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The structures of the MLLT1 YEATS domain with two inhibitors generated were solved and deposited into the PDB (Table 3). SGC-iMLLT was shown to occupy the acyllysine binding site previously identified in the literature, making a number of contacts.
PDB ID |
Structure Details |
Crystal structure of MLLT1 (ENL) YEATS domain in complexed with compound 94 |
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Crystal structure of MLLT1 (ENL) YEATS domain in complexed with SGC-iMLLT (compound 92) |
Table 3: Structures solved
Figure 4: Structure of SGC-iMLLT bound to the YEATS domain of MLLT1 (6HT1) with compound contacting residues highlighted in blue. (A) Complete view, N- and C- termini marked. (B) Detail view of binding pocket. Blue arrows: hydrogen bonds between SGC-iMLLT and side-chains (E85, S58) and backbone (Y78); Red arrows: p -p stacking of SGC-iMLLT and side chains (F28, F59, Y78, H56); Black arrow denotes “flipped” conformations ofY78.
Proteins Purified
All four human members of the YEATS domain (Table 4) were recombinantly expressed in E. coli BL21(DE3) cells upon induction with 0.4 mM IPTG and purified using standard protocols for centrifugal harvest. Cells were lysed via sonication and the lysate clarified via centrifugation. Immobilised metal affinity chromatography (IMAC) was was performed with GE HisTrapFF columns (5 ml) to isolate target proteins from lysate, followed by size exclusion chromatography with GE HiLoad 16/60 Superdex 75pg columns. For buffer, 20-50 mM Tris at pH 7.5, 500 mM NaCl and 2 mM DTT were used, supplemented for IMAC with 20 mM imidazole for lysis and wash, and 300 mM imidazole for elution.
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Protein |
UniProtKB |
Boundaries |
6His tag |
|
Start |
Stop |
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MLLT1 |
Q03111 |
M1 |
M148 |
C-terminal |
MLLT3 |
P42568 |
M1 |
A138 |
C-terminal |
YEATS2 |
Q9ULM3 |
S202 |
E345 |
N-terminal |
YEATS4 |
O95619 |
V16 |
K225 |
C-terminal |
Table 4: Proteins purified for this TEP
Peptide displacement assay
Peptide displacement assays were set up with biotinylated peptides (chosen based on ChIPseq data from the literature and purchased from LifeTein, Table 5) and 6His tagged protein. 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.
YEATS domain |
Peptide shorthand |
Position |
Peptide sequence |
MLLT1 |
H3K18ac |
12-30 |
GGKAPR(K-acetyl)QLATKAARKSAPY(K-biotin) |
MLLT3 |
H3K9ac |
2-20 |
ARTKTAR(K-acetyl)STGGKAPRKQLY(K-biotin) |
YEATS2 |
H3K27cro |
15-32 |
biotin-GKPRKQLATAAR(K-crotonyl)SAPAT |
YEATS4 |
H3K27cro |
15-32 |
biotin-GKPRKQLATAAR(K-crotonyl)SAPAT |
Table 5: 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 (DTm) 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.
SAR by catalogue
Additional compounds were purchased to perform ‘SAR by catalogue’. The compounds were analysed in the peptide displacement assay and differential scanning fluorimetry as described above (Figure 3, Table 6).
Figure 5: Structures of compounds purchased for ‘SAR by catalogue’.
