Homer

The probe is available from Sigma and Cayman Chemicals.

The control may be requested here.

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

 

 

Homer

 

nc_WDR5

 

nc_VHL

The Histone Lysine Methyltransferase (HMT) complex MLL1 is a crucial epigenetic writer to control DNA accessibility and promote gene transcription [1,2]. The complex incorporates WDR5 (WD40-repeat containing protein 5) that acts as scaffolding component and enhances HMT activity of MLL1. [2] The propeller-shaped WDR5 protein contains two binding sites that interact not only with oncogenic drivers like MLL1, but also with various other proteins like the oncoprotein MYC. [3]

Here, we present a new chemical degrader probe for the WDR5 protein, Homer, that was initially designed by the OICR and the SGC Toronto [4, 5] and then further advanced into a PROTAC (Proteolysis targeting chimera) chemical probe by the SGC Frankfurt [6]. This scaffold represents the first chemical degrader probe for WDR5.

Homer binds potently to WDR5 with a K D (ITC) of 18 nM. It is selective as shown in quantitative proteomic studies. This compound is non-toxic as demonstrated by the AlarmarBlue assay in U2OS cells. Homer shows remarkably cellular activity in MV4-11 cells by inducing WDR5 degradation in a catalytic manner a DC 50 of 53 nM. The DC max of Homer is 58% and observed at cellular concentrations of 1 µM.

In addition, two negative control compounds, nc_WDR5 and nc_VHL, are provided, which showed no degradational activity.

Summary of Potency and efficacy of Homer

WDR5Homer KD
DSF13.2 K 
ITC18 nM 
NanoBRET13.6 µM 
HiBit

DCmax: 58% at 1 µM 

DC50: 53 nM 

quantitative Proteomics (-log10p > 3, log2FC < -0.5) 

Selectivity 

Selectivity of the first WDR5 probe OICR-9429 was shown previously [5, 6]. Selectivity for the Homer probe was confirmed by quantitative proteomics.

Dosage 

For optimal degradation and to overcome the Hook effect, we recommend that a concentration of 1 µM should be used in cell-based assays. 

Cellular Activity 

Homer induces depletion of endogenous WDR5 in various cancer cell lines by inducing protein ubiquitylation and degradation. 

properties
Probe Negative control Negative control

 

 

Homer

 

nc_WDR5

 

nc_VHL

Homer
Physical and Chemical Properties
Molecular weight1012.16
Molecular formulaC52H60F3N9O7S
IUPAC nameN-(4'-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)carbamoyl)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide
clogPo/w (SwissADME)5.31
TPSA (SwissADME)237.41 Å2
No. of chiral centres3
No. of rotatable bonds23
No. of hydrogen bond acceptors12
No. of hydrogen bond donors6
Storagestable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble in DMSO at 50 mM

 

nc_VHL
Physical and Chemical Properties
Molecular weight1012.16
Molecular formulaC52H60F3N9O7S
IUPAC nameN-(4'-((5-(((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)carbamoyl)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 
clogPo/w (SwissADME)5.25
TPSA (SwissADME)237.41 Å2
No. of chiral centres3
No. of rotatable bonds23
No. of hydrogen bond acceptors12
No. of hydrogen bond donors6
Storagestable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble in DMSO at 50 mM
nc_WDR5 
Physical and Chemical Properties
Molecular weight999.12
Molecular formulaC51H57F3N8O9S
IUPAC nameN-(4'-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)carbamoyl)-4-morpholino-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 
clogPo/w (SwissADME)5.41
TPSA (SwissADME)243.40 Å2
No. of chiral centres3
No. of rotatable bonds23
No. of hydrogen bond acceptors12
No. of hydrogen bond donors6
Storagestable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble in DMSO at 50 mM

SMILES: 

Homer: CC1=C(SC=N1)C2=CC=C(C=C2)CNC([C@@H]3C[C@H](CN3C([C@@H](NC(CCCCNC(C4=CC=C(C=C4)C5=CC=C(C(NC(C6=CNC(C=C6C(F)(F)F)=O)=O)=C5)N7CCN(CC7)C)=O)=O)C(C)(C)C)=O)O)=O

nc_VHL: CC1=C(C2=CC=C(CNC([C@@H]3C[C@H](O)CN3C([C@H](C(C)(C)C)NC(CCCCNC(C4=CC=C(C5=CC=C(N6CCN(C)CC6)C(NC(C7=CNC(C=C7C(F)(F)F)=O)=O)=C5)C=C4)=O)=O)=O)=O)C=C2)SC=N1 

nc_WDR5: CC1=C(C2=CC=C(CNC([C@@H]3C[C@@H](O)CN3C([C@H](C(C)(C)C)NC(CCCCNC(C4=CC=C(C5=CC=C(N6CCOCC6)C(NC(C7=CNC(C=C7C(F)(F)F)=O)=O)=C5)C=C4)=O)=O)=O)=O)C=C2)SC=N1 

InChI: 

Homer: InChI=1S/C52H60F3N9O7S/c1-31-45(72-30-59-31)34-11-9-32(10-12-34)27-58-49(70)42-25-37(65)29-64(42)50(71)46(51(2,3)4)61-43(66)8-6-7-19-56-47(68)35-15-13-33(14-16-35)36-17-18-41(63-22-20-62(5)21-23-63)40(24-36)60-48(69)38-28-57-44(67)26-39(38)52(53,54)55/h9-18,24,26,28,30,37,42,46,65H,6-8,19-23,25,27,29H2,1-5H3,(H,56,68)(H,57,67)(H,58,70)(H,60,69)(H,61,66)/t37-,42+,46-/m1/s1 

nc_VHL: InChI=1S/C52H60F3N9O7S/c1-31-45(72-30-59-31)34-11-9-32(10-12-34)27-58-49(70)42-25-37(65)29-64(42)50(71)46(51(2,3)4)61-43(66)8-6-7-19-56-47(68)35-15-13-33(14-16-35)36-17-18-41(63-22-20-62(5)21-23-63)40(24-36)60-48(69)38-28-57-44(67)26-39(38)52(53,54)55/h9-18,24,26,28,30,37,42,46,65H,6-8,19-23,25,27,29H2,1-5H3,(H,56,68)(H,57,67)(H,58,70)(H,60,69)(H,61,66)/t37-,42-,46+/m0/s1 

nc_WDR5: InChI=1S/C51H57F3N8O8S/c1-30-44(71-29-58-30)33-10-8-31(9-11-33)26-57-48(68)41-24-36(63)28-62(41)49(69)45(50(2,3)4)60-42(64)7-5-6-18-55-46(66)34-14-12-32(13-15-34)35-16-17-40(61-19-21-70-22-20-61)39(23-35)59-47(67)37-27-56-43(65)25-38(37)51(52,53)54/h8-17,23,25,27,29,36,41,45,63H,5-7,18-22,24,26,28H2,1-4H3,(H,55,66)(H,56,65)(H,57,68)(H,59,67)(H,60,64)/t36-,41+,45-/m1/s1 

InChIKey: 

Homer: OFNZESNEBSQKSE-BQGOKDIQSA-N 

nc_VHL: OFNZESNEBSQKSE-CCVFZADKSA-N 

nc_WDR5: BFXBMIZHEIFXOC-LTJJNQLXSA-N 

selectivity profile
Quantitative Proteomics revealed that only WDR5 is significally and substantially depleted by Homer treatment. 
Homer binds potently to WDR5 as determined by ITC. 
in vitro potency
cell based assay data

Homer is cell permeable and degrades WDR5 in MV4-11 cells. The control compounds nc_WDR5 and nc_VHL are not active. 

NanoBRET data of Homer in HEX293 cells show Homer is cell permeable. 

(up) HiBiT assay curves from Homer (black) and both negative controls (red) as well as from parent WDR5 compound (blue) show that Homer degrades WDR5 in MV4-11 cells after 24 h. The most effective degradation (58% WDR5 depletion) is observed at 1 µM. (down) WesternBlots validate HiBiT data and show degradation of WDR5 by Homer treatment in MV4-11 cells after 24 h. 

