CBFβ SMMHC-RUNX1

To develop a targeted inhibitor of CBFβ-SMMHC function, we used a previously described fluorescence resonance energy transfer (FRET) assay with Venus-CBFβ-SMMHC replacing Venus-CBFb (fig. S1) to screen the National Cancer Institute, NIH, Diversity Set for compounds that inhibit the binding of CBFβ-SMMHCto theRUNX1 Runt domain. This screen identified the active compound AI-4-577 with a 50% inhibitory concentration (IC50) of 22 μM, whereas AI-4-88, a derivative lacking the methoxy functionality, is inactive (Table 1). Changes in the chemical shifts in a nuclear magnetic resonance (NMR) spectrum of a protein upon binding of a small molecule are a powerful method to confirm binding to a protein. We recorded two-dimensional 2D 15N-1H heteronuclear single quantum coherence (HSQC) spectra and 1D saturation transfer difference (STD) NMR experiments of AI-4-57 with CBFβ and the Runt domain. No interaction was observed for the Runt domain, but we can demonstrate chemical shift perturbations in the HSQC spectrumof CBFβ upon addition of AI-4-57 (Fig. 1A) and no changes upon addition of the inactive derivative AI-4-88 (fig. S2), which establishes that the compound binds to CBFβ. Chemical shift perturbations in the backbone and in two aromatic side chains [tryptophan at position 113 (W113) and tyrosine at position 96 (Y96)] indicate that the compound binds in a site spatially close to CBFβ but not on the protein-protein interaction surface on CBFβ, that is, it acts in an allosteric manner to inhibit binding [1].

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Initial Lead for Small Molecule Inhibitors of CBFβ-RUNX Binding Which Bind to CBFβ [2]
We recently reported the 2-pyridyl benzimidazole AI-4-57 as a compound which binds to the CBFβ portion of the CBFβ-SMMHC fusion protein and inhibits its binding to the Runt domain of RUNX proteins (Illendula et al., 2015). Using our previously described FRET assay for binding of the amino acid 1-141 portion of CBFβ to the Runt domain (Gorczynski et al., 2007), we showed this compound is also a modest potency inhibitor of the binding of wildtype CBFβ to the RUNX1 Runt domain (see Fig. 1, Table 3). In order to develop more potent analogs for use as tool compounds to probe RUNX and CBFβ protein function, we synthesized a library of analogs of AI-4-57 and characterized their activity.

Analysis of the pharmacokinetic properties of AI-4-57 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, 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 [1].

Docking [2]
In Grid Generation, under docking tab we have used the site as a centroid of binding site residues in the protein. The active site residues were determined by chemical shift perturbations in 15N-1H and 13C-1H HSQC NMR experiments of protein binding to AI-4-57. The following residues were selected for grid generation: V86, L88, R90, E91, Y96, K98, A99, K111, G112, W113, M122, G123, C124. Docking was carried out using the Virtual Screening Workflow framework. All the compounds were docked flexibly and after docking 100% of best compounds with all good states were scored by MM-GBSA.

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CBFβ Mutant Proteins [2]
Wildtype CBFβ (1-141) and CBFβ (1-141) mutants R90E, K98E and K111E were expressed at 15 °C in 15N labeled minimal media. Proteins were purified using a Ni-NTA column, cleaved with rTev protease digestion overnight followed by size exclusion chromatography to remove the affinity tag and impurities. Protein samples at 150 μM were dialyzed, inserted into an NMR buffer, and titrated with 600 μM AI-4-57. All 15N-1H HSQCs were recorded on a Bruker 800 MHz NMR spectrometer equipped with a cryoprobe.

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NMR Spectroscopy [2]
All NMR-based experiments were acquired using CBFβ (1-141) solutions in buffer containing 50 mM KPi, 0.1 mM EDTA, 1 mM DTT, 0.01%(w/v) sodium azide, 5%(v/v) DMSO and 5%(v/v) D2O at a final pH of 7.5. All experiments were recorded on samples of uniformly labeled 15N CBFβ concentrated to 0.5 mM at 25 °C on a Bruker 18.8 T spectrometer equipped with a CryoProbe™. All NMR data were processed using NMRPipe. Samples containing compound were made by adding AI-4-57 in 100% DMSO to yield an equimolar protein-compound solution.

15N and 13C chemical shift perturbations were determined from 15N- and 13C-HSQC experiments, respectively, collected in the presence and absence of AI-4-57. Peaks were assigned using previously known resonances, and spectra were overlaid and compared using CcpNMR software suite (Vranken et al., 2005). Weighted chemical shift changes in parts per million were calculated by using the equation ∆ 15N + 1HN) = |∆ δHN | + (|∆ δN |/4.69) as described in Yuan et al., (2002). A resonance shift of 0.1 ppm or more was considered to be significant.

