Discovery of the DNA-PKcs inhibitor DA-143 which exhibits enhanced solubility relative to NU7441

Abstract

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a vital role in DNA damage repair and lymphocyte function, presenting a significant target in cancer and immune diseases. Current DNA-PKcs inhibitors are undergoing Phase I/II trials as adjuncts to radiotherapy and chemotherapy in cancer. Nevertheless, clinical utility is limited by suboptimal bioavailability. This study introduces DNA-PKcs inhibitors designed to enhance bioavailability. We demonstrate that a novel DNA-PKcs inhibitor, DA-143, surpasses NU7441 in aqueous solubility as well as other available inhibitors. In addition, DA-143 displayed an improvement in DNA-PKcs inhibition relative to NU7441 achieving an IC₅₀ of 2.5 nM. Consistent with current inhibitors, inhibition of DNA-PKcs by DA-143 resulted in increased tumor cell sensitivity to DNA-damage from chemotherapy and inhibition of human T cell function. The improved solubility of DA-143 is critical for enhanced efficacy at reduced doses and facilitates more effective evaluation of DNA-PKcs inhibition in both preclinical and clinical development.

Introduction

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is crucial for genomic stability, orchestrating the repair of DNA double-strand breaks (DSBs) primarily through the non-homologous end-joining (NHEJ) pathway. Unlike homologous recombination (HR) which predominates during the S and G2 phases, NHEJ, facilitated by DNA-PKcs, repairs DSBs throughout the cell cycle. DNA-PKcs partners with the Ku70/Ku80 heterodimer, forming the active DNA-PK holoenzyme at DSB sites. This complex initiates a series of reactions that, while sometimes error-prone, are essential for the reconnection of DNA ends, leveraging the exonuclease Artemis among other factors for end processing¹. As a kinase, DNA-PKcs phosphorylates several crucial proteins involved in DNA repair, cell cycle progression, and apoptosis, modulating the cell's response to DNA damage. Its key role in DNA repair pathways makes DNA-PKcs a compelling target for therapeutic intervention^2–6, and it is currently being investigated for its synergy with anticancer agents like doxorubicin. Inhibitors of DNA-PKcs enhance the efficacy of radiation and chemotherapy by targeting its central function in the NHEJ repair mechanism. Beyond DNA repair, DNA-PKcs is essential to the development of the adaptive immune system^7,8, particularly in the recombination processes of antibody and receptor genes, further underscoring its therapeutic potential in immune system-related pathologies. For example, mice lacking functional DNA-PKcs exhibit a SCID (severe combined immunodeficiency) phenotype⁹. Recent studies have identified DNA-PKcs as a crucial element in T cell signaling and the cytoplasmic detection of foreign DNA^10,11. Our research is particularly focused on the role of DNA-PKcs in T cell activation. Beyond its implications in cancer therapy, we are investigating its utility in modulating the immune response, especially in mature T cells. Our findings indicate that DNA-PKcs is vital for mature T cell activation¹²; inhibitors of DNA-PKcs impede proper T cell response post-stimulation, highlighting its essential role in this process. Given the advancement of two DNA-PKcs inhibitors to phase I trials for safety assessment, there is promising potential for these inhibitors in treating immunological conditions. Particularly in transplantation, where therapeutic innovation has been limited, DNA-PKcs presents a new target for preventing tissue rejection but more preclinical testing is required. As our work with DNA-PKcs inhibitors continues, we have observed a significant problem with currently available inhibitors that greatly limits effective preclinical and clinical evaluation.

Belonging to the PI3K kinase family, DNA-PKcs is inadvertently affected by traditional PI3K inhibitors like Wortmannin and LY294002. However, specific inhibitors such as NU7026¹³ were among the first developed to target DNA-PKcs, enhancing cellular sensitivity to anticancer agents. Among the notable inhibitors are ATP-competitive agents like NU7441¹⁴, AZD7648¹⁵, and M3814¹⁶. NU7441, in particular, is a potent DNA-PKcs inhibitor with an IC₅₀ of approximately 14 nM, demonstrating specificity and potential in augmenting anti-cancer therapeutic strategies. However, NU7441 as well as other DNA-PKcs inhibitors face significant challenges with water solubility, complicating their preparation for in vivo studies, especially in the larger animal models required for clinical assessments. To overcome this limitation, we have synthesized and assessed a new inhibitor, DA-143, which maintains structural similarity to NU7441 but exhibits improved solubility. In our study, we provide an in-depth characterization of DA-143 in vitro, demonstrating its efficacy in cells undergoing DNA damage and its impact on activated T cells.

