UNC3866

Reagent Authentication Certificate:

Chemical Probes Portal UNC3866.pdf

Protein target name:

CBX7, CBX4

Mechanism of action:

Antagonist

Recommended Concentration for use in cells:

30 uM

Probe Availability

MedChem Express (1872382-47-2)

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Methyl-lysine reader

HUGO ID:

HGNC:1557 HGNC:1554

Entrez gene ID:

23492 8535

UNIPROT ID:

O95931 O00257

Target alias:

NBP16, Chromobox 7, Chromobox Homolog 7, Chromobox 4, Chromobox Homolog 4, NS5ATP1-binding protein 16, NBP16, Polycomb 2 Homolog, PC2

Probe Details

Chemical Probes Portal ID:

CPP661

PubChem CID:

101043861

ChEMBL ID:

CHEMBL3939958

SMILES string:

CC(C)(C)C1=CC=C(C(N[C@@H](CC2=CC=CC=C2)C(N[C@@H](C)C(N[C@@H](CC(C)C)C(N[C@@H](CCCCN(CC)CC)C(N[C@H](C(OC)=O)CO)=O)=O)=O)=O)=O)C=C1

InChi Key:

UMRRDXVUROEIKJ-JCXBGQGISA-N

Control compound:

UNC4219

Physico-chemical properties

Solubility - preferred solvent:

water or DMSO

Solubility - concentration:

up to 1 mM in water, up to 50 mM in DMSO

Potential reactivities:

Possible susceptibility to hydrolysis of the methyl ester in cells. The resultant metabolite (UNC4007) is still capable of target engagement (CBX7 Kd = 0.41 ± 0.16 µM) and is not the dominant species in cells.

NMR:

1H NMR (400 MHz, CD3OD): δ 7.75 – 7.68 (m, 2H), 7.52 – 7.44 (m, 2H), 7.35 – 7.25 (m, 4H), 7.24 – 7.18 (m, 1H), 4.74 (dd, J = 9.3, 5.5 Hz, 1H), 4.50 (t, J = 4.2 Hz, 1H), 4.46 (dd, J = 8.7, 5.4 Hz, 1H), 4.38 (dd, J = 10.0, 4.7 Hz, 1H), 4.32 (q, J = 7.2 Hz, 1H), 3.91 (dd, J = 11.4, 4.6 Hz, 1H), 3.79 (dd, J = 11.4, 3.9 Hz, 1H), 3.74 (s, 3H), 3.26 (dd, J = 13.9, 5.5 Hz, 1H), 3.17 (q, J = 7.3 Hz, 4H), 3.14 – 3.01 (m, 3H), 2.02 – 1.89 (m, 1H), 1.86 – 1.75 (m, 1H), 1.75 – 1.59 (m, 5H), 1.56 – 1.42 (m, 2H), 1.38 (d, J = 7.2 Hz, 3H), 1.33 (s, 9H), 1.27 (t, J = 7.3 Hz, 6H), 0.98 – 0.86 (m, 6H).

Storage Recommendations

Storage (dry powder):

Store in freezer to be safe (-20C), although there is no current evidence of instability at room temperature

Storage (solution):

Store in freezer to be safe (-20C), no evidence of significant decomposition over time

Storage sensitivities:

none known

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

CBX7: 97 nM; CBX4: 94 nM

Potency assay:

Isothermal titration calorimetry (ITC)

PDB ID for probe-target interaction (3D structure):

5EPJ

Structure-activity relationship:

Detailed SAR data are available in a J. Med. Chem. publication that should be available shortly. SAR data were determined using an AlphaScreen assay, which is a bead-based competition assay that utilizes His-tagged proteins and biotinylated-peptide bait ligands. The SAR study primarily explored ligand length, unnatural methyl-lysine mimetics, and N-terminal modifications, as well as modifications to the phenylalanine side chain, alanine side chain, and C terminus. We found that certain tertiary amine mimetics of trimethyl-lysine are able to maintain similar affinities for chromodomains as Kme3 and Kme3 derivatives are not active in cells. A thorough examination of the N terminus of our inhibitors has also begun to establish the requirements for optimizing potency and selectivity for the Pc, HP1 and CDY families of chromodomains. CBX4/7 show a strong preference for alkyl substituents located at the para-position of the N-terminal benzoyl cap.

Off target activity

Potency (off target):

Off target protein and potency:

CBX2: 1.8 uMCBX6: 0.610 uMCBX8: 1.2 uMCDY1: 6.3 uMCDYL1b: 0.91 uMCDYL2: 0.85 uM

Potency assay (off target):

Isothermal titration calorimetry (ITC). We also profiled the compound on a microarray containing 96 epigenetic reader proteins across multiple domain types.

