CBFβ-SMHHC

AI-10-49, with an IC50 value of 0.26 μM, blocks CBFβ-SMMHC from binding to the RUNX1 Runt domain [1]. AI-10-49 (1 μM; 3, 6, 12 hours) reduces CBFβ-SMMHC binding to RUNX1 in a specific manner[1].

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Measurements of stability in liver microsomes showed that AI-10-47 reduced the metabolic liability and so justified the synthesis of the bivalent derivative AI-10-49 (Table 1).AI-10-49 is potent (FRET IC50=260nM) (Table 1) [isothermal titration calorimetry (ITC) measurements yielded a dissociation constant (KD) = 168 nM] (fig. S6), has improved in vivo pharmacokinetic properties (t½ = 380min) (fig. S5), and has enhanced inhibitory activity on ME-1 cell growth (IC50 = 0.6 mM) (Fig. 1F) compared with the parent protonated bivalent compound AI-4-83 (IC50 of ~3 μM) (Fig. 1E). Note that AI-10-49 showed negligible activity (IC50 > 25 μM) in normal human bone marrow cells (Fig. 1G), which indicated a robust potential therapeutic window. In a panel of 11 human leukemia cell lines, ME-1 cells were the only cell line highly sensitive to AI-10-49 (fig. S7).
The specificity of AI-10-49 in disrupting endogenous RUNX1 binding to CBFβ-SMMHC versus CBFβ was assessed in ME-1 cells. AI-10-49 effectively dissociatedRUNX1 from CBFβ-SMMHC, with 90% dissociation after 6 hours of treatment, whereas it had only a modest effect on CBFβ-RUNX1 association (Fig. 2A). The stability of RUNX1, CBFβ, andCBFβ-SMMHCwas not affected by AI-10-49 (fig. S8A). Expression of the RUNX1-regulated genes RUNX3, CSF1R, and CEBPA is repressed by CBFβ-SMMHC in inv(16)AML. Previous studies have shown decreased RUNX1 binding to target genes in the presence of CBFβ-SMMHC, which suggests that CBFβ-SMMHC represses RUNX1 target genes by blocking binding ofRUNX1 to targetDNAsites (Fig. 2B). Consistent with this model, chromatin-immunoprecipitation (ChIP) assays showed that treatment of ME-1 cells for 6 hours with AI-10-49 increased RUNX1 occupancy 8-, 2.2-, and 8-fold at the RUNX3, CSF1R, and CEBPA promoters, respectively, whereas no enrichment was observed at control loci (Fig. 2C and fig. S8, B and C). In accordance with this, treatment of ME-1 cells for 6 or 12 hours with AI-10-49 increased expression of RUNX3, CSF1R, and CEBPA but had no effect on control gene PIN1 (Fig. 2D).Neither of these effectswas observed in inv(16)-negative U937 cells. These data establish AI-10-49 selectivity in inhibiting CBFβ-SMMHC binding to RUNX1 and validate our approach of using bivalent inhibitors to achieve this specificity [1].
To test the potential utility of AI-10-49 for use in human inv(16) leukemia treatment, we evaluated the survival of four primary inv(16) AML cell samples treated for 48 hours with a dose range ofmonovalent AI-10-47 and bivalent AI-10-49. As shown in Fig. 3B, the viability of inv(16) patient cells was reduced by treatment with AI-10-49 at 5 and 10 μM concentrations (individual dose-response experiments are shown in fig. S12). Note that the bivalent AI-10-49 was more potent than the monovalent compound AI-10-47 and so recapitulated the effects observed in the human inv(16) cell line ME-1. In contrast, the viability of normal karyotype AML sampleswas not affected by AI-10-49 treatment (Fig. 3C). Analysis of an additional set of five AML samples revealed that AI-10-49 treatment specifically reduces the viability of inv(16) leukemic cells without having an apparent effect on their differentiation (fig. S13). AI-10-49 specificity was also evident when we assessed the ability of AML cells to form colonies by evaluating colony-forming units (CFUs) after compound exposure. The ability of inv(16) AML cells to form CFUs was selectively reduced by AI-10-49 when compared with normal karyotype and t(8;21) AML patient samples (Fig. 3D). This inhibitory effect was dose-dependent (40 and 60% at 5 and 10 μM, respectively) (Fig. 3E), whereas there was no change in CFUs of AML cells treated with AI-10-47, AML cells with normal karyotype (Fig. 3F), or CD34+ cord blood cells (Fig. 3G). These studies show that AI-10-49 selectively inhibits viability and CFU capacity in inv(16) AML blasts, whereas it has negligible effects on AML blasts with normal karyotype or, importantly, on normal human hematopoietic progenitors [1].