Methods

All experimental protocols were approved by the University of Arkansas for Medical Sciences/Arkansas Children’s Research Institute’s Institutional Biosafety Committee.

Chemicals synthesis

Details describing the synthesis of DA-143 can be found in the supplemental information.

Salt preparation (Fig. S1)

DA-143 hydrochloride: The HCl salt of DA-143 was prepared by dissolving DA-143 in ether and adding excess HCl until a precipitate formed. The precipitate was collected and dried.

Organic Salts (DA-143 acetate, tosylate, succinate, and tartrate): One equivalent of an organic acid was dissolved in diethyl ether, and one equivalent of DA-143 was slowly added to the stirring solution. After adding DA-143, the solution was left stirring for 45 min. The precipitate was collected and dried.

DA-143 phosphate: One equivalent of phosphoric acid was dissolved in diethyl ether, and one equivalent of DA-143 was slowly added to the stirring solution. The solution was left stirring for one hour, and the precipitate was collected and dried.

Solubility measurement

DA-143, NU7441 and salt forms of DA-143 were tested for aqueous solubility in phosphate buffered saline, pH7.2 (PBS). 10 mM DMSO stock solutions of the compounds were used to prepare a twofold serial dilution of each in PBS at a concentration range 100–0.1 μM. The absorbance was then measured at 600 nM to determine the solubility limit^17,18. DMSO percentage did not exceed 1% in the serial dilutions.

Cell lines and culture

Jurkat E6.1 human T cell leukemia and MC38 murine colon cancer cell lines were obtained from American Type Culture Collection (ATCC). Mice used in the study were housed in the Arkansas Children’s Research Institute Animal Facility. All animal studies were approved by and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences/Arkansas Children’s Research Institute. Mice were anesthetized by inhalation of 1–5% isoflurane and euthanized by cervical dislocation. Study is reported in accordance with The Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. Mouse T cells were isolated from the spleens of C56BL/6 mice using StemCell EasySep Mouse CD8 + T cell isolation kit #19853A and stimulated using 5 µg/ml plate-bound anti-CD3 (Biolegend #100340) and 5 µg/ml of anti-CD28 (Biolegend #102116). Human PBMCs were received frozen from a healthy donors (StemCell #70025) and stimulated with plate-bound anti-CD3 (Biolegend #317347) and anti-CD28 (Biolegend #302934) both at 5 µg/ml. T cells and PBMCs were cultured in RPMI with 10% FBS and 1X Penstrep (Corning #30-002-CL). MC38 cells were cultured in DMEM with 10% FBS and Penstrep.

Inhibitors

NU7441 and DA-143 were dissolved and stored frozen in DMSO at 5 mM, − 80 °C. DA-143 was synthesized in-house as described. NU7441 was purchased from Selleckchem.

Assays

In vitro DNA-PK kinase assay

The DNA-PK Kinase Enzyme System (Cat #V4106) and ADP-Glo Assay (Cat #V6930) from Promega were used to perform this assay. Inhibitors were used at 100 nM concentration and equivalent amounts of DMSO were added to samples without inhibitor.

IC₅₀ determinations

The IC₅₀ for DA-143 against DNA-PK, mTOR, ATM, and PI3KΔ was measured by Reaction Biology Corp. A Kinase HotSpot Assay was utilized in the case of DNA-PK and mTOR, the ADP-Glo Assay was utilized for PI3KΔ, and the HTRF (Homogeneous time-resolved fluorescence) assay was utilized for determining the IC₅₀ value for ATM. Three-fold serial dilutions of inhibitor starting at 10 µM were used to generate curves, and ATP was used at 10 µM.

