2.1. Assays

qHTS ATAD5 Assay (AID 504467). ATAD5-luc cells were dispensed at 2,000/4 μL/well into a tissue culture treated 1,536-well white/solid bottom assay plates (Greiner Bio-One) using a Multidrop Combi dispenser (Thermo Scientific). After the assay plates were incubated for 3–4 hr at 37 °C for the cell adherence, 23 nL of each compound was transferred via Pin Tool (Kalypsys) to columns 5–48 of the assay plates, resulting in the final concentrations ranging from 1.0 μM to 46 μM. DMSO was only included in columns 1 to 4. For antagonist screening, the compound transfer was followed by the addition of either 1 μL of culture medium (columns 1 and 3) or 5-FUrd (10 μM final concentration in rest of the columns), a known stabilizer of ATAD5. The assay plates were incubated for 16 hr at 37 °C, followed by the addition of Amplite Luciferase reagent (AAT Bioquest, Inc.) at 5 μL/well using a Flying Reagent Dispenser (FRD) (Aurora Discovery). After 30 min incubation at room temperature, the luminescence intensity was quantified using a ViewLux CCD-based plate reader (Perkin Elmer). Raw plate reads for each titration point were first normalized relative to FUrd control (10 μM, 100%) and DMSO only wells (basal, 0%) and then corrected by applying a pattern correction algorithm using compound free control plates (DMSO) plates.

Biochemical Luciferase Counter Screen (AID 686933). A biochemical was used to validate the inhibitor identified from the primary screen. Three μL of 10 μM substrate (50 mM Tris acetate, 13.3 mM Magnesium acetate, 0.01 mM D-Luc, 0.01 mM ATP, 0.01% Tween, 0.05% BSA and dH₂O) was dispensed into 1,536-well white/solid bottom assay plates (Greiner Bio-One) using a Flying Reagent Dispenser (FRD) (Aurora Discovery). Twenty three nanoliter of each compound was transferred via Pin Tool (Kalypsys) to rows 1 – 30 of the assay plates resulting in the final concentrations ranging from 0.2 nM to 46 μM and DMSO only was transferred to rows 31 – 32. Then compound addition was followed by adding 1 μL of buffer (row 32 only) or enzyme; “Pyralis Luciferase” (0.04 μM, final concentration) for rest of the plate. After 5 min of incubation at room temperature, the luminescence intensity was quantified using a ViewLux CCD-based plate reader (Perkin Elmer). Raw plate reads for each titration point were first normalized relative to Pyralis luciferase control (0.04 μM, 100%) and DMSO only wells (basal, 0%) and then corrected by applying a pattern correction algorithm using compound free control plates (DMSO) plate.

Cell Viability Assay (AID 686921). To evaluate the cytotoxic effect of the inhibitors, a cell viability assay using HCT116 cell line was developed. 1 × 10⁴ HCT116 or PARP-1 deficient cells were seeded into each well of a 96-well. After allowing the cells to attach to the bottom of the plate for 24 hours, compounds were added at serial dilutions from starting concentration of 40 μM. 48 hours following treatment, cell viability was determined using Cell Titer-Glo (Promega) according to the manufacturer’s protocol and quantified on a Fluoroskan Ascent Luminometer (Thermo Scientific).

FLAG-ATAD5 Transfections and Western Blotting. To evaluate the cellular activity of the probe, a secondary assay using a different cell line was developed. HEK293T cells were transfected with FLAG-tagged ATAD5 using Lipofectamine 2000 (Life Technologies), according to the manufacturer’s protocol. 48 hours post-transfection, the cells were treated with the indicated compounds for 16 hours. To obtain total lysate, the cells were resuspended in lysis buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, protease inhibitors (Roche)] and lysed on ice for 30 min. Proteins were separated by SDS-PAGE using a 4–15% Tris-glycine gel (Bio-Rad) and transferred to a Polyvinylidene difluoride membrane. FLAG-ATAD5 protein levels were detected by the ECL Western Blotting Detection System (GE Healthcare) using an HRP-conjugated antibody against FLAG (Sigma). Equal protein loading was confirmed using an antibody against tubulin (Abcam). The ratio of FLAG/Tubulin was quantified using ImageJ.

2.2. Probe Chemical Characterization

Probe ML367 (CID 921541)

*Purity > 98% as determined by LC/MS and ¹H NMR analyses.

N-(3,4-difluorophenyl)-2-(pyridin-4-yl)quinazolin-4-amine (ML367): LC-MS Retention Time: t₁ (Method 1) = 4.368 min and t₂ (Method 2) = 2.845 min; ¹H NMR (400 MHz, DMSO-d₆) δ 10.20 (s, 1H), 8.90 – 8.83 (m, 2H), 8.59 (d, J = 8.3 Hz, 1H), 8.46 – 8.40 (m, 2H), 8.09 (ddd, J = 13.2 Hz, 7.5 Hz & 2.6 Hz, 1H), 8.01 – 7.87 (m, 2H) and 7.80 – 7.44 (m, 3H); ¹³C (400 MHz, DMSO-d₆) δ 158.18, 158.03, 155.98, 150.10, 149.97, 149.81, 148.74, 147.68, 147.55, 147.16, 147.10, 146.99, 144.74, 144.61, 135.88, 135.85, 135.78, 133.95, 128.42, 127.59, 123.17, 123.09, 122.98, 122.92, 122.83, 119.00, 117.53, 117.20, 114.36, 111.86, 111.81, 111.66 and 111.60; HRMS: m/z (M+H)⁺ = 335.1086 (Calculated for C₁₉H₁₃F₂ N₄ = 335.1103).

Figure 2Structures of ML367 analogs with their corresponding Compound IDs listed in Table 1

Table 1

List of the ML367 probe and related analogs.

2.3. Probe Preparation

Preparation of N-(3,4-difluorophenyl)-2-(pyridin-4-yl)quinazolin-4-amine (ML367) is a two-step process described below and illustrated in Scheme 1.

Scheme 1

Synthetic route to ML367 is a 2 step process.

1. A mixture of 2,4-dichloroquinazoline, 3,4-difluoroaniline and diisopropylethylamine ((iPr)₂NEt) in 2-propanol was heated at reflux with stirring for 16 hr. The solvent was removed and the crude product was triturated with water and sonicated, which caused the brown oil to become a tan solid. The solid was removed by filtration and was washed with water. 2-chloro-N-(3,4-difluorophenyl)quinazolin-4-amine was isolated as a tan solid and used in the next step without further purification.
2. A mixture of 2-chloro-N-(3,4-difluorophenyl)quinazolin-4-amine, pyridin-4-ylboronic acid, and 2 molar solution of sodium carbonate and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) in dimethoxyethane was degassed with argon for 5 min then heated in a microwave for 30 min at 150 °C. The solvent was removed by forced air and the crude product was dissolved in dimethyl sulfoxide (DMSO) then stirred with palladium scavenger for 30 min. The solution was passed through a thiol cartridge and finally purified in preparative high-performance liquid chromatography (HPLC) to provide N-(3,4-difluorophenyl)-2-(pyridin-4-yl)quinazolin-4-amine as a trifluoroacetate (TFA) salt.

3.1. Dose Response Curves for Probe

Figure 3Dose response curves for the probe ML367 against the ATAD5-Luc primary screen (green) and the cell viability assay (red)

Results showed ML367’s dose response inhibition of ATAD5 activity without any significant cytotoxic effect.

3.2. Cellular Activity

Figure 4Inhibition of FLAG-ATAD5 stabilization by ML367

HEK293T cells were transiently transfected with FLAG-tagged ATAD5 and treated with the indicated amount of ML367 for 16 hours in the presence or absence of 20 μM 5-FUrd. ATAD5 protein levels were visualized by western blotting using an antibody against FLAG (top panel), and quantified using ImageJ (bottom panel). Results showed inhibition of ATAD5 expression upon treatment of the probe.

3.3. Profiling Assays

The preliminary absorption, distribution, metabolism, excretion (ADME) profile of ML367 supports its use as a valuable probe for ATAD5 destabilization. While both its microsomal stability (in rat and human) and solubility (in PBS buffer) are moderate, the latter was above the IC₅₀ determined in the cell based assay. ML367 has good PAMPA permeability, and its overall ADME profile is consistent with its measured Log D of 1.58 (Table 2). Additionally, ML367 showed good stability in mouse plasma as well as a series of aqueous stability assessments including pH 2 and pH 10 buffers and aqueous 5 mM glutathione (Figure 1). While the overall ADME profile may limit the utility of ML367 in vivo, results of solubility, permeability and biological activity showed good results.

Table 2

ADME profile of ML367.

Figure 1

Stability of ML367 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at room temperature over 48 hr in (A) DPBS buffer pH 7.4, (B) assay buffer, (C) pH 2 and (D) pH 10 buffers; and (E) 5 mM solution of (more...)

4.1. Comparison to Existing Art and How the New Probe is an Improvement

As no prior art for inhibitors of ATAD5 stabilization exist, ML367 represents an important tool for the scientific community to begin to understand the protein’s role in DNA repair as well as other biological modalities. First, ML367 can be used to dissect initial events in the DNA damage response. The molecular mechanism of action in which ML367 destabilizes TEL2 will unveil how DNA damage can activate TEL2 and its downstream targets. Many of these targets have been suggested to play important roles in tumorigenesis. Additionally, a number of genetic disorders are the result of mutations in these genes. For instance, ataxia telansiectasia (AT) is caused by a mutation in ATM, Seckel syndrome is caused by a hypomorphic mutation in ATR, and severe combined immunodeficiency is caused by a mutation in DNA-PKcs. As no prior art exists for inhibitors of ATAD5 or TEL2 stabilization, ML367 is positioned to be a novel tool to study the molecular mechanism of DNA damage response and the resultant signal cascade with a potentially wide application to cancer and other genetic diseases.