The prevailing view in biomedicine and drug discovery is that we need more “innovation”.
At the SGC, we (narrowly) interpret “innovation” to mean novel targets and mechanisms because we believe this sort of innovation will have the greatest impact on unmet medical needs.
The Structural Genomics Consortium (SGC) recently launched a new scientific public-private partnership in tuberculosis and malaria drug discovery: the Structure-guided Drug Discovery Coalition, SDDC. Participants to the Coalition include the Seattle Structural Genomics Center for Infectious Disease, the Midwest Center for Structural Genomics, the Center for Structural Genomics of Infectious Diseases, the Tuberculosis Structural Genomics Consortium, leading academic researchers in North America and Europe, and drug discovery teams from academia and industry.
This compound is available from Sigma, Tocris and Cayman Chemical
| Probe |
 |
A-366 |
A collaboration between the SGC and Abbvie has resulted in the discovery of A-366, a chemical Probe for G9a/GLP. A-366 is a potent inhibitor of G9a/GLP with IC50 3 nM and > 100-fold selectivity over other methyltransferases and other non-epigenetic targets. A-366 has been shown to inhibit H3K9 methylation in cells with an IC50 of 100 nM and exhibits minimal cellular toxicity compared with previous quinazoline-based probes.
A recent study by Wagner et al. (PMID: 26893353) showed that A-366 displaces H3K4me3 from Spindlin1 (2nd Tudor domain) with IC50 = 182.6 ± 9.1 nM.
| A-366 |
|
| 5'-Methoxy-6'-[3-(1-pyrrolidinyl)propoxy]spiro[cyclobutane-1,3'-indol]-2'-amine |
| Physical and chemical properties for A-366 | |
| Molecular weight | 329.2 |
| Molecular formula | C19H27N3O2 |
| IUPAC name | 5'-Methoxy-6'-[3-(1-pyrrolidinyl)propoxy]spiro[cyclobutane-1,3'-indol]-2'-amine |
| MollogP | 3.924 |
| PSA | 49.37 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 6 |
| No. of hydrogen bond acceptors | 4 |
| No. of hydrogen bond donors | 2 |
Discovery and development of potent and selective inhibitors of histone methyltransferase g9a. ACS Med Chem Lett. 2014 Jan 2;5(2):205-9
Sweis RF, Pliushchev M, Brown PJ, Guo J, Li F, Maag D, Petros AM, Soni NB, Tse C, Vedadi M, Michaelides MR, Chiang GG, Pappano WN.
The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia. PLoS ONE 10(7): e0131716. doi:10.1371/journal. pone.0131716. William N. Pappano, Jun Guo, Yupeng He, Debra Ferguson, Sujatha Jagadeeswaran, Donald J. Osterling, Wenqing Gao, Julie K. Spence, Marina Pliushchev, Ramzi F. Sweis, Fritz G. Buchanan, Michael R. Michaelides, Alexander R. Shoemaker, Chris Tse, Gary G. Chiang.
Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform. Nucleic Acids Res. 2016 Feb 17.
Wagner T, Greschik H, Burgahn T, Schmidtkunz K, Schott AK, McMillan J, Baranauskienė L, Xiong Y, Fedorov O, Jin J, Oppermann U, Matulis D,Schüle R, Jung M.
Main features
INGELHEIM, Germany (6th May, 2013) – Today Boehringer Ingelheim announced that it has joined the Structural Genomics Consortium (SGC). The consortium intends to promote research into protein structures and epigenetics that could pave the way for the development of novel therapies for previously uncurable diseases.As a member of the SGC, Boehringer Ingelheim will help fund precompetitive drug research aimed at bringing new, more effective medicines to patients faster. The SGC is based at the Universities of Toronto, Canada, and Oxford, England.
This probe hydrochloride) is available from Sigma (including its negative control), Tocris (including its negative control) and Cayman Chemical (including its negative control).
| Probe | Negative control | |
 |
|  |
(R)-PFI-2 |
| (S)-PFI-2 |
A collaboration between the SGC and Pfizer has resulted in the discovery of (R)-PFI-2, a chemical Probe for SETD7. PFI-2 is a potent inhibitor of SETD7 with IC50 2 nM and 1000-fold selectivity over other methyltransferases and other non-epigenetic targets. PFI-2 has been shown to bind to SETD7 by ITC (Kd=18nM) and biotinylated PFI-2 interacts with SETD7 in pull-down studies. Its enantiomer (S)-PFI-2 is 500-fold less active making it an excellent negative control. Treatment of low-density MEFs with (R)-PFI-2 resulted in higher nuclear YAP levels indicating an effect on the Hippo pathway.
