SGC707 A Chemical Probe for PRMT3

SGC707 is available from Cayman Chemical and Sigma and from Tocris
XY1 (the negative control of SGC 707) is available from Tocris and from Cayman Chemical.

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

overview

Probe		Negative control

SGC707 (IC₅₀=31 nM)		XY1 (IC₅₀>100,000 nM)

A collaboration between the SGC, China Novartis Institutes for Biomedical Research, and the center for Integrative Chemical Biology and Drug Discovery (CICBDD) at the University of North Carolina has resulted in the discovery of SGC707, a chemical probe for PRMT3, a protein which catalyzes the asymmetrical di-methylation of arginine residues. SGC707 is a potent allosteric inhibitor of PRMT3 (IC₅₀ = 50 nM) with >100-fold selectivity over other methyltransferases, and other non-epigenetic targets. SGC707 has been shown to bind to PRMT3 with K_D of 50 nM (ITC), and inhibits the methylation of histones in cells with IC₅₀ value below 1 µM.

properties

Probe		Negative control

SGC707 (IC₅₀=31 nM)		XY1 (IC₅₀>100,000 nM)

Click here to download SDF file

Physical and chemical properties
Molecular weight	298.1430
Molecular formula	C₁₆H₁₈N₄O₂
IUPAC name	2-((3-aza-bicyclo[4.4.0]deca-1,3,5,7,9-pentaen-8-ylamino)-formylamino)-1-(pyrrolidin-1-yl)-ethanone
logP	1.8
PSA	59.9 A

SMILES:
SGC707: O=C(N1CCCC1)CNC(NC2=CC3=CC=NC=C3C=C2)=O
XY1: O=C(N1CCCC1)CNC(NC2=CC3=CC=CC=C3C=C2)=O
InChI:
SGC707: InChI=1S/C16H18N4O2/c21-15(20-7-1-2-8-20)11-18-16(22)19-14-4-3-13-10-17-6-5-12(13)9-14/h3-6,9-10H,1-2,7-8,11H2,(H2,18,19,22)
InChIKey:
SGC707: DMIDPTCQPIJYFE-UHFFFAOYSA-N
XY1: GSQHGSRPQHBTTP-UHFFFAOYSA-N

selectivity profile

(C) Effect of SGC707 on the activity of 27 protein methyltransferases as well as DNMT1, DNMT3A-3L, DNMT3B-3L and BCDIN3D (an RNA-methyltransferase) were assessed.

(D) IC50 values were determined at saturating concentration of peptide substrate (1.5 µM) and SAM concentrations equal to 0.25, 0.5, 1, 2, 4, 8, 10 and 12x of the Km for SAM. No change in IC50 values at varying SAM concentrations was consistent with a noncompetitive pattern of inhibition with respect to SAM. (E) To determine the competition with peptide, the SAM concentration was kept at saturation (70 µM) and IC50 values were determined at different peptide concentrations (0.5, 1, 2, 4, 8, 12, 16, and 20 ×Km). A similar noncompetitive pattern was observed for SGC707 with respect to peptide confirming the allosteric mode of inhibition.

in vitro potency

cell based assay data

Western blot analysis of H4R3me2a levels. HEK293 cells were co-transfected with FLAG tagged PRMT3 (WT) or its catalytically dead mutant E335Q (Mut) and treated with different concentrations of SGC707, as indicated. Total cell lysates were collected 24 h post inhibitor treatment and analysed for H4R3me2a levels. The total levels of exogenous histone H4 and overexpressed PRMT3 were determined with anti-GFP, anti-H4 and anti-FLAG antibodies, respectively

references

Kaniskan, H. Ü., Szewczyk, M. M., Yu, Z., Eram, M. S., Yang, X., Schmidt, K., Luo, X., Dai, M., He, F., Zang, I., Lin, Y., Kennedy, S., Li, F., Dobrovetsky, E., Dong, A., Smil, D., Min, S.-J., Landon, M., Lin-Jones, J., Huang, X.-P., Roth, B. L., Schapira, M., Atadja, P., Barsyte-Lovejoy, D., Arrowsmith, C. H., Brown, P. J., Zhao, K., Jin, J. and Vedadi, M. (2015), A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3).

Angew. Chem.. doi: 10.1002/ange.201412154

pk properties

co-crystal structures

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Main features

synthetic schemes

materials and methods

07.05.2014

SGC Oxford wins Royal Society of Chemistry 2014 Rita and John Cornforth Award

by: SGC

The Group led by Professor Chas Bountra, Chief Scientist of SGC Oxford, has been awarded the Royal Society of Chemistry's Rita and John Cornforth Award 2014 for "... world leading collaborative research across the disciplines of structural biology, medicinal chemistry, chemical biology and enzymology towards understanding and exploiting the potential of epigenetics as a target family for future drug discovery".