MLLT1 | MLLT3 | YEATS2 | YEATS4 | |||||
---|---|---|---|---|---|---|---|---|
Compound | IC50 [µM] | DTm [°C] | IC50 [µM] | DTm [°C] | IC50 [µM] | DTm [°C] | IC50 [µM] | DTm [°C] |
XS043798c | 8.8 | 1.0 | 27.6 | 1.8 | 134.0 | 0.2 | >200 | 0.0 |
XS043798d | 7.1 | 1.2 | >200 | 2.1 | >200 | 0.2 | >200 | 0.1 |
XS096172b | 162.9 | 0.2 | >200 | 0.4 | >200 | 0.2 | >200 | 0.0 |
XS098176b | 49.9 | 0.0 | >200 | 0.0 | >200 | 0.2 | >200 | 0.1 |
XS102315b | 13.8 | 0.3 | 52.2 | 0.6 | 83.3 | 0.2 | >200 | 0.1 |
XS102728c | 18.0 | 0.2 | 51.8 | 0.0 | 18.6 | 0.2 | >200 | 0.1 |
XS171208c | 5.6 | 0.0 | 47.1 | -0.1 | 6.7 | 0.2 | 44.0 | -0.4 |
YT000270a | 18.2 | 0.0 | 59.3 | 0.3 | 64.9 | 0.1 | >200 | 0.1 |
YT000272a | 43.8 | 0.0 | 70.1 | 0.1 | 33.9 | 0.1 | 52.0 | 0.1 |
YT000289a | 30.1 | 0.0 | >200 | 0.3 | >200 | 0.5 | 52.0 | 0.4 |
YT000290a | 16.1 | 0.0 | >200 | 0.6 | >200 | 0.5 | >200 | 1.4 |
YT000291a | 19.3 | 0.3 | >200 | 0.6 | >200 | 0.5 | >200 | 0.2 |
YT000294a | 44.3 | 0.2 | >200 | 1.0 | >200 | 1.3 | >200 | 1.1 |
YT000295a | >200 | 0.0 | >200 | 0.6 | >200 | 0.5 | 57.6 | 0.9 |
YT000309a | 9.7 | 0.3 | >200 | 0.6 | >200 | 0.8 | >200 | 1.1 |
YT000330a | 0.7 | -0.2 | 0.9 | 0.1 | 1.0 | 0.1 | 0.9 | -0.3 |
YT000333a | 26.4 | 0.0 | >200 | -0.1 | >200 | 0.1 | >200 | 0.1 |
Table 6: Results of peptide displacement assays and differential scanning fluorimetry assays with the compounds purchased for ‘SAR by catalogue’.
Chemistry
Substitutions in positions R1 and R2 around the central motif of the library screen hit (Figure 6) were performed and the resulting compounds tested in the peptide displacement assay (Table 7). Detailed information of chemical synthesis can be found in Moustakim, Christott et al., 2018 (11)
Figure 6: Synthesis of the central motif of the library screen hit. a ethyl 2-chloroacetate (1.2 eq), 4N HCl (0.6 M), 16 h, 100°C, 4-95%; b amine (1.2-1.5 eq), Na2CO3 (1.5 eq), 23°C, 3-82; c H2 Pd/C (10%), 17-88%; d sulfonyl/acid chloride (1.2 eq), PS-DIPEA (2 eq), 16 h; e acid (1.2 eq), HATU (1.5 eq), PS-DIPEA (2 eq), 16 h, 8-100%.
Table 7: Selected binding affinity characterisation data of benzimidazole series for MLLT1 YD
NanoLuciferase Bioluminescent Resonance Energy Transfer (NanoBRET) Assay
Cellular activity against MLLT3 was assessed using a NanoBRET assay. HEK293 cell (8 x 105) were plated in each well of 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 and 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 media (in the presence of SAHA 2.5 µM) at final concentrations 0-10 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.
Crystallography
Crystallization was performed using protein at ~0.45 mM concentration and sitting drop vapor diffusion method at 20C. Co-crystals with compounds grew in various conditions containing either PEG3350 or PEG Smear Medium (12), of which other compositions were reported in the publications (11, 13). Alternatively, the inhibitor-complexed crystals were used for preparing seeds, which was then used for crystallization of apo crystals. Soaking was performed using fragments or inhibitors at ~5-40 mM. All crystals were cryo-protected with mother liquor supplemented with 25% ethylene glycol prior to flash-cooled in liquid nitrogen.
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