Cellular target Engagement

In the NanoBRET cellular target engagement assay, NVS-MLLT-1 displayed potent inhibition, with an average IC50 value of 0.5 μM (±0.12) (Figure 1). In comparison, NVS-MLLT-C 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 NVS-MLLT-1  (Figure 2) . 

Figure 1: A NanoBRET assay was used in HEK293 cells to determine target engagement in cells. All probes were tested for 24h in the presence of 2.5 μM SAHA (24h). Graph represents Mean±SEM, n=3 independent replicates, with n=4 technical replicates.  mBU: BRET units

Figure 2: Further confirmation of MLLT1 target engagement in cells.  Fluorescence Recovery After Photobleaching analysis shows NVS-MLLT-1 decreases the half-life recovery time of cells. n=3 independent experiments, Mean±SEM, fold change from SAHA treatment alone. One way ANOVA, Dunnett's multiple comparisons.

Materials and Methods

NanoLuciferase Bioluminescent Resonance Energy Transfer (NanoBRET) Assay
Cellular activity against MLLT3 was assessed using a NanoBRET assay. HEK293 cell (8 x 10⁵) were plated a 6-well plate, after 6h cells were co-transfected with C-terminal HaloTag-Histone 3.3 (NM_002107) and an N-terminal NanoLuciferase fusion of MLLT3 (original MLLT3 WT sequences from Promega HaloTag® human ORF in pFN21A or MLLT3 MUT - Y78A Tyrosine is changed to an Alanine) at a 1:10 (NanoLuc® to HaloTag®) ratio respectively with FuGENE HD transfection reagent. Sixteen hours post-transfection, cells were collected, washed with PBS, and exchanged into media containing phenol red-free DMEM and 4% FBS in the absence (control sample) or the presence (experimental sample) of 100 nM NanoBRET 618 fluorescent ligand (Promega). Cells were then re-plated in a 96-well assay white plate (Corning Costar #3917) at 2x10⁴ cells per well. Compounds were then added directly to the cells (in the presence of SAHA 2.5 µM) at final concentrations 0-30 μM or an equivalent amount of DMSO as a vehicle control, and the plates were incubated for 24 h at 37°C in the presence of 5% CO2. NanoBRET Nano-Glo substrate (Promega) was added to both control and experimental samples at a final concentration of 10 µM. Readings were performed within 10 minutes using a ClarioSTAR (BMG Labtech) equipped with 460 nm and 610 nm filters. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/460 nm for experimental samples minus the emission at 610 nm/460 nm for control samples (without NanoBRET fluorescent ligand). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000.

Peptide Displacement Assay
Peptide displacement assays were set up with biotinylated peptides (chosen based on ChIPseq data from the literature and purchased from LifeTein) and 6His tagged protein (Table 2).  For detection, two orthogonal technologies were used, i. AlphaScreen® technology from Perkin Elmer and ii. HTRF from Cisbio.  Compounds were dispensed in duplicate at single concentration (100 µM) for the initial screen and as 11-point dose response curves starting from 200 µM for IC₅₀ value determination.

Table 2: Peptides used for the peptide displacement assays

To determine optimal assay conditions for each protein prep, proteins and peptides were titrated against each other in a 16 by 16 matrix in 1:1 dilutions, starting from 3.2 µM. For the final ratio of protein and peptide to use in the assay, the point representing the EC₉₀ in the two-dimensional titration was chosen.  Typically, final assay concentrations for protein and peptide fell between 25 and 200 nM.  For AlphaScreen®, AlphaScreen Histidine (Nickel Chelate) Detection Kit donor and acceptor beads were used at a 1:2500 dilution from purchased stock; for HTRF, SA-XL665 and anti-6His antibody were used at 1:2000 and 1:10000 dilution from purchased stock, respectively.  Assays were performed on 384 well ProxiPlates (Perkin Elmer) at a final volume of 20 µl and plates were read using a Pherastar FSX plate reader (BMG Labtech).

Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) was carried out using a TA NanoITC (standard volume) instrument.  Protein was prepared by dialysis (overnight at 4°C) against a ~1000 times excess of buffer (20 mM Tris at pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, 2 mM DTT) using SnakeSkin® Dialysis Tubing with a 7 kDa MWCO and then concentrated to 300 µM.  The experiment was carried out at 20°C in reverse mode with the compound in the cell at 50 µM and the protein in the syringe at 300 µM due to the solubility of the compound with the first injection at 4 µl and the following 30 at 8 µl. Data was analysed using the NanoAnalyze software package by TA Instruments.

Differential Scanning Fluorimetry
Differential scanning fluorimetry (DSF) to determine the effect of compounds on the thermal stability of proteins (DT_m) was carried out on 384 well PCR plates using a LightCycler 480 (Roche).  Protein at 10 µM was buffered in 10 mM HEPES at pH 7.5 and 500 mM NaCl.  The experiment was carried out from 25 to 95°C with three acquisitions per degree.  Compounds were added at 50 µM final concentration and DMSO reference and no-addition controls were also collected.