Leukemia in mice is delayed in its progression by AI-10-49 (200 mg/kg; daily) [1].
Up to 90% of inv AML patients have cooperating mutations in components of the receptor tyrosine kinase pathway, including N-RAS and c-Kit . We have recently developed an efficient mouse model of inv AML, by combining the conditional NrasLSL-G12D and CbfbMYH11 alleles. To test the effects of AI-10-49 administration in vivo, we transplantedmice with Cbfb+/MYH11:Ras+/G12D leukemic cells, waited 5 days for engraftment, and then treated mice with vehicle [dimethyl sulfoxide (DMSO)] or 200 mg/kg of body weight AI-10-49 for 10 days, and assessed the effect on disease latency. As shown in Fig. 3A, vehicle-treatedmice succumbed to leukemia with a median latency of 33.5 days, whereas AI-10-49-treated mice survived significantly longer (median latency = 61 days; P = 2.7 × 10−6). Thus, transient treatment with AI-10-49 reduces leukemia expansion in vivo. Although we have not assessed toxicity after longterm exposure, after 7 days of administration of AI-10-49, we observe no evidence of toxicity [1].

FRET assays. [1]
Cerulean-Runt domain was expressed and purified as described previously. Venus-CBFβ-SMMHC was constructed by inserting 6xHis tag and Venus into pET22b vector between NdeI and NcoI sites, and by inserting CBFβ-SMMHC (the CBFβ-SMMHC construct contains 369 amino acids, 1-166 from CBFβ and 166-369 from MYH11 (amino acids 1526-1730)) between the NcoI and BamHI sites. The fusion protein was purified by standard Ni-affinity chromatography with an on column benzonase treatment to remove residual DNA contaminants. Proteins were dialyzed into FRET buffer (25mM Tris-HCl, pH 7.5, 150mM KCl, 2mM MgCl2) prior to use. Protein concentrations were determined by UV absorbance of the Cerulean and Venus at 433 and 513 nm, respectively. Cerulean-Runt domain and Venus-CBFβ-SMMHC were mixed 1:1 to achieve a final concentration of 10 nM in 96 well black COSTAR plates. DMSO solutions of compounds were added to a final DMSO concentration of 5% (v/v) and the plates incubated at room temperature for one hour in the dark. A PHERAstar microplate reader was used to measure fluorescence (excitation at 433 nm and emission measured at 474 and 525 nm). For IC50 determinations, the ratios of the fluorescence intensities at 525 nm and 474 nm were plotted versus the log of compound concentration, and the resulting curve was fit to a sigmoidal curve using Origin7.0. Three independent measurements were performed and their average and deviation were used for IC50 data fitting.[1]

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Protein NMR spectroscopy [1]
All NMR experiments were performed at 30 °C on a Bruker 800 MHz instrument equipped with a cryogenic probe. All NMR samples were prepared in 50 mM potassium phosphate, 0.1 mM EDTA, 0.1 mM NaN3, 1 mM DTT, and 5% (v/v) D2O at a final pH of 7.5. 15N1 H HSQC experiments utilized a 500 µM sample and 13C1 H HSQC experiments were conducted on a 1 mM sample. All NMR data was processed using NMRPipe and Sparky. Weighted chemical shift changes in parts per million were calculated by using the equation: ∆( 15N + 1 HN) = |∆�HN |+(|∆�N|/4.69).[1]

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Isothermal titration calorimetry [1]
The 369 amino acid CBFß-SMMHC construct consisting of CBFß residues 1-166 and 166-369 from MYH11 (MYH11 amino acids 1526-1730) was cloned into a modified pET22b vector with an N-terminal 6xHis tag and a Tev protease site and was expressed in Rosetta2(DE3) cells in Terrific Broth media with 1mM IPTG induction for 8 hours at 30°C. CBFß-SMMHC was purified via Niaffinity chromatography followed by benzonase treatment and Tev protease digestion overnight. The fusion protein was then passaged a second time through the Ni-NTA column followed by Q-Sepharose ion exchange chromatography to remove residual nucleic acid. Additional purification was achieved via size exclusion chromatography using a Sephacryl S300 column. Purified CBFß-SMMHC was dialyzed into 1 L of ITC buffer (12.5 mM KPi (pH 6.5), 150 mM NaCl, 2 mM MgCl2, 1 mg/mL NaN3, 1 mM DTT, and 0.25% DMSO) for 4 hours. AI-10-49 was added separately to 10 mL of ITC buffer to a final concentration of 2 µM. The concentration of AI-10-49 was verified using 1 H NMR on a Bruker 600 MHz NMR spectrometer using DSS as a standard. All ITC measurements were carried out at 30 °C on a MicroCalorimetry System. CBFß-SMMHC and AI-10-49 samples were degassed for 20 min and 8 µL injections of 2µM AI-10-49 were made to a 400 nM solution of CBFßSMMHC in the calorimetric cell. A biphasic transition was consistently observed in the ITC data indicative of more than one process contributing to the observed heats. As a result of this and because of the relatively small heats observed, this data could not be analyzed using the standard software available on the calorimeter which uses the heat values to determine a KD. Rather, data were analyzed utilizing Origin 7.5 and were fit to a one-site sigmoidal binding curve after correction for dilution enthalpy to derive apparent KD values. The measurement was repeated three times and values are reported as the average ± standard deviation.

