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A public-private partnership that supports the discovery of new medicines through open access research.
PFI-6 A novel chemical probe for MLLT1/3The probe and control are available from Sigma. For any inquiries please contact proberequests@thesgc.org.group newOverview 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 ProfilePFI-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 HTRF IC50 (µM) 0.14 0.16 >40 >40 BLI Kd (µM) 0.11 0.11 n.d. n.d. ITCKD (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 DataIn 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) . Figure 1: A NanoBRET assay was used to determine MLLT3 target engagement in cells. Figure 2: Confirmation of MLLT1 target engagement in cells by Fluorescence Recovery After Photobleaching analysis. Co-crystal structuresThe figure below shows PFI-6 is soaked into the YEATS domain. PDB codes will be added when they become available.