Homer decreases WDR5 protein stability as shown in the Cycloheximide chase assay. 

Rescue experiments show that WDR5 depletion requires binding of Homer. WDR5 levels can be restored by excess of WDR5 ligand. 

qPCR shows that Homer does not affect WDR5 transcription.

references

[1] Wu, Shu, “MLL1/WDR5 complex in leukemogenesis and epigenetic regulation”, Chin. J. Cancer 2011, 30(4), 240-246.
[2] Jiang, “The complex activities of the SET1/ MLL complex core subunits in development and disease“, BBA – Gene Regulatory Mechanisms 2020, 1863, 194560.
[3] Thomas et al., “Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC”, Mol. Cell 2015, 58, 440-452.
[4] Grebien et al., “Pharmacological targeting of the Wdr5-MLL interaction in C/EBPa N-terminal leukemia”, Nat Chem Biol. 2015, 11, 571.
[5] Getlik et al., “Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1)”, J Med Chem. 2016, 59, 2478.
[6] Dölle, Adhikari et al., “Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders”, J Med Chem. 2021, May 13. doi: 10.1021/acs.jmedchem.1c00146. Epub ahead of print. PMID: 33980013.
[7] A.C. Wallace, R.A. Laskowski, J.M. Thornton, “LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions”, Protein Eng. 8, 1995, 127-134.

pk properties
co-crystal structures

PDB ID 7Q2J

Left panel: Quaternary complex of WDR5:VHL:ElonginB:ElonginC:Homer bound to chemical probe PROTAC Homer in surface representation.

Right panel: Surface slice with focus on the binding pocket of WDR5 and VHL. Homer is shown in stick representation

Observed electron density of Homer countered at 1 sigma.

Left panel: Contact surface area between WDR5 (wheat) and VHL (green) in surface representation

Right panel: Close up on the detailed interaction between WDR5 and VHL in cartoon/stick representation. The orientation is the same as in the left Panel.

WDR5 (D)       
VHL (C)

Detailed interaction between Homer and VHL:WDR5. The figure was created with LIGPLOT [7]

synthetic schemes
materials and methods
12.02.2021

Promising new approach to stop growth of brain cancer cells

by: SGC

Inhibiting a key enzyme that controls a large network of proteins important in cell division and growth paves the way for a new class of drugs that could stop glioblastoma, a deadly brain cancer, from growing.

Researchers at Princess Margaret Cancer Centre, the Hospital for Sick Children (SickKids) and University of Toronto, showed that chemically inhibiting the enzyme PRMT5 can suppress the growth of glioblastoma cells.

SGC-SMARCA-BRDVIII A Chemical Probe for SMARCA2/4 and PB1(5)

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

The control is available from Sigma.

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

 

SGC-SMARCA-BRDVIII

 

SGC-BRDVIII-NC

The SWI/SNF (switch/sucrose non-fermenting) chromatin remodelling complexes, BAF (BRG1/BRM-associated factor) and PBAF (polybromo-associated BAF), are crucial epigenetic regulators to control DNA accessibility, and thus largely contribute to cell proliferation and differentiation mechanisms [1]. The complexes incorporate either the related subunit SMARCA2 or SMARCA4 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 2/4), which both consist of a mutually exclusive catalytic ATPase domain and a bromodomain that reads acetylated histones on the chromatin. A unique feature of PBAF is the subunit PBRM1 (polybromo-1) containing six tandem-acting bromodomains. SWI/SNF complexes are known to be strong tumor suppressors and their dysfunctions trigger substantial oncogenic programs or deregulate cell lineage differentiation mechanisms [1].

Here, we present a new chemical probe for the SMARCA2/4 and PB1(5) bromodomains of BAF/PBAF, SGC-SMARCA-BRDVIII, that was initially designed by Genentech/Constellation [2] and then further advanced into the chemical probe platform by the SGC Frankfurt [3]. This scaffold represents the second chemical probe for the SMARCA2/4 and PB1(5) bromodomains that is based on a different chemotype than our first generation bromodomain inhibitor PFI-3 [4,5]. Moreover, this inhibitor has also recently been used to develop the PROTAC (proteolysis targeting chimera) ACBI-1 [6].

SGC-SMARCA-BRDVIII binds potently to the SMARCA2/4 and PB1(5) bromodomains with a KD(ITC) of 35, 36 and 13 nM. It is selective within the other bromodomain families and shows no off-targets activity on 85 protein kinases screened using temperature shift binding assays. This compound is non-toxic as demonstrated by the NCI-60 human tumor cell lines screen, but it shows remarkably cellular activity in an adipogenesis cell differentiation assay with an EC50 of < 1.0 µM.

Of special note is that SGC-SMARCA-BRDVIII outperformed the chemical probe PFI-3 in that particular assay, and therefore, we advise to use SGC-SMARCA-BRDVIII and PFI-3 to confirm the results, when elucidating the biological role of the SWI/SNF bromodomains.

In addition, a negative control compound, SGC-BRDVIII-NC, is provided, which showed no cellular activity. If interested, a chemogenomic tool compound, SGC-pan-BRDVIII, that additionally hits the PB1(2,3) members with a KD(ITC) of 200-400 nM can be inquired [3].

Potency Against Target Family

Bromodomain SGC-SMARCA-BRDVIII KD (nM)
SMARCA235
SMARCA436
PB1(5)13
PB1(2)3655
PB1(3)1963

Table 1: Screening SGC-SMARCA-BRDVIII against selected targets.

Selectivity 
Selectivity of the SGC-SMARCA-BRDVIII probe within the bromodomain families was confirmed by an in-house thermal shift panel containing 25 bromodomains. No activity was also observed on 85 protein kinases screened in an in-house DSF panel.

Selectivity Dosage
To minimize the chance of off-target effects, we recommend that a concentration of no higher than 10 µM should be used in cell-based assays.

Cellular Activity
The formation from 3T3-L1 mouse fibroblasts into adipocytes was impaired with an EC50 below 1.0 µM upon treatment with SGC-SMARCA-BRDVIII.

properties
Probe Negative control

 

SGC-SMARCA-BRDVIII

 

SGC-BRDVIII-NC

 

 

 

 

 

SGC-SMARCA-BRDVIII
Physical and chemical properties
Molecular weight380.45 g/mol
Molecular formulaC19H25N5O3
IUPAC nametert-butyl 4-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]piperazine-1-carboxylate
logP1.82
TPSA104.8
No. of chiral centres0
No. of rotatable bonds5
No. of hydrogen bond acceptors5
No. of hydrogen bond donors2
StorageStable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
DissolutionDMSO (up to 50 mM)
SGC-BRDVIII-NC
Physical and chemical properties
Molecular weight385.21 g/mol
Molecular formulaC20H27N5O3
IUPAC nametert-butyl-4-[3-amino-6-(2-methoxyphenyl)yridazine-4-yl]piperazine-1-carboxylate
clogP2.02
TPSA93.8
No. of chiral centres0
No. of rotatable bonds6
No. of hydrogen bond acceptors5
No. of hydrogen bond donors1
StorageStable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
DissolutionDMSO (up to 50 mM)

SMILES:
SGC-SMARCA-BRDVIII: OC1=CC=CC=C1C2=CC(N3CCN(CC3)C(OC(C)(C)C)=O)=C(N=N2)N
SGC-BRDVIII-NC: NC(N=NC(C1=CC=CC=C1OC)=C2)=C2N3CCN(CC3)C(OC(C)(C)C)=O

InChI:
SGC-SMARCA-BRDVIII: InChI=1S/C19H25N5O3/c1-19(2,3)27-18(26)24-10-8-23(9-11-24)15-12-14(21-22-17(15)20)13-6-4-5-7-16(13)25/h4-7,12,25H,8-11H2,1-3H3,(H2,20,22)
SGC-BRDVIII-NC:InChI=1S/C20H27N5O3/c1-20(2,3)28-19(26)25-11-9-24(10-12-25)16-13-15(22-23-18(16)21)14-7-5-6-8-17(14)27-4/h5-8,13H,9-12H2,1-4H3,(H2,21,23)

InChIKey:
SGC-SMARCA-BRDVIII: AQTNUGRRZDRZIA-UHFFFAOYSA-N
SGC-BRDVIII-NC: YOBVMDSDYHOOLN-UHFFFAOYSA-N
 

selectivity profile

In-house DSF panel revealed no off-targets for SGC-SMARCA-BRDVIII outside the BRD subfamily VIII. The negative control compound, SGC-BRDVIII-NC is completely inactive on all tested targets.

