SGC-AAK1-1 A dual inhibitor of AAK1 and BMP2K/BIKE

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

Its negative control (SGC-AAK1-1N) is available for purchase from Tocris and Sigma.

  • overview
  • properties
  • references
overview
Probe Negative control

 

SGC-AAK1-1

 

SGC-AAK1-1N

AAK1 (adaptor protein 2-associated kinase) and BMP2K/BIKE (BMP-2 inducible kinase) comprise half of the numb-associated kinase (NAK) family, which also includes cyclin G associated kinase (GAK) and STK16/MPSK1 (serine/threonine kinase 16/myristoylated and palmitoylated serine/threonine kinase 1).1

AAK1 is a 104 kDa serine/threonine kinase with broad tissue expression. Within the cell AAK1 localizes to the cell membrane and cytoplasm.2 AAK1 is involved in clathrin-mediated endocytosis (CME), both by direct binding to clathrin and by phosphorylation of the medium subunit of AP-2 (adaptor protein 2).3-5 In this manner, AAK1 has been identified as a negative regulator of Wnt signaling via mediation of LRP6 internalization. Studies have implicated multiple roles for AAK1 in influencing Notch signaling, including priming and redistribution of Numb as well as Notch activation.6-7

Relatively less is known about the highly understudied kinase BIKE. BIKE is broadly expressed, and in the cell, it localizes to nuclear speckles.2 BIKE was originally identified as its expression was observed to increase upon bone morphogenic protein (BMP-2)-induced differentiation of a prechondroblastic cell line.8 The same study provided evidence for BIKE having an important regulatory role in attenuating the program of osteoblast differentiation. Proteomic studies identified BIKE as a clathrin vesicle-associated protein and have also identified interaction between BIKE and Numb.9-10

NAK family domain structures

Location of AAK1 and BIKE on kinome tree

Snapshot of crystal structure of acylaminoindazole bound to BIKE

SGC-AAK1-1 is a chemical probe for AAK1 and BIKE that potently targets the ATP-binding site (AAK1 Ki =  9.1 nM; BIKE Ki = 17 nM). Regarding kinase selectivity, only three kinases were observed to bind SGC-AAK1-1 within 30-fold of the KD of AAK1 in a KINOMEscan assay (DiscoverX) at 1 μM followed by KD determinations: RIOK1 (KD = 72 nM), RIOK3 (KD = 290 nM), and PIP5K1C (KD = 260 nM). In a live cell NanoBRET assay (Promega) SGC-AAK1-1 has potency for ectopically expressed full-length AAK1- and BIKE-Nluc fusion proteins (AAK1 IC50 = 230 nM; BIKE IC50 = 1.5 μM).

A chemically related negative control compound, SGC-AAK1-1N, is provided.

properties
Probe Negative control

 

SGC-AAK1-1

 

SGC-AAK1-1N

Physical and chemical properties for BAY-876
Molecular weight427.52
Molecular formulaC21H25N5O3S
IUPAC nameN-(6-(3-((N,N-diethylsulfamoyl)amino)phenyl)-1H-indazol-3-yl)cyclopropanecarboxamide
MollogP4.185
PSA89.47
No. of chiral centres0
No. of rotatable bonds9
No. of hydrogen bond acceptors8
No. of hydrogen bond donors3
Storage-20 °C as DMSO stock
DissolutionSoluble in DMSO at least up to 10 mM



 

Physical and chemical properties for BAY-588 (Negative Control)
Molecular weight398.48
Molecular formulaC20H22N4O3S
IUPAC nameN-(6-(3-(cyclopropanesulfonamido)phenyl)-1H-indazol-3-yl)isobutyramide
MollogP3.381
PSA87.62
No. of chiral centres0
No. of rotatable bonds7
No. of hydrogen bond acceptors7
No. of hydrogen bond donors3
Storage-20 °C as DMSO stock
DissolutionSoluble in DMSO at least up to 10 mM

SMILES:
SGC-AAK1-1: O=C(NC1=NNC2=C1C=CC(C3=CC(NS(N(CC)CC)(=O)=O)=CC=C3)=C2)C4CC4
SGC-AAK1-1N: O=C(NC1=NNC2=C1C=CC(C3=CC(NS(C4CC4)(=O)=O)=CC=C3)=C2)C(C)C