Western blotting

Samples for Western blots of MC38 cells were pre-treated for 1 h with inhibitor, then treated with Doxorubicin for the indicated time. Samples were lysed in Cell Extraction Buffer (Thermo FNN0011) with protease inhibitors (Thermo Scientific #1860932) and phosphatase inhibitors (Thermo Scientific #78428) followed by sonication in a QSonica Q800R3 with the settings 30% amplitude, 30 s on/off, and 15 min sonication time. Jurkat Western blot samples for Fig. 4a were pre-treated with inhibitors, stimulated with 5 µg/ml anti-human CD3 and anti-human CD28 for 15 min, and lysed in 0.1% NP40 in water with protease and phosphatase inhibitors. Jurkat Western blot samples for Fig. 4b were pre-treated with inhibitors, stimulated with 1 µg/ml PHA (phytohaemagglutinin) and 50 ng/ml PMA (phorbol 12-myristate 13-acetate), and lysed similarly to the MC38 cells. Lysed samples were pelleted at 10,000 xg for 10 min, and supernatants were normalized by protein concentration determined using the bicinchoninic assay (BCA) (Thermo Scientific #23225). Samples were heated in LDS (lithium dodecyl sulfate) loading buffer (Thermo Scientific #B0007) then loaded into 4–12% bis–tris gels (Thermo Scientific #NW04122BOX). Transfer to a PVDF membrane was completed using a Pierce Power Blotter system run at 25 V for 10 min. Images were acquired as 8 bit TIF files from a GE ImageQuant LAS 4000. All uncropped images can be found in Supplemental File 2.

Fig. 4.

DA-143 sensitizes cancer cells to doxorubicin. (a) MC38 mouse colon cancer cells were pre-treated with inhibitors (5 µM) then exposed to a low dose (25 nM) of doxorubicin for 48 h. Annexin V staining was evaluated by flow cytometry, quantified in the bar graph. Dot plots are the corresponding flow cytometry plots with Annexin V staining on the X axis and 7-AAD staining on the Y axis. Error bars = s.d. of the mean of three replicates. *p < 0.001 by Student’s T test. (b) MC38 cells were treated with the indicated combination of doxorubicin and inhibitor for 24 h. The indicated proteins were detected from whole cell lysates by Western blotting to evaluate H2AX phosphorylation. (c) Both MC38 and Jurkat T cells were treated with doxorubicin for 3 h and whole cell lysates were probed by Western blotting to evaluate ATM autophosphorylation. The blots have been cropped to show only relevant proteins to improve the clarity and conciseness of the data. All samples were run on the same gel. Antibodies used a listed in the methods. Uncropped blots can be found in Supplemental File 2.

Primary antibodies

Anti-DNA-PKcs (Cell Signaling #38168), Anti-phospho-DNA-PKcs ser2056 (Invitrogen #PA5-78130), Anti-AKT (Cell Signaling #2938S), Anti-phospho-AKT ser473 (Cell Signaling #4058S), Anti-Kap1 (abcam #ab10483), Anti-phospho-Kap1 ser824 (Abcam #ab70369), Anti-Beta-actin (Thermo Scientific #MA1-140) Anti-GAPDH (Thermo Scientific #MA5-15738), Anti-ATM (Abcam #ab78), Anti-phospho-ATM ser1981 (Abcam # ab81292), anti-H2A (Cell signaling #12349S) and anti-phospho-H2AX (Cell signaling #25775). Secondary antibodies are as follows: Thermo Scientific goat anti-mouse IgG (H + L) Alexa Fluor Plus 647 (#A32728) and GE Healthcare donkey anti-rabbit HRP (#NA934V). Imaging was done with a GE ImageQuant LAS4000.

Apoptosis assay

Trypsinized MC38 cells were plated at 10,000 cells/well in a 96 well plate. The next day, 1000X stocks of each inhibitor was made in DMSO such that each well would receive the same amount of DMSO. Aliquots of each drug at assay concentration were then made in culture media. The media from the plated cells was removed by vacuum and the indicated inhibitor concentration in 100 µl of media was added to each well. Cells were pre-treated this way for 1 h before adding another 100 µl of media containing inhibitor and a 2X concentration of doxorubicin (from a 1 mM frozen stock). Cells grew at 37C with 5% CO₂ for 48 h. Cells were harvested by trypsinization and transferred to a 96 well V-bottom plate, and an eBiosciences Annexin V efluor 450 Apoptosis detection kit (#88-8006-74) was used to stain the cells. Flow cytometry was performed on an Attune NxT flow cytometer. The gating strategy used for flow cytometry analysis is as follows: Doublet discrimination was used based on forward scatter area and height profiles to gate on single cells.

Proliferation assay

After isolation or thawing, T cells were counted by flow cytometry to measure total CD8 + (mouse) or CD3 + (human) cells within the total population. The cells were stained with CellTrace Violet (Thermo Scientific #C34557) according to the kit. Cells were then plated at 1 million cells/ml and stimulated with anti-CD3 and anti-CD28 antibodies after being pre-treated with inhibitor. They were allowed to grow in culture for the indicated times at 37 °C/5% CO₂. Proliferation of mouse colon MC38 cells was measured similarly, where cells were plated in a 24 well plate at 50,000 cells/well the day before treatment and allowed to grow for two days post-treatment with inhibitors and/or doxorubicin. Analysis was done by flow cytometry on an Attune NxT flow cytometer.