OICR-9429 A chemical probe for WDR5

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

The control is available from Cayman Chemical.

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

overview

Probe		Negative control

OICR-9429		OICR-0547

A collaboration between the SGC and The Drug Discovery Program at the Ontario Institute for Cancer Research (OICR) has resulted in the discovery of OICR-9429, a chemical probe for WDR5, a component of several chromatin regulatory complexes including the MLL1 (Mixed Lineage Leukemia 1) complex. OICR-9429 is a potent antagonist of the interaction of WDR5 with peptide regions of MLL and Histone 3 with >100-fold selectivity over other chromatin “reader” domains, methyltransferases, and other non-epigenetic targets. OICR-9429 has been shown to bind to WDR5 with K_D values of 24 nM (Biacore) and 52 nM (ITC). OICR-9429 also disrupts the interaction of WDR5 with MLL1 and RbBP5 in cells with IC₅₀ values below 1 µM.

A closely-related compound, OICR-0547, does not show WDR5 antagonism, and can therefore be used as a negative control in phenotypic assays.

properties

Probe		Negative control

OICR-9429		OICR-0547

Physical and chemical properties for OICR-9429
Molecular weight	555.2
Molecular formula	C29H32F3N5O3
IUPAC name	5-((2-(4-methyl-piperazin-1-yl)-5-(3-((morpholin-4-yl)-methyl)-phenyl)-phenylamino)-formyl)-4-(trifluoro-methyl)-1,2-dihydro-pyridin-2-one
MollogP	3.382
PSA	65.34
No. of chiral centres	0
No. of rotatable bonds	8
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	2

Physical and chemical properties for OICR-0547 (Negative Control)
Molecular weight	542.2
Molecular formula	C28H29F3N4O4
IUPAC name	5-((2-(morpholin-4-yl)-5-(3-((morpholin-4-yl)-methyl)-phenyl)-phenylamino)-formyl)-4-(trifluoro-methyl)-1,2-dihydro-pyridin-2-one
MollogP	3.331
PSA	69.77
No. of chiral centres	0
No. of rotatable bonds	8
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	2

SMILES:
OICR-9429: CN1CCN(C2=CC=C(C3=CC(CN4CCOCC4)=CC=C3)C=C2NC(C5=CNC(C=C5C(F)(F)F)=O)=O)CC1
OICR-0547: FC(F)(F)C(C(C(NC1=CC(C2=CC(CN3CCOCC3)=CC=C2)=CC=C1N4CCOCC4)=O)=CN5)=CC5=O
InChI:
OICR-9429: InChI=1S/C29H32F3N5O3/c1-35-7-9-37(10-8-35)26-6-5-22(21-4-2-3-20(15-21)19-36-11-13-40-14-12-36)16-25(26)34-28(39)23-18-33-27(38)17-24(23)29(30,31)32/h2-6,15-18H,7-14,19H2,1H3,(H,33,38)(H,34,39)
OICR-0547: InChI=1S/C28H29F3N4O4/c29-28(30,31)23-16-26(36)32-17-22(23)27(37)33-24-15-21(4-5-25(24)35-8-12-39-13-9-35)20-3-1-2-19(14-20)18-34-6-10-38-11-7-34/h1-5,14-17H,6-13,18H2,(H,32,36)(H,33,37)
InChIKey:
OICR-9429: DJOVLOYCGXNVPI-UHFFFAOYSA-N
OICR-0547: RFHOOFYUTGZPFH-UHFFFAOYSA-N

selectivity profile

in vitro potency

cell based assay data

references

Pharmacological targeting of the Wdr5-MLL interaction in C/EBPa N-terminal leukemia ,Florian Grebien, Masoud Vedadi, Matthäus Getlik, Roberto Giambruno, Amit Grover, Roberto Avellino, Anna Skucha, Sarah Vittori, Ekaterina Kuznetsova, David Smil, Dalia Barsyte-Lovejoy, Fengling Li, Gennadiy Poda, Matthieu Schapira, Hong Wu, Aiping Dong, Guillermo Senisterra, Alexey Stukalov, Kilian V M Huber,Andreas Schönegger, Richard Marcellus, Martin Bilban, Christoph Bock, Peter J Brown, Johannes Zuber, Keiryn L Bennett, Rima Al-awar, Ruud Delwel, Claus Nerlov, Cheryl H Arrowsmith & Giulio Superti-Furga. Nature Chemical Biology. 10.1038/nchembio.1859

