Discovery, Structure–Activity Relationship and In Vitro Anticancer Activity of Small-Molecule Inhibitors of the Protein–Protein Interactions between AF9/ENL and AF4 or DOT1L

Simple Summary Chromosomal translocations involving the mixed lineage leukemia (MLL) gene generate potent fusion oncogenes and cause acute myeloid leukemia or lymphocytic leukemia, which account for ~75% infant and 5–10% child/adult acute leukemia cases with a poor prognosis (5-year survival rates < 45%). Protein–protein interactions between the most frequent MLL fusion partner proteins AF9/ENL and AF4 or histone methyltransferase DOT1L are critical to malignant gene expression and are therefore a potential drug target for cancer. Compound screening followed by medicinal chemistry studies identified several novel small-molecule inhibitors showing strong inhibition of these protein–protein interactions, significant suppression of characteristic gene expression, and robust cellular anticancer activities with negligible cytotoxicity. These compounds are useful chemical probes for biological studies of these protein–protein interactions, as well as pharmacological leads for further drug development against MLL-rearranged and other leukemias. Abstract Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause 5–10% acute leukemias with poor clinical outcomes. Protein–protein interactions (PPI) between the most frequent MLL fusion partner proteins AF9/ENL and AF4 or histone methyltransferase DOT1L are drug targets for MLL-rearranged (MLL-r) leukemia. Several benzothiophene-carboxamide compounds were identified as novel inhibitors of these PPIs with IC50 values as low as 1.6 μM. Structure–activity relationship studies of 77 benzothiophene and related indole and benzofuran compounds show that a 4-piperidin-1-ylphenyl or 4-pyrrolidin-1-ylphenyl substituent is essential for the activity. The inhibitors suppressed expression of MLL target genes HoxA9, Meis1 and Myc, and selectively inhibited proliferation of MLL-r and other acute myeloid leukemia cells with EC50 values as low as 4.7 μM. These inhibitors are useful chemical probes for biological studies of AF9/ENL, as well as pharmacological leads for further drug development against MLL-r and other leukemias.

The C-terminal AHD domain (~70 residues) of AF9 and highly homologous ENL is a novel and validated drug target for MLL-r leukemia [11,26].Although it is disordered by itself, AF9/ENL AHD forms a structured protein complex with a consensus peptide segment of LxVxIxLxxV/L in AF4 or its paralog AFF4 with a high affinity [27,28].Such proteinprotein interaction (PPI) is essential for the oncoprotein MLL-AF9/-ENL or MLL-AF4 to recruit SEC for aberrant gene expression.Moreover, similar interactions between AF9/ENL AHD and histone-H3 lysine-79 (H3K79) methyltransferase DOT1L (which also contains several LxVxIxLxxV/L sequences) can recruit DOT1L to MLL target gene loci, causing genome-wide H3K79 hypermethylation, which has been observed and characteristic to MLL-r leukemia [29].Knockdown or pharmacological inhibition of DOT1L was found to selectively inhibit MLL-r leukemia in cells, animal models and clinical trials [30][31][32].Therefore, disruption of the PPIs between AF9/ENL and AF4/AFF4 or DOT1L is a potentially useful therapy for MLL-r leukemia by suppressing SEC-mediated gene expression and DOT1L-caused H3K79 methylation [26].
Two series of compounds have been reported to inhibit the AF9/ENL-DOT1L interactions, including our previously disclosed compound SYC-1456 [26] and a series of 7-mer peptidomimetic compounds derived from DOT1L, such as Cpd-10 [33,34] (Chart 1).These compounds can inhibit AF9-DOT1L and other related PPIs and selectively suppress aberrant gene expression and cell proliferation of MLL-r leukemia, showing on-target activities, as well as a need for additional inhibitor discovery and development.Here, we report the discovery, synthesis, structure-activity relationships (SAR) and biological activities of a new chemo-type of inhibitors of the PPIs between AF9/ENL and DOT1L or AF4.