 

InChI:
SGC-AAK1-1: InChI=1S/C21H25N5O3S/c1-3-26(4-2)30(28,29)25-17-7-5-6-15(12-17)16-10-11-18-19(13-16)23-24-20(18)22-21(27)14-8-9-14/h5-7,10-14,25H,3-4,8-9H2,1-2H3,(H2,22,23,24,27)

SGC-AAK1-1N: InChI=1S/C20H22N4O3S/c1-12(2)20(25)21-19-17-9-6-14(11-18(17)22-23-19)13-4-3-5-15(10-13)24-28(26,27)16-7-8-16/h3-6,9-12,16,24H,7-8H2,1-2H3,(H2,21,22,23,25)

InChIKey:
SGC-AAK1-1: UCBIQZUJJSVQHL-UHFFFAOYSA-N
SGC-AAK1-1N: RAIAORGFMNXPOV-UHFFFAOYSA-N

selectivity profile
in vitro potency
cell based assay data
references
  1. Sorrell, F. J.; Szklarz, M.; Abdul Azeez, K. R.; Elkins, J. M.; Knapp, S., Family-wide Structural Analysis of Human Numb-Associated Protein Kinases. Structure 2016, 24 (3), 401-11.
  2. Thul, P. J.; Akesson, L.; Wiking, M.; Mahdessian, D.; Geladaki, A.; Ait Blal, H.; Alm, T.; Asplund, A.; Bjork, L.; Breckels, L. M.; Backstrom, A.; Danielsson, F.; Fagerberg, L.; Fall, J.; Gatto, L.; Gnann, C.; Hober, S.; Hjelmare, M.; Johansson, F.; Lee, S.; Lindskog, C.; Mulder, J.; Mulvey, C. M.; Nilsson, P.; Oksvold, P.; Rockberg, J.; Schutten, R.; Schwenk, J. M.; Sivertsson, A.; Sjostedt, E.; Skogs, M.; Stadler, C.; Sullivan, D. P.; Tegel, H.; Winsnes, C.; Zhang, C.; Zwahlen, M.; Mardinoglu, A.; Ponten, F.; von Feilitzen, K.; Lilley, K. S.; Uhlen, M.; Lundberg, E., A subcellular map of the human proteome. Science 2017, 356 (6340).
  3. Conner, S. D.; Schmid, S. L., Differential requirements for AP-2 in clathrin-mediated endocytosis. J Cell Biol 2003, 162 (5), 773-9.
  4. Henderson, D. M.; Conner, S. D., A novel AAK1 splice variant functions at multiple steps of the endocytic pathway. Mol Biol Cell 2007, 18 (7), 2698-706.
  5. Neveu, G.; Ziv-Av, A.; Barouch-Bentov, R.; Berkerman, E.; Mulholland, J.; Einav, S., AP-2-associated protein kinase 1 and cyclin G-associated kinase regulate hepatitis C virus entry and are potential drug targets. J Virol 2015, 89 (8), 4387-404.
  6. Gupta-Rossi, N.; Ortica, S.; Meas-Yedid, V.; Heuss, S.; Moretti, J.; Olivo-Marin, J. C.; Israel, A., The adaptor-associated kinase 1, AAK1, is a positive regulator of the Notch pathway. J Biol Chem 2011, 286 (21), 18720-30.
  7. Sorensen, E. B.; Conner, S. D., AAK1 regulates Numb function at an early step in clathrin-mediated endocytosis. Traffic 2008, 9 (10), 1791-800.
  8. Kearns, A. E.; Donohue, M. M.; Sanyal, B.; Demay, M. B., Cloning and characterization of a novel protein kinase that impairs osteoblast differentiation in vitro. J Biol Chem 2001, 276 (45), 42213-8.
  9. Borner, G. H.; Antrobus, R.; Hirst, J.; Bhumbra, G. S.; Kozik, P.; Jackson, L. P.; Sahlender, D. A.; Robinson, M. S., Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles. J Cell Biol 2012, 197 (1), 141-60.
  10. Krieger, J. R.; Taylor, P.; Gajadhar, A. S.; Guha, A.; Moran, M. F.; McGlade, C. J., Identification and selected reaction monitoring (SRM) quantification of endocytosis factors associated with Numb. Mol Cell Proteomics 2013, 12 (2), 499-514.
pk properties
co-crystal structures
synthetic schemes
materials and methods

SGC-GAK-1 A chemical probe for GAK

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

Its negative control (SGC-GAK-1N) is available for purchase from Sigma.