pk properties

co-crystal structures

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Main features

synthetic schemes

materials and methods

BAZ2-ICR A chemical probe for BAZ2A/B bromodomains

The probe is available from Tocris , Cayman Chemical and Sigma

overview
properties
selectivity profile
cell based assay data
references
pk properties
co-crystal structures
materials and methods

overview

Probe

BAZ2-ICR

Biology of the BAZ2 Bromodomains

BAZ2A/B belong to a family of ubiquitously expressed bromodomain containing proteins. Proteins of the BAZ (bromodomain adjacent zinc finger) family are characterized by a carboxy-terminal bromodomain adjacent to a PHD finger and a WACZ motif. In addition four other conserved motifs are typically found in the N-terminus of BAZ family members, namely the LH motif (a leucine-rich helical domain), the ZB2 motif (conserved between ZK783.4 and BAZ2 proteins) and the BAZ 1 and BAZ 2 motifs (conserved between all BAZ proteins, Acf, and ZK783.4).

Little is known about the biological function of BAZ2B but it has been suggested to regulate nucleosome mobilization by the ATP-dependent chromatin remodeling factor ISWI. Interaction of BAZ2B with ISWI is mediated by the BAZ1 motif [1]. The BAZ2B locus has also been identified as associated with sudden cardiac death [2]. The related BAZ2A (also called TIP5) protein plays a central role in the NoRC (nucleolar remodeling complex). BAZ2A is essential for heterochromatin formation leading to transcriptional silencing of certain rRNAs [3].

BAZ2-ICR

A collaboration between the SGC and Swen Hoelder’s group at the Institute of Cancer Research, London (ICR) has resulted in the discovery of BAZ2-ICR, a chemical probe for BAZ2A/B [4]. This is the second probe identified against this target, GSK2801, has previously been reported [5].

Co-crystal structure

The co-crystal structure of BAZ2-ICR and BAZ2B has been solved (pdb id 4XUB)

Potency Against Target Family

BAZ2-ICR has been shown by ITC to bind to BAZ2A with a KD of 109 nM (ITC) and to BAZ2B with a KD of 170 nM.

Selectivity

Click to enlarge

The inhibitor was screened at 10 μM concentration against 47 bromodomains using a temperature shift assay. The screened targets are labelled on the phylogenetic tree; targets that have not been screened are shown in grey. Temperature shifts are represented as spheres as indicated in the figure.

electivity Beyond Target Family

BAZ2-ICR (10 µm) displayed no activity against a panel of 55 receptors and ion channels

Cellular Activity

BAZ2-ICR shows accelerated FRAP recovery at 1 µM in the BAZ2A FRAP assay

properties

BAZ2-ICR

4-(4-(1-methyl-1H-pyrazol-3-yl)-1-(2-(1-methyl-1H-pyrazol-4-yl)ethyl)-1H-imidazol-5-yl)benzonitrile

For SDF click here

An inactive, negative control, compound for BAZ2-ICR has yet to be identified

Physical and chemical properties
Molecular weight	357.42
Molecular formula	C₂₀H₁₉N₇
IUPAC name	4-(4-(1-methyl-1H-pyrazol-3-yl)-1-(2-(1-methyl-1H-pyrazol-4-yl)ethyl)-1H-imidazol-5-yl)benzonitrile
logP	2.54
PSA	70.59
No. of chiral centres	0
No. of rotatable bonds	5
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	0
Storage	Stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolution	25 mM in D₂O

selectivity profile

Potency against Target

ITC

BAZ2-ICR has been shown by ITC to bind to BAZ2A with a KD of 109 nM (ITC) and to BAZ2B with a KD of 170 nM. The strong negative binding enthalpy possibly due to aromatic stacking interaction of pyrozole/phenyl

Click to enlarge

Selectivity Within Target family

DSF screen

BAZ2-ICR showed significant thermal shifts of 5.2 and 3.8 °C for BAZ2A and BAZ2B respectively. BAZ2-ICR did not show significant thermal shifts against all other bromodomains, except for Cat Eye syndrome chromosome region, candidate 2 (CECR2), where a smaller but significant shift (ΔTm: 2.0 °C) was observed. The Kd of BAZ2-ICR binding to CERC2 was determined to be 1.55 μM by ITC, giving a 15-fold and 10-fold selectivity over BAZ2A and BAZ2B respectively.

Click the images to enlarge them

AlphaScreen Assay

To confirm selectivity over BRD4, BAZ2-ICR assay was studied in a BRD4 AlphaScreen assay, where it did not show significant inhibition up to 50 μM, translating in greater than 100-fold window.