Discovery of Novel Inhibitors of the AF9-DOT1L Interaction
An AlphaLisa assay we previously developed [26] was used for compound screening for inhibitors of the AF9-DOT1L interaction.The PPI takes the DOT1L-peptide coated donor beads and AF9 AHD coated acceptor beads together.The donor beads are illuminated with a laser beam (680 nm) to generate singlet oxygen radicals, which activate the adjacent acceptor beads to produce luminescence at 615 nm.When a compound can disrupt such PPI and separate the donor and acceptor beads, the highly unstable radicals are rapidly quenched by water without generating luminescence.With this method, indolecarboxamide compounds 1 and 2 were found to be novel inhibitors of the AF9-DOT1L interaction, which can inhibit the PPI dose-dependently with an IC 50 value of 3.3 and 4.5 µM, respectively (Table 1 and Supplementary Materials Figure S1), which showed comparable activities to SYC-1456 (IC 50 : 3.5 µM) [26].Moreover, another reported inhibitor, Cpd-10 (Chart 1), was synthesized and found to exhibit an IC 50 of 2.1 µM in our AlphaLisa assay conditions (Figure S1).These results support medicinal chemistry optimization based on the novel chemical scaffold in compounds 1 and 2.

Synthesis
Medicinal chemistry based on the structures of these two compounds was next performed to investigate structure-activity relationships, as well as to find compounds with improved potency.Three series of indole-and closely related benzothiophene-and benzofuran-carboxamide compounds were synthesized for the study.

