZAXHBP-UHFFFAOYSA-N

selectivity profile

Selectivity of UNC1215 against a panel of Kme reader proteins.

Alphascreen
IC50 [µM]a		MBT domainsChromodomainTudor DomainsPHD Fingers
L3MBTL1L3MBTL3L3MBTL4SFMBTMBTD1CBX753BP1UHRF1PHF23JARID1A
UNC121520.0411>306>304>30>30>30
(R2 = 0.93,
n = 17)		(R2 = 0.93,
n = 17)		(R2 = 0.63,
n = 21)		(n = 18)(R2 = 0.71,
n = 11)		(n = 19)(R2 = 0.90,
n = 12)		(n = 18)(n = 15)(n = 15)

a - The data for the IC50 values was calculated from n replicate runs; datapoints for each compound concentration were averaged and plotted using 4-parameters curve fitting. R2 is a statistical estimate of goodness-of-fit.

Molecular profiling of UNC1215

Target CategoryTarget Activity

Within methyl-lysine

reader target family

TargetIC50 (µM)aKd (µM)b
L3MBTL30.040.12
L3MBTL129.4
53BP14> 31
PHF20 TudorNT5.6
SPF30 (S.p.) TudorNTWeak binding
PHF20L1 TudorNT> 13
Histone methyltransferases< 50% inhibition at 250 mM versus 10 targetsc
BromodomainsTm shift < 0.5 °C at 10 mM versus 12 targetsd
Histone demethylasesTm shift < 0.1 °C at 50 mM versus 15 targetsd

NIMH Psychoactive Drug

Screening Program

Selectivity Panel  

< 50% inhibition at 10 mM versus 35 targets

> 50% inhibition at 10 mM versus 8 targets

TargetKi (µM)eIC50 (µM)
Alpha 2C0.86NT
DAT> 10NT
KOR4.0NT
M10.0973.6f
M20.07230% at 30 µMg
M30.89NT
M40.40NT
M54.3NT
Kinase Selectivity Panel< 15% inhibition at 10 mM versus 49 kinases
Target% Inhibition at 10 mM
FLT364%

aAlphascreen assays results. bITC results. cRadioactive SAM methyl transfer assay results. dDifferential scanning fluorimetry results. eRadioligand binding assay results. fCa2+ mobilization assay results. gcAMP biosensor assay results.

in vitro potency
cell based assay data

UNC1215 potently antagonizes 3MBT localization in cells.  (a) Recovery time of a photobleached area in GFP-3MBT expressing cells is reduced upon treatment with UNC1215 in a dose response manner, whereas inactive compound UNC1079 shows no effect.  Inset shows time lapse images of photobleached nuclei.  (b, c) GFP fusions of 3MBT and FLMBT localize to the nucleus and form distinct foci in HEK293 cells. UNC1215 inhibits the foci formation of GFP-3MBT in a dose response fashion, whereas UNC1079 has no effect on the foci. In contrast, UNC1215 is relatively ineffective at inhibiting foci formation of GFP-FLMBT (scale bar, 10 µm). (d) The GFP-3MBT D274A MBT1 mutant shows a reduction in foci formation, while the GFP-3MBT D381A MBT2 mutant does not form nuclear foci.

references

Discovery of a chemical probe for a methyl-lysine reader domain: L3MBTL3

James, Lindsey I.; Barsyte-Lovejoy, Dalia; Zhong, Nan; Krichevsky, Liubov; Korboukh, Victoria K.; Herold, J. Martin; MacNevin, Christopher J.; Norris, Jacqueline L.; Sagum, Cari A.; Tempel, Wolfram; Marcon, Edyta; Guo, Hongbo; Gao, Cen; Huang, Xi-Ping; Duan, Shili; Emili, Andrew; Greenblatt, Jack F.; Kireev, Dmitri B.; Jin, Jian; Janzen, William P.; Brown, Peter J.; Bedford, Mark T.; *Arrowsmith, Cheryl H. *Frye, Stephen V. Nature Chemical Biology 9, 184–191 (2013) -  DOI 10.1038/nchembio.1157

pk properties
co-crystal structures

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Main features

  • L3MBTL3 Dimer: UNC1215 binds to L3MBTL3 with a 2:2 stoichiometry. Each "arm" of UNC1215 binds to a different molecule of L3MBTL3.
  • Pocket Key interactions of pyrrolidine A with YYF aromatic cage and pyrrolidine B with YFW aromatic cage. Both interactions are augmented by a hydrogen bond with an Asp.
synthetic schemes
materials and methods
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