PFI-6 A novel chemical probe for MLLT1/3

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Overview


PFI-6	PFI-6N (Negative control)

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

Physical and chemical properties for PFI-6
Molecular weight	391.43
Molecular formula	C₂₂H₂₁N₃O₄
IUPAC name	(R0-N-(2,3-dihydro-1H-inden-1-yl)-5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide
MollogP	2.6
PSA	91.23
No. of chiral centres	0
No. of rotatable bonds	6
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	2
Storage	store as a dry poweder or as DMSO stock solutions (10mM) at -20 °C
Dissolution	Soluble in DMSO up to 10mM

Physical and chemical properties for PFI-6N
Molecular weight	357.41
Molecular formula	C₁₉H₂₃N₃O₄
IUPAC name	N-cyclopentyl-5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-methylisoxazole-3-carboxamide
MollogP	1.86
PSA	82.44
No. of chiral centres	0
No. of rotatable bonds	6
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	1
Storage	Store as a dry powder or as DMSO stock solutions (10mM) at -20 °C
Dissolution	Soluble in DMSO up to 10mM

SMILES:

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

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.

	MLLT1	MLLT3	YEATS2	YEATS4
	MLLT1	MLLT3	YEATS2	YEATS4
HTRF IC50 (µM)	0.14	0.16	>40	>40
BLI K_d (µM)	0.11	0.11	n.d.	n.d.
ITC_KD(µM)	0.082	0.076	n.d.	n.d.
Tm Shift (°C)	3.61	5.13	0.00	-0.02

Table 1: Potency Against Target Family

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

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) .