Structure-Activity Relationships
Because of the high costs for the AlphaLisa assay, inhibitory activities of all compounds were first screened at 5 µM and the IC 50 values for those exhibiting >50% inhibition were determined.
The structures and inhibitory activities of 6-substituted indole compounds, including compounds 1 and 2 (IC 50 = 3.3 and 4.5 µM) with a piperidin-1-ylphenyl group, are summarized in Table 1.Ring-contracted compound 3 with a pyrrolidin-1-ylphenyl substituent was found to be equally potent, with an IC 50 of 2.8 µM.Compound 4 without an N atom in the 6-substituent exhibited reduced activity (31% inhibition at 5 µM) and compound 5 having two N atoms is also weaker (40% inhibition).Replacing the piperidin-1-yl group with a thiophene in compound 6 (14% inhibition at 5 µM) significantly decreased activity.Changing to a 2-hydroxyethyl in compound 7 or to an acetyl group in 8 also resulted in a lowered potency.Compounds 9 and 10 with meta-substituted tert-butyland cyano-phenyl R 6 , respectively, are very weak (17% and 0% inhibition at 5 µM).Compound 11 with a smaller thiophene group at this position is almost inactive.In addition, three analogs of compound 2 with a 3-carboxylamide sidechain were synthesized.Compound 12 with a 4-pyrrolidin-1-ylphenyl R 6 group was found to exhibit slightly improved activity with an IC 50 of 2.9 µM.Compound 13 with another similar morpholin-4-yl group showed reduced activity (29% inhibition at 5 µM), while compound 14 with a polar aminomethyl group was almost inactive (11% inhibition at 5 µM).
Compounds 15-23 (Table 2) with a variety of groups at the 5-position of the indole core were investigated.Compounds 15 and 16 with the 4-piperidin-1-ylphenyl and 4-pyrrolidin-1-ylphenyl R 5 group, respectively, showed comparable activities (IC 50 = 3.6 and 2.7 µM) to compounds 1-3 and 12.Other groups in compounds 17-22 showed less potent activities.While having a 4-piperidin-1-ylphenyl R 5 group, compound 23 with a 3-carboxylamide sidechain showed reduced activity (43% inhibition at 5 µM).Results of the above indole compounds show that in most cases, 4-piperidin-1ylphenyl or similar 4-pyrrolidin-1-ylphenyl group contributes favorably to inhibition of the AF9-DOT1L interaction, regardless whether they are in the 6-or 5-position.However, despite their strong biochemical activities, these indole-containing inhibitors showed only modest activities to inhibit the proliferation of MLL-r leukemia cells (described below), which might be due to poor cell permeability or uptake of these compounds.
Next, a structurally similar, less polar benzothiophene core was used to replace indole.The structures and inhibitory activities of 6-substituted benzothiophene compounds are shown in Table 3. Compound 24 with a 4-piperidin-1-ylphenyl group was found to be a strong inhibitor of the AF9-DOT1L interaction with an IC 50 of 1.6 µM, showing ~2× activity compared to the indole analog 1. Compound 25 with a 4-pyrrolidin-1-ylphenyl had weaker activity (IC 50 = 5.3 µM).Changing to a morpholine in compound 26 (40% inhibition at 5 µM) or a diethylamino group in 27 (25% inhibition at 5 µM) considerably reduced inhibitory activity.Adding a -CH 2 -(in compound 28) or a carbonyl (in 29 and 30) between the piperidinyl/pyrrolidine and phenyl groups lost activity (0-8% inhibition at 5 µM).Replacing the phenyl group with a non-aromatic piperidine ring yielded very weak compounds 31 and 32 (14% and 20% inhibition at 5 µM).Inserting a carbonylphenyl into these two compounds produced inactive compounds 33 and 34.Compound 35 with an additional -NH-between the 4-piperidin-1-ylphenyl group and benzothiophene core was found to be a strong inhibitor (IC 50 = 2.3 µM).Compound 36 having a 4-(4-chlorophenoxy)phenoxy R 6 group is a moderate inhibitor (IC 50 ~5 µM).In addition, a variety of substituted phenyl R 6 groups in compounds 37-49 with different steric and electronic properties resulted in inactive to moderate inhibitors.5-Substituted benzothiophene compounds 50-56 were synthesized and tested for SAR studies.As shown in Table 4, similar SAR results were observed with compound 50 having a 4-piperidin-1-ylphenyl R 5 group showing the strongest activity (IC 50 = 2.5 µM), while other compounds showed no to moderate activities.Moreover, benzofuran compounds 57-77, which possess subtle structural (e.g., angles between substituents), electronic or hydrophobic differences from indole or benzothiophene analogs, were synthesized for the SAR studies.As shown in Table 5, similar SARs were observed, but benzofuran inhibitors appeared to be less potent.The strongest benzofuran compounds 57 with a 4-piperidin-1-ylphenyl and 74 with a 4-piperidin-1-ylphenylamino R 6 group had IC 50 values of 7.2 and 4.6 µM, respectively.Replacing the 4-piperidin-1ylphenyl group with a closely related morpholine (in compound 58), piperazine (in 59/60), cyclohexyl (in 61), or diethylamino group (in 62) significantly reduced the inhibitory activity.Activities of compounds 63-66 indicate that an amide-containing R 6 group is disfavored.Other R 6 groups in compounds 67-73 and 75-77 also showed no to modest activities against the AF9-DOT1L interaction.

Activity Confirmation by a Pull-Down Assay
A pull-down assay [26] was used to confirm that the strong inhibitors could disrupt the AF9-DOT1L interaction.Streptavidin agarose beads coated with biotinylated DOT1L peptide were used to pull-down the protein AF9 AHD in the solution, followed by thorough washing, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blotting to visualize and quantitate the protein.As shown in Figure 1, inhibitors 24 and 50 (IC 50 = 1.6 and 2.5 µM) can dose-dependently reduce AF9 AHD, while inactive compound 37 did not significantly affect the binding of the protein to the DOT1L-coated beads even at 50 µM.This alternative, non-optical method confirmed that compounds 24 and 50 can inhibit the PPI between AF9 and DOT1L.