  • overview
  • properties
  • references
overview
Probe Negative control

 

SGC-GAK-1

 

SGC-GAK-1N

Cyclin G associated kinase (GAK) is a 160 kDa serine/threonine kinase originally identified and so named as a direct association partner with cyclin G.1 GAK is a member of the numb-associated kinase (NAK) family, which includes AAK1 (adaptor protein 2-associated kinase), STK16/MPSK1 (serine/threonine kinase 16/myristoylated and palmitoylated serine/threonine kinase 1), and BMP2K/BIKE (BMP-2 inducible kinase).2 In addition to its kinase domain, the C-terminus of GAK protein bears high homology to a domain found in auxilin and tensin.3 GAK has ubiquitous tissue expression and within the cell localizes to the Golgi complex, cytoplasm, and nucleus.

NAK family domain structures

Location of GAK on kinome tree

Multiple biological roles for GAK and disease associations have been made despite it being a relatively understudied kinase. GAK is involved in membrane trafficking and sorting of proteins, including as an essential cofactor for HSC70-dependent uncoating of clathrin coated vesicles in the cytoplasm.6-7 GAK is required for maintenance of centrosome maturation and progression through mitosis.8 GAK is over-expressed in osteosarcoma cell lines and tissues where it contributes to proliferation and survival.9 Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms in the GAK gene associated with susceptibility to Parkinson’s disease.10 Emerging evidence suggests that GAK may be a therapeutic target in prostate cancer (PCa): GAK expression levels increase upon prolonged androgen treatment and during the progression of cells to hormone independence, and analysis of patient-derived tissue samples demonstrated that GAK expression levels positively correlated with the Gleason score, a quantitative scale of PCa aggressiveness.11 GAK interacts directly with the androgen receptor (AR) and potentiates its transcriptional activity.12 Further elucidation of the role of GAK in these and other biological processes would be facilitated by access to a potent and selective inhibitor of the kinase.

SGC-GAK-1 is a chemical probe for GAK that potently targets the ATP-binding site (KD = 1.9 nM). Regarding kinase selectivity, no kinases were observed to bind SGC-GAK-1 within 30-fold of the KD of GAK in a KINOMEscan assay (DiscoverX) at 1 μM followed by KD determinations. In a live cell NanoBRET assay (Promega) SGC-GAK-1 had an IC50 of 110 nM against ectopically expressed full-length GAK-Nluc fusion.

A chemically related negative control compound, SGC-GAK-1N, is provided.

Despite weakly binding RIPK2 in weakly binding RIPK2 in vitro (DiscoverX RIPK2 KD = 110 nM; 58-fold of GAK KD), SGC-GAK-1 potently engaged RIPK2 in a live cell NanoBRET assay (RIPK2 IC50 = 360 nM); accordingly, we have identified a control compound that is a highly cell-potent RIPK2 ligand, HY-19764, (IC50 = 2.2 nM) that lacks GAK affinity (IC50 > 10 µM).

properties
Probe Negative control

 

SGC-GAK-1

 

SGC-GAK-1N

Physical and chemical properties for BAY-876
Molecular weight389.25
Molecular formulaC38H36BrF3N4O6
IUPAC name6-Bromo-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine
MollogP4.65
PSA40.19
No. of chiral centres0
No. of rotatable bonds5
No. of hydrogen bond acceptors4
No. of hydrogen bond donors1
Storage-20 °C as DMSO stock
DissolutionSoluble in DMSO at least up to 10 mM
Physical and chemical properties for BAY-588 (Negative Control)
Molecular weight392.38
Molecular formulaC20H19F3N2O3
IUPAC nameN-methyl-6-(trifluoromethyl)-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine
MollogP5.157
PSA33.85
No. of chiral centres0
No. of rotatable bonds6
No. of hydrogen bond acceptors4
No. of hydrogen bond donors0
Storage-20 °C as DMSO stock
DissolutionSoluble in DMSO at least up to 10 mM

