GSK6853 is available for purchase from Cayman Chemical, Sigma and Tocris.
Its negative control (GSK9311) is available for purchase from Sigma and Tocris (hydrochloride).
| 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.
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.
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.
| Chrom logD pH 7.4 | 2.0 |
| CLND solubility (μg/mL) | 140 |
| iv CLb (mL/min/kg)/t1/2 (h) | 107/1.7 |
| F% ip/po (3 mg/kg) | 85/22 |
| Probe | Negative control | |
|
| |
GSK6853 |
| GSK9311 |
| Physical and chemical properties for GSK-6853 | |
| Molecular weight | 409.2 |
| Molecular formula | C22H27N5O3 |
| IUPAC name | 3-((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 |
| MollogP | 1.779 |
| PSA | 62.28 |
| No. of chiral centres | 1 |
| No. of rotatable bonds | 5 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 2 |
| Physical and chemical properties for GSK-9311 (Negative Control) | |
| Molecular weight | 437.2 |
| Molecular formula | C24H31N5O3 |
| IUPAC name | 3-(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 |
| MollogP | 2.369 |
| PSA | 54.93 |
| No. of chiral centres | 1 |
| No. of rotatable bonds | 6 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 1 |
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 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).
Work on this probe has been published in 'GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain’.
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)
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.
| 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>
| Probe | Negative control | |
|
| |
GSK8814 |
| GSK8815 |
| Physical and chemical properties for GSK8814 | |
| Molecular weight | 527.3 |
| Molecular formula | C28H35F2N5O3 |
| IUPAC name | 10-(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 |
| MollogP | 3.212 |
| PSA | 78.42 |
| No. of chiral centres | 3 |
| No. of rotatable bonds | 7 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 3 |
| Physical and chemical properties for GSK8815 (Negative Control) | |
| Molecular weight | 527.3 |
| Molecular formula | C28H35F2N5O3 |
| IUPAC name | 10-(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 |
| MollogP | 3.212 |
| PSA | 78.42 |
| No. of chiral centres | 3 |
| No. of rotatable bonds | 7 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 3 |
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.
This probe is available from Cayman Chemical and Sigma (trihydrochloride).
| Probe | Negative control | |
 |
|  |
BAY-850 |
| BAY-460 |
ATAD2 and ATAD2B are epigenetic regulators known to function as co-factors for oncogenic transcription 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.
BAY-850 is a chemical probe for the ATAD2A bromodomain which displaces acetylated H4 peptide from the ATAD2 bromodomain with an IC50 value of 20 nM (HTRF). MST and BROMOscan measurements indicate a Kd of 84.9 nM and 120 nM, respectively. BAY-850 displays exquisite selectivity over all other bromodomains (no hits in BROMOscan at 10 µM) and displaces full-length ATAD2 from chromatin in live cells at a concentration of 1 µM as determined by FRAP. To avoid potential unspecific off-target effects concentrations of ≥ 5 µM are not recommended.
BAY-460 is a companion control compound with strongly reduced potency against ATAD2 (HTRF IC50 = 16 µM, no activity in BROMOscan at 10 µM test concentration).
Properties
| ATAD2H4AcK5/8/12/16 IC50 (HTRF) | 0.02 μM |
| LLElogD | 2.6 |
| ATAD2 TSA ΔT(100μM) | 13.9°C |
| BRD4 BD1 IC50 (HRTF) | >20 μM |
| BRD4BD2 IC50 (HTRF) | >20 μM |
| DiscoverX BromoScan | ATAD2A @ 10 μM |
| DiscoverX ATAD2A KD | 0.12 μM |
| MST KD | 0.08 μM |
| Sw pH 6.5 [mg/L] | >500* |
| log D (pH 7.5) | 2.9 |
*HCl salt
| Probe | Negative control | |
|
| |
BAY-850 |
| BAY-460 |
| Physical and chemical properties for BAY-850 | |
| Molecular weight | 653.3 |
| Molecular formula | C38H44ClN5O3 |
| IUPAC name | 4-(3-(4-amino-cyclohexylamino)-2-((2-chloro-4-methoxy-5-(5-((1-p-tolyl-ethylamino)-methyl)-furan-2-yl)-phenyl)-formylamino)-propyl)-benzonitrile |
| MollogP | 6.17 |
| PSA | 98.59 |
| No. of chiral centres | 2 |
| No. of rotatable bonds | 15 |
| No. of hydrogen bond acceptors | 8 |
| No. of hydrogen bond donors | 5 |
| Physical and chemical properties for BAY-460 (Negative Control) | |
| Molecular weight | 568.2
|
| Molecular formula | C33H33ClN4O3 |
| IUPAC name | 4-(2-((2-chloro-5-(5-((1-p-tolyl-ethylamino)-methyl)-furan-2-yl)-phenyl)-formylamino)-4-methylamino-4-oxo-butyl)-benzonitrile |
| MollogP | 5.533 |
| PSA | 84.53 |
| No. of chiral centres | 2 |
| No. of rotatable bonds | 14 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 3 |
BAY-460: HBEUJCCOUJWRIC-AMGIVPHBSA-N
Fernández-Montalván, A., Berger, M., Kuropka, B., Koo, S., Badock, V., & Weiske, J. et al. (2017). Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action. ACS Chemical Biology. http://dx.doi.org/10.1021/acschembio.7b00708
Oxford, UK, December 19, 2016, Scientists from the Structural Genomics Consortium (SGC) and Oxford University report a three-dimensional view of the Polycystin-2 (PC2), a protein that causes autosomal dominant polycystic kidney disease (ADPKD) when mutated, in an article published today in Nature Structural and Molecular Biology.