Activity to Block the AF9/ENL-AF4 Interactions
Transcription cofactor AF4, which is the most frequent (~35%) fusion protein in MLL1-r leukemia, is another binding partner of AF9/ENL [35][36][37].The AF9/ENL-AF4 interactions are essential for the formation of SEC, which causes aberrant gene expression in MLL-r leukemia and other AMLs [20,28].Using our previously developed AlphaLisa assays [26], selected compounds were tested for their ability to inhibit the PPIs between AF9 or ENL and AF4.As summarized in Table 6, inhibitors 16, 24, 25 and 50 of the AF9-DOT1L interaction (IC 50 = 1.6-5.3µM) were found to exhibit comparable activities to block the AF9-AF4 and ENL-AF4 interactions with IC 50 values of 1.9-7.9µM, while inactive compounds 22 and 37 did not inhibit these PPIs.These results are consistent with our previous finding that an inhibitor of the AF9-DOT1L interaction is broadly active against PPIs between AF9/ENL and AF4 [26].

Activity to Suppress Oncogenic Gene Expression
The major function of the SEC is to promote transcription elongation for its bound genes [20][21][22].Because of the high affinity of the PPIs between AF9/ENL and AF4, onco-MLL (e.g., MLL-AF4 or MLL-AF9/-ENL) recruits SEC to MLL target genes, facilitates their expression, and eventually causes leukemia initiation [27,28].Next, we investigated whether selected inhibitors of these PPIs can inhibit the expression of HoxA9, Meis1 and Myc, which are MLL target genes and their high expression is characteristic of MLL-r leukemia.In addition, high expression of Myc is also observed in a broad range of AMLs and other cancers and is considered a driving factor for oncogenesis [38][39][40][41].To this end, RNAs from the control and compound-treated Molm-13 cells (harboring MLL-AF9) were extracted and subjected to quantitative PCR.As shown in Figure 2, potent inhibitors 24 and 50 were able to reduce the expression of HoxA9, Meis1 and Myc.However, inactive compound 37 did not significantly affect the expression of these MLL target genes.

Antitumor Activity
The antitumor activity of selected compounds was evaluated against the proliferation of a panel of MLL-r leukemia and other cancer cells.The results are shown in Table 7. MV4;11 and Molm-13 leukemia cells contain MLL-AF4 and MLL-AF9 fusion oncogenes, respectively.NB4 and HL60 are AML cells without MLL translocation.Hela (cervical cancer) is a solid tumor cell line.Indole-containing compounds 2, 12 and 15 were found to exhibit modest to no activities against proliferation of MLL-r leukemia MV4;11 and Molm-13 cells, despite their strong inhibitory activities against the AF9-DOT1L interaction (IC 50 = 2.9-4.5 µM) (Figure S2).They did not significantly affect the growth of other cancerous cells.Presumably due to improved cell permeability or uptake, benzothiophene-containing inhibitor 24 (IC 50 = 1.6 µM) showed more potent activities against proliferation of MLL-r leukemia MV4;11 and Molm-13 and AML NB4 and HL60 cells with EC 50 values of 9.6, 4.7, 5.3 and 10 µM.Benzothiophene compound 25 had generally reduced antitumor activities with EC 50 s of 8.7-26 µM, in line with its reduced biochemical activity (IC 50 = 5.3 µM).Another benzothiophene inhibitor 50 (IC 50 = 2.5 µM) exhibited comparable antitumor activities (EC 50 = 5.1-9.4µM) to compound 24.The anti-proliferation activity of these compounds against other AML cells (i.e., NB4 and HL60) might be due to their inhibition of SEC-mediated gene expression (e.g., Myc), as observed in our previous studies [26].In addition, these three compounds were found to have weak to no activities against the proliferation of Hela cells, showing good selectivity.Benzofuran compound 57 (IC 50 = 7.2 µM) moderately inhibited the proliferation of MLL-r leukemia and AML cells with EC 50 s of 9.7-21 µM.It did not affect the growth of Hela cells.Moreover, inactive indole, benzothiophene and benzofuran compounds 22, 37 and 72 were included in this assay as negative controls.None of these three compounds inhibited tumor cell proliferation, largely excluding possible off-target effects for these series of compounds.

Materials and Methods
All chemicals for synthesis were purchased from Aldrich (Milwaukee, WI, USA) or Alfa Aesar (Ward Hill, MA, USA).Unless otherwise stated, all solvents and reagents were used as received.All reactions were performed using a Teflon-coated magnetic stir bar at the indicated temperature and were conducted under an inert atmosphere when stated.The identity of the synthesized compounds was characterized by 1 H and 13 C NMR on a Varian (Palo Alto, CA, USA) 400-MR spectrometer and mass spectrometer (Shimadzu LCMS-2020, Shimadzu Kyoto, Japan).Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane.Proton coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br).The identity of the potent inhibitors was confirmed with high resolution mass spectra (HRMS) using an Agilent 6550 iFunnel quadrupole-time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with electrospray ionization (ESI).The purities of the final compounds were determined to be >95% with a Shimadzu Prominence HPLC (Shimadzu, Tokyo, Japan) using a Zorbax C18 (or C8) column (4.6 × 250 mm) (Agilent Technologies, Santa Clara, CA, USA) monitored by UV at 254 nm.To a solution of 5-or 6-bromoindole-2-carboxylic ester 78 (2.0 g, 7.46 mmol) in THF/H 2 O (10/10 mL), sodium hydroxide (0.90 g, 22.4 mmol) was added slowly.The resulting mixture was stirred and heated at 50 • C for 5 h.After cooling down to room temperature, the solution was removed in a vacuum.The resulting residues were diluted with H 2 O (10 mL) and then acidified with 3 N hydrochloride acid solution to give a white precipitate, which was filtered, washed with cold H 2 O and dried under a vacuum overnight to give the corresponding acid as a white solid, which was used directly for the next step.
The reaction mixture containing indole-2-carboxamide 79 (0.2 mmol), aryl boronic acid or aryl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.24 mmol), tetrakis(triphenylphosphine)palladium (11.6 mg, 0.01 mmol), and sodium carbonate (42 mg, 0.4 mmol) in p-dioxane/H 2 O (5/1, v/v, 6 mL) was heated at 100 • C for 16 h.Upon cooling, it was diluted with brine (5 mL), and the crude product was extracted with ethyl acetate (3 × 15 mL).The organic layers were washed with brine (3 × 10 mL), dried over Na 2 SO 4 , and concentrated.The residue was purified with column chromatography (silica gel, hexanes: ethyl acetate from 8:1 to 1:1) to give the corresponding Suzuki coupling product (58-83% yield), which was deprotected in a solution of dichloromethane (3 mL) containing 4N HCl in p-dioxane (0.2 mL) at 0 • C to room temperature for 6 h.Upon filtration, the powder was washed with cold water to give the final compounds 3, 4, 8, 9, 10 and 20 as a hydrochloric salt (~100% yield).To a solution of 5-or 6-substituted indole 80 (3 mmol) in DMF (10 mL) was slowly added to trifluoroacetyl anhydride (0.61 mL, 4.5 mmol) at 0 • C. The resulting mixture was stirred at room temperature for 2 h before quenched with H 2 O (10 mL) to give a white powder, which was filtered and washed with cold water to give compound 81, which was stirred in a 20% NaOH solution (20 mL) at 50 • C for 2 days.The reaction was slowly acidified with 3 N HCl solution to give a white powder, which was filtered and washed with cold water to give the corresponding indole-3-carboxylic acid, which was directly used without further purification as the starting compound for the general synthetic procedure-A (described above) to give the final compounds 2, 12, 13 and 23.      13  An amidation and a Suzuki coupling reaction, as described in the General synthetic procedure-A, were used for synthesis of benzothiophene-or benzofuran-2-carboxamide 85.Compounds 29, 30, 33, 34, 51-52, and 63-66 were prepared using an additional amidation reaction followed by BOC deprotection, following the General synthetic procedure-A, as a hydrochloric salt.

Protein Expression and Purification
The AF9 or ENL expression plasmid was used to transform E. coli BL21(DE3) strain (Novagen, Madison, WI, USA), and protein expression was induced by adding 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 • C overnight.The cells were collected and lysed using a French press (GlenMills, Clifton, NJ, USA) in a lysis buffer (50 mM HEPES, 200 mM NaCl, 1 mM DTT, pH 7.4).Upon centrifugation, the supernatant was applied to an amylose resin column (GE Healthcare) and the recombinant protein with a N-terminal maltose-binding protein (MBP) tag was eluted with 20 mM HEPES, 10 mM Maltose, pH 7.4, which was further purified to be > 95% (SDS-PAGE) using a size exclusion column (HiLoad 16/60 Superdex 200, GE Healthcare, Chicago, IL, USA).

AlphaLisa Assays
Alpha assays were developed in our previous publication [26], using a Perkin-Elmer AlphaLisa anti-MBP kit, which contains streptavidin donor beads and anti-MBP acceptor beads.Briefly, the assay was performed using a MBP-protein (5 nM), a biotinylated peptide (40 nM), and increasing concentrations of a compound in 25 µL buffer (PBS with 0.5% BSA, pH 7.5) in 384-well plates according to the manufacturer's protocol and measured with a Tecan SPARK microplate reader.Data were imported into Prism (version 5.0), and IC 50 values from 3 independent experiments with standard deviation were obtained by using a standard dose-response curve fitting.

Antiproliferation Assay
Proliferation inhibition assays were performed using an XTT assay kit (Biotium, Fremont, CA, USA) following our previous methods [26].The antiproliferation EC 50 values were determined using Prism 5 and the reported results were the mean values of at least three independent experiments.The incubation time for all compounds was 7 days.

Statistical Analysis
At least three independent experiments were carried out to generate each dataset.The significance of experimental differences was evaluated using the Student's t test (Prism 5.0, GraphPad Software, Boston, MA, USA).The results are expressed as the mean ± SEM.

Conclusions
PPIs between AF9/ENL and AF4 or DOT1L are a potential drug target for MLL-r leukemia, as well as other cancers (e.g., AML) driven by SEC-mediated aberrant gene expression.Several indole-carboxamide compounds were identified as novel inhibitors of the AF9-DOT1L interaction.Synthesis and structure-activity relationship studies of 77 compounds show that a 4-piperidin-1-ylphenyl or 4-pyrrolidin-1-ylphenyl R 5 or R 6 substituent is essential for these indole-and closely related benzothiophene-and benzofurancarboxamide compounds to have strong inhibitory activity.The activities of these inhibitors with IC 50 values as low as 1.6 µM were further confirmed using a pull-down assay.Inhibitors of the AF9-DOT1L interaction also blocked the PPIs between AF9/ENL and AF4 with comparable IC 50 s.Selected inhibitors were found to suppress expression of MLL target genes HoxA9, Meis1 and Myc and inhibit proliferation of MLL-r leukemia and other AML cells with EC 50 s as low as 4.7 µM, while they were inactive against solid tumor Hela cells.This antitumor activity profile is consistent with the roles of AF9/ENL in these tumors, suggesting that their activities are on-target.In conclusion, the identified benzothiophene compounds 24 and 50 are not only useful chemical probes for biological studies of AF9/ENL or SEC, but they also represent novel lead compounds for further drug development against MLL-r leukemia and related cancers.

Figure 1 .
Figure 1.Pull-down assay indicated that compounds 24 and 50 significantly reduced the amounts of AF9 AHD bound to DOT1L peptide-coated resins in a dose-dependent manner, while inactive compound 37 did not significantly affect the AF9 AHD levels.The uncropped bolts are shown in Supplementary Materials.

Table 1 .
Structures and activities of 6-substituted indole compounds against the AF9-DOT1L interaction.

Table 2 .
Structures and activities of 5-substituted indole compounds against the AF9-DOT1L interaction.