SMILES:
SGC-GAK1-1: BrC1=CC2=C(N=CC=C2NC3=CC(OC)=C(C(OC)=C3)OC)C=C1
SGC-GAK1-1N: CN(C1=C2C(C=CC(C(F)(F)F)=C2)=NC=C1)C3=CC(OC)=C(C(OC)=C3)OC

 

InChI:
SGC-GAK1-1: InChI=1S/C18H17BrN2O3/c1-22-16-9-12(10-17(23-2)18(16)24-3)21-15-6-7-20-14-5-4-11(19)8-13(14)15/h4-10H,1-3H3,(H,20,21)

SGC-GAK1-1N: InChI=1S/C20H19F3N2O3/c1-25(13-10-17(26-2)19(28-4)18(11-13)27-3)16-7-8-24-15-6-5-12(9-14(15)16)20(21,22)23/h5-11H,1-4H3

InChIKey:
SGC-GAK1-1: AUOSKLDNVNGKRR-UHFFFAOYSA-N
SGC-GAK1-1N: PVTQCCFMFWASHK-UHFFFAOYSA-N

selectivity profile
in vitro potency
cell based assay data
references
  1. Kanaoka, Y.; Kimura, S. H.; Okazaki, I.; Ikeda, M.; Nojima, H., GAK: a cyclin G associated kinase contains a tensin/auxilin-like domain. FEBS Lett 1997, 402 (1), 73-80.
  2. Sorrell, F. J.; Szklarz, M.; Abdul Azeez, K. R.; Elkins, J. M.; Knapp, S., Family-wide Structural Analysis of Human Numb-Associated Protein Kinases. Structure 2016, 24 (3), 401-11.
  3. Kimura, S. H.; Tsuruga, H.; Yabuta, N.; Endo, Y.; Nojima, H., Structure, expression, and chromosomal localization of human GAK. Genomics 1997, 44 (2), 179-87.
  4. Sato, J.; Shimizu, H.; Kasama, T.; Yabuta, N.; Nojima, H., GAK, a regulator of clathrin-mediated membrane trafficking, localizes not only in the cytoplasm but also in the nucleus. Genes Cells 2009, 14 (5), 627-41.
  5. Thul, P. J.; Akesson, L.; Wiking, M.; Mahdessian, D.; Geladaki, A.; Ait Blal, H.; Alm, T.; Asplund, A.; Bjork, L.; Breckels, L. M.; Backstrom, A.; Danielsson, F.; Fagerberg, L.; Fall, J.; Gatto, L.; Gnann, C.; Hober, S.; Hjelmare, M.; Johansson, F.; Lee, S.; Lindskog, C.; Mulder, J.; Mulvey, C. M.; Nilsson, P.; Oksvold, P.; Rockberg, J.; Schutten, R.; Schwenk, J. M.; Sivertsson, A.; Sjostedt, E.; Skogs, M.; Stadler, C.; Sullivan, D. P.; Tegel, H.; Winsnes, C.; Zhang, C.; Zwahlen, M.; Mardinoglu, A.; Ponten, F.; von Feilitzen, K.; Lilley, K. S.; Uhlen, M.; Lundberg, E., A subcellular map of the human proteome. Science 2017, 356 (6340).
  6. Zhang, C. X.; Engqvist-Goldstein, A. E.; Carreno, S.; Owen, D. J.; Smythe, E.; Drubin, D. G., Multiple roles for cyclin G-associated kinase in clathrin-mediated sorting events. Traffic 2005, 6 (12), 1103-13.
  7. 7.         Eisenberg, E.; Greene, L. E., Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis. Traffic 2007, 8 (6), 640-6.
  8. Chaikuad, A.; Keates, T.; Vincke, C.; Kaufholz, M.; Zenn, M.; Zimmermann, B.; Gutierrez, C.; Zhang, R. G.; Hatzos-Skintges, C.; Joachimiak, A.; Muyldermans, S.; Herberg, F. W.; Knapp, S.; Muller, S., Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies. Biochem J 2014, 459 (1), 59-69.
  9. Susa, M.; Choy, E.; Liu, X.; Schwab, J.; Hornicek, F. J.; Mankin, H.; Duan, Z., Cyclin G-associated kinase is necessary for osteosarcoma cell proliferation and receptor trafficking. Mol Cancer Ther 2010, 9 (12), 3342-50.
  10. Dzamko, N.; Zhou, J.; Huang, Y.; Halliday, G. M., Parkinson's disease-implicated kinases in the brain; insights into disease pathogenesis. Front Mol Neurosci 2014, 7, 57.
  11. Sakurai, M. A.; Ozaki, Y.; Okuzaki, D.; Naito, Y.; Sasakura, T.; Okamoto, A.; Tabara, H.; Inoue, T.; Hagiyama, M.; Ito, A.; Yabuta, N.; Nojima, H., Gefitinib and luteolin cause growth arrest of human prostate cancer PC-3 cells via inhibition of cyclin G-associated kinase and induction of miR-630. PLoS One 2014, 9 (6), e100124.
  12. Ray, M. R.; Wafa, L. A.; Cheng, H.; Snoek, R.; Fazli, L.; Gleave, M.; Rennie, P. S., Cyclin G-associated kinase: a novel androgen receptor-interacting transcriptional coactivator that is overexpressed in hormone refractory prostate cancer. Int J Cancer 2006, 118 (5), 1108-19.
pk properties
co-crystal structures
synthetic schemes
materials and methods
10.01.2018