Selectivity Beyond Target Family

CEREP screen

BAZ2-ICR (10 µm) displayed no activity against a panel of 55 receptors and ion channels

Click to enlarge

in vitro potency

cell based assay data

FRAP

To investigate whether BAZ2-ICR can displace BAZ2 bromodomains from chromatin in living cells, we performed a fluorescence recovery after photobleaching (FRAP) assay utilizing GFP-tagged BAZ2A full length protein transfected into human osteosarcoma cells (U2OS). Mutant BAZ2A (N1873F) that does not bind acetylated lysine containing peptides and therefore mirrors the behaviour of inhibitor bound BAZ2A was used as a control. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) was used to increase overall levels of histone acetylation, resulting in a sufficient assay window to enable the measurement of differences in recovery time and demonstrating the acetylation dependence of the FRAP experiments. BAZ2-ICR at 1 μM reduced the recovery time of the wt construct to a level similar to the mutant, confirming that BAZ2-ICR inhibits BAZ2A in cells. Attempts to clone full length BAZ2B for this assay were unsuccessful.

Click the images to enlarge them

To confirm selectivity in the cellular setting, a similar FRAP assay for BRD4 selectivity was performed. Cells were transfected with GFP-tagged BAZ2A or BRD4 and treated with 1 μM PFI-1 (a selective probe for BET Bromodomains) or BAZ2-ICR. Light bars depict assay with SAHA addition, and dark bars depict assays without SAHA addition (2.5 μM). Fluorescence recovery (t½) expressed as a percentage of the relevant wild-type control cells without inhibitor; the dotted line demarks the point equivalent to 100% of the relevant wild-type control cells without inhibitor. Error bars depict the standard error of the mean. Bars marked with an asterisk indicate a significant difference from controls (P < 0.05).

references

A Novel Family of Bromodomain Genes,
Jones, M. H.; Hamana, N.; Nezu, Ji.; Shimane, M., Genomics, 63, 40– 45 (2000)
Identification of a sudden cardiac death susceptibility locus at 2q24.2 through genome-wide association in European ancestry individualsArking, D. E.; Junttila, M. J.; Goyette, P.; Huertas-Vazquez, A.; Eijgelsheim, M.; Blom, M. T.; Newton-Cheh, C.; Reinier, K.; Teodorescu, C.; Uy-Evanado, A.; Carter-Monroe, N.; Kaikkonen, K. S.; Kortelainen, M. L.; Boucher, G.; Lagace, C.; Moes, A.; Zhao, X.; Kolodgie, F.; Rivadeneira, F.; Hofman, A.; Witteman, J. C.; Uitterlinden, A. G.; Marsman, R. F.; Pazoki, R.; Bardai, A.; Koster, R. W.; Dehghan, A.; Hwang, S. J.; Bhatnagar, P.; Post, W.; Hilton, G.; Prineas, R. J.; Li, M.; Kottgen, A.; Ehret, G.; Boerwinkle, E.; Coresh, J.; Kao, W. H.; Psaty, B. M.; Tomaselli, G. F.; Sotoodehnia, N.; Siscovick, D. S.; Burke, G. L.; Marban, E.; Spooner, P. M.; Cupples, L. A.; Jui, J.; Gunson, K.; Kesaniemi, Y. A.; Wilde, A. A.; Tardif, J. C.; O’Donnell, C. J.; Bezzina, C. R.; Virmani, R.; Stricker, B. H.; Tan, H. L.; Albert, C. M.; Chakravarti, A.; Rioux, J. D.; Huikuri, H. V.; Chugh, S. S., PLoS Genet., 7, e1002158 (2011)
The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats Guetg, C.; Lienemann, P.; Sirri, V.; Grummt, I.; Hernandez‐Verdun, D.; Hottiger, M.O.; Fussenegger, M.; Santoro, R., The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats, Embo J., 29, 2135-2146, (2010)
Structure Enabled Design of BAZ2-ICR, A Chemical Probe Targeting the Bromodomains of BAZ2A and BAZ2BDrouin, L.; McGrath, S.; Vidler, L.R.; Chaikuad, A.; Monteiro, O.; Tallant, C.; Philpott, M.; Rogers, C.; Fedorov, O.; Liu, M.; Akhtar, W.; Hayes, M.; Raynaud, F.; Müller, S.; Knapp, S.; Hoelder. S., J. Med. Chem., 58, 2553–2559, (2015)
Discovery and Characterization of GSK2801, a Selective Chemical Probe for the Bromodomains BAZ2A and BAZ2BChen, P.; Chaikuad, A.; Bamborough, P.; Bantscheff, M.; Bountra, C.; Chung, C.; Fedorov, O.; Grandi, P.; Jung, D.; Lesniak, R.; Lindon, M.; Müller, S.; Philpott, M.; Prinjha, R.; Rogers, C.; Selenski, C.; Tallant, C.; Werner, T.; Willson, T.M.; Knapp, S.; Drewry, D.H., J. Med. Chem., 2015, DOI: 10.1021/acs.jmedchem.5b00209
Kinetic Stabilization of an Oligomeric Protein by a Single Ligand Binding EventWiseman, R. L.; Johnson, S. J.; Kelker, M. S.; Foss, T.; Wilson, I. A.; and Kelly, J. W., J. Am. Chem. Soc. 127, 5540-5551, (2005)
Selective inhibition of BET bromodomainsFilippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W.B.; Fedorov, O.; Morse, E.M.; Keates, T.; Hickman, T.T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown, M.R.; Wang, Y.; Christie, A.L.; West, N.; Cameron, M.J.; Schwartz, B.; Heightman, T.D.; La Thangue, N.; French, C.A.; Wiest, O.; Kung, A.L.; Knapp, S.; Bradner, J. E., Nature 468, (7327) 1067-1073 ( 2010)

