Structure-Based Design of Ester Compounds to Inhibit MLL Complex Catalytic Activity by Targeting Mixed Lineage Leukemia 1 (MLL1)-WDR5 Interaction

Dong-Dong Li, Zhi-Hui Wang, Wei-Lin Chen, Yi-Yue Xie, Qi-Dong You, Xiao-Ke Guo

Abstract

WDR5 is an essential protein for enzymatic activity of MLL1. Targeting the protein-protein interaction (PPI) between MLL1 and WDR5 represents a new potential therapeutic strategy for MLL leukemia. Based on the structure of reported inhibitor WDR5-0103, a class of ester compounds were designed and synthetized to disturb MLL1-WDR5 PPI. These inhibitors efficiently inhibited the histone methyltransferase activity in vitro. Especially, WL-15 was one of the most potent inhibitors, blocking the interaction of MLL1-WDR5 with IC50 value of 26.4nM in competitive binding assay and inhibiting the catalytic activity of MLL1 complex with IC50 value of 5.4μM. Docking model indicated that ester compounds suitably occupied the central cavity of WDR5 protein and recapitulated the interactions of WDR5-0103 and the hydrophobic groups and key amino greatly increased the activity in blocking MLL1-WDR5 PPI.

Keywords: Docking; Ester compounds; Histone methyltransferase; Leukemia; MLL1–WDR5 interaction.

1. Introduction

Histone lysine methyltransferases (HMTs) play a critical role in the regulation of gene expression, cell cycle, genome stability, and nuclear architecture [1]. Dysregulation or mutation of HMTs involved in the development of a wide range of human diseases, including cancer [2-3].
MLL1 is one member of six known SET1 family that catalyzes H3K4 mono-, di-, and trimethylation through its evolutionarily conserved SET domain [4]. In normal hematopoiesis, MLL1 regulates the expression level of Hoxa9 and Meis-1 genes, which are important for self-renewal of hematopoietic stem cells [5-7]. Dysregulation of MLL1 is associated with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) [8]. MLL fusion proteins (MLL-FPs) were observed in leukemia resulted from MLL allele translocating, and MLL1-N terminal fusing in frame with one of more than 70 partners [9]. Lacking C-terminal SET domain, MLL-FPs cooperated with wild-type MLL1 complex to activate MLL1 targeted Hox and Meis-1 genes, leading to leukemogenesis [10-11].
H3K4me2/3 markers were necessary for MLL1 and MLL-FPs to be recruited stably to the targeted Hox gene in leukemogenesis [12]. MLL1 alone can partially catalyze monomethylation of H3K4, but the enzymatic activity is weak [13]. In complex with WDR5, RbBP5 and Ash2L, MLL1 greatly increased the HMTs activity and the complex is necessary for dimethylation of H3K4 [14]. As a bridge between MLL1 and the reminder of the complex, WDR5 is required to maintain the integrity and the catalytic activity of MLL complex [15]. Thus, targeting MLL1WDR5 PPI to inhibit MLL1 H3K4 HMT activity represents a new strategy for the treatment of leukemia carrying MLL-FPs [10].
Recently, series of small molecule inhibitors and peptidomimetics were identified to disturb MLL1-WDR5 PPI [16-22]. Three inhibitors were disclosed through screening compounds libraries using fluorescence polarization assay (FP assay) [16]. Based on the co-crystal structure of inhibitor-WDR5 protein, a more potent antagonist WDR5-47 (Figure 1a) was acquired from optimization of WDR5-0102 [17]. Then an aromatic ring was introduced at the 5-position of N-(2-(4methylpiperazin-1-yl) phenyl) benzamide to defined compound W-23 [18]. Further modification of this structure, OICR-9429 was obtained to explore the mechanism of p30-dependent transformation in CEBPA-mutant AML [19-20]. With high affinity binding to WDR5, the minimal motif of MLL1 protein, 3-mer peptide Ac-ARA-NH2, was determined based upon MLL1 sequences [21]. Then linear peptidomimetic MM-102 and cyclic peptidomimetic MM-401 were acquired by modification of AcARA-NH2 [10, 22] (Figure 1b).
Here, we reported a series of ester compounds optimized from WDR5-0103 based on the co-crystal structure of WDR5-0103 and WDR5 protein (Figure 2). These compounds designed to block MLL1-WDR5 PPI effectively inhibited the histone methyltransferase activity in vitro. Especially, WL-15 (IC50 = 26.4 nM) was one of the most potent inhibitors. Docking study was applied to elucidate the binding model of ester compounds, which may stimulate more potent inhibitors in future.