SGC wins Oxford AHSN Public-Private Collaboration Award
Shown left-to-right: John Harris (CEO of OBN), Dr. Rab Prinjha (VP of GSK Medicines Research Centre), Chas Bountra (Director SGC Oxford), Dr. Paul Durrands (COO Oxford AHSN), and Dr. Wen Hwa Lee (Director Disease Foundations Network & Strategic Alliances SGC).
Target-enabling packages provide a crucial link between human genetics and disease biology to accelerate drug discovery in cancer and malaria
The SGC has released its first set of Target-Enabling Packages (TEPs) to the scientific community.
We announce today a groundbreaking partnership between the Alzheimer’s Research UK Drug Discovery Alliance (DDA) and the Structural Genomics Consortium (SGC), an international group of academic and industrial researchers with a strong track record in uncovering new drug targets and enabling new clinical trials, quickly.
We are pleased to announce that an updated version of The Chemical Probes Portal is now live. Please visit the portal and look around. In addition to the site’s new look, we’ve added new content, including a lot more data about the existing probes, reviewer ratings and their comments.
NVS-PAK1-1 is available from Sigma, Cayman Chemical and Tocris.
Its negative control (NVS-PAK1-C) is available for purchase from Tocris and Cayman Chemical.
| Probe | Negative control | |
 |
|  |
NVS-PAK1-1 |
| NVS-PAK1-C |
Biology of the PAK1 kinase
PAK kinases belong belong to the family of STE kinases, named after the yeast STE7, STE11 and STE20 genes, which form the MAPK cascade, transducing signals from the surface of the cell to the nucleus. The PAK family itself consist of 6 members and can be subdivided into 2 groups Group I PAKs (PAK1-3) and Group II PAKs (PAK 4-6). Group I PAKs (1-3) contain a N-terminal PBD-AIN domain and a kinase domain as well as two SH3-domains and one non-classical SH3-domain.
PAK1 is involved in many intracellular signaling pathways downstream of integrins and receptor-type kinases and plays an important role in in cell adhesion, cytoskeleton dynamics, migration, proliferation, apoptosis, mitosis, and in vesicle-mediated transport processes. It also plays a role in the regulation of insulin secretion in response to elevated glucose levels. PAKs have been shown to activate components of ERK, AKT and WNT signaling pathway and functions as GTPase effector that links the Rho-related GTPases CDC42 and RAC1 to the JNK MAP kinase pathway. PAK1 phosphorylates and activates MAP2K1, and thereby mediates activation of downstream MAP kinases.
PAK1 has been found to be overexpressed and/or activated in many tumor types and its expression levels have been correlated with patient prognosis. In addition Group I PAKs have been implicated in neurodegenerative disorders like Alzheimer and Huntington diseases as well as FXS (fragile X syndrome).
We recommend using NVS1-PAK1-1 at 2.5 uM for PAK1/2 inhibition or 0.25 uM for PAK1 inhibition.
The inactive control at similar concentration has no effect on PAK1/2
NVS-PAK1-1: A Chemical Probe for PAK1
NVS1-PAK-1, a specific allosteric PAK1 inhibitor, has been developed by Novartis [1]
Chemical structure of NVS-PAK1-1 and its inactive control compound NVS-PAK1-C
Co-Crystal Structure
Details of the co-crystal structure of the hit compound and a related compound with the PAK1 kinase domain, click on the 'Co-Crystal structures' tab above for more details
Potency Against Target Family
NVS-PAK1-1 is a potent allosteric inhibitor of PAK1 with an IC 50 of 5 nM for dephosphorylated PAK1 and 6 nM for phosphorylated PAK1 as assessed in a Caliper assay. This data is in good agreement with KD of 7 nM assessed in DiscoverX kinome scan. Inhibition is ATP-competitive, most likely due to indirect competition due to incompatibility of ATP binding with the DFG-out binding conformation of the allosteric compound. The control compound NVS-PAK1-C has more than 100 fold less activity in the respective Caliper assays.