Enamine supplies DSI poised fragment and analogue libraries to Diamond Light Source XChem facility and SGC Oxford screening efforts

by: SGC

Kiev, Ukraine and Oxford, UK, 10 January 2018: Diamond Light Source (Diamond) and the Structural Genomic Consortium (SGC) Oxford announced today that Enamine, a chemical company and producer of novel chemical building blocks and screening libraries, will become a key supplier of poised fragment and analogue libraries to its XChem facility. Enamine will offer a new generation of the hit-finding library, Diamond-SGC-iNEXT (DSI) Poised Library to enable fast and productive fragment-based lead discovery(FBLD).

BI01383298 A chemical probe for SLC13A5

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

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

 

BI01383298

 

BI01372674

Biology of SLC13A5

SLC13A5 (NaCT, INDY) is a sodium-citrate co-transporter that is highly expressed in the liver.  It is a member of the SLC13 family of which there are 4 other members. SLC13A5 transports citrate from the circulatory system into hepatocytes where it is used in the synthesis of sterols and fatty acids.  SLC13A5 was first identified in Drosophila where the name I’m Not Dead Yet or INDY was coined. A study in 2000(1) showed that lower expression of SLC13A5 in Drosophila led to increased lifespan.  Mouse models have demonstrated the potential of this protein as a target for obesity and diabetes with knockout mice protected from adiposity (2), reduced lipid concentrations in a siRNA study (3) and a substrate analogue used to lower blood glucose levels (4).  More recently mutations in SLC13A5 have been linked to early-infantile epileptic encephalopathy (5) while silencing of the SLC13A5 gene inhibits proliferation of human hepatocarcinoma cells (6).

BI1383298 is a potent inhibitor of SLC13A5 which unlike previously published inhibitors (4,7) has no structural homology to the substrate (citrate).  It is selective over other family members and other transporters.  A chemically related negative control (BI01372674) is also provided.

 

BI01383298: a chemical probe for SLC13A5

In addition to the chemical probe (BI01383298) we also include a negative control (BI01372674) which is chemically analogous to the probe molecule.

The probe molecule (BI01383298) and control (BI01372674) are soluble, after visual inspection, at 10 µM and 200µM in the presence of 0.1% DMSO in media and assay buffer.

Aliquots of stock Solutions were prepared as follows: 10mM BI01383298 in DMSO (Stored -20oC), 5mM BI01383298 in DMSO (Stored -20oC, need agitation upon thaw and gentle warming to 30oC).