Results

Design of new DNA-PKcs inhibitors

Due to poor aqueous solubility of the DNA-PKcs inhibitor NU7441, our objective was to create a new DNA-PKcs inhibitor featuring enhanced water solubility. The intention behind developing such an inhibitor with improved solubility characteristics is to streamline drug administration through various routes, including intravenous, while also enhancing potency by reducing desolvation penalties. This advancement may enhance the feasibility of studying DNA-PKcs across diverse animal models and may also simplify the adaptation of DNA-PKcs inhibitors in a clinical setting.

A co-crystal structure of NU7441 bound to DNA-PKcs was employed to identify critical binding regions and areas suitable for modification to enhance aqueous solubility¹⁹. Analysis of the co-crystal structure revealed that the dibenzothiophene segment of NU7441 occupies a solvent-exposed pocket that is non-essential for binding, suggesting this region could be altered to enhance solubility (Fig. 1a). The morpholine segment in NU7441 establishes a crucial hydrogen bond with the DNA-PKcs hinge, and any modifications at this site would jeopardize binding interactions. Consequently, water-soluble groups were incorporated into the dibenzothiophene motif by introducing a pyrazole (DA-138) and subsequently linking the pyrazole to pyrrolidine (DA-143) (Fig. 1b–d). Pyrrolidine, characterized by an ionizable functional group with a pKa range of 9–11, is readily ionizable at physiological pH, thereby offering a substantial potential to enhance aqueous solubility. Additionally, we explored another derivative of NU7441, incorporating an N-methylpiperazine at the solvent-exposed region (DA-147). However, this particular derivative was not given priority due to predictions indicating a suboptimal pharmacokinetic profile, attributed to the rapid N-dealkylation of the methyl group.

Fig. 1.

Design of DA-143. (a) The morpholine segment of NU7441 plays a crucial role in binding to the DNA-PKcs hinge, and any modification at this region will result in a loss of binding affinity. (b) In the case of DA-138 and DA-143, the morpholine segment remains unchanged, while modifications were made to the solvent-accessible region. The pyrazole on DA-138 serves as a tetherable site to introduce an aliphatic basic amine, enhancing solubility. (c, d) Predicted orientation of the pyrazole in both DA-138 and DA-143 is towards the solvent. Additionally, the pyrrolidine on DA-143, being highly ionizable, has the potential to improve water solubility. PDB ID: 7OTM.

The synthesis of DNA-PKcs inhibitors DA-138, DA-143, and DA-147 is outlined in Scheme 1 and Scheme 2. Initially, starting materials 1-(3-bromo-2-hydroxyphenyl)ethan-1-one (1) and N,N-dimethylformamide dimethyl acetal were dissolved in DMF and heated to 75 °C for one hour, yielding 1-(3-bromo-2-hydroxyphenyl)-3-(dimethylamino)prop-2-en-1-one (2). Compound 2 underwent reflux in DCM and HCl, resulting in 8-bromo-4H-chromen-4-one (3).

Scheme 1.

Synthesis of 2-morpholino-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one. Reagents and conditions: (a) DMF acetal, DMF, 75 °C; (b) DCM, HCl, reflux; (c) triazole, iodine, K₂CO₃, DMF, 80 °C; (d) morpholine, K₂CO₃, DMF, 80 °C; (e) bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, dioxane, 90 °C.

Scheme 2.

Synthesis of DNA-PKcs inhibitors. Reagents and conditions: (a) Iodine, iodobenzene diacetate, acetic anhydride, acetic acid, H₂SO₄; (b) Various boronic ester, Na₂CO₃, Pd(dppf)Cl2, 5:1 DMF:H2O, 80 °C or N-methylpiperazine, potassium tert-butoxide, BINAP, Pd(dppf)Cl2, toluene, 80 °C; (c) 2-morpholino-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one, K₂CO₃, tetrakis, dioxane, 90 °C.