pk properties

The physicochemical and mouse pharmacokinetic properties of BAZ2-ICR were assessed. BAZ2-ICR showed very high solubility (25 mM in D₂O), a measured log D of 1.05, high stability in mouse microsomes, and permeation in the CaCo-2 model and thus a suitable profile for oral and intravenous gavage. We therefore performed a full mouse pharmacokinetic experiment. In agreement with the in vitro data, BAZ2-ICR showed 70% bioavailability and moderate clearance (∼50% of mouse liver blood flow) and volume of distribution, suggesting that BAZ2-ICR is suitable for modulating BAZ2A and BAZ2B in vivo.

3 mice were dosed IV and 3 mice PO. Key parameters and the curves are shown for each animal.

Click to enlarge

co-crystal structures

The co-crystal structure of BAZ2-ICR and BAZ2B has been solved (pdb id 4XUB)

The key interactions are illustrated below.

Click to enlarge

synthetic schemes

materials and methods

Isothermal Titration Calorimetry

Experiments were carried out on a VP-ITC titration microcalorimeter from MicroCalTM, LLC (Northampton, MA), at 15 °C while stirring at 295 rpm, in ITC buffer (50 mM HEPES, 150 mM NaCl). All titrations were conducted using an initial injection of 2 µl followed by 34 identical injections of 8 µl with a duration of 16 sec/injection and a spacing of 250 sec between injections. The heat of dilution was determined by independent titrations (protein into buffer) and was subtracted from the experimental data. MicroCal^TM Origin software was used to calculate enthalpies of binding (ΔH) and binding constants (KB). Thermodynamic parameters were calculated (ΔG = ΔH - TΔS = -RTlnKB, where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding respectively). In all cases a single binding site model was employed.

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. Compounds were 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. Data was analysed as previously reported [7]

CEREP assay

BAZ2-ICR (10 µm) was screened against a panel of 55 ligand receptors, ion channels and transports using an established and widely utilized commercial assay platform (ExpresSProfile; CEREP, Paris, FRANCE).

Fluorescence Recovery After Photobleaching (FRAP) Assay

FRAP studies were performed using U20S cells expressing full-length BAZ2A or BRD4 protein fused with an N-terminal eGFP as previously described [4]. Six hours after transfection 2.5uM SAHA (to increase global histone acetylation) was added and BAZ2-ICR was added 1 hour before imaging, which was carried out 24 hours after transfection. Percent inhibition was calculated between the DMSO treated (0%) and N1873F expressing mutant (100%)

03.03.2014

SGC and DiscoveRx® Corporation Publish Study Identifying Clinical Kinase Inhibitors that Potently Cross-React with Bromodomain Epigenetic Reader Proteins

by: SGC

Oxford, UK and Fremont, US - March 3, 2014 – The Structural Genomics Consortium (SGC) and DiscoveRx Corporation announced the publication of findings demonstrating that several clinical kinase inhibitors also potently inhibit diverse bromodomain epigenetic reader proteins.

