CBFβ SMMHC-RUNX1

The binding of CBFβ-RUNX is weakly inhibited by AI-10-47 (10 μM) [2]. ME-1, TUR, M0-91, THP-1, and U937 cell growth is dramatically inhibited by AI-10-47 [2].
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 [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-10-47and 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].

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]

Western Blot Analysis[2]
Cell Types: 4 × 106 SEM cells.
Tested Concentrations: 10μM.
Incubation Duration: 6 hrs (hours).
Experimental Results: The binding of CBFβ to RUNX1 in cells was weakly diminished (probably due to its poor solubility).

---

Co-immunoprecipitation Assays [2]
4 × 106 SEM cells were treated with DMSO or 10 μM of AI-4-88, AI-10-47, AI-10-104, AI-12-126 and AI-14-91 for 6 h. Cells were lysed in modified RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate and 1 mM EDTA). RUNX1 was immunoprecipitated from cell lysates using anti-RUNX1 antibody and protein-A Agarose beads as follows: cell lysates were mixed with protein A agarose beads and 2 μg RUNX1 antibody in IP buffer I (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP40, 0.25% sodium deoxycholate) and rotated at 10 rpm for 5 h. Agarose beads were washed twice with IP Buffer I followed by washing with IP buffer II (50 mM Tris pH 7.5, 0.1% NP40, 0.05% sodium deoxycholate). All lysis, immunoprecipitation, and washing steps included DMSO/corresponding inhibitor (10 μM). The beads were heated at 95 °C for 12 min in Western blot loading buffer (100 mM Tris-HCL pH 6.8, 200 mM DTT, 4% SDS, 0.2% Bromophenol-blue, 20% glycerol). The eluted protein was resolved in a 12% polyacrylamide gel. CBFβ was detected using anti-CBFβ antibody. The membrane was re-probed with anti-RUNX1 antibody and detected using Clean-Blot IP Detection Reagents.

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]

[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.
[2]. Small Molecule Inhibitor of CBFβ-RUNX Binding for RUNX Transcription Factor Driven Cancers. EBioMedicine. 2016 Jun;8:117-131.

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]

---

Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFβ binding partner. CBFβ enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFβ are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFβ and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers. [2]

C13H8F3N3O

279.2173

279.061

C, 55.92; H, 2.89; F, 20.41; N, 15.05; O, 5.73

1256094-31-1

63053-14-5

49804932

Off-white to gray solid powder

3.3

1

6

2

20

339

0

FC(OC1C([H])=C([H])C2=C(C=1[H])N([H])C(C1=C([H])C([H])=C([H])C([H])=N1)=N2)(F)F

JSNWHSDKJSOXET-UHFFFAOYSA-N

InChI=1S/C13H8F3N3O/c14-13(15,16)20-8-4-5-9-11(7-8)19-12(18-9)10-3-1-2-6-17-10/h1-7H,(H,18,19)

2-pyridin-2-yl-6-(trifluoromethoxy)-1H-benzimidazole

AI-10-47; AI-10 47; 1256094-31-1; 2-(Pyridin-2-yl)-6-(trifluoromethoxy)-1H-benzo[d]imidazole; 1H-Benzimidazole, 2-(2-pyridinyl)-6-(trifluoromethoxy)-AI-10-47; 2-(pyridin-2-yl)-5-(trifluoromethoxy)-1H-1,3-benzodiazole; SCHEMBL179143; 2-pyridin-2-yl-6-(trifluoromethoxy)-1H-benzimidazole; CHEMBL3675778; AI10-47; AI 10-47

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~358.14 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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