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.

For any inquiries please contact proberequests@thesgc.org.

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Overview

Figure 1: Chemical structure of SGC-SMARCA-BRDVIII (Left) and the inactive SGC-BRDVIII-NC (Right).

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 K_D(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 K_D(ITC) of 200-400 nM can be inquired [3].

Potency Against Target Family

Bromodomain	SGC-SMARCA-BRDVIII K_D(nM)
SMARCA2	35
SMARCA4	36
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

SGC-SMARCA-BRDVIII
Physical and chemical properties
Molecular weight	380.45 g/mol
Molecular formula	C₁₉H₂₅N₅O₃
IUPAC name	tert-butyl 4-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]piperazine-1-carboxylate
logP	1.82
TPSA	104.8
No. of chiral centres	0
No. of rotatable bonds	5
No. of hydrogen bond acceptors	5
No. of hydrogen bond donors	2
Storage	Stable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolution	DMSO (up to 50 mM)

SGC-BRDVIII-NC
Physical and chemical properties
Molecular weight	385.21 g/mol
Molecular formula	C₂₀H₂₇N₅O₃
IUPAC name	tert-butyl-4-[3-amino-6-(2-methoxyphenyl)yridazine-4-yl]piperazine-1-carboxylate
clogP	2.02
TPSA	93.8
No. of chiral centres	0
No. of rotatable bonds	6
No. of hydrogen bond acceptors	5
No. of hydrogen bond donors	1
Storage	Stable as powder at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolution	DMSO (up to 50 mM)

SMILES:
SGC-SMARCA-BRDVIII: OC1=CC=CC=C1C2=CC(N3CCN(C(OC(C)(C)C)=O)CC3)=C(N)N=N2
SGC-BRDVIII-NC:NC(N=N1)=C(N2CCN(C(OC(C)(C)C)=O)CC2)C=C1C3=CC=CC=C3OC

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 (ΔT_m°C)^a,b
TAF1L(1)	-0.2± 0.1	ATAD2	0.4± 0.1
TAF1L(2)	-0.1± 0.1	EP300	0.0± 0.1
BRD2(1)	0.0± 0.2	BRD4(1)	0.7± 0.1
PB1(1)	0.9± 0.1	BRD3(2)	-0.1± 0.1
PB1(2)	2.5± 0.1	BRPF1A	0.2± 0.1
PB1(3)	2.7± 0.2	BRD1	0.2± 0.1
PB1(4)	0.7± 0.1	TRIM33B	0.1± 0.1
PB1(5)	11.3± 0.3	SP110A	-0.1± 0.1
PB1(6)	0.3± 0.1	PCAF	0.0± 0.1
SMARCA2	7.7± 0.2	WDR9	-0.4± 0.1
SMARCA4	7.4± 0.1	BRDT(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).

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.

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.