The chromenone (3) was dissolved in DMF. Iodine and triazole were added, and the mixture was heated for 17 h, producing 8-bromo-2-(1H-1,2,4-triazol-1-yl)-4H-chromen-4-one (4). Subsequent nucleophilic substitution of compound 4 with morpholine yielded 8-bromo-2-morpholino-4H-chromen-4-one (5). Compound 5 underwent Miyaura borylation cross-coupling, resulting in 2-morpholino-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one (6), which resulted in the essential hinge region motif of the scaffold.

After synthesizing the hinge-binding motif, the synthesis of the solvent-front region was completed. 4-bromodibenzo[b,d]thiophene (7) was reacted with iodine and iodobenzene diacetate in the presence of acetic acid, acetic anhydride, and H₂SO₄, which furnished 6-bromo-2-iododibenzo[b,d]thiophene (8). Suzuki or Buchwald-Hartwig cross-coupling of compound 8 with boronic esters or amines, followed by Suzuki coupling, ultimately yielded final compounds DA-138, DA-143, and DA-147.

Biochemical evaluation of new DNA-PKcs inhibitors

From the synthesis outlined, three candidate DNA-PKcs inhibitors were generated: DA-138, DA-143, and DA-147 (Fig. 2a). To assess their capability to inhibit DNA-PKcs kinase activity, a comparative analysis with known DNA-PKcs inhibitors NU7441 and M3814 was completed. An in vitro phosphorylation assay (Fig. 2b) was employed, which indirectly measured kinase activity by monitoring ATP to ADP conversion. Treatment with NU7441, M3814, DA-143, and DA-147 led to an almost complete loss of detectable kinase activity compared to the vehicle-treated sample. NU7441, DA-147, and DA-143 demonstrated statistically comparable inhibitory effects in relation to each other. DA-138 exhibited inhibition of DNA-PKcs, but to a much lesser amount.

Fig. 2.

DA-143 exhibits comparable activity to NU7441 in vitro with improved solubility. (a) DNA-PK inhibitors tested in this study. (b) Inhibitor activity with an in vitro assay measuring ATP to ADP conversion by DNA-PKcs enzyme in the presence of a polypeptide substrate. (c) Solubility of NU7441 and DA-143 in phosphate buffered saline (PBS) at pH 7.2 determined via turbidimetric solubility measurement. Error bars = s.d. of the mean of three replicates. *Indicates p < 0.001 by Student’s T test. ***Indicates p > 0.1 by T test between each sample pairing.

Solubility studies of DA-143

Having confirmed that the new inhibitors exhibited a comparable impact on DNA-PKcs kinase activity to inhibitors in clinical studies, our next objective was to assess their solubility in comparison to NU7441. Despite both DA-143 and DA-147 exhibiting similar activity, we opted to advance with DA-143 due to considerations of metabolic stability. The N-methyl group in the solvent front region of DA-147 is susceptible to N-dealkylation through CYP enzymes. In contrast, the presence of steric bulk around the pyrrolidine nitrogen in DA-143 is expected to impede N-dealkylation, making it a more favorable choice from a metabolic stability standpoint.

We conducted a comparative solubility analysis between the DNA-PKcs inhibitor NU7441 and DA-143 (Fig. 2c). The key distinction between the two inhibitors is the 1-(2-(pyrrolidine-1-yl)ethyl)-1H-pyrazole motif at the solvent front region of DA-143, which is hypothesized to enhance solubility due to the ionizable nature of the pyrrolidine ring at physiological pH. We prepared stock solutions of both inhibitors in DMSO and N-methyl pyrrolidine (NMP). The maximum concentration of NU7441 achievable without precipitation in dimethyl sulfoxide (DMSO) and N-methyl-2-pyrrolidone (NMP) was 16.5 mg/mL (40 mM) and 42 mg/mL (100 mM), respectively. Beyond these concentrations, NU7441 was insoluble despite sonication or heating. In contrast, DA-143 exhibited solubility at 200 mg/mL (~ 350 mM) in both solvents. This indicates that DA-143 possesses approximately 9 times better solubility in DMSO and 4 times better solubility in NMP compared to NU7441.

After determining that DA-143 exhibited improved solubility in both DMSO and NMP, we further investigated the solubility of both compounds in an intravenous formulation containing PEG/NMP/EtOH/H₂O at 50/12/10/28 v/v that has been successfully utilized in large animals²⁰. We prepared stock solutions of both inhibitors in NMP and then added the stock into PEG/EtOH/H₂O to achieve a final concentration of 10 mg/mL. It is noteworthy that NU7441 exhibited immediate precipitation upon the addition of the NMP stock into the formulation, while DA-143 remained soluble within the formulation. This showcases the superior solubility of DA-143 compared to NU7441 and raises concerns about the potential formation of hazardous embolisms when NU7441 is administered intravenously. Emphasizing the importance of considering solubility characteristics, especially for intravenous use, this observation underscores the enhanced solubility of DA-143 in the tested formulation suggesting its suitability for intravenous use, which will maximize applications in both pre-clinical and clinical settings.

