UNC0638 Selective chemical probe for G9a/GLP methyltransferases

Selectivity Within Target Family

(nt=not tested, nd=not detected, ¹ singlicate determination @ 100 µM)

**Screened at BPS Bioscience using different format

Blank box indicates Tm shift of <2 °C

Selectivity Beyond Target Family

Radioligand binding performed at Ricerca

Materials and Methods

Activity Assay

Histone methyltransferase assay was performed using a coupled assay originally developed by Collazo et al. 2005. In this assay SAHH (S-adenosylhomocysteine hydrolase) and adenosine deaminase convert the methyltransferase reaction product (S-adenosylhomocysteine) to homocysteine and inosine. Homocysteine can be quantified using Thioglo-1 (Calbiochem). Substrate peptides used in this assay were: the first 25 residues of histone 3 [H3 (1-25)] for G9a, EHMT1 and SETD7 at 10, 20 and 100 µM respectively; the first 24 residues of histone 4 [H4 (1-24)] at 10 and 500 µM for PRMT3 and SETD8 respectively; and H3K9Me1 [H3 (1-15), monomethylated at lysine 9] at 200 µM for SUV39H2. The assay mixtures were prepared in 25 mM potassium phosphate buffer pH 7.5, 1 mM EDTA, 2 mM MgCl₂, 0.01% Triton X-100 with 5 µM SAHH , 0.3 U/ml of adenosine deaminase from Sigma, 25 µM SAM, and 15 µM Thioglo-1. G9a (25 nM), EHMT1 (100 nM), SUV39H2 (100 nM), SETD7 (200 nM) and PRMT3 (1 µM) were assayed in the presence of UNC0638 at concentrations ranging from 4 nM to 16 µM. After 2 min incubation, reactions were initiated by the addition of above mentioned histone peptides. The methylation reactions were followed by monitoring the increase in fluorescence using BioTek Synergy2 plate reader with 360/40 nm excitation filter and 528/20 nm emission filter for 20 min in 384-well format. SETD8 (250 nM) was assayed under the same conditions; however the Thioglo-1 was added at the end of the reaction for quantification. The peptide and protein background were subtracted. IC50 values were calculated using Sigmaplot and the standard deviations were calculated from two independent experiments. SAHH clone was provided by Dr. Trievel, University of Michigan.

Differential Scanning Fluorimetry (DSF)

DOT1L, PRDM1, PRDM10, PRDM12, SMYD3, HIATIP, G9a, EHMT1 and SETD7 were screened for binding to UNC0638 by DSF. A real-time PCR device (RTPCR 480 II) from Roche was used to monitor protein unfolding by monitoring the increase in the fluorescence of the fluorophor SYPRO Orange (Invitrogen, Carlsbad, CA) as described before (Niesen et al. 2007; Vedadi et al. 2006). Protein samples ranging from 0.05 to 0.2 mg/mL in 100 mM Hepes buffer (pH 7.5) containing 150 mM NaCl, and 0, 1, 10 and 100 µM of the compound were screened. Compound dilutions were made from stock solutions of 100% DMSO. The final concentration of DMSO was kept at 0.2% throughout the dilutions. All these solutions contained 5x Sypro Orange. 20 µL aliquots were transferred to a 384-well PCR plate and scanned at a heating rate of 1 °C/min from 20 to 95 °C. Fluorescence intensities were plotted as a function of temperature by using an internally developed software package (Vedadi et al. 2006).

Differential Static Light Scattering (DSLS)

SETD8 and PRMT3 were screened for binding to UNC0638 by DSLS. Temperature-dependent aggregation was measured by using static light scattering (StarGazer) (Vedadi et al. 2006, Senisterra et al. 2006). Fifty microliters of protein (0.4 mg/ml) was heated from 27°C to 85°C at a rate of 1°C per min in each well of a clear-bottom 384-well plate (Nunc, Rochester, NY) in the presence of 0, 1, 10 and 100 µM of UNC0638. Incident light was shone on the protein drop from beneath at an angle of 30°. Protein aggregation was monitored by measuring the intensity of the scattered light every 30 s with a CCD camera. The pixel intensities in a preselected region of each well were integrated to generate a value representative of the total amount of scattered light in that region. These total intensities were then plotted against temperature for each sample well and fitted to the Boltzman equation by nonlinear regression. The resulting point of inflection of each resulting curve was defined as the Tagg.

Peptide Displacement

Fluorescence polarization (FP) measurements were performed in 384 well-plates, using Synergy 4 microplate reader from BioTek. H3 (1-15) peptide (ARTKQTARKSTGGKA) was synthesized, N-terminally labeled with fluorescein [F-H3 (1-15)] and purified by Tufts University Core Services (Boston, MA, USA). Displacement of F-H3 (1-15) peptide was monitored using the fluorescence polarization signal obtained upon peptide binding to G9a protein. G9a (4 µM in 20 mM Tris pH 8.0, 250 mM NaCl, 500 µM SAH, and 0.01% Triton) was incubated with 40 nM F-H3 (1-15) peptide, and different concentrations of BIX-01294 (purchased from Sigma-Aldrich), UNC0224, UNC0638 and unlabeled H3 (1-25) peptide from 0.1 to 100 µM were added. Displacement of peptide was monitored by following the decrease in FP signal. Data were normalized and plotted as percentage, and fit to a hyperbolic function using Sigma Plot software.