2. Results and discussion

Ester derivatives (WL-1~5 and WL-8~20) were synthetized following the synthetic route depicted in Scheme 1. 4-Fluoro-3nitrobenzoyl chloride reacted with different alcohols giving ester intermediates 3a~d. Compounds 4a~d were formed by substituting the fluoro of 3a~d with N-methyl piperazine. Reducing 4a~d to provide amines 5a~d. 5a~d were treated with substituted acyl chlorides to afford target compounds. Nitro compounds WL-1, 4, 8, 11, 14, 16 and 19 were reduced by SnCl2.2H2O to access compounds WL-2, 5, 9, 12, 15, 17 and 20, respectively. In Scheme 2, 3-benzamido-4-(4-methylpiperazin-1yl) benzoic acids WL-21 and WL-22 were generated by hydrolysing methyl ester of compounds WL-1 and WL-3, respectively. Then benzoyl chlorides reacted with ethyl alcohol to provide ethyl ester compounds WL-6 and WL-7. All of the target compounds were verified by 1H NMR, HRMS, which in accordance with their depicted structures.

2.2. Competitive Binding Assay.

All synthetized compounds were determined the inhibition of MLL1-WDR5 PPI with FP assay, and the reported compound MM-102 was selected as positive control. As illustrated in Table1, compound WL-3, the combination of WDR5-0103 and WDR5-47, kept the activity in blocking MLL1-WDR5 PPI. Substituting the methyl of WL-3 with various ester groups (WL7, 10, 13) were tolerated except the cyclohexyl ester (WL-18).
Introduction of larger hydrophobic substitutes seemed to be more potent than a small one (WL-13, 10, 7, 3), while removing of ester groups (WL-21, 22) leading to complete loss in activity. It may because that the hydrophobic ester groups occupy the hydrophobic cleft surrounded by Phe149, Pro173and Tyr191, but the polar group such as carboxyl was not tolerated within the pocket (Figure 4). Phenyl substitute may form an additional π-π stacking interaction with the aromatic ring of residues such as Tyr191 of WDR5 protein, which resulted in higher activity of compounds with a phenol ester (WL-13 versus WL-3, 7 and 10, WL-15 versus WL-5).
To explore whether electron withdrawing groups strengthened the amide-to-Ser91 hydrogen bond interaction [17], another electron withdrawing group-nitro was introduced at the oposition of F of the benzamide moiety. The effects of nitro were various in different esters. The nitro led to a slight increase of activity in compounds bearing a methyl (WL-4 versus WL-3), while resulting in 4-fold loss of potency in phenol ester (WL-14 versus WL-13). Introduction of nitro decreased the electron density of benzamide to strengthen the amide-to-Ser91 hydrogen bond interaction, but the hydrophobic effect may account for the loss of activity. Such a query required further investigation.
Removing methyl and chloro group form corresponding compounds, WL-1 and 11 with 4-fluoro-3-nitro group kept potency, but compounds WL-2 and 12 only with amino showed loss in activity. That proved the criticality of hydrophobic groups in benzamide moiety.
Great gain in potency was achieved when reducing the nitro compounds (WL-4, 14 and 19) to amino compounds (WL-5, 15 and 20). The key amino of compounds WL-5, 15 and 20 played an important role in increasing activity versus parent compounds WL-3, 13 and 18, respectively. Asp107 of WDR5 was vital in driving binding MLL1 to WDR5 through interacting with Arg3765 of MLL peptide [23]. Compounds with an amino may form strong hydrogen bonds interaction with the residue of Asp107 of WDR5 protein, which led to the great gain of potency (Figure 4b and 4d). Overall, combining the modification with a phenol ester and 5-amino-2-chloro-4-fluoro-3-methyl benzamide, WL-15 effectively blocked MLL1-WDR5 interaction (IC50 = 26.4 nM) in FP assay (Figure 3).

