This assay allows us not only to quantitatively measure the dissociation constants of several JHDM probes, but also to validate the inhibitory mechanism of methylstat

This assay allows us not only to quantitatively measure the dissociation constants of several JHDM probes, but also to validate the inhibitory mechanism of methylstat. RESULTS AND DISCUSSION The synthesis of fluorophore 3 began with conversion of commercially available 4-cyanobenzaldehyde to aldehyde 4 (Plan 2),11 which then underwent a reductive amination with amine 57 to afford secondary amine 6. methyltransferases and histone demethylases. Since 2004, two families of enzymes have been reported to exhibit demethylation activities: FAD-dependent monoamine oxidases (LSD1 and 2) and jumonji C domain-containing histone demethylases (JHDMs).2 Compared with LSDs, JHDMs have a much broader substrate scope and can modify lysine residues at all methylation states. Expression of JHDMs plays crucial functions in both development and diseases, such as malignancy and mental retardation.3 Overproduction of 2-hydroxy-glutarate, a natural JHDM inhibitor, due to mutation of isocitrate dehydrogenases has been identified in multiple cancers.4 This development has led to numerous efforts to develop ZM-241385 chemical probes targeting JHDMs. Several classes of -ketoglutarate (KG) mimics have been developed to inhibit JHDM activity,5 since all JHDMs use KG as a cofactor. In addition, a substrate-mimicking small molecule was recently reported to selectively inhibit H3K9-demethylase KIAA1718. 6 Our group recently discovered a selective, cell-permeable, small-molecule inhibitor methylstat (1, Plan 1), which was designed as a bivalent substrate-cofactor conjugate.7 Its corresponding acid, 2 (Plan 1), selectively inhibits JHDMs in vitro. This bivalent strategy has also confirmed successful in two very recent reports on JMJD2 class-selective peptidic inhibitors.8 Open in a separate window Scheme 1 Structures of methylstat (1), methylstat acid (2), and its fluorescent analogue methylstatfluor (3) Although several classes of JHDM inhibitors have been discovered, determining the selectivity of these inhibitors against various JHDM classes remains a major challenge. This is mainly due to the lack of a uniformed biochemical assay for numerous JHDM isoforms. Most established JHDM biochemical assays are enzyme inhibition assays.5a,9 Due to the self-destructive nature of JHDMs under biochemical reaction conditions,10 these assays typically require optimization for different JHDM isoforms. In addition, they do not allow for accurate measurement of the dissociation constants of the JHDM probes. Thus, the IC50 values derived from these assays cannot be compared directly. Here, we statement the synthesis of a fluorescent JHDM probe, 3 (Plan 1), and the development of a fluorescence polarization (FP)-based binding assay. This assay allows us not only to quantitatively measure the dissociation constants of several JHDM probes, but also to validate the inhibitory mechanism of methylstat. RESULTS AND DISCUSSION The synthesis of fluorophore 3 began with conversion of commercially available 4-cyanobenzaldehyde to aldehyde 4 (Plan 2),11 which then underwent a reductive amination with amine 57 to afford secondary amine 6. The 120 ZM-241385 mP, Physique 1A); however, the 300 mP, Physique 1A). The factor value (0.78) was calculated,17 which further demonstrates the robustness of this assay and its suitability for high-throughput screening. CONCLUSION We have discovered a fluorescent analogue of methyl-stat, 3, and have used it to develop an FP binding assay. 3 selectively binds JHDM1A with high affinity (300 mP). Ni2+ ion was found not only to be a good surrogate to the native cofactor Fe2+, but it also stabilizes the ZM-241385 protein. The binding of 3 to JHDM1A can be displaced by several known JHDM probes, including its cofactor (KG), substrate (H3K36me2), and methylstat acid (2). These results confirm that methylstat acid is usually a bivalent competitive inhibitor of JHDMs. In addition, this FP competition Rabbit Polyclonal to NCAPG2 assay allows quantitative measurement of Ki values of non-fluorescent JHDM1A active site binding molecules. It is also noteworthy that this Ki of KG and JHDM1A was decided, which has previously been impossible and illustrates the power of our FP binding assay for quantifying the binding affinities of native JHDM substrates quickly and ZM-241385 easily. Furthermore, we were able to use our FP system to develop a highly strong and miniaturized assay appropriate for high-throughput screening of large compound libraries (Z: 0.78). Further optimization of the fluorophore for the development of FP assays appropriate for other JHDMs is usually ongoing and will be reported in due course. EXPERIMENTAL SECTION Synthetic procedure for the preparation of fluorophore 3, characterization data, NMR spectra of all new compounds are in supporting information. Protein expression and purification Recombinant JHDM1A (1-517) and JMJD3 (1018-1590) were expressed as 6XHis fusion proteins using the pNIC28 and the pNH-TrxT expression vectors, respectively. The coding regions were verified by sequencing and the plasmids were transfected into BL21 E. coli. Following expression, JHDM1A was purified using Ni SepharoseTM 6 Fast Circulation beads (GE) by gravity chromatography according to the manufacturers instructions. JMJD3 was purified using cobalt (high density) agarose beads (Platinum Biotechnology) according to the manufacturers protocol. JMJD2A was expressed and purified as explained previously.7 The purified proteins were exchanged into assay buffer, flash frozen in liquid nitrogen and stored at ?80 C. FP binding Assay FP binding experiments were performed in black, low-binding, half-area 96-well plates (Corning 3993). 80 L.