Bromodomains 

SGC-SMARCA-BRDVIII

(ΔTm°C)a,b

TAF1L(1)-0.2± 0.1ATAD20.4± 0.1
TAF1L(2)-0.1± 0.1EP3000.0± 0.1
BRD2(1)0.0± 0.2BRD4(1)0.7± 0.1
PB1(1)0.9± 0.1BRD3(2)-0.1± 0.1
PB1(2)2.5± 0.1BRPF1A0.2± 0.1
PB1(3)2.7± 0.2BRD10.2± 0.1
PB1(4)0.7± 0.1TRIM33B0.1± 0.1
PB111.3± 0.3SP110A-0.1± 0.1
PB10.3± 0.1PCAF0.0± 0.1
SMARCA27.7± 0.2WDR9-0.4± 0.1
SMARCA47.4± 0.1BRDT(1_2)0.1± 0.1
CREBBP-0.1± 0.1  

Table 1: Screening SGC-SMARCA-BRDVIII against a panel of bromodomains. 

Figure 1: SGC-SMARCA-BRDVIII (22) binds potently to SMARCA2/4 and PB1(5) as determined by ITC and shows weak affinity for the highly conserved homologues PB1(2) and PB1(3).

in vitro potency
cell based assay data

SGC-SMARCA-BRDVIII (22) is cell active and it impairs the formation of adipocytes from 3T3-L1 fibroblasts by reducing the expression levels of adipocyte-related genes. The control compound SGC-BRDVIII-NC (35) is not active.

Figure 1: Cell based assay data for SGC-SMARCA-BRDVIII

SGC-SMARCA-BRDVIII is non-toxic and does not influence cell growth at a dose of 10 µM as indicated in the NCI-60 human tumor cell lines screen, and can therefore ideally used in cell differentiation assays.

references
  1. Kadoch, C., Crabtree, GR. Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics. Sci. Adv., 2015, 1, 5, e1500447.
  2. Albrecht, BK., Cote, A., Crawford, TD., Duplessis, M., Good, AC., LeBlanc, Y., Magnuson, SR., Nasveschuk, CG., Romero, AF., Tang, Y., Taylor, AM. Therapeutic pyridazine compounds and uses thereof. US Patent, WO 2016/138114 A1.
  3. Wanior, M., Preuss, F., Ni, X., Krämer, A., Mathea, S., Göbel, T., Heidenreich, D., Simonyi, S., Kahnt, AS., Joerger, AC., Knapp, S. Pan-SMARCA/PB1 bromodomain inhibitors and their role in regulating adipogenesis. J. Med. Chem., 2020, 63, 23, 14680–14699.
  4. Gerstenberger, B.S., Trzupek, JD., Tallant, C., Fedorov, O., Filippakopoulos, P., Brennan, PE., Fedele, V., Martin, S., Picaud, S., Rogers, C., Parikh, M., Taylor, A., Samas, B., O'Mahony, A., Berg, E., Pallares, G., Torrey, AD., Treiber, DK., Samardjiev, IJ., Nasipak, BT., Padilla-Benavides, T., Wu, Q., Imbalzano, AN., Nickerson, JA., Bunnage, ME., Müller, S., Knapp, S., Owen, DR. Identification of a chemical probe for family VIII bromodomains through optimization of a fragment hit. J. Med. Chem. 2016, 59, 4800–4811.
  5. Fedorov, O., Castex, J., Tallant, C., Owen, DR., Martin, S., Aldeghi, M., Monteiro, O., Filippakopoulos, P., Picaud, S., Trzupek, JD., Gerstenberger, BS., Bountra, C., Willmann, D., Wells, C., Philpott, M., Rogers, C., Biggin, PC., Brennan, PE., Bunnage, ME., Schüle, R., Günther, T., Knapp, S., Müller, S. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance. Sci. Adv. 2015, 1, 10, e1500723.
  6. Farnaby, W., Koegl, M., Roy, MJ., Whitworth, C., Diers, E., Trainor, N., Zollman, D., Steurer, S., Karolyi-Oezguer, J., Riedmueller, C., Gmaschitz, T., Wachter, J., Dank, C., Galant, M., Sharps, B., Rumpel, K., Traxler, E., Gerstberger, T., Schnitzer, R., Petermann, O., Greb, P., Weinstabl, H., Bader, G., Zoephel, A., Weiss-Puxbaum, A., Ehrenhöfer-Wölfer, K., Wöhrle, S., Boehmelt, G., Rinnenthal, J., Arnhof, H., Wiechens, N., Wu, MY., Owen-Hughes, T., Ettmayer, P., Pearson, M., McConnell, DB., Ciulli, A. BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design. Nat. Chem. Biol. 2019, 15, 672–680.
pk properties
co-crystal structures

Binding mode of SGC-SMARCA-BRDVIII in complex with PB1(5)-BRD (PDB-ID 6ZS4). The inhibitor binds to the highly conserved Asn739 and Tyr696. The methylation of the hydroxy group in SGC-BRDVIII-NC blocks its binding to the bromodomain K(ac) binding pocket.

synthetic schemes
materials and methods
10.09.2020

Cheryl Arrowsmith of SGC-Toronto elected a Fellow of the Royal Society of Canada

by: SGC

On September 8th, 2020, the Royal Society of Canada (RSC) elected Dr. Cheryl Arrowsmith, SGC-Toronto's Chief Scientist, as an RSC Fellow, the highest honour an individual can achieve in the Arts, Social Sciences and Sciences.

NR162 A chemical probe for CASK

Probe and control are available from Sigma; the probe is available from Tocris.

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

 

NR162

 

NR187

CASK (Ca2+/calmodulin-dependent Ser/Thr kinase) is a multidomain scaffolding protein, containing several protein-protein interaction domains. From the N-to the C-terminus CASK consists of a CAMK-like domain, two L27 domains, a PDZ domain, a SH3 domain and a guanylate kinase domain1. CASK is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as a Mg2+-independent neurexin kinase with implicated roles in neuronal synapses and trafficking2. Furthermore, CASK has been categorized as a pseudokinase as it lacks the typical DFG motif and shows a GFG-motif instead. Pseudokinases carry a mutation at least in one of three highly conserved motifs within the catalytic kinase domain (VAIK-, DGF and Y/HRD motif) resulting in inactivation of catalytic activity. All in all 48 kinases of the kinome are pseudokinases3. However, CASK has been shown to maintain some catalytic activity in the absence of metal ions. High expression levels and mutations in CASK have been linked to diverse diseases, including colorectal cancer4, Parkinson's disease5 and X-linked mental retardation6, making CASK a potential drug target.


Phylogenetic kinase tree, CASK highlighted with red circle. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

SGC has developed NR162, a potent and selective CASK inhibitor with a Kd of 22 nM in ITC and IC50 of 80 nM in NanoBRET assay. NR162 has been developed based on a series of TYRO3 inhibitors first published by Pfizer7. TYRO3 is a weak off target, but NR162 is 47-fold selective against TYRO3 (IC50 of 3.8 µM (NanoBRET). In addition, the chemical probe (NR162) is accompanied by a negative control (NR187), which is structurally similar to the probe molecule.

Potency Against Target Family
NR162 is a chemical probe for CASK with a Kd of 22 nM in ITC and an IC50 of 80 nM in NanoBRET assay. The closest off-target within the target family is ERBB3 with an IC50 of 18.2 µM (227 fold selective).

Kinase NR162 (µM)
CASK0.08
ERBB318.2

 

Selectivity
NR162 has been shown to be selective in an in vitro kinase panel followed by cellular NanoBRET assays. Selectivity has been also confirmed by a DiscoverX KINOMEScan at 1000 nM. Off-targets were assessed as false positive in a follow-up NanoBRET. The selectivity outside target family revealed TYRO3 with an IC50 of 3.8 µM (47-fold) as closest off-target.

Dosage
To minimize the chance of off-target effects, we recommend a concentration of no higher than 10 µM for cell-based assays.

Cellular Activity
In NanoBRET assay using HEK923T cells NR162 shows an IC50 of 80 nM against on target.

properties
NR162

Click here to download the SDF file.

NR162
Physical and chemical properties
Molecular weight608.07
Molecular formulaC24H30Br2N6O3
IUPAC name4-(cyclopentylamino)-2-((2,5-dibromo-4-methylbenzyl)amino)-N-(3-(2-oxooxazolidin-3-yl)propyl)pyrimidine-5-carboxamide
logP4.2
TPSA107.42
No. of chiral centres0
No. of rotatable bonds10
No. of hydrogen bond acceptors6
No. of hydrogen bond donors3
Storagestable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble in DMSO
NR187

Click here to download the SDF file.

NR187
Physical and chemical properties
Molecular weight499.06
Molecular formulaC26H35ClN6O2
IUPAC name2-((4-chlorobenzyl)(ethyl)amino)-4-(cyclopentylamino)-N-(3-(2-oxopyrrolidin-1-yl)propyl)pyrimidine-5-carboxamide
clogP3.53
TPSA89.4
No. of chiral centres0
No. of rotatable bonds11
No. of hydrogen bond acceptors6
No. of hydrogen bond donors2
Storagestable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolutionsoluble in DMSO

SMILES:
NR162: O=C(C1=CN=C(N=C1NC2CCCC2)NCC3=CC(Br)=C(C=C3Br)C)NCCCN4CCOC4=O
NR187: O=C(C1=CN=C(N=C1NC2CCCC2)N(CC3=CC=C(C=C3)Cl)CC)NCCCN4CCCC4=O

InChI:
NR162: InChI=1S/C24H30Br2N6O3/c1-15-11-20(26)16(12-19(15)25)13-28-23-29-14-18(21(31-23)30-17-5-2-3-6-17)22(33)27-7-4-8-32-9-10-35-24(32)34/h11-12,14,17H,2-10,13H2,1H3,(H,27,33)(H2,28,29,30,31)
NR187: InChI=1S/C26H35ClN6O2/c1-2-32(18-19-10-12-20(27)13-11-19)26-29-17-22(24(31-26)30-21-7-3-4-8-21)25(35)28-14-6-16-33-15-5-9-23(33)34/h10-13,17,21H,2-9,14-16,18H2,1H3,(H,28,35)(H,29,30,31)

InChIKey:
NR162: BPLPIBNWDLPUKP-UHFFFAOYSA-N
NR187: AYRUNBYNKCLGFI-UHFFFAOYSA-N

selectivity profile

A DiscoverX KINOMEScan of NR162 at 1000 nM revealed very few off-targets.

TargetNR162
(% Control)
CASK0
AURKC0
ABL10
RPSKA615
PIKFYVE30
ERBB335
RPS6KA535
LRRK241
STK38L42

Hits from the KINOMEScan were assessed in a cellular NanoBRET assay in HEK293T cells and IC50 of 18.2 µM for ERBB3 (227 fold selective) was determined. All other off-targets were not confirmed in the follow-up NanoBRET assay.

The negative control NR187 with its blocked hinge-binding amine showed no activity in NanoBRET and KINOMEScan was also clean.

in vitro potency
cell based assay data

In NanoBRET assay perfomed in HEK923T cells NR162 displayed the following IC50 value against CASK 80 nM and 3.8 µM against TYRO3.

In the NCI-60 screen, which is a human tumor cell line screen, NR162 showed no significant cell toxicity as well as growth inhibition. It was tested in a single high dose of 10 µM in the full NCI-60 panel. https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm

references
  1.  Y.-P. Hsueh, Annals of neurology 2009, 66, 438.
  2. K. Mukherjee, M. Sharma, H. Urlaub, G.P. Bourenkov, R. Jahn, T.C. Südhof, M.C. Wahl, Cell 2008, 133, 328.
  3. J.Bourdeau, D. Miranda-Saavedra, G.J. Barton, D.R. Alessi, Trends in cell biology 2006, 16, 443.
  4. J.-L. Wei, Z.-X. Fu, M. Fang, Q.-Y. Zhou, Q.-N. Zhao, J.-B. Guo, W.-D. Lu, H. Wang, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2014, 35, 9185.
  5. L. Fallon, F. Moreau, B. G. Croft, N. Labib, W.-J. Gu, E. A. Fon, The Journal of biological chemistry 2002, 277, 486.
  6. Y.-P. Hsueh, Annals of neuro[4] N. A. Powell, J. K. Hoffman, F. L. Ciske, M. D. Kaufman, J. T. Kohrt, J. Quin, D. J. Sheehan, A. Delaney, S. M. Baxi, C. Catana et al., Bioorganic & medicinal chemistry letters 2013, 23, 1046.
  7. N.A. Powell, J.K.Hoffman, F.L.Ciske, M.D.Kaufman, et al., Highly selective 2,4-diaminopyrimidine-5-carboxamide inhibitors of Sky kinase. Bioorganic & Medicinal Chemistry Letters. 2013 https://doi.org/10.1016/j.bmcl.2012.12.013
pk properties
co-crystal structures

Crystallography revealed that due to the missing aspartate in CASK the inhibitor shows an improved selectivity pattern and is no longer active on MERTK, AXL and ABL1 and affinity is reduced to TYRO3 (off-targets of the lead structure PFE-PKIS12).

Figure 1: Overview of NR162 in complex with CASK (right) and details of the interaction of NR162 with the CASK hinge region (left).

Figure 2: Co-crystal structure of NR26_162 in complex with CASK (GFG-pocket) and alignment with MERTK (DFG-pocket).

synthetic schemes
materials and methods

DSF assay
Recombinant proteins were assessed as described in (Fedorov O, Niesen FH, Knapp S. Kinase inhibitor selectivity profiling using differential scanning fluorimetry. Methods Mol Biol. 2012;795:109-18.)

ITC
The ITC measurement was performed on a NanoITC (TA Instruments) at 25°C in buffer containing 30 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP and 5% Glycerol. CASK at 141 µM was injected into the cell, containing compounds at 2-6 µM. The integrated heat of titration was calculated and fitted to a single, independent binding model using the software provided by the manufacture. The thermodynamic parameters (ΔH and TΔS), equilibrium association and dissociation constants (Ka and KD), and stoichiometry (n) were calculated.

NanoBRET (Promega)
N-terminal NanoLuc and C-terminal NanoLuc/ kinase fusions, encoded in pFC32K expression vectors (Promega), were used. For cellular BRET target engagement experiments, HEK-293T were transfected with NLuc/target fusion constructs using FuGENE HD (Promega) according to the manufacturer’s protocol. Briefly, Nluc/target fusion constructs were diluted into Transfection Carrier DNA (Promega) at a mass ratio of 1:10 (mass/mass), diluted with OptiMEM media to a twentieth part of the volume of the HEK cells. FuGENE HD was added at a ratio of 1:3 (μg DNA: μL FuGENE HD). One part (vol) of FuGENE HD complexes was combined with 20 parts (vol) of HEK-293 cells suspended at a density of 2 x 105 cells/ml and afterwards the mixture was incubated in a humidified, 37°C/5% CO2 incubator for 24 h.