 

SLC13A5 engagement in vitro

BI01383298 but not BI01372674 demonstrates target engagement in vitro with purified human SLC13A5 protein as shown using a thermostabilisation assay with the Prometheus label-free system from Nanotemper  (figure 2).

Figure 2: Thermostabilisation of human SLC13A5 by BI01383298 and BI01372674 between 0.1µM and 10µM. SLC13A5 assay concentration of 1µM. Data summarises 3 biological samples with between 4 and 8 replicates for each.

properties
Probe Negative control

 

BI01383298

 

BI01372674

Physical and chemical properties for BI01383298
Molecular weight444.0
Molecular formulaC19H19Cl2FN2O3S
IUPAC name(1-(3,5-dichloro-phenylsulfonyl)-piperidin-4-yl)-((4-fluoro-phenyl)-methylamino)-methanone
MollogP4.64
PSA56.3
No. of chiral centres0
No. of rotatable bonds6
No. of hydrogen bond acceptors7
No. of hydrogen bond donors1
Physical and chemical properties for BI1372674 (Negative Control)
Molecular weight502.1
Molecular formulaC21H25Cl2N2O4PS
IUPAC name 
MollogP3.54
PSA69.5
No. of chiral centres0
No. of rotatable bonds7
No. of hydrogen bond acceptors9
No. of hydrogen bond donors1


SMILES:
BI01383298: FC(C=C1)=CC=C1CNC(C2CCN(S(C3=CC(Cl)=CC(Cl)=C3)(=O)=O)CC2)=O
BI1372674: ClC1=CC(Cl)=CC(S(=O)(N2CCC(CC2)C(NCC3=CC=C(C=C3)P(C)(C)=O)=O)=O)=C1

InChI:
BI01383298: InChI=1S/C19H19Cl2FN2O3S/c20-15-9-16(21)11-18(10-15)28(26,27)24-7-5-14(6-8-24)19(25)23-12-13-1-3-17(22)4-2-13/h1-4,9-11,14H,5-8,12H2,(H,23,25)

BI1372674: InChI=1S/C21H25Cl2N2O4PS/c1-30(2,27)19-5-3-15(4-6-19)14-24-21(26)16-7-9-25(10-8-16)31(28,29)20-12-17(22)11-18(23)13-20/h3-6,11-13,16H,7-10,14H2,1-2H3,(H,24,26)

InChIKey:
BI01383298: VUOYAALVGSMUHC-UHFFFAOYSA-N
BI1372674: GSNNWZZIEKEEQF-UHFFFAOYSA-N

 SolventBI01383298BI01372674
Solubility (200uM, 4% DMSO)Assay Buffer*- 0hrSolubleSoluble
Assay Buffer*- 12hrsSolubleSoluble
Freestyle Media- 0hrSolubleSoluble
Freestyle Media- 12hrSolubleSoluble
Water- 0hrsppt. observedppt. observed
Solubility (10uM, 0.1% DMSO)Assay Buffer*- 0hrSolubleSoluble
Assay Buffer*- 12hrsSolubleSoluble
Freestyle Media- 0hrSolubleSoluble
Freestyle Media- 12hrSolubleSoluble
Water- 0hrsSolubleSoluble

*50mM HEPES (pH 7.5), 200mM NaCl, 0.024% n-Dodecyl-β-D-Maltopyranoside, 0.0024% Cholesterol Hemisuccinate.

selectivity profile

Selectivity against SLC13 family members and other transporters in cellular assays

BI01383298 is more than 1000-fold selective (table 1) over the closest family members:  human SLC13A2 / SLC13A3 that share physiological substrates citrate and succinate.

Table 1:  Citrate uptake inhibition was measured for all citrate transporters and glycine uptake measured to GLYT2. Potency was assessed for the probe candidate and the negative control on uptake of 14C-citrate into cells over-expressing SLC13A5, SLC13A2, SLC13A3, mouse SLC13A5 and in HEPG2 cells.