IC Activity against the closest PAK member, PAK2 is over 54 fold. NVS-PAK1-1 has an IC50 of 270 nM against dephosphorylated PAK2 and 720 nM against phosphorylated PAK2 respectively in Caliper assay.
Selectivity
Selectivity of NVS-PAK1-1 was assessed against a panel of 442 kinases (DiscoverX kinome scan) at 10 µM and the compound was found to be exquisitely selective.
Selectivity Beyond Target Family
NVS-PAK1-1 was found to be inactive vs 27 receptors (Novartis panel) at 10µM, with the most potent activity measured of 13 uM against H1 and M1. No activity at 10µM was found in a panel of 48 BRDs.
Cellular Activity
NVS-PAK1-1 potently inhibits autophosphorylation of PAK1 (S144) at 0.25 µM in the Su86.86 cell line and MEK S289 phosphorylation with an IC50 = 0.21 in Su86.86 cells in which PAK2 is downregulated.
| NVS-PAK1-1 |
|
(S)-3-((2-chloro-5-(2,2-difluoroethyl)-8-fluoro-5H-dibenzo[b,e][1,4]diazepin-11-yl)amino)-N-isopropylpyrrolidine-1-carboxamide |
Selectivity Within Target family
KINOMEscan
NVS-PAK1-1 is highly selective against 442 kinases tested at 10 µM with a selectivity score (S10-score) of 0.003.
442 Kinases tested at 10 uM
S(35), 3 hits, Sscore = 0.008
S(10), 1 hit, Sscore = 0.003
Selectivity Beyond Target Family
NVS-PAK1-1 shows no cross-reactivity in a panel of 53 proteases (tested at 10 µM), 22 receptors (IC50> 13 µM against closest off-target Phosphodiesterase 4D) and a panel of 28 bromodomains.
Materials and Methods
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 [3]
KINOMEscan
NVS-PAK1-1 was profiled by KINOMEscan™, a platform that measures the interactions between test compounds and a panel of kinase assays (http://www.discoverx.com), against 442 kinases.
Potency against Target
Caliper Assay
NVS-PAK1-1 is a potent allosteric inhibitor of PAK1 with an IC50 of 0.005 µM for dephoshorylated PAK1 and 0.006 µM for phosphorylated PAK1 as shown in a Caliper in vitro dephosphorylation assay.
KINOME scan PAK1 Binding Assay.
NVS-PAK1-1 binds to PAK1 with a KD of 7 nM.
Materials and Methods
Caliper assay
Inhibition of PAK1 kinase activity was measured using the Caliper (Caliper LC3000, PerkinElmer) assay. The assay was performed using 384-well microtiter plates. Compounds were tested as 8-point dose responses using PAK1 expressed in E.coli. IC50 values were derived from percent inhibition values at different compound concentrations by non-linear regression analysis.
Binding Assay
Binding assays were performed at DiscoverX coporation in the KINOMEscan assay. Inhibitor 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.
The cellular efficacy of NVS-PAK-1-1 on PAK1 was investigated assessing autophosphorylation in the pancreatic duct carcinoma cell line Su86.86 expressing high levels of PAK1 and PAK2. PAK1 and PAK2 are activated by autophosphorylation on PAK1 (S144) and PAK2 (S141), respectively due to blockage of PDB-domain autoinhibition.
NVS-PAK-1-1 blocks autophosphorylkation at both sites in dose-dependent manner.
Substrate phosphorylation
NVS-PAK-1-1 also inhibits phosphorylation of the downstream substrate MEK1 at Ser289, but only at concentrations that inhibit both PAK1 and PAK2 (6-20 µM).
Antiproliferative effect
Pancreatic duct carcinoma Su86.86 cells treated with NVS-PAK1-1 for 5 days showed inhibition of proliferation with an IC50 of 2 μM due to inhibition of both PAK1 and partial inhibition of PAK2. NVS-PAK1-1 shows growth inhibition with an IC50 of 0.21 μM in SU86.86 cells treated with shPAK2.
We recommend using NVS1-PAK1-1 at 2.5 uM for PAK1/2 inhibition or 0.25 uM for PAK1 inhibition.