2.3. Molecular docking

To better understand the binding model of these compounds, we docked WL-5 and WL-15 into WDR5 protein by GOLD 5.1. The binding model of compounds WL-5, 15 and WDR5 protein was depicted in Figure 4. The best fitness scores of compounds WL-5 and WL-15 were 83.8 and 89.2, respectively. From the docking model, WL-5 and 15 recapitulated interactions of WDR5-0103 with WDR5 protein, including direct and water mediated hydrogen bonds interaction with Ser91 and Cys261 and π-π stacking with Phe133. But the 2-chloro-4-fluoro-3-methyl benzamide moiety of both compounds occupied the hydrophobic groove surrounded by side-chains of Ala 65, Ala47 and Ile90, and the additional amino formed a direct and a water mediated hydrogen bonds with Asp107, which may account for the great increase in potency. What’s more, in the binding model of WL-5 and WL-15, the hydrophobic cleft surround by Phe149, Pro173and Tyr191 was occupied by methyl and phenyl. Interestingly, the phenyl of WL-15 form an additional π-π stacking with Tyr191 (Figure 4d), which may lead to an appreciable potency gain of WL-15. The molecular docking results, along with the competitive binding assay data, suggested that compounds WL-5 and WL-15 were potential inhibitors in disturbing MLL1-WDR5 protein-protein interaction.

2.4. Inhibition of MLL complex methyltransferase activity in vitro

WDR5 was essential for the integrity of MLL core complex and the catalytic activity of methyltransferase [15]. Disturbing the interaction of MLL1-WDR5 with small molecule should inhibit the histone methyltransferase activity. To explore the inhibition of ester compounds for MLL complex methyltransferase activity, the most two potent compounds (WL5 and 15, Figure 3) were determined the IC50 value in a recombinant MLL complex Alpha Screen assays in vitro. MM102 was selected as the positive control. As shown in Figure 5, compounds WL-5 and WL-15 designed to block MLL1-WDR5 PPI efficiently inhibited the catalytic activity of the MLL1 complex (IC50 = 4.6 and 5.4 µM, respectively).

2.5. Anti-proliferative activity

Active compounds in FP assay were selected to test their our delight, compounds WL-1 and WL-4 with moderate activity ability in inhibiting proliferation of two leukemia cell lines with in FP assay, effectively inhibited leukemia cells growth in MV4-or without MLL1 fusion protein. 11 (IC50 = 4.7, 3.4 µM, respectively).

3. Conclusion

Based on the co-crystal structure of WDR5-0103 and WDR5 protein, a series of ester compounds were designed and synthetized to disturb the interaction of MLL1-WDR5. With ester groups occupied the hydrophobic groove surrounded by Phe149, Pro173 and Tyr191, compounds WL-3, 7, 10, and 13 kept the affinity to WDR5 and the anti-proliferation activity of MV4-11 cells. More potent inhibitors were acquired by introducing a nitro or an amino into benzamide moiety. The binding models of WL-5 and 15 to WDR5 protein were elucidated by docking study. WL-5 and 15 recapitulated interactions of WDR5-0103 with WDR5 protein, but the additional amino formed hydrogen bonds interaction with Asp107, which increased activity greatly. Further, disturbing MLL1-WDR5 PPI with nanomolar activity (IC50 = 46.5 and 26.4 nM, respectively) in FP assay, WL-5 and 15 efficiently inhibited MLL complex HMT activity in vitro (IC50 = 4.6 and 5.4 µM, respectively). Introducing a nitro at the benzamide moiety, WL1, 4, and 14 kept the affinity to WDR5, but effectively and selectively inhibited the growth of MV4-11 cells harboring MLLAF4 fusion protein. However, the anti-proliferation activity of amino compounds were not consistent with the high affinity, which reminded that the physicochemical properties of ester compounds would be promoted in future study.

4. Experiments

4.2 Competitive Binding assay.

The binding affinities of all synthesized compounds were tested using a fluorescence polarization (FP) based competitive binding assay. 10 Peptidomimetic of Win motif (ARTEVHLRKS) for WDR5 was synthesized, C-terminallabeled with 5-carboxy fluorescein (5-FAM) tagged tracers linked through the side chain of a lysine residue next to the two 6-amino hexanoic acid with the serine [13]. Competitive binding assays were performed in a 60 µL volume at a constant labeled peptide concentration of 3 nM and WDR5 concentration of 8 nM in 100 mM potassium phosphate (pH 6.5), 25 mM KCl and 0.01% Triton X-100. Fluorescence polarization assays were performed in 384-well Corning plates using a SpectraMax paradigm reader (Molecular Device). An excitation wavelength of 485 nm and an emission wavelength of 535 nm were used. IC50 values were calculated by nonlinear regression analysis using Graphpad software.

4.3 Molecular docking

Docking study was carried out using GOLD5.1. The protein structure of WDR5 was downloaded from PDB (3UR4) and was edited by adding hydrogen, deleting unnecessary waters and ligands. Then the binding sites were defined according the endogenous ligand WDR5-0103. Gold score was chosen as the score function of binding interaction energy for ranking. Compounds WL-5 and WL-15 were prepared by DS3.0 with CHARMm. The high fitness score model was selected to analyze binding model.