GSK-LSD1 A chemical probe for LSD1

The probe GSK-LSD-1 (hydrochloride) is available from Cayman Chemical, Sigma and Tocris.

overview
properties

overview

Probe

GSK-LSD1

GSK-LSD1 (trans-racemic)
MW 216.3
FW 289.2 (di-HCl)

Lysine specific demethylase 1 (LSD1) is a histone demethylase found in various transcriptional co-repressor complexes. LSD1 is involved in ES cell differentiation, hematopoiesis, and has been described as having a role in Acute Myeloid Leukemia (AML). GlaxoSmithKline has developed an irreversible, mechanism based inhibitor of LSD1, GSK-LSD1, and has made this available as a chemical probe as part of the SGC epigenetics initiative. GSK-LSD1 inhibits LSD1 with an IC₅₀ of 16 nM and is > 1000 fold selective over other closely related FAD utilizing enzymes (i.e. LSD2, MAO-A, MAO-B). GSK-LSD1 induces gene expression changes in cancer cell lines (average EC₅₀ < 5 nM) and inhibits cancer cell line growth (average EC₅₀< 5 nM).

LSD-1 at 10 µM showed no activity against 55 human recombinant receptors in the CEREP ExpresSProfile selectivity/specificity profile (GPCR, transporters, ion channels), except 5-HT1A (49% inhibition) 5-HT transporter (74% inhibition) and dopamine transporter (39 % inhibition).

properties

Probe

GSK-LSD1

Physical and chemical properties for GSK-LSD1
Molecular weight	216.2
Molecular formula	C14H20N2
IUPAC name	N-(2-phenyl-cyclopropyl)-piperidin-4-amine
MollogP	2.639
PSA	23.66
No. of chiral centres	2
No. of rotatable bonds	3
No. of hydrogen bond acceptors	2
No. of hydrogen bond donors	2

SMILES:
C1(N[C@@H]2C[C@H]2C3=CC=CC=C3)CCNCC1
InChI:
InChI=1S/C14H20N2/c1-2-4-11(5-3-1)13-10-14(13)16-12-6-8-15-9-7-12/h1-5,12-16H,6-10H2/t13-,14+/m0/s1
InChI Key:
BASFYRLYJAZPPL-UONOGXRCSA-N

selectivity profile

in vitro potency

cell based assay data

references

pk properties

co-crystal structures

synthetic schemes

materials and methods

18.12.2013

Bayer HealthCare to Join Not-For-Profit SGC to Accelerate Research in Epigenetics

by: SGC

Toronto, December 18, 2013 – Bayer Inc. is pleased to announce that Bayer HealthCare has joined the Structural Genomics Consortium (SGC), a not-for-profit, public-private partnership with active research facilities at the Universities of Toronto and Oxford, UK. Bayer will support funding the consortium to accelerate precompetitive drug research in the areas of protein sciences and epigenetics. Furthermore, Bayer will provide a subset of its compound library for screening to the SGC and will also conduct the chemical work to identify probes.

PFI-3 A selective chemical probe for SMARCA bromodomains

This probe is available from Cayman Chemical, Sigma and Tocris

overview
properties
selectivity profile
cell based assay data
references

overview

Probe		Negative control

PFI-3		PFI-3oMet

In vitro Potency
Assay	K_d/IC₅₀ (nM)
SMARCA4 (ITC)	89
PB1(5) (ITC)	48

SMARCA4 (SWI/SNF related, Matrix associated, Actin dependent Regulator of Chromatin, subfamily A, member 4), also known as BRG1 (Brahma-Related Gene 1), is part of the SWI/SNF (SWItch/Sucrose NonFermentable) family of proteins. Multiple transcript variants encoding different isoforms have been found for this gene in normal tissue as well as in cancer. SMARCA4 and the related protein SMARCA2 (BRM, BRahMa) contain a bromo and helicase domain and both proteins are central components of the large ATP-dependent SNF/SWI chromatin remodelling complex, which plays a key role in chromatin remodelling and transcription control. PB1 (PolyBromo 1) can also be part of the SWI/SNF complex.

Loss of function of SMARCA4 and components of SWI/SNF has been linked to cancer development suggesting a tumour suppressor function for BRG1. SMARCA4 has also been shown to interact with the breast cancer gene BRCA1 (BReast CAncer 1, early onset), as well as to regulate the expression of the tumorigenic protein CD44.

