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].

---

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]

---

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]

---

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.

---

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.

---

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.

Pharmacokinetic analysis of AI-4-57 (an analog of AI-10-49) revealed a short half-life in mouse plasma (t½ = 37 min) (Figure S5), with demethylation of the methoxy group being the major metabolite. Trifluoromethoxy (CF3O) substitution has been shown to result in lower reactivity (18, 19), therefore we synthesized AI-10-47 with this substituent. FRET measurements showed that this substituent actually enhanced the activity of the monovalent compound (Table 1). Liver microsomal stability assays indicated that AI-10-47 reduced metabolic risk, thus justifying the synthesis of the divalent derivative AI-10-49 (Table 1). AI-10-49 exhibits potent activity (FRET IC50 = 260 nM) (Table 1) [dissociation constant (KD) determined by isothermal titration calorimetry (ITC) = 168 nM] (Figure S6), improved in vivo pharmacokinetic properties (t½ = 380 min) (Figure S5), and enhanced inhibitory activity against ME-1 cell growth (IC50 = 0.6 mM) compared to the parent protonated divalent compound AI-4-83 (IC50 ~3 μM) (Figure 1E) (Figure 1F). Notably, AI-10-49 shows extremely low activity in normal human bone marrow cells (IC50 > 25 μM) (Figure 1G), indicating a promising potential therapeutic window. Among 11 human leukemia cell lines, ME-1 cells are the only cell line highly sensitive to AI-10-49 (Figure S7). [1]

---

Pharmacokinetic Study[1]
Before the study began, mice were fasted for at least 3 hours and allowed free access to water. Animals were housed in an environment with a 12-hour light/12-hour dark cycle, a temperature of 72-74°C, and a relative humidity of 30-50%. For intraperitoneal administration, 24-28 g male C57BL/6 mice were artificially fixed and intraperitoneal injection was performed at the midpoint between the sternum and pubis, slightly off the midline of the mouse. A 1 mL syringe and a 27-gauge needle were used for each injection. Blood was collected from the animals at predetermined time points. Animals were anesthetized with isoflurane and blood was collected by cardiac puncture. The blood was immediately transferred to a 1.5 mL heparinized microcentrifuge tube and centrifuged at 4000 rpm for 10 minutes. The plasma was then transferred to a clean centrifuge tube and frozen. Animals died from blood loss without waking from anesthesia and were ensured to die under anesthesia by thoracotomy. This method conforms to the American Veterinary Medical Association (AVMA) guidelines on euthanasia regarding the use of bloodletting as a means of euthanasia. Non-compartmental pharmacokinetic analysis of plasma concentration-time data for the test compounds was performed using PK Solutions 2.0 software. [1] AI-10-49 – High-performance liquid chromatography (HPLC) analysis was performed using a liquid chromatography system consisting of a Shimadzu SCL-10Avp controller, SIL-10A autosampler, LC-10ADvp pump, SPD-10Avp detector, and CTO-10Avp column oven. Data acquisition, peak integration, and calculations were all performed using LabSolutions software. Separation was performed using a 4.6 mm × 13 mm × 150 mm Atlantis T3 5 μm column. The mobile phases were phase A and phase B, with acetonitrile/water/trifluoroacetic acid ratios of 5/95/0.1 and 95/5/0.1, respectively. The gradient elution program was as follows: the proportion of phase B increased from 50% to 95% within 4 minutes, held at 95% phase B for 2 minutes, decreased from 95% phase B to 50% within 0.5 minutes, and finally held at 50% phase B for 4.5 minutes. The flow rate was 1 mL/min, and the temperature was 45°C. The injection volume was 40 μL, and the detection wavelength was 325 nm. Quantitative analysis was performed using external standards prepared from blank plasma. The linear range was 25–750 ng/mL (R² > 0.995). The plasma sample extraction procedure is as follows: Take 100 μL of sample, add 0.5 mL of methyl tert-butyl ether (MtBE), and add 10 μL of AI-10-49 spiking solution (10X). Vortex to mix and let stand at room temperature for 5 min. Vortex the two-phase mixture for 5 min, and then centrifuge at 12,000 rpm for 5 min. Transfer 450 μL of the MtBE layer to a clean centrifuge tube and evaporate to dryness. Resuspend the resulting residue in 100 µL of 50/50/0.1 acetonitrile/water/trifluoroacetic acid solution, vortex, and centrifuge at 12,000 rpm for 5 min. Transfer 90 µL of the supernatant to an autosampler vial with a polypropylene insert for analysis.

Although we have not assessed the toxicity following long-term exposure, we did not observe any signs of toxicity after 7 consecutive days of administration of AI-10-49 (Figures S9 to S11).

---

Toxicology Studies[1]
We assessed the cumulative toxicity following 7 days of daily administration of AI-10-49. These studies did not include maximally tolerated dose or long-term (>30 days) toxicity. Six-week-old female C57BL/6 mice were intraperitoneally injected daily with either DMSO or 200 mg/kg AI-10-49 for 7 days (n=3 to 4 per group). We analyzed the appearance of the mice to assess signs of toxicity, including grooming, activity level, and body weight. Peripheral blood cells were analyzed by flow cytometry and quantified using a hematology counter 4 hours after the last injection. The mice were then euthanized to collect tissue samples. Bone marrow hematopoietic progenitor cells were analyzed by flow cytometry, including hematopoietic stem cells and multilineage progenitor cells [LSK+= Lin(-), kit(+), Sca1(+)], myeloid progenitor cells [CMP=Lin(-)Sca1(-)kit(+)CD34(+)CD16/32(-)], granulocyte/monocyte progenitor cells [GMP=Lin(-)Sca1(-)kit(+)CD34(+)CD16/32(+)], and megakaryocyte/erythroid progenitor cells [MEP=Lin(-)Sca1(-)kit(+)CD34(-)CD16/32(-)]. Paraffin sections of bone marrow, liver, lung, spleen, intestine, and brain were prepared and stained with hematoxylin and eosin for tissue structure analysis.

[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 type of leukemia in adults. In AML with the chromosomal inversion inv(16)(p13q22), the transcription factor fusion CBFβ-SMMHC (core-binding factor β and smooth muscle myosin heavy chain) expresses competitively with wild-type CBFβ for binding to the transcription factor RUNX1, thereby disrupting RUNX1 activity during hematopoiesis and inducing AML. Currently, non-selective cytotoxic chemotherapy for inv(16) AML achieves good initial efficacy, but long-term survival is limited. This article reports the development of a protein-protein interaction inhibitor, AI-10-49, which selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, exhibits good pharmacokinetic properties, and delays the progression of leukemia in mice. Treatment of primitive cells from patients with primary inv(16) AML with AI-10-49 induced selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective approach for treating inv(16) AML and support transcription factor-targeted therapy for 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.

---

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.

---

View More

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

---

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