4.4 MLL complex Alpha Screen assays

MLL1 methyltransferase inhibition assays were performed by the HUAWEI PHARMA, Shanghai, China, using the Histone methyltransferase Assays platform (www.huajianpharma.com). The MLL1 enzymatic reactions were conducted in duplicate at room temperature for 60 minutes in a 50 µL mixture containing proper methyltransferase assay buffer (50 mM Tris, pH 8.8, 5 mM MgCl2, 4M DTT), S-adenosylmethionine (1µM), recombinant enzyme (MLL1, WDR5, Ash2L and RbBP5, 150 ng), and the test compounds in wells of a Histone substrate precoated plate. Compounds and control compound MM-102 were dissolved in DMSO and tested in 10-dose IC50 mode with 3-fold serial dilution starting at 100 µM. After enzymatic reactions, the reaction mixtures were discarded and each of the wells was washed three times with TBST buffer, and slowly shaken with Blocking Buffer for 10 minutes. Wells were emptied, and 100 µL of diluted primary antibody was added. The plate was then slowly shaken for 60 minutes at room temperature. As before, the plate was emptied and washed three times, and shaken with Blocking Buffer for 10 minutes at room temperature. After discarding the Blocking Buffer, 100 µL of diluted secondary antibody was added. The plate was then slowly shaken for 30 minutes at room temperature. As before, the plate was emptied and washed three times, and shaken with Blocking Buffer for 10 minutes at room temperature. Blocking Buffer was discarded and a mixture of the HRP chemiluminescent substrates was freshly prepared. 100 µL of this mixture was added to each empty well. Immediately, the luminescence of the samples was measured in a BioTek SynergyTM 2 microplate reader. Data were normalized to the no enzyme control and the IC50 values were calculated using nonlinear regression with normalized dose−response fit using Prism GraphPad software.

4.5 Anti-proliferative activity

MV4-11and K562 cells were bought from Cell Bank of Chinese Academy of Sciences. Cell viabilities of the synthetized compounds were evaluated using CCK8 (WST-8, 2-(2-methoxy 4-nitro phenyl)-3-(4-nitro phenyl)-5- (2, 4-disulfonic acid benzene)-2H-tetrazolium monosodium salt)-based colorimetric assay. Cell lines were cultured to log phase in IMDM supplemented with 10% fetal bovine serum, under a humidified atmosphere of 5% CO2 at 37 oC. Cells were seeded in 96- well white opaque cell culture plates at a density of 5000 cells/ well in 100 µL of culture medium. The plates were returned to the incubator for 24 h to allow the cells to reattach. Subsequently, cells were treated with the target compounds diluted in 10 µL IMDM at increasing concentrations for 72 h. Then, cell viability was assessed by the conventional (CCK8) reduction assay and the absorption was measured at 450 nm using Thermo Multiskan Spectrum. IC50 was taken as the concentration that caused 50 % inhibition of cell viabilities and calculated by nonlinear regression analysis using Graphpad software.

4.6 Permeability determination

Permeability (Pe) was determined by a standard parallel DSP5336 artificial membrane permeability assay (PAMPA by pION). PAMPA was conducted on a PAMPA Explorer instrument (pION Inc., Woburn, MA) with PAMPA Explorer command software (Version 3.7.4.1) as follows: test compound stock was dissolved in DMSO at 10 mM concentration. Then 5µL DMSO diluted with compounds was added to 1 mL Prisma HT buffer (pH 7.4) in deep well plate to make sample solution. Then 150 µL of diluted test compound was transferred to a UV plate (pION Inc., Woburn, MA), and the UV spectrum was read as the reference plate. The membrane on a preloaded PAMPA sandwich (pION Inc., Woburn, MA) was painted with 5 µL of GIT lipid (pION Inc., Woburn, MA). The acceptor chamber was then filled with 200 µL of acceptor solution buffer (pION Inc., Woburn, MA), and the donor chamber was filled with 200 µL of diluted test compound. The PAMPA sandwich was assembled at 25 °C for 4 hours. The UV spectrum (240−500 nm) of the donor and the acceptor were read. The permeability coefficient was calculated using PAMPA Explorer command software (Version 3.7.4.1) based on the AUC of the reference plate, the donor plate, and the acceptor plate. All compounds were tested in quadruplicate, and the data were presented as an average value. Standards for this assay included Ketoprofen (2.2 × 10−6 cm/s) and propranolol (98.7 × 10−6 cm/s).

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