We have developed an inhibitor, PFI-3, against the SMARCA2/4 and PB1(5) bromodomains.

properties

(2E)-1-(2-hydroxyphenyl)-3-[(1R,4R)-5-(pyridin-2-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl]prop-2-en-1-one
Click here to download SDF file

Physical and chemical properties
Molecular weight	321.4
Molecular formula	C ₁₉H ₁₉N ₃O ₂
IUPAC name	(2E)-1-(2-hydroxyphenyl)-3-[(1R,4R)-5-(pyridin-2-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl]prop-2-en-1-one
logP (ChemBioDraw Ultra)	2.39
PSA (ChemBioDraw Ultra)	56.14
Storage	-20°C as powder. NB making aliquots rather than freeze-thawing is recommended
Dissolution	Soluble in DMSO at least up to 10mM
Stability* at 37°C	t _1/2 > 15 days
Stability* at 20°C	t _1/2 > 200 days

*as measured by LCMS

SMILES:
PFI-3: OC1=CC=CC=C1C(/C=C/N2[C@@H]3C[C@H](C2)N(C4=NC=CC=C4)C3)=O
PFI-3oMet: COC1=CC=CC(C(/C=C/N2[C@H](C3)CN(C4=NC(NCC#C)=CC=N4)[C@H]3C2)=O)=C1O
InChI:
InChI=1S/C19H19N3O2/c23-17-6-2-1-5-16(17)18(24)8-10-21-12-15-11-14(21)13-22(15)19-7-3-4-9-20-19/h1-10,14-15,23H,11-13H2/b10-8+/t14-,15-/m1/s1
InChIKey:
PFI-3: INAICWLVUAKEPB-QSTFCLMHSA-N
PFI-3oMet: SLDSPTPDLGRIQU-ZVYLKZBJSA-N

selectivity profile

PFI-3 only shows a significant Tm shift with PB1(5) and SMARCA2/4. No interaction was observed with PB1(2-4), and there was no cross-reactivity in a kinase panel of 36 kinases.

Selectivity
Bromodomains
Target	Tm shift °C @ 10 µM
SMARCA2	6.4
SMARCA4	5.1
PB1 1st Bromodomain	-0.5
PB1 2nd Bromodomain	1.1
PB1 3rd Bromodomain	0.9
PB1 5th Bromodomain	7.5
PB1 6th Bromodomain	0.4
PCAF	0.5
BRD4 1st Bromodomain	0.3
BRD4 2nd Bromodomain	0.4
CREBBP	-0.0
BRD1	0.0
TIF1alpha	0.7
TRIM2B	0.3
TAF1L 1st Bromodomain	0.2
TAF1L 2nd Bromodomain	0.3
Other proteins
Invitrogen kinase panel	No activity

in vitro potency

cell based assay data

Selectivity
SMARCA2 (Frap Assay)	Accelerated FRAP recovery at 1 uM
SMARCA2 (Frap Assay) *	Accelerated FRAP recovery at 1 uM
SMARCA2 (Frap Assay) #	Accelerated FRAP recovery at 1 uM

*24 hour compound incubation on cells
^1 hour compound incubation on cells preceded by 24 hour pre-incubation of the compound in medium

references

Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance. Oleg Fedorov, Josefina Castex, Cynthia Tallant, Dafydd R. Owen, Sarah Martin, Matteo Aldeghi, Octovia Monteiro, Panagis Filippakopoulos, Sarah Picaud, John D. Trzupek, Brian S. Gerstenberger, Chas Bountra, Dominica Willmann, Christopher Wells, Martin Philpott, Catherine Rogers, Philip C. Biggin, Paul E. Brennan, Mark E. Bunnage, Roland Schüle, Thomas Günther, Stefan Knapp, Susanne Müller

Science Advances 13 Nov 2015:
Vol. 1, no. 10, e1500723
DOI: 10.1126/sciadv.1500723

pk properties

co-crystal structures

synthetic schemes

materials and methods

LLY-507 (multiple off-targets) A chemical probe for SMYD2 protein lysine methyltransferase

This probe is available from Cayman Chemical and Sigma.

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

overview

Probe		Negative control

LLY-507 (IC₅₀ < 15 nM)		SGC705 (IC₅₀> 10,000 nM)

A collaboration between the SGC and Eli Lilly and Company has resulted in the discovery of LLY-507, a chemical probe for SMYD2 (a protein lysine methyltransferase). LLY-507 is a potent inhibitor of SMYD2 with in vitro IC₅₀ <15 nM and >100-fold selectivity over other methyltransferases and other non-epigenetic targets. LLY-507 has been shown to inhibit p53K370 monomethylation in cells with an IC₅₀ ~600 nM.

Note: After LLY-507 was released, off-target screening (in addition to those presented in the publication, doi:10.1074/jbc.M114.626861) was done. Here is a copy of the report from a functional screen of LLY-507 and its control SGC705 in the NIMH Psychoactive Drug Screening Program (PDSP). This report clearly shows more than 50% inhibition of a number of targets.

properties

Probe		Negative control

LLY-507 (IC₅₀ < 15 nM)		SGC705 (IC₅₀> 10,000 nM)

Physical and chemical properties for LLY-507
Molecular weight	574.3
Molecular formula	C36H42N6O
IUPAC name	5-(2-(4-(2-(9-methyl-7-aza-bicyclo[4.3.0]nona-1(6),2,4,8-tetraen-7-yl)-ethyl)-piperazin-1-yl)-phenyl)-3-((3-(pyrrolidin-1-yl)-propylamino)-formyl)-benzonitrile
MollogP	5.891
PSA	54.28
No. of chiral centres	0
No. of rotatable bonds	12
No. of hydrogen bond acceptors	5
No. of hydrogen bond donors	1

Physical and chemical properties for SGC705 (Negative Control)
Molecular weight	573.3
Molecular formula	C37H43N5O
IUPAC name	3-((3-cyclopentyl-propylamino)-formyl)-5-(2-(4-(2-(9-methyl-7-aza-bicyclo[4.3.0]nona-1(6),2,4,8-tetraen-7-yl)-ethyl)-piperazin-1-yl)-phenyl)-benzonitrile
MollogP	7.504
PSA	50.49
No. of chiral centres	0
No. of rotatable bonds	12
No. of hydrogen bond acceptors	4
No. of hydrogen bond donors	1

SMILES:
LLY-507: CC1=CN(C2=C1C=CC=C2)CCN3CCN(C4=CC=CC=C4C5=CC(C(NCCCN6CCCC6)=O)=CC(C#N)=C5)CC3
SGC705: CC1=CN(C2=C1C=CC=C2)CCN3CCN(C4=C(C5=CC(C(NCCCC6CCCC6)=O)=CC(C#N)=C5)C=CC=C4)CC3
InChI:
LLY-507: InChI=1S/C36H42N6O/c1-28-27-42(34-11-4-2-9-32(28)34)22-19-40-17-20-41(21-18-40)35-12-5-3-10-33(35)30-23-29(26-37)24-31(25-30)36(43)38-13-8-16-39-14-6-7-15-39/h2-5,9-12,23-25,27H,6-8,13-22H2,1H3,(H,38,43)
SGC705: InChI=1S/C37H43N5O/c1-28-27-42(35-14-6-4-12-33(28)35)22-19-40-17-20-41(21-18-40)36-15-7-5-13-34(36)31-23-30(26-38)24-32(25-31)37(43)39-16-8-11-29-9-2-3-10-29/h4-7,12-15,23-25,27,29H,2-3,8-11,16-22H2,1H3,(H,39,43)
InChIKey:
LLY-507: PNYRDVBFYVDJJI-UHFFFAOYSA-N
SGC705: UZLKKMNSZHUBDI-UHFFFAOYSA-N

selectivity profile

Effect of LLY-507 on the activity of 27 protein methyltransferases as well as DNMT1.

Mechanism of Action

in vitro potency

cell based assay data

Cellular Activity

Dose dependent inhibition of p53 K370 me1 by LLY-507 in KYSE-150 cells stably expressing SMYD2 as measured by a Meso Scale Discovery sandwich ELISA assay (IC50 = 0.6 microM).

references

LLY-507, a Cell-Active, Potent and Selective Inhibitor of Protein Lysine Methyltransferase SMYD2 J. Biol. Chem. doi:10.1074/jbc.M114.626861
Hannah Nguyen, Abdellah Allali-Hassani, Stephen Antonysamy, Shawn Chang, Lisa Hong Chen, Carmen Curtis, Spencer Emtage, Li Fan, Tarun Gheyi, Fengling Li, Shichong Liu, Joseph R. Martin, David Mendel, Jonathan B. Olsen, Laura Pelletier, Tatiana Shatseva, Song Wu, Feiyu Fred Zhang, Cheryl H. Arrowsmith, Peter J. Brown, Robert M. Campbell, Benjamin A. Garcia, Dalia Barsyte-Lovejoy, Mary Mader and Masoud Vedadi.

pk properties

co-crystal structures

Please wait whilst the interactive viewer is loaded!

PDB: 4WUY
Main features

synthetic schemes

materials and methods

25.10.2013

SGC Announces Janssen has joined Public-Private Drug Discovery Partnership

by: SGC

Toronto, ON (October 25, 2013) – The Structural Genomics Consortium (SGC) announced today that Janssen Pharmaceuticals NV has joined its group to partner in the investigation of epigenetics-based drug research, a new and burgeoning field in the development of future medicines.

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