After this, the cells were washed and resuspendend in OptiMEM medium. For Target engagement assays 4 x 103 cells/well were plated out in a 384-well plate (Greiner). For all experiments the recommended energy transfer probes (Promega) were used if possible, at a final concentration of the Kd of the tracer on the target. In some cases, higher tracer concentrations were used for better signal-to-noise ratios. Compounds and the energy transfer probe were added to the cells and incubated for 2h in humidified, 37°C/5% CO2 incubator. The chemical inhibitors were prepared as concentrated stock solutions in DMSO (Sigma-Aldrich) and diluted with OptiMEM for this experiment. Straight before the measurement NanoGlo Substrate and Extracellular NanoLuc Inhibitor (Promega) were mixed carefully with the supernatant.

Luminescence was measured on a BMP PheraStar with 450 nm (donor) and 600 nm filters (acceptor) using 0.5 s integration time. Milli-BRET units (mBU) are calculated by multiplying the raw BRET values by 1000. Tracer and DMSO controls were used to calculate a normalized signal.

mBU=1000*I[600 nm]/I[450 nm]

Inhibitory constants were calculated by using the sigmoidal dose-response (four parameters) equation in GraphPad Prism.
Y=Bottom+((Top-Bottom) )/(1+〖10〗^(X-LogIC50*HillSlope) ) 

SGC-CK2-1 A chemical probe for CK2

This probe is available from Tocris, Sigma and Cayman

This control is available from Sigma

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

 

SGC-CK2-1

 

SGC-CK2-1N

Based upon a pyrazolopyrimidine scaffold with potent CK2 inhibition exemplified by AstraZeneca, the SGC has developed a high-quality chemical probe and its negative control for protein kinase CK2.1 This compound shows equal potency in binding to both catalytic subunits of CK2: CK2α (CSNK2A1) and  CK2α' (CSNK2A2). CK2 is a serine/threonine kinase that is part of the larger CMGC family, which is named after the initials of its subfamily members including cyclin-dependent kinases (CDK), mitogen-activated protein kinase (MAPK), glycogen synthase kinase (GSK), and CDC-like kinase (CLK). A plethora of biological functions have been ascribed to CK2 ranging from cell survival and proliferation to inflammation. CK2 phosphorylates more than 300 proteins and, via genetic and biochemical studies in a variety of experimental models, has been found to be both constitutively active and ubiquitously expressed.2-5 While CK2 inhibition has been widely studied in the context of cancer, pervasive use of non-selective CK2 inhibitors has confounded interpretation of results in many oncological studies.

Figure 1: Phylogenetic kinase tree with CK2α and CK2α' highlighted with red circles. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com)

Key profiling data:

  • Enzymatic assays (Eurofins): CK2α IC50 = 4.2 nM; CK2α' IC50 = 2.3 nM at 10µM ATP
  • Cellular data (nanoBRET): CK2α IC50 = 36 nM; CK2α' IC50 = 16 nM
  • Only 11/403 kinases with PoC <35 when screened at 1 μM
properties
Probe

SGC-CK2-1

SMILES:
CC1=CC=C(C=C1NC(CC)=O)NC2=NC3=C(C=NN3C(NC4CC4)=C2)C#N
InChI:
InChI=1S/C20H21N7O/c1-3-19(28)25-16-8-15(5-4-12(16)2)23-17-9-18(24-14-6-7-14)27-20(26-17)13(10-21)11-22-27/h4-5,8-9,11,14,24H,3,6-7H2,1-2H3,(H,23,26)(H,25,28)
InChIKey: YKDZIFFKQUNVHH-UHFFFAOYSA-N

Physical and chemical properties
Molecular weight375.44
Molecular formulaC20 H21 N7 O
IUPAC nameN-(5-((3-cyano-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-2-methylphenyl)propionamide
clogP1.94
PSA104.91
No. of chiral centres0
No. of rotatable bonds7
No. of hydrogen bond acceptors4
No. of hydrogen bond donors3
StorageStable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
DissolutionSoluble up to 10mM in DMSO
Negative control

SGC-CK2-1N

SMILES:

CC1=CC=C(C=C1NC(C2CC2)=O)N(C3=NC4=C(C=NN4C(N(C5CC5)C)=C3)C#N)C
InChI:
InChI=1S/C23H25N7O/c1-14-4-7-18(10-19(14)26-23(31)15-5-6-15)28(2)20-11-21(29(3)17-8-9-17)30-22(27-20)16(12-24)13-25-30/h4,7,10-11,13,15,17H,5-6,8-9H2,1-3H3,(H,26,31)
InChIKey: LGMBLPOARZRSKR-UHFFFAOYSA-N

Physical and chemical properties
Molecular weight415.2
Molecular formulaC23 H25 N7 O
IUPAC name4-((3-(cyclopropyl-formylamino)-4-methyl-phenyl)-methyl-amino)-2-(cyclopropyl-methyl-amino)-1,5,9-triaza-bicyclo[4.3.0]nona-2,4,6,8-tetraene-7-carbonitrile
clogP3.67
PSA63.46
No. of chiral centres0
No. of rotatable bonds8
No. of hydrogen bond acceptors5
No. of hydrogen bond donors1
StorageStable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
DissolutionSoluble up to 10mM in DMSO
selectivity profile

SGC-CK2-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM. Only 11 kinases showed PoC <35 giving an S(35) at 1 μM = 0.027. Potential off-targets were tested in the nanoBRET target engagement assay (DYRK2) and via Eurofins radiometric and LANCE kinase assays. Data corresponding with off-target kinase activity is shown in the table below.

 

Kinase% Control at 1µMEnzymatic IC50 (nM)NanoBRET IC50 (nM)
CK2α'/CSNK2A202.316
DRAK10>10000NT
CK2α/CSNK2A10.54.236
DYRK2144403700
PLK423>10000NT
HIPK2263400NT
MEK5280% AT 1µMNT
HIPK1323700NT
HIPK3348100NT
RIOK232NTNT
SGK334>10000NT

Figure 1: SGC-CK2-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM and off-target kinases inhibited PoC <35 were tested in an orthogonal assay.
 

SGC-CK2-1N was also tested in the DiscoverX panel and 1 kinase had a PoC <35. The negative control was sent to Eurofins for testing in enzyme assays for CSNK2A1 and CSNK2A2. The results are in the table below.

Kinase% Control at 1µMEnzymatic IC50 (nM)NanoBRET IC50 (nM)
RIOK123NTNT
RET35NTNT
FLT340NTNT
BRAF55NTNT
IKK-alpha58NTNT
MKNK158NTNT
STK3361NTNT
TNK162NTNT
EPHA263NTNT
CK2α'/CSNK2A265>10000>10000
CK2α/CSNK2A1100>10000NT

Figure 2: SGC-CK2-1N was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM and follow-up CK2α and CK2α' enzymatic assays were done to confirm no activity.

in vitro potency
cell based assay data

A NanoBRET assay was utilized to assess the binding affinity of SGC-CK2-1 to CK2α and CK2α'. The negative control shows no binding affinity for CK2α'.

Figure 1: SGC-CK2-1 and SGC-CK2-1N were profiled in the CK2 NanoBRET assays.

references
  1. Wells, C., Drewry, D. H., Pickett, J. E. & Axtman, A. D. SGC-CK2-1: the first selective chemical probe for the pleiotropic kinase CK2. ChemRxiv, 10.26434/chemrxiv.12296180.v12296181 (2020).
  2. Rabalski, A. J., Gyenis, L. & Litchfield, D. W. Molecular Pathways: Emergence of Protein Kinase CK2 (CSNK2) as a Potential Target to Inhibit Survival and DNA Damage Response and Repair Pathways in Cancer Cells. Clinical Cancer Research 22, 2840-2847, doi:10.1158/1078-0432.Ccr-15-1314 (2016).
  3. Meggio, F. & Pinna, L. A. One-thousand-and-one substrates of protein kinase CK2? FASEB journal : official publication of the Federation of American Societies for Experimental Biology 17, 349-368, doi:10.1096/fj.02-0473rev (2003).
  4. Nuñez de Villavicencio-Diaz, T., Rabalski, A. J. & Litchfield, D. W. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks. Pharmaceuticals 10, 27 DOI: 10.3390/ph10010027 (2017).
  5. Ahmed, K., Gerber, D. A. & Cochet, C. Joining the cell survival squad: an emerging role for protein kinase CK2. Trends in Cell Biology 12, 226-230, doi:10.1016/S0962-8924(02)02279-1 (2002).