 Substrate uptake inhibition IC50 [nM]
BI01383298BI01372674
HEK cells- hSLC13A556>100,000
HepG2 cells24>100,000
HEK cells- mSLC13A5>100,00088,000
HEK cells- hSLC13A2>100,000n.d.
HEK cells- hSLC13A3>100,000n.d.
HEK cells – GLYT2>100,000>100,000

Selectivity panel (% inhibition @ 10μM): 35/38 targets<50%; CB1(h): 78%; K(KOP): 81%; Na+channel: 52%

in vitro potency
cell based assay data

Potency against SLC13A5 in cellular assays

BI01383298 is a potent inhibitor of SLC13A5 with an apparent IC50 value of 56nM in HEK cells overexpressing SLC13A5 and 24nM in HepG2 cell expressing endogenous SLC13A5. BI01383298 was not found to inhibit citrate transport in HEK cells over-expressing mouse SLC13A5 and thus we do not recommend this compound for use in mouse models. BI01372674 was not found to inhibit citrate uptake in a  HEPG2 model expressing endogenous levels of SLC13A5 or in HEK cellular models over-expressing human SLC13A5, mouse SLC13A5 or GLYT2.

Figure 1:  Measured IC50 of hSLC13A5-mediated 14C-citrate  in (A) overexpressed, HEK293-Flp-In-hSLC13A5 and  (B) endogenous, HepG2 celluar models.

references
  1. Rogina, B., et al. (2000). Science 290, 2137-2140
  2. Birkenfeld, A. L., et al. (2011). Cell Metab 14, 184-195
  3. Brachs, S., et al. (2016). Mol Metab 5, 1072-1082
  4. Huard, K., et al. (2015). Sci Rep 5, 17391
  5. Bainbridge, M. N., et al. (2017). Mol Genet Metab 121, 314-319
  6. Li, Z., et al. (2017). The Journal of biological chemistry 292, 13890-13901
  7. Huard, K., et al. (2016). Journal of medicinal chemistry 59, 1165-1175
pk properties
co-crystal structures
synthetic schemes
materials and methods
11.12.2017

Drug discovery must change to urgently address global health needs

by: SGC

Oxford researchers call for a new Pharmaceutical Commons

Pharmaceutical research and development (R & D) is one of the best examples of human ingenuity, attracting vast funding, employing brilliant minds, and deploying the most advanced technologies. Over the past century, it has enabled unprecedented advances for human health. Yet the pharmaceutical R & D system is struggling to keep up with society’s medical needs.

06.12.2017

SGC and The Neuro Form Open Science Partnership

by: SGC

A new partnership between the Structural Genomics Consortium (SGC) and the Montreal Neurological Institute and Hospital (The Neuro) will use a unique open science framework to help scientists discover new targets for drug development for neurological diseases.

04.12.2017

FFAR Awards $1 Million Grant to Create Open Source Technology for Gene Discovery in Plants

by: SGC

The Foundation for Food and Agriculture Research (FFAR), a nonprofit established in the 2014 Farm Bill with bipartisan congressional support, awarded a $1 million Seeding Solutions grant to University of California, Davis (UC Davis) to study the genetics of rice plants. Together with researchers at the University of North Carolina and collaborators, the team will develop and implement a chemistry-driven gene discovery approach to identify genes that modulate root traits. 

L-Moses A chemical probe for PCAF and GCN5 Bromodomains

This probe (dihydrochloride) is available from Tocris and Cayman Chemical. 

  • overview
  • properties
  • references
  • synthetic schemes
overview
Probe Negative control

 

L-Moses

 

D-Moses

p300/CBP-associated factor (PCAF/KAT2B) and general control non-derepressible 5 (GCN5/KAT2A) are members of subfamily 1 of the bromodomain phylogenetic tree. These multi-domain proteins that have been implicated in retroviral infection, inflammation pathways and cancer development. However, outside of viral replication, little is known about the dependence of these effects on the C-terminal bromodomain.  L-Moses is as a chemical probe for the PCAF/GCN5 bromodomain and D-Moses is the enantiomeric negative control. Rational inhibitor design and biophysical characterization led to the discovery of L-Moses. The probe was optimized from the non-selective pan-bromodomain inhibitor, bromosporine to generate a potent, selective (>4500-fold selective over BRD4), permeable and cell-active PCAF/GCN5 bromodomain chemical probe.

Potency

PCAF Ki 47 nM in a HTRF binding competition assay using PCAF bromodomain and a biotin tagged bromodomain ligand.