Western Blot Analysis[1]
Cell Types: ME-1 cells
Tested Concentrations: 1 μM
Incubation Duration: 3, 6 hrs (hours)
Experimental Results: Effectively dissociated RUNX1 from CBFβ-SMMHC.

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RT-PCR[1]
Cell Types: ME-1 and U937 cells
Tested Concentrations: 1 μM
Incubation Duration: 6, 12 hrs (hours)
Experimental Results: Increased expression of RUNX3, CSF1R, and CEBPA.

Animal/Disease Models: Mice (Cbfb+/MYH11:Ras+/G12D leukemic cells)[1]
Doses: 200 mg/kg
Route of Administration: per day
Experimental Results: decreased leukemia expansion in vivo and survived Dramatically long.

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Leukemia transplantation studies in mice [1]
Leukemic cells carrying Cbfb+/MYH11 and Nras+/G12D oncogenic alleles were generated in CD45.2 C57BL/6 mice, as previously described. Briefly, 2x103 Cbfb+/MYH11;Nras+/G12D leukemic cells were transplanted into each of 22 sublethally irradiated six to eight week old CD45.1 C57BL/6 female mice. The number of mice per group was selected in preliminary assays to achieve statistical power under the established experimental conditions. At day five post-transplantation, mice were randomized into two groups, and injected intraperitoneally for ten days with 50 µL DMSO or AI-10-49 (200 mg/kg) in DMSO. Mice were kept under observation by more than one person to determine the median leukemia latency, and were euthanized once signs of disease were detected, including reduced motility and grooming activity, hunched back, pale paws (anemia), and hypothermia. At time of euthanasia, peripheral blood and spleen cell were extracted and analyzed as previously described. Leukemia burden was analyzed in peripheral blood by measuring the total white blood cell counts and the number of cells in the c-kit(+)-gated population.

Analysis of the pharmacokinetic properties of AI-4-57 (analog of AI-10-49) showed that the compound has a short half-life (t½ = 37 min) in mouse plasma (fig. S5) and that loss of the methyl group from the methoxy functionality is the primary metabolite. Trifluoromethoxy (CF3O) substitutions have been shown to be less reactive (18, 19), so we synthesized AI-10-47 with this substitution. FRET measurements show that this substitution actually enhances the activity of the monovalent compound (Table 1). Measurements of stability in liver microsomes showed that AI-10-47 reduced the metabolic liability and so justified the synthesis of the bivalent derivative AI-10-49 (Table 1).AI-10-49 is potent (FRET IC50=260nM) (Table 1) [isothermal titration calorimetry (ITC) measurements yielded a dissociation constant (KD) = 168 nM] (fig. S6), has improved in vivo pharmacokinetic properties (t½ = 380min) (fig. S5), and has enhanced inhibitory activity on ME-1 cell growth (IC50 = 0.6 mM) (Fig. 1F) compared with the parent protonated bivalent compound AI-4-83 (IC50 of ~3 μM) (Fig. 1E). Note that AI-10-49 showed negligible activity (IC50 > 25 μM) in normal human bone marrow cells (Fig. 1G), which indicated a robust potential therapeutic window. In a panel of 11 human leukemia cell lines, ME-1 cells were the only cell line highly sensitive to AI-10-49 (fig. S7). [1]

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Pharmacokinetic Studies [1]
Prior to the study, mice were fasted at least three hours and water was available ad libitum. Animals were housed on a 12-hour light/dark cycle at 72-74°C and 30-50% relative humidity. For intraperitoneal dosing 24 – 28 gm male C57BL/6 mice were manually restrained and injected in the peritoneal cavity midway between the sternum and pubis and slightly off the midline of the mouse. A 1-cc syringe with a 27-gauge needle was used for each injection. Blood was collected from the animals according to scheduled time points. Animals were anesthetized with isoflurane and blood drawn via cardiac puncture. Blood was immediately transferred to 1.5 mL heparinized microcentrifuge tubes and centrifuged at 4000 rpm for ten minutes. Plasma was then transferred to clean tubes and frozen. Due to exsanguination, the animals did not wake from the anesthesia and death was insured while under anesthesia by thoracotomy. This method is consistent with the recommendations of the AVMA Guidelines on Euthanasia for use of exsanguination as a means of euthanasia. Noncompartmental pharmacokinetic analysis of the test compound plasma concentration-time data was conducted using PK Solutions 2.0. [1]

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AI-10-49– HPLC analysis was performed with an LC system consisting of a Shimadzu SCL-10Avp controller, SIL-10A autosampler, LC-10ADvp pumps, SPD-10Avp detector and CTO-10Avp column oven. Data acquisition, peak integration and calculation were accomplished with LabSolutions software. A 4.6 13 x 150 mm Atlantis T3 5 micron column was used for fractionation using a gradient mobile phase consisting of solvents A and B with ratios of 5/95/0.1 and 95/5/0.1 acetonitrile/water/TFAacid, respectively, at 50 -95% B in four minutes, 95% B for 2 min, 95 – 50% B in 0.5 min, and 50% B for 4.5 min. Flow rate was 1 mL/min at 45°C. The injection volume was 40 microliters with detection at 325 nm. Quantiation was versus external standards prepared in blank plasma over a linear range of 25 – 750 ng/mL (R2 > 0.995) Extraction of plasma samples was conducted on 100 µL of the respective samples using 0.5 mL of MtBE after adding 10 µL of AI-10-49 spiking solution (10X), vortexing, and allowing the sample sit at room temperature for 5 min. The two phase mixture was vortexed for five min then centrifuged at 12,000 rpm for five min. 450 µL of the MtBE layer was transferred to clean tubes and evaporated to dryness. The resulting residue was reconstituted in 100 µL of 50/50/0.1, ACN/H2O/TFA and vortexed followed by centrifugation at 12,000 rpm for five min. 90 µL of the supernatant was transferred to autosampler vials with polypropylene inserts and analyzed.

Although we have not assessed toxicity after longterm exposure, after 7 days of administration of AI-10-49, we observe no evidence of toxicity (figs. S9 to S11).

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Toxicology Studies [1]
The cumulative toxicity of AI-10-49 was evaluated after daily administration for seven days. These studies did not include maximum tolerated dose or long term (> 30 days) toxicity. Six week old C57BL/6 female mice were treated with daily intraperitoneal injection of DMSO or 200mg/kg AI-10-49 for seven days (n=3 to 4 per group). The appearance of mice was analyzed for signs of toxicity, including grooming, motility, and weight. Four hours after last injection, peripheral blood cells were analyzed by flow cytometry and quantified with hemocytometer. Mice were then euthanized for tissue harvesting. Bone marrow hematopoietic progenitors were analyzed by flow cytometry, including hematopoietic stem and multilineage progenitors [LSK+= Lin(-), kit(+), Sca1(+)], common myeloid progenitors [CMP=Lin(-)Sca1(-)kit(+)CD34(+)CD16/32(-)], granulocyte/monocyte progenitor [GMP=Lin(-)Sca1(-)kit(+)CD34(+)CD16/32(+)], and megakaryocyte/erythroid progenitors [MEP=Lin(-)Sca1(-)kit(+)CD34(-)CD16/32(- )]. Parafin-sections of bone marrow, liver, lung, spleen, intestine, and brain were prepared and stained with hematoxylin & eosin for the analysis of tissue architecture.

[1]. Chemical biology. A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice. Science. 2015 Feb 13;347(6223):779-84.

Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers. [1]

C30H22F6N6O5

660.5233

660.155

C, 54.55; H, 3.36; F, 17.26; N, 12.72; O, 12.11

1256094-72-0

49806644

Off-white to yellow solid powder

1.5±0.1 g/cm3

790.3±70.0 °C at 760 mmHg

431.8±35.7 °C

0.0±2.8 mmHg at 25°C

1.611

7

2

15

12

47

913

0

FC(OC1C([H])=C([H])C2=C(C=1[H])N([H])C(C1C([H])=C([H])C(=C([H])N=1)OC([H])([H])C([H])([H])OC([H])([H])C([H])([H])OC1=C([H])N=C(C([H])=C1[H])C1=NC3C([H])=C([H])C(=C([H])C=3N1[H])OC(F)(F)F)=N2)(F)F

WJBSSBFGPKTMQQ-UHFFFAOYSA-N

InChI=1S/C30H22F6N6O5/c31-29(32,33)46-17-1-5-21-25(13-17)41-27(39-21)23-7-3-19(15-37-23)44-11-9-43-10-12-45-20-4-8-24(38-16-20)28-40-22-6-2-18(14-26(22)42-28)47-30(34,35)36/h1-8,13-16H,9-12H2,(H,39,41)(H,40,42)

6-(trifluoromethoxy)-2-[5-[2-[2-[6-[6-(trifluoromethoxy)-1H-benzimidazol-2-yl]pyridin-3-yl]oxyethoxy]ethoxy]pyridin-2-yl]-1H-benzimidazole

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.78 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.78 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10% DMSO +90%PEG400: 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)