Materials and Methods
PAK1/2 SER141/144 TARGET MODULATION ASSAY
Su86.86 pancreatic cancer cell line was grown and seeded in 6-well plate at 500,000 cell density. 10 mM compound diluted in DMSO was further diluted to appropriate concentration (3 fold dilution for 8 concentrations) using growth media and added to each well with cells grown 1 day before. Cells were harvested after 30 minutes of compound treatment and lysed with RIPA buffer (Sigma cat# R0278). Protein concentration of lysates was normalized before loading on SDS-PAGE. Western blot was done as described. pPAK1/2 Ser144/141 antibodies were generated as described. PAK1 and PAK2 antibodies were purchased from Cell Signaling Technology (PAK1 Cat#2602; PAK2 Cat#2608) and were mixed in equal amounts for generation of PAK1/2 signal in western blot.
[1] Karpov AS, Amiri P, Bellamacina C, Bellance MH, Breitenstein W, Daniel D, Denay R, Fabbro D, Fernandez C, Galuba I, Guerro-Lagasse S, Gutmann S, Hinh L, Jahnke W, Klopp J, Lai A, Lindvall MK, Ma S, Mobitz H, Pecchi S, Rummel G, Shoemaker K, Trappe J, Voliva C, Cowan-Jacob SW, Marzinzik AL. Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor. ACS medicinal chemistry letters. 2015;6:776-81.
[2] Radu M, Semenova G, Kosoff R, Chernoff J. PAK signalling during the development and progression of cancer.Nature reviews Cancer. 2014;14:13-25.
[3] Wang G, Zhang Q, Song Y, Wang X, Guo Q, Zhang J, Li J, Han Y, Miao Z, Li F. PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion. Cell death & disease. 2015;6:e1682.
[4] Fang F, Pan J, Li YP, Li G, Xu LX, Su GH, Li ZH, Feng X, Wang J. p21-activated kinase 1 (PAK1) expression correlates with prognosis in solid tumors: A systematic review and meta-analysis. Oncotarget. 2016.
[5] Pinto VI, Mohammadi H, Lee WS, Cheung AH, McCulloch CA. PAK1 is involved in sensing the orientation of collagen stiffness gradients in mouse fibroblasts. Biochimica et biophysica acta. 2015;1853:2526-38.
The co-crystal structure of a related compound with PAK1 has been solved with a resolution of 1.99Å. The key features are
This probe (as hydrochloride) and its inactive control are available from Sigma (A-395 and A-395N) and Cayman (A-395).
| Probe | Negative control | |
 |
|  |
A-395 |
| A-395N |
A collaboration between Abbvie and the SGC has resulted in the discovery of A-395, the first potent and selective chemical probe for EED. The in vitro activity of A-395 includes potent binding to EED with Ki = 0.4 nM, inhibits the PRC2 complex with IC50 = 34 nM for methylation of H3K27 and greater than 100-fold selectivity over other histone methyltransferases and non-epigenetic targets. In cellular assays, A-395 inhibits the PRC2 complex (thus inhibiting the formation of H3K27me3) with IC50 = 90 nM (RD rhabdoid tumor cell line; 3 days).
A closely related compound, A-395N, exhibits no activity in the biochemical and cellular assays, and is an ideal control compound for cellular studies.
A-395 potently inhibited the formation of H3K27me3 (via antagonizing EED in the trimeric PRC2 complex (EZH2:EED:SUZ12)) with IC50 = 34 ± 2 nM (Hill Slope = 0.7)
| Probe | Negative control | |
|
| |
A-395 |
| A-395N |
| Physical and chemical properties for A-395 | |
| Molecular weight | 486.2 |
| Molecular formula | C26H35FN4O2S |
| IUPAC name | 1-(5-fluoro-bicyclo[4.3.0]nona-1,3,5-trien-7-yl)-N,N-dimethyl-4-(4-(4-methylsulfonyl-piperazin-1-yl)-phenyl)-pyrrolidin-3-amine |
| MollogP | 4.425 |
| PSA | 39.69 |
| No. of chiral centres | 3 |
| No. of rotatable bonds | 5 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 0 |
| Physical and chemical properties for A-395N | |
| Molecular weight | 442.2
|
| Molecular formula | C24H34N4O2S |
| IUPAC name | 1-benzyl-N,N-dimethyl-4-(4-(4-methylsulfonyl-piperazin-1-yl)-phenyl)-pyrrolidin-3-amine |
| MollogP | 3.158 |
| PSA | 40.58 |
| No. of chiral centres | 2 |
| No. of rotatable bonds | 6 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 0 |
Selectivity of A-395 against 32 methyltransferase enzymes
Selectivity of a) A-395 and b) A-395N relative to epigenetic readers using a thermal shift assay
 |  |
A-395 inhibits the activity of the PRC2 complex in cells – high-content screening assay of RD Rhabdoid tumor cell line treated for 72 hours
Main features