PCAF KD 48 nM in a BROMOscan assay run at DiscoverX.
GCN5 KD 220 nM in a BROMOscan assay run at DiscoverX.

PCAF KD 126 nM (ITC) using PCAF bromodomain.
GCN5 KD 600 nM (ITC) using GCN5 bromodomain.

Non-family targets

GPCR/Eurofins Panel: In an panel of 130 potential off targets, L-Moses showed no binding (>60% at 10 μM) to all targets except the opioid receptors (mu 100 nM, OPRL1 840 nM, kappa 1,100 nM,) and the 5-HT transporter (220 nM).

Cellular Potency

PCAF:  IC50 220 nM in Promega NanoBRET assay, measuring displacement of NanoLuc-tagged truncated bromodomain PCAF from Halo-tagged histone H3.3 in HEK293 cells.
IC50 1.2 μM in NanoBRET assay, measuring displacement of NanoLuc-tagged full-length PCAF from Halo-tagged histone H3.3 in HEK293 cells.

IC50 660 nM for competing pull-down of full-length PCAF from cell lysates using immobilized L-Moses

GCN5: IC50 220 nM for competing pull-down of full-length PCAF from cell lysates using immobilized L-Moses.

Cytoxicity assay:

Toxicity of D-Moses and L-Moses was assessed on peripheral blood mononuclear cells  (PBMC) obtained   from   5   healthy   donors.   PBMC   were   cultured   either   with D-Moses or L-Moses at concentrations of 0.1, 1 and 10 μM or with a control (DMSO) for 24 hours. Viability of PBMC were then checked using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific). No observed cytotoxicity was observed at any concentration.

properties
Probe Negative control

 

L-Moses

 

D-Moses

Physical and chemical properties for L-Moses
Molecular weight360.2
Molecular formulaC21H24N6
MollogP2.38
PSA46.14
No. of chiral centres2
No. of rotatable bonds5
No. of hydrogen bond acceptors4
No. of hydrogen bond donors1
Physical and chemical properties for D-Moses (Negative Control)
Molecular weight360.2
Molecular formulaC21H24N6
MollogP2.38
PSA46.14
No. of chiral centres2
No. of rotatable bonds5
No. of hydrogen bond acceptors4
No. of hydrogen bond donors1
  • SMILES:
  • L-Moses: C[C@@H]([C@H](C1=CC=CC=C1)N(C)C)NC(C2=CC=CC=C23)=NN4C3=NN=C4C
  • D-Moses: CC1=NN=C2C3=CC=CC=C3C(N[C@@H]([C@@H](C4=CC=CC=C4)N(C)C)C)=NN12
  • InChI:
  • D-Moses: InChI=1S/C21H24N6/c1-14(19(26(3)4)16-10-6-5-7-11-16)22-20-17-12-8-9-13-18(17)21-24-23-15(2)27(21)25-20/h5-14,19H,1-4H3,(H,22,25)/t14-,19+/m1/s1
  • InChIKey:
  • L-Moses: MSFPLTWUFWOKBX-IFXJQAMLSA-N
  • D-Moses: MSFPLTWUFWOKBX-KUHUBIRLSA-N
selectivity profile
in vitro potency
cell based assay data
references

M. Moustakim, P. G. K. Clark, L. Trulli, A. L. Fuentes de Arriba, M. T. Ehebauer, A. Chaikuad, E. J. Murphy, J. Mendez-Johnson, D. Daniels, C.-F. D. Hou, Y.-H. Lin, J. R. Walker, R. Hui, H. Yang, L. Dorrell, C. M. Rogers, O. P. Monteiro, O. Fedorov, K. V. M. Huber, S. Knapp, J. Heer, D. J. Dixon, P. E. Brennan Discovery of a PCAF Bromodomain Chemical Probe. Angew. Chem. Int. Ed. 2017, 56, 827.

pk properties
co-crystal structures
synthetic schemes

Selectivity Bromodomains

>40-fold selectivity over other bromodomain targets. (>4500-fold selective over BRD4). No observable activity on any other bromodomain target <6 µM in a BROMOscan assay run at DiscoverX.

materials and methods
28.09.2017

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

by: SGC

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

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