Related Links: https://www.tocris.com/products/sgc-ck2-1_7450#product-literature

pk properties
co-crystal structures
synthetic schemes
materials and methods

PFI-6 A novel chemical probe for MLLT1/3

The probe PFI-6 is available from Sigma and Cayman Chemicals.

The negative control PFI-6N is available from Sigma.

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

 

PFI-6

 

PFI-6N

Increasing numbers of papers describing YEATS biology are emerging, and there is a growing need for high quality chemical probes that can be used to study YEATS domains. A collaboration between Pfizer and the SGC has resulted in the discovery of PFI-6, a potent inhibitor of MLLT1/3. PFI-6 has a unique chemotype relative to the current MLLT1/3 chemical probe NVS-MLLT-1.  PFI-6N has also been developed as a structurally similar negative control compound.

properties
Probe

PFI-6

Physical and chemical properties for PFI-6
Molecular weight391.43
Molecular formulaC22H21N3O4
IUPAC name(R0-N-(2,3-dihydro-1H-inden-1-yl)-5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide
MollogP2.6
PSA91.23
No. of chiral centres0
No. of rotatable bonds6
No. of hydrogen bond acceptors7
No. of hydrogen bond donors2
Storagestore as a dry poweder or as DMSO stock solutions (10mM) at -20 °C
DissolutionSoluble in DMSO up to 10mM
Negative control

PFI-6N

Physical and chemical properties for PFI-6N
Molecular weight357.41
Molecular formulaC19H23N3O4
IUPAC nameN-cyclopentyl-5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-methylisoxazole-3-carboxamide
MollogP1.86
PSA82.44
No. of chiral centres0
No. of rotatable bonds6
No. of hydrogen bond acceptors7
No. of hydrogen bond donors1
StorageStore as a dry powder or as DMSO stock solutions (10mM) at -20 °C
DissolutionSoluble in DMSO up to 10mM

SMILES:

PFI-6: O=C(C1=C(C=C(C=C1)C2=CC(C(N[C@@H]3CCC4=C3C=CC=C4)=O)=NO2)O)N(C)C
PFI-6N: O=C(C1=C(C=C(C=C1)C2=CC(C(N(C3CCCC3)C)=O)=NO2)O)N(C)C

InChI:

PFI-6: 1S/C22H21N3O4/c1-25(2)22(28)16-9-7-14(11-19(16)26)20-12-18(24-29-20)21(27)23-17-10-8-13-5-3-4-6-15(13)17/h3-7,9,11-12,17,26H,8,10H2,1-2H3,(H,23,27)/t17-/m1/s1
PFI-6-N: 1S/C19H23N3O4/c1-21(2)18(24)14-9-8-12(10-16(14)23)17-11-15(20-26-17)19(25)22(3)13-6-4-5-7-13/h8-11,13,23H,4-7H2,1-3H3

InChIKey:

PFI-6: IXWUILRSNIQHDM-QGZVFWFLSA-N
PFI-6-N:CKEICVFLYGXFOP-UHFFFAOYSA-N

selectivity profile

PFI-6 shows potent activity on MLLT1/3 (Table 1). PFI-6 is universally inactive in the SGC Bromodomain panel, and showed no activity in an Invitrogen panel of 40 kinases (screening conducted at 10µM). Additionally, PFI-6 showed no activity in a panel of 25 PDEs, ion channels and GPCRs >50µM. The negative control, PFI-6N, was not reactive against MLLT1 >30µM, MLLT3 >30µM, YEATS2 >30µM and YEATS4 >30µM.

 

 MLLT1MLLT3YEATS2YEATS4
HTRF IC50 (µM)0.140.16>40>40
BLI Kd (µM)0.110.11n.d.n.d.
ITCKD (µM)0.0820.076n.d.n.d.
Tm Shift (°C)3.615.130.00-0.02

Table 1: Potency Against Target Family 

Figure 1: PFI-6 specfically inhibits MLLT1 and MLLT3.

in vitro potency
cell based assay data

In the NanoBRET cellular target engagement assay, PFI-6 displayed potent inhibition, with an average IC50 value of 0.76 μM (±0.1) (Figure 1). In comparison, PFI-6N had no inhibitory properties (up to 30 μM) (Figure 1). Further in cell validation using a Fluorescence Recovery After Photobleaching (FRAP) assay was used to confirm target inhibition by PFI-6  (Figure 2) . 

Figure 1: A NanoBRET assay was used to determine MLLT3 target engagement in cells.

Figure 2: Confirmation of MLLT1 target engagement in cells by Fluorescence Recovery After Photobleaching analysis.

 

references
pk properties
co-crystal structures

The figure below shows PFI-6 is soaked into the YEATS domain. PDB codes will be added when they become available. 

 

 

synthetic schemes
materials and methods
15.05.2020

The Viral Interruption Medicines Initiative (VIMI): Open Science coalition in Canada to prevent future pandemics

by: SGC

In an opinion piece to Canada's The Hill Times, Drs Richard Gold, Tania Bubela, Aled Edwards and John Bamforth announced the creation of the Viral Interruption Medicines Initiative (VIMI). The partnership will leverage open science to develop anti-viral drugs in anticipation for future pandemics. 

07.04.2020

Open science drug discovery partnership, READDI, aims to invest $125 million to prevent future pandemics

by: SGC

(Chapel Hill, N.C.— April 7, 2020) — Today, the Structural Genomics Consortium (SGC), the University of North Carolina at Chapel Hill and the Eshelman Institute for Innovation, announce the launch of the Rapidly Emerging Antiviral Drug Development Initiative (READDI), a global organization formed to discover and develop drugs to put “on the shelf” for clinical trial testing in anticipation of future viral pandemics.

NVS-MLLT-1 A Potent and Selective inhibitor of YEATS proteins

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

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

 

NVS-MLLT-1

 

NVS-MLLT-C

MLLT1 (also known as ENL, LTG19 and YEATS1) is a fusion gene partner within the MLL oncogene, involved in chromosome translocation [1]. In MLL1 rearranged mixed lineage leukaemia, MLLT1 acts as fusion partner for the transcriptional co-activation of MLL1. This interaction leads to linkage of MLL1 with the super elongation complex, and histone methyl transferase DOT1L, ultimately driving the dysregulation of oncogenes [2-6].
Accumulating evidence suggests that MLLT1 can also be a driver in non-MLLT1 rearranged acute myeloid leukaemia (AML). The MLLT1 epigenetic reader domain (YEATS domain) recognises acylated lysine residues on histone 3 (mainly acetyl lysine) (Figure 1). In AML, the MLLT1 YEATS domain directly links the epigenetic readout to the oncogenic dysregulation of gene expression [7,8]

MLLT1 CRISPR/Cas9 studies have shown significant reductions in cell proliferation, and invasiveness of AML cell lines. Subsequent rescue with ectopically expressed native MLLT1 restores the disease phenotype, while rescue with mutants that are deficient in the binding pocket (where MLLT1 recognises acyl lysine residues) is unable to restore the phenotype [8]. These studies, combined with the knowledge of known epigenetic readers being used as tractable targets for small molecule inhibitors, indicate that inhibition of MLLT1 may be a viable approach for the treatment of AML. The SGC therefore develped SGC-iMLLT, a first in class potent and selective inhibitor of YEATS proteins [9]. Expanding this work, a probe with improved in cell activity was developed with Novartis. Here, we present NVS-MLLT-1 as a potent and selective inhibitor of YEATS proteins. 