All 15N relaxation measurements were carried out with samples of CBFβ + AI-4-57 and CBFβ alone. 15N T1 experiments were conducted using relaxation delays of 10, 180, 300, 500, 1300, 1800, and 2300 ms. 15N T2 experiments used relaxation delays of 10, 25, 50, 75, 100, 150, 200, 225, and 250 ms. Peak intensities were fit to y = Ae− Bx, where B is the relaxation rate (R), using CcpNMR to determine T1 and T2 relaxation times. R1 and R2 relaxation rates, the inverses of T1 and T2, were used to calculate R1 ∗ R2 and R2/R1 values for protein with compound and protein with DMSO alone. Differences in R1, R2, R1 ∗ R2 and R2/R1 were calculated between the values for the CBFβ + AI-4-57 and CBFβ alone samples. Residues with changes in R1 ∗ R2 greater than two standard deviations above or below a trimmed mean consisting of the median 60% of the data were considered significantly different.

Saturation transfer difference NMR samples were composed of 200 μM CBFβ, 2 mM AI-4-57, 10% D2O, and 5% DMSO in 50 mM KPi, 1 mM DTT, 0.1 mM EDTA, 001% w/v NaN3, pH 7.5 in a final volume of 200 μl. All STD experiments were performed using a 600 MHz Bruker NMR spectrometer at 25 °C with saturation times of 500, 750, 1000, 1500, and 2000 ms. Samples were irradiated at 0.4 ppm (protein) and 30 ppm (off-resonance control) and the difference spectra calculated using MestReNova.

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]

Inhibitors With Favorable ADMET Properties Development of a useful tool compound which can be utilized for in vivo studies requires optimization not only of the activity of the compound but also its metabolic stability in vivo. In the context of our previous work on development of small molecule inhibitors that are specific for CBFβ-SMMHC (Illendula et al., 2015), we showed that AI-4-57 has a short half-life in mice with loss of the methyl group on the methoxy functionality being the resulting metabolite. Introduction of trifluoromethoxy abrogated the metabolic liability. Introduction of a trifluoromethoxy substitution into AI-4-57 yielded a compound (AI-10-47) with improved activity in the FRET assay (see Table 1) as well as in assays of cellular activity (see below). Introduction of this substitution into 7a (Supplementary Fig. 1), yielded the inhibitor AI-10-104 with IC50 = 1.25 μM (Table 1). However, administration of AI-10-104 to mice via intraperitoneal (IP) injection of a captisol formulation at 178 mg/kg resulted in significant sedative effects within 30 s, from which the mice recovered in approximately 1 h, whereas administration of a nanoparticle formulation at 200 mg/kg was lethal in approximately ~ 3.5 h. Hypothesizing that this effect is driven by an off-target activity, we have engineered additional analogs (AI-12-126, AI-14-55, and AI-14-91, see Table 1) where we have appended morpholine ring substituents to the pyridine ring, thereby altering the structure as well as polarity of the compounds. These compounds retain similar activity in the FRET assay to the parent compounds (see Table 3). Importantly, AI-12-126 and AI-14-91, when formulated as the HCl salts with captisol and administered IP at 100 mg/kg do not induce the sedative effects seen with AI-10-104 and are well-tolerated by mice. Measurements of the pharmacokinetic properties of these compounds in mice (see Supplementary Fig. 2) showed that at a dose of 100 mg/kg, we can achieve useful concentrations of the compounds with reasonable half-lives in vivo (AI-14-91, oral gavage, t1/2 = 203 min). These derivatives are, therefore, viable options for in vivo studies of CBFβ and RUNX function.

[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]

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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]

C13H12CLN3O

261.71

225.09

C, 59.66; H, 4.62; Cl, 13.55; N, 16.06; O, 6.11

63053-14-5

186762

Typically exists as solid at room temperature

1.268g/cm3

459.9ºC at 760mmHg

164.7ºC

2.633

1

3

2

17

261

0

COC1=CC2=C(C=C1)N=C(N2)C3=CC=CC=N3

FXXLTIFEBMOGOJ-UHFFFAOYSA-N

InChI=1S/C13H11N3O/c1-17-9-5-6-10-12(8-9)16-13(15-10)11-4-2-3-7-14-11/h2-8H,1H3,(H,15,16)

6-methoxy-2-pyridin-2-yl-1H-benzimidazole

AI-4-57 Hydrochloride; AI457 Hydrochloride; 63053-14-5; 6-methoxy-2-pyridin-2-yl-1H-benzimidazole; 5-methoxy-2-(pyridin-2-yl)-1h-benzimidazole; 5-Methoxy-2-pyridin-2-yl-1H-benzoimidazole; MLS001208923; SMR000503813; 5-Methoxy-2-(pyridin-2-yl)-1H-benzo[d]imidazole; 1H-Benzimidazole, 6-methoxy-2-(2-pyridinyl)-; AI 4 57 Hydrochloride; AI 4 57 HCl; AI457 HCl; AI-4-57 HCl

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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