| Probe | Negative control | |
|
| |
(R)-PFI-2 |
| (S)-PFI-2 |
| Physical and chemical properties for (S)-PFI-2 | |
| Molecular weight | 499.2 |
| Molecular formula | C23H25F4N3O3S |
| MollogP | 3.504 |
| PSA | 70.67 |
| No. of chiral centres | 1 |
| No. of rotatable bonds | 8 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 2 |
| Physical and chemical properties for (R)-PFI-2 | |
| Molecular weight | 499.2 |
| Molecular formula | C23H25F4N3O3S |
| MollogP | 3.504 |
| PSA | 70.67 |
| No. of chiral centres | 1 |
| No. of rotatable bonds | 8 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 2 |
Barsyte-Lovejoy D, Li F, Oudhoff MJ, Tatlock JH, Dong A, Zeng H, Wu H, Freeman SA, Schapira M, Senisterra GA, Kuznetsova E, Marcellus R, Allali-Hassani A, Kennedy S, Lambert JP, Couzens AL, Aman A, Gingras AC, Al-Awar R, Fish PV, Gerstenberger BS, Roberts L, Benn CL, Grimley RL, Braam MJ, Rossi FM, Sudol M, Brown PJ, Bunnage ME, Owen DR, Zaph C, Vedadi M, Arrowsmith CH. (R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells. Proc Natl Acad Sci U S A. 2014 Sep 2;111(35):12853-8. doi: 10.1073/pnas.1407358111. Epub 2014 Aug 18.
The probe UNC1999 is available from Cayman Chemical, Sigma and Tocris.
| 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.
| 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 weight | 569.74 |
| Molecular formula | C33H43N7O2 |
| IUPAC name | 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 |
| logP | 4.01 |
| PSA | 95.39 |
UNC1999 is selective for EZH2 over 15 other methyltransferases and proteins in other target classes
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
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
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.
This probe is available from Sigma and Cayman Chemical.
This probe (as hydrochloride) is available from Cayman Chemical.
| 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 | |
|---|---|
| Assay | IC50/Kd |
| CREBBP (Alphascreen) | 0.170 |
| CREBBP (BLI) | 0.170 |
| CREBBP (ITC) | 0.151 |
| EP300 (ITC) | 0.625 |
| 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 weight | 468.3 |
| clogP | 4.5 |
| PSA | 50.2 |
| Storage | 2-8°C as powder. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | Soluble in DMSO at least up to 50 mM |
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.
| Probe |
|
I-CBP112 |
CREBBP (3x bromodomain) (FRAP assay) - Accelerated FRAP recovery at 1 µM.
No significant cytotoxicity up to 50 μM in U2OS cells.
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.
I-CBP112 has been co-crystallised with CREBBP and is deposited in the PDB - 4NR6
This probe is available from Tocris, Sigma and Cayman Chemical
| Probe | Negative control | |
 |
|  |
SGC-CBP30 |
| BDOIA513 |
| In vitro Potency | |
|---|---|
| Assay | IC50/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)
| 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 weight | 508.2 |
| Molecular formula | C28H33ClN4O3 |
| IUPAC name | 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 |
| logP | 4.89 |
| PSA | 51.8 |
| Storage | 2-8°C as powder. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | Soluble in DMSO at least up to 50mM |
Measured Properties
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
(click for larger version)
(click for larger version)
(click for larger version)
| Activity In cells | |
| CREBBP (3x bromodomain) (FRAP assay) | Accelerated FRAP recovery at 1 µM |
Moderate cytotoxicity in U2OS cells and HeLa cells.
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
SGC-CBP30 has been co-crystallised with CREBBP and is deposited in the PDB - 4NR7
This compound is available from Tocris, Sigma and Cayman Chemical
| 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)
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 weight | 404.44 |
| Molecular formula | C17H20N6O4S |
| IUPAC Name | ethyl N-[6-(3-methanesulfonamido-4-methylphenyl)-3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl]carbamate |
| clogP | 0.78 |
| PSA | 105.64 |
| Storage | 2-8°C as powder. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | Soluble in DMSO at least up to 50mM |
CC1=NN=C2N1N=C(C3=CC=C(C)C(NS(=O)(C)=O)=C3)C=C2NC(OCC)=O
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)
UYBRROMMFMPJAN-UHFFFAOYSA-N
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 | |
|---|---|
| Bromodomain | Thermal 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 |
| CECR2 | 8.3 |
| PB1(5) | 0.4 |
| TAF1(1) | 1.3 |
| TAF1(2) | 5.2 |
| TAF1L(1) | 0.9 |
| TAF1L(2) | 0.9 |
| BAZ2A | 1.2 |
| TIF1α | 0.4 |
| ATAD2 | -0.2 |
| BRD9 | 3.9 |
| CREBBP | 3.4 |
| Potency in Cells | |
|---|---|
| BRD4 | Accelerated FRAP recovery at 1 µM |
| CREBBP | Accelerated FRAP recovery at 1 µM |
| TIF1α | Inactive at 10 µM |
| BAZ2A | Inactive at 10 µM |
| SMARCA2 | Inactive at 10 µM |
Bromosporine shows moderate cytotoxicity in HeLa cells at 18 µM
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
Scheme 1
Scheme 2