We subsequently evaluated the aqueous solubility of both DA-143 and NU7441. Stock solutions of the inhibitors were serially diluted and added to phosphate-buffered saline (PBS, pH 7.2) and DA-143 exhibited approximately five times higher aqueous solubility compared to NU7441 (Fig. 1C and Table 1). This enhancement in solubility is likely due to the introduction of an ionizable pyrrolidine functional group in DA-143, which ionizes readily at physiological pH, a feature absent in NU7441. To further examine solubility, we exploited the basicity of the pyrrolidine group by preparing various salt forms of DA-143, including hydrochloric, acetate, tosylate, succinate, tartrate, and phosphate salts. These salt forms were then assessed for aqueous solubility relative to DA-143 and NU7441 (see Fig. S2). Notably, the tartrate and phosphate salts exhibited a 14- and 13-fold increase in solubility compared to NU7441 (Table S1). This significant improvement in solubility is crucial as it can lead to enhanced absorption and bioavailability of the inhibitor.

Table 1.

Solubility of DNA-PKcs inhibitors in phosphate buffered saline at pH 7.2

Compound	MW	Log P	Solubility limit (μM)	Solubility limit (μg/ml)
NU7441	413.11	5.02	6.25	2.58
DA-143	576.71	5.35	25	14.42

IC₅₀ determination for DA-143

NU7441 is reported to have an IC₅₀ of 14 nM and highly selective for DNA-PKcs. Therefore, to determine if the improved solubility of DA-143 impacted potency, we determined the IC₅₀ of DA-143. Here we show DA-143 potently inhibited DNA-PKcs with an IC₅₀ of 2.5 nM, a significant improvement compared to the reported IC₅₀ of NU7441. In comparison, DA-143 displayed an IC₅₀ for mTOR, PI3KΔ, and ATM at 280 nM, 106 nM and 6,594 nM respectively. These results show significant selectivity of DA-143 for DNA-PKcs over other closely related kinases (Fig. 3).

Fig. 3.

IC₅₀ values for DA-143. DA-143 was screened against DNA-PKcs, mTOR/FRAP1, PI3KΔ, and ATM. To generate IC₅₀ curves, DA-143 was tested using a 10-point, threefold serial dilution starting at 10 µM in triplicate. Percent activity for each enzyme at each concentration was determined, and the data was analyzed using GraphPad Prism with a one-phase decay model.

DA-143 sensitizes cancer cells to doxorubicin

DNA-PKcs inhibitors increase the sensitivity of cancer cells to DNA-damaging chemotherapies by preventing the ability of cancer cells to rapidly repair DNA damage. Therefore, to confirm that DA-143 maintained the same affects as its parent compound, we tested the ability of DA-143 to increase MC38 cancer cell chemosensitivity to doxorubicin by measuring the level of apoptosis following treatment with the DNA DSB-inducing chemotherapy. As a popular anti-cancer therapy, doxorubicin administration in combination with DNA-PKcs inhibitors has been explored as a combination therapy to enhance cancer treatment regimens. As expected, MC38 cancer cells were largely resistant to doxorubicin treatment as evident by a low level of annexin V positive cells following 48 h of treatment. When MC38 cells were treated with either inhibitor alone at 5 µM concentration, very little differences in annexin V staining were observed compared to vehicle treated controls (No Treatment, 0 µM inhibitor) suggesting that the inhibitors alone did not induce apoptosis at 48 h. However, combining doxorubicin treatment (25 nM) with NU7441 or DA-143 (5 µM) resulted in a 2.5-fold increase in annexin V staining (> 50% of cells), suggesting an increased vulnerability to DNA damage as would be expected in the absence DNA-PKcs activity (Fig. 4a). Consistent with these findings, DA-143 elicited an increase in phosphorylation of s139 of the histone variant H2AX after 24 h of doxorubicin exposure, suggesting that the inhibitor efficiently blocks DNA-PKcs activity and therefore the ability to repair double-strand breaks (Fig. