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 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

Probe		Negative control

SGC-SMARCA-BRDVIII		SGC-BRDVIII-NC

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(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 (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	11.3± 0.3	SP110A	-0.1± 0.1
PB1	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).

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

Kadoch, C., Crabtree, GR. Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics. Sci. Adv., 2015, 1, 5, e1500447.
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.
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.
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.
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.
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