Probe Selectivity in Vitro:

UNC3866 is selective over a broad range of chromatin-associated proteins (profiled versus 100+ Kme readers, 52 bromodomains, 33 lysine methyltransferases, and 7 demethylase enzymes). Additionally, UNC3866 was profiled against 49 GPCRs, 5 ion channels and 3 transporter proteins. In the Nature Chemical Biology publication, see Figures 2-3, Supplementary Figures 3-8, and Supplementary Tables 2-5.

In cell validation

On target activity in cells

Potency in cells:

IC50

7.6 uM

Potency assay (cells):

Consistent with inhibition of PRC1 chromodomains (Bernard, D. et al. CBX7 controls the growth of normal and tumor-derived prostate cells by repressing the Ink4a//Arf locus. Oncogene 24, 5543-51 (2005)), UNC3866 inhibits PC3-cell proliferation in a dose-dependent manner and induces senescence after exposure for 6 days while a negative control, UNC4219, has no affect.

Target engagement assay (cells):

Whole-cell treatment of PC3 cells with UNC3866 inhibits UNC4195 (biotinylated-UNC3866)-chemiprecipitation of CBX7 from cell lysates in a dose-dependent fashion. UNC4195 chemiprecipitates endogenous CBX4/7 and other PRC1 components from cell lysates, suggesting that UNC3866 engages 'canonical' PRC1 without disrupting the complex's composition.

Off target activity in cells

Potency in cells, off target:

Potency assay, off target (cells):

Assays to assess the cellular consequences of engaging the other non-PRC1 chromodomain targets (CDYs) of UNC3866 have not been performed since these are relatively uncharacterized targets.

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

Treatment with UNC3866 up to 100 uM did not induce toxic effects as assessed by CellTiter-Glo in PC3 and HEK293T cells.

Primary Reference

Reference (doi):

10.1038/nchembio.2007

Reference (PMID):

26807715

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

no rating

SAB Members

SAB Comments

UNC3866 is a first in class inhibitor for CBX7 and CBX4, as well as other related family members. It's highly potent in vitro and represents a great advancement in the CBX protein biology field. The compound was shown to inhibit cell proliferation at 7.6 uM, and at 30 uM, to induce a cell senescence phenotype that was linked to the target by data published elsewhere. A biotinylated version of the compound can pull down the PRC1 complex components, an effect that is inhibited if the PC3 cells were incubated with 30 uM of UNC3866 for 24 h prior to lysis and pulldown. Data on the cellular compound pulldowns was focused on the known targets instead of a broader proteomic profiling of the targets bound to UNC3866. Additional data on UNC3866's effect on more specific cellular activity readout such as nanoBRET or ChIP would be very informative.

Oct 24 2016 - 12:24pm

This probe is well-characterized and somewhat selective for CBX4 and CBX7 in biochemical assays, though it is only 6-fold less active against CBX6 and about 10-fold less active against CDYL1b and CDYL2. It also has low cell permeability, necessitating high concentrations to be used in cell assays. This means that any biological effects observed in cell assays can not be attributed solely to inhibition of CBX4 and CBX7.

Jan 12 2017 - 12:10pm

The probe UNC3866 has been well characterized for its interaction with the desired target CBX7 using ITC, a biotinylated analog, and co-crystal stuctures to provide strong evidence of interactions. Cellular penetration is low (5% of external concentration), meaning high concentrations are needed. A closely related, inactive analog provides evidence that the probe's biological effects are target related. However, the probe is active against other members of the CBX chromodomains (equipotent at CDX4) and also CDY choromodomains so attributing effects to a specific interaction in cells is not possible. The probe has been assessed for pharmacokinetics in mice (IP injection) and shows evidence of moderate clearance that may allow for use in vivo. The high concentrations required for cellular assays would be difficult to achieve in vivo, but the authors propose to examine this possible use in the future.

Mar 20 2017 - 6:41pm

UNC3866

Probe Availability

Probe Information

Target Details

NBP16, Chromobox 7, Chromobox Homolog 7, Chromobox 4, Chromobox Homolog 4, NS5ATP1-binding protein 16, NBP16, Polycomb 2 Homolog, PC2

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

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UNC3866

Probe Availability

Probe Information

Target Details

NBP16, Chromobox 7, Chromobox Homolog 7, Chromobox 4, Chromobox Homolog 4, NS5ATP1-binding protein 16, NBP16, Polycomb 2 Homolog, PC2

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

Interested in supporting the portal?