UNC1215 A potent, selective antagonist of L3MBTL3 with cellular activity

This probe is available from Cayman Chemical, Sigma and Tocris
Its control compound UNC1079 is available from Cayman Chemical

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

overview

Methyl-lysine (Kme) recognition domains play a central role in epigenetic regulation during cellular differentiation, development, and gene transcription with more than 200 known “reader” domains in the human proteome. In collaboration with the Center for Integrative Chemical Biology and Drug Discovery (CICBDD) at The University of North Carolina at Chapel Hill, we have developed the first chemical probe for a Kme-binding protein. UNC1215 is a potent and selective chemical probe for the Kme reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds the MBT domains of L3MBTL3 with a K_d of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides. This probe is greater than 50-fold selective versus other members of the human MBT family and also demonstrates selectivity against more than 200 other Kme reader domains examined. UNC1079 is a structurally similar but substantially less potent antagonist and negative control.

properties

N-phenyl-2,5-bis[4-(pyrrolidin-1-yl)piperidine-1-carbonyl]aniline
Click here to download SDF file

Physical and chemical properties
Molecular weight	529.3
Molecular formula	C32H43N5O2
IUPAC Name	N-phenyl-2,5-bis[4-(pyrrolidin-1-yl)piperidine-1-carbonyl]aniline
logP	4.17
PSA	59.13

SMILES:

C1CCN(C1)C1CCN(CC1)C(c1ccc(C(N2CCC(CC2)N2CCCC2)=O)c(c1)Nc1ccccc1)=O

InChI:

InChI=1S/C32H43N5O2/c38-31(36-20-12-27(13-21-36)34-16-4-5-17-34)25-10-11-29(30(24-25)33-26-8-2-1-3-9-26)32(39)37-22-14-28(

InChIKey:

PQOOIERVZAXHBP-UHFFFAOYSA-N

selectivity profile

Selectivity of UNC1215 against a panel of Kme reader proteins.

Alphascreen IC₅₀ [µM]^a	MBT domains					Chromodomain	Tudor Domains		PHD Fingers
Alphascreen IC₅₀ [µM]^a	L3MBTL1	L3MBTL3	L3MBTL4	SFMBT	MBTD1	CBX7	53BP1	UHRF1	PHF23	JARID1A
UNC1215	2	0.04	11	>30	6	>30	4	>30	>30	>30
UNC1215	(R² = 0.93, n = 17)	(R² = 0.93, n = 17)	(R² = 0.63, n = 21)	(n = 18)	(R² = 0.71, n = 11)	(n = 19)	(R² = 0.90, n = 12)	(n = 18)	(n = 15)	(n = 15)

^a - The data for the IC₅₀ values was calculated from n replicate runs; datapoints for each compound concentration were averaged and plotted using 4-parameters curve fitting. R2 is a statistical estimate of goodness-of-fit.

Molecular profiling of UNC1215

Target Category	Target Activity
Within methyl-lysine reader target family	Target	IC₅₀ (µM)^a		K_d (µM)^b
	L3MBTL3	0.04		0.12
	L3MBTL1	2		9.4
	53BP1	4		> 31
	PHF20 Tudor	NT		5.6
	SPF30 (S.p.) Tudor	NT		Weak binding
	PHF20L1 Tudor	NT		> 13
Histone methyltransferases	< 50% inhibition at 250 mM versus 10 targets^c
Bromodomains	T_m shift < 0.5 °C at 10 mM versus 12 targets^d
Histone demethylases	T_m shift < 0.1 °C at 50 mM versus 15 targets^d
NIMH Psychoactive Drug Screening Program Selectivity Panel	< 50% inhibition at 10 mM versus 35 targets > 50% inhibition at 10 mM versus 8 targets
	Target		K_i (µM)^e		IC₅₀ (µM)
	Alpha 2C		0.86		NT
	DAT		> 10		NT
	KOR		4.0		NT
	M1		0.097		3.6^f
	M2		0.072		30% at 30 µM^g
	M3		0.89		NT
	M4		0.40		NT
	M5		4.3		NT
Kinase Selectivity Panel	< 15% inhibition at 10 mM versus 49 kinases
	Target		% Inhibition at 10 mM
	FLT3		64%

^aAlphascreen assays results. ^bITC results. ^cRadioactive SAM methyl transfer assay results. ^dDifferential scanning fluorimetry results. ^eRadioligand binding assay results. ^fCa²⁺ mobilization assay results. ^gcAMP biosensor assay results.

in vitro potency

cell based assay data

UNC1215 potently antagonizes 3MBT localization in cells. (a) Recovery time of a photobleached area in GFP-3MBT expressing cells is reduced upon treatment with UNC1215 in a dose response manner, whereas inactive compound UNC1079 shows no effect. Inset shows time lapse images of photobleached nuclei. (b, c) GFP fusions of 3MBT and FLMBT localize to the nucleus and form distinct foci in HEK293 cells. UNC1215 inhibits the foci formation of GFP-3MBT in a dose response fashion, whereas UNC1079 has no effect on the foci. In contrast, UNC1215 is relatively ineffective at inhibiting foci formation of GFP-FLMBT (scale bar, 10 µm). (d) The GFP-3MBT D274A MBT1 mutant shows a reduction in foci formation, while the GFP-3MBT D381A MBT2 mutant does not form nuclear foci.

references

Discovery of a chemical probe for a methyl-lysine reader domain: L3MBTL3

James, Lindsey I.; Barsyte-Lovejoy, Dalia; Zhong, Nan; Krichevsky, Liubov; Korboukh, Victoria K.; Herold, J. Martin; MacNevin, Christopher J.; Norris, Jacqueline L.; Sagum, Cari A.; Tempel, Wolfram; Marcon, Edyta; Guo, Hongbo; Gao, Cen; Huang, Xi-Ping; Duan, Shili; Emili, Andrew; Greenblatt, Jack F.; Kireev, Dmitri B.; Jin, Jian; Janzen, William P.; Brown, Peter J.; Bedford, Mark T.; *Arrowsmith, Cheryl H. *Frye, Stephen V. Nature Chemical Biology 9, 184–191 (2013) - DOI 10.1038/nchembio.1157

pk properties

co-crystal structures

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Main features

L3MBTL3 Dimer: UNC1215 binds to L3MBTL3 with a 2:2 stoichiometry. Each "arm" of UNC1215 binds to a different molecule of L3MBTL3.
Pocket Key interactions of pyrrolidine A with YYF aromatic cage and pyrrolidine B with YFW aromatic cage. Both interactions are augmented by a hydrogen bond with an Asp.

synthetic schemes

materials and methods