Figure 1: Biology of the Targets.

 

Potency Against Target Family
An AlphaScreen competition assay was used to assess the potency of SGC-iMLLT against all four human YEATS domains. NVS-MLLT-1 showed inhibition of both MLLT1 and MLLT3 at 0.15 µM and 0.254 µM respectively (Table 1). Conversely, the negative control molecule NVS-MLLT-C did not show any inhibition in the assay. More information on MLLT1 as a target can be found in our TEPs repository: https://www.thesgc.org/tep/mllt1.

In vitro assaysNVS-MLLT-1 IC50 (µM)NVS-MLLT-C IC50 (µM)
TR-FRET MLLT10.15> 20
TR-FRET MLLT30.254> 20 
TR-FRET YEATS2> 20> 20
TR-FRET YEATS4> 20> 20
KD BLI MLLT10.075-
KD ITC MLLT10.109-

Table 1: Potency Against Target Family and biophysical properties

Selectivity
NVS-MLLT-1 shoes potent activity on MLLT1/3 whilst being selective for YEATS2/4. NVS-MLLT-1 shows selectivity across all bromodomains (CERC2 IC50 > 40 µM) and no activity was shown in a kinase panel. NVS-MLLT-1 has some activity on ACES and H3 binding. 

Dosage
Use between 1 and 10 µM for cellular assays, and 1 µM for screening at a single shot.

In vitro Activity
In the NanoBRET cellular target engagement assay, NVS-MLLT-1 displayed potent inhibition, with an average IC50 value of 0.5 μM (±0.12) (Figure 2). In comparison,NVS-MLLT-C had no inhibitory properties (up to 30 μM). Further in cell validation was used to confirm target inhibition by NVS-MLLT-1.

 
properties
Probe

NVS-MLLT-1

Physical and chemical properties for NVS-MLLT-1
Molecular weight385.47
Molecular formulaC23H23N5O
IUPAC name(S)-N-(2-((-methylpyrrolidin-1-yl)methyl-1H-benzo[d]imidazel-5-yl)isoquinoline-6-carboxamide
MollogP3.6
PSA69.09
No. of chiral centres1
No. of rotatable bonds5
No. of hydrogen bond acceptors5
No. of hydrogen bond donors2
StorageDMSO stock solutions (up to 50mM) are stable at -20 °C
DissolutionSoluble in DMSO up to 50mM
Negative control

NVS-MLLT-C

Physical and chemical properties for NVS-MLLT-C
Molecular weight385.43
Molecular formulaC22H19N5O2
IUPAC nameN-(2-((2-oxopyrrolidin-1-yl)methyl)-1Hbenzo[d]imidazol-5-yl)isoquinoline-6-carboxamide
MollogP 
PSA86.16
No. of chiral centres0
No. of rotatable bonds5
No. of hydrogen bond acceptors6
No. of hydrogen bond donors2
StorageDMSO stock solutions (up to 50mM) are stable at -20 °C
DissolutionSoluble in DMSO up to 50mM

SMILES:

NVS-MLLT-1: C[C@H]1CCCN1CC2=NC3=CC(NC(C4=CC5=CC=NC=C5C=C4)=O)=CC=C3N2
NVS-MLLT-C: O=C(C1=CC2=CC=NC=C2C=C1)NC3=CC=C(N4)C(N=C4CN5C(CCC5)=O)=C3

InChI:

NVS-MLLT-1: 1S/C23H23N5O/c1-15-3-2-10-28(15)14-22-26-20-7-6-19(12-21(20)27-22)25-23(29)17-4-5-18-13-24-9-8-16(18)11-17/h4-9,11-13,15H,2-3,10,14H2,1H3,(H,25,29)(H,26,27)/t15-/m0/s1
NVS-MLLT-C: 1S/C22H19N5O2/c28-21-2-1-9-27(21)13-20-25-18-6-5-17(11-19(18)26-20)24-22(29)15-3-4-16-12-23-8-7-14(16)10-15/h3-8,10-12H,1-2,9,13H2,(H,24,29)(H,25,26)

InChIKey:

NVS-MLLT-1: ZRTFTZCKJUNZIU-HNNXBMFYSA-N
NVS-MLLT-C: GMSVIEOZPBLYCQ-UHFFFAOYSA-N

selectivity profile

The AlphaScreen assays were employed to screen NVS-MLLT-1 and NVS-MLLT-C against a panel of bromodomains. NVS-MLLT-1 shows potent activity on MLLT1/3 whilst being selective for YEATS2/4. NVS-MLLT-1 shows selectivity across all bromodomains (CERC2 IC50 > 40 μM). NVS-MLLT-1 shows no activity on kinases tested, NVS-MLLT-1 has some activity on ACES and E3 binding. NVS-MLLT-C shows no activity in a panel of kinases, and no activties on other targets tested. 

In vitro assaysNVS-MLLT-1 IC50 (µM)NVS-MLLT-C IC50 (µM)
TR-FRET MLLT10.15> 20
TR-FRET MLLT30.254> 20 
TR-FRET YEATS2> 20> 20
TR-FRET YEATS4> 20> 20
KD BLI MLLT10.075-
KD ITC MLLT10.109-

Table 1: Potency Against Target Family and biophysical properties

General Selectivity- NVS-MLLT-1

NVS-MLLT-1 shows no activity on kinases tested. NVS-MLLT-1 has some activity on ACES and H3 binding.

General Selectivity- NVS-MLLT-C

NVS-MLLT-C shows no activity on kinases tested. NVS-MLLT-C shows no activities on other targets tested

in vitro potency
cell based assay data

Cellular target Engagement

In the NanoBRET cellular target engagement assay, NVS-MLLT-1 displayed potent inhibition, with an average IC50 value of 0.5 μM (±0.12) (Figure 1). In comparison, NVS-MLLT-C had no inhibitory properties (up to 30 μM) (Figure 1). Further in cell validation using a Fluorescence Recovery After Photobleaching (FRAP) assay was used to confirm target inhibition by NVS-MLLT-1  (Figure 2) . 

Figure 1: A NanoBRET assay was used in HEK293 cells to determine target engagement in cells. All probes were tested for 24h in the presence of 2.5 μM SAHA (24h). Graph represents Mean±SEM, n=3 independent replicates, with n=4 technical replicates.  mBU: BRET units

Figure 2: Further confirmation of MLLT1 target engagement in cells.  Fluorescence Recovery After Photobleaching analysis shows NVS-MLLT-1 decreases the half-life recovery time of cells. n=3 independent experiments, Mean±SEM, fold change from SAHA treatment alone. One way ANOVA, Dunnett's multiple comparisons.

Materials and Methods

NanoLuciferase Bioluminescent Resonance Energy Transfer (NanoBRET) Assay 
Cellular activity against MLLT3 was assessed using a NanoBRET assay. HEK293 cell (8 x 105) were plated a 6-well plate, after 6h cells were co-transfected with C-terminal HaloTag-Histone 3.3 (NM_002107) and an N-terminal NanoLuciferase fusion of MLLT3 (original MLLT3 WT sequences from Promega HaloTag® human ORF in pFN21A or MLLT3 MUT - Y78A Tyrosine is changed to an Alanine) at a 1:10 (NanoLuc® to HaloTag®) ratio respectively with FuGENE HD transfection reagent. Sixteen 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). Cells were then re-plated in a 96-well assay white plate (Corning Costar #3917) at 2x104 cells per well. Compounds were then added directly to the cells (in the presence of SAHA 2.5 µM) at final concentrations 0-30 μM or an equivalent amount of DMSO as a vehicle control, and the plates were incubated for 24 h at 37°C in the presence of 5% CO2. NanoBRET Nano-Glo substrate (Promega) was added to both control and experimental samples at a final concentration of 10 µM. Readings were performed within 10 minutes using a ClarioSTAR (BMG Labtech) equipped with 460 nm and 610 nm filters. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/460 nm for experimental samples minus the emission at 610 nm/460 nm for control samples (without NanoBRET fluorescent ligand). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000.

Peptide Displacement Assay
Peptide displacement assays were set up with biotinylated peptides (chosen based on ChIPseq data from the literature and purchased from LifeTein) and 6His tagged protein (Table 2).  For detection, two orthogonal technologies were used, i. AlphaScreen® technology from Perkin Elmer and ii. HTRF from Cisbio.  Compounds were dispensed in duplicate at single concentration (100 µM) for the initial screen and as 11-point dose response curves starting from 200 µM for IC50 value determination.

YEATS domainPeptide shorthandPositionPeptide sequence
MLLT1H3K18ac12-30GGKAPR(K-acetyl)QLATKAARKSAPY(K-biotin)
MLLT3H3K9ac2-20ARTKTAR(K-acetyl)STGGKAPRKQLY(K-biotin)
YEATS2H3K27cro15-32biotin-GKPRKQLATAAR(K-crotonyl)SAPAT
YEATS4H3K27cro15-32biotin-GKPRKQLATAAR(K-crotonyl)SAPAT

Table 2: Peptides used for the peptide displacement assays

To determine optimal assay conditions for each protein prep, proteins and peptides were titrated against each other in a 16 by 16 matrix in 1:1 dilutions, starting from 3.2 µM. For the final ratio of protein and peptide to use in the assay, the point representing the EC90 in the two-dimensional titration was chosen.  Typically, final assay concentrations for protein and peptide fell between 25 and 200 nM.  For AlphaScreen®, AlphaScreen Histidine (Nickel Chelate) Detection Kit donor and acceptor beads were used at a 1:2500 dilution from purchased stock; for HTRF, SA-XL665 and anti-6His antibody were used at 1:2000 and 1:10000 dilution from purchased stock, respectively.  Assays were performed on 384 well ProxiPlates (Perkin Elmer) at a final volume of 20 µl and plates were read using a Pherastar FSX plate reader (BMG Labtech).

Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) was carried out using a TA NanoITC (standard volume) instrument.  Protein was prepared by dialysis (overnight at 4°C) against a ~1000 times excess of buffer (20 mM Tris at pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, 2 mM DTT) using SnakeSkin® Dialysis Tubing with a 7 kDa MWCO and then concentrated to 300 µM.  The experiment was carried out at 20°C in reverse mode with the compound in the cell at 50 µM and the protein in the syringe at 300 µM due to the solubility of the compound with the first injection at 4 µl and the following 30 at 8 µl. Data was analysed using the NanoAnalyze software package by TA Instruments.

Differential Scanning Fluorimetry
Differential scanning fluorimetry (DSF) to determine the effect of compounds on the thermal stability of proteins (DTm) was carried out on 384 well PCR plates using a LightCycler 480 (Roche).  Protein at 10 µM was buffered in 10 mM HEPES at pH 7.5 and 500 mM NaCl.  The experiment was carried out from 25 to 95°C with three acquisitions per degree.  Compounds were added at 50 µM final concentration and DMSO reference and no-addition controls were also collected.

references

References

1. Doty, R.T., et al., The leukemia-associated gene Mllt1/ENL: characterization of a murine homolog and demonstration of an essential role in embryonic development. Blood Cells Mol Dis, 2002. 28(3): p. 407-17.https://www.ncbi.nlm.nih.gov/pubmed/12367585

2. Mueller, D., et al., A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. Blood, 2007. 110(13): p. 4445-54.DOI: 10.1182/blood-2007-05-090514.https://www.ncbi.nlm.nih.gov/pubmed/17855633

3. Mueller, D., et al., Misguided transcriptional elongation causes mixed lineage leukemia. PLoS Biol, 2009. 7(11): p. e1000249.DOI: 10.1371/journal.pbio.1000249.https://www.ncbi.nlm.nih.gov/pubmed/19956800

4. Yokoyama, A., et al., A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell, 2010. 17(2): p. 198-212.DOI: 10.1016/j.ccr.2009.12.040.https://www.ncbi.nlm.nih.gov/pubmed/20153263

5. Biswas, D., et al., Function of leukemogenic mixed lineage leukemia 1 (MLL) fusion proteins through distinct partner protein complexes. Proc Natl Acad Sci U S A, 2011. 108(38): p. 15751-6.DOI: 10.1073/pnas.1111498108.https://www.ncbi.nlm.nih.gov/pubmed/21896721

6. Monroe, S.C., et al., MLL-AF9 and MLL-ENL alter the dynamic association of transcriptional regulators with genes critical for leukemia. Exp Hematol, 2011. 39(1): p. 77-86 e1-5.DOI: 10.1016/j.exphem.2010.09.003.https://www.ncbi.nlm.nih.gov/pubmed/20854876

7. Erb, M.A., et al., Transcription control by the ENL YEATS domain in acute leukaemia. Nature, 2017. 543(7644): p. 270-274.DOI: 10.1038/nature21688.https://www.ncbi.nlm.nih.gov/pubmed/28241139

8. Wan, L., et al., ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia. Nature, 2017. 543(7644): p. 265-269.DOI: 10.1038/nature21687.https://www.ncbi.nlm.nih.gov/pubmed/28241141

9. Christott, T. et al., Discovery of a selective inhibitor for the YEATS domains of ENL/AF9. SLAS Discov, 2019. 24(2):133-141. DOI: 10.1177/2472555218809904. https://www.ncbi.nlm.nih.gov/pubmed/30359161

10. Chaikuad, A., S. Knapp, and F. von Delft, Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens. Acta Crystallogr D Biol Crystallogr, 2015. 71(Pt 8): p. 1627-39.DOI: 10.1107/S1399004715007968.https://www.ncbi.nlm.nih.gov/pubmed/26249344

11. Moustakim, M., et al., Discovery of an MLLT1/3 YEATS Domain Chemical Probe. Angew Chem Int Ed Engl, 2018. 57(50): p. 16302-16307.DOI: 10.1002/anie.201810617.https://www.ncbi.nlm.nih.gov/pubmed/30288907

12. Heidenreich, D., et al., Structure-Based Approach toward Identification of Inhibitory Fragments for Eleven-Nineteen-Leukemia Protein (ENL). J Med Chem, 2018. 61(23): p. 10929-10934.DOI: 10.1021/acs.jmedchem.8b01457.https://www.ncbi.nlm.nih.gov/pubmed/30407816

pk properties

In vitro Metabolic stability of NVS-MLLT-1

To establish the in cell half life of NVS-MLLT-1, metabolic stability studies were carried out exposing nominated compounds to samples of primary human hepatocytes. Results from ADME and phys chem experiments are summarised below. 

In vitro Metabolism (Liver microsomes)NVS-MLLT-1
[CL int(µL/min/mg)])
Human 89
Rat 68
Mouse 90

Table 1: In vitro metabolism

Permeability

NVS-MLLT-1
PAMPA:
Log PAMPA (cm/s) -5.1
% FA  49
Low Efflux MDCK:
Papp(A-B)(x10-6 )11
% FA80-100
% Recovery 81

Table 2: PhysChem Properties: Permeability 

Solubility and pKa

NVS-MLLT-1
Equilibrium Solubility 
pH 6.8 (g/L)0.375
pKa14.6
pKa2 2.1

Table 3: PhysChem Properties: Solubility and pKa

Solubility and pKa

NVS-MLLT-1
clogP3.6
logP (octanol)2.8

Table 4: PhysChem Properties: Lipophilicity

co-crystal structures

The figures below shows MLLT1 co-crystal strutcure at 1.7 Å. NVS-MLLT-1 is soaked into the YEATS domain. PDB codes will be added when they become available. 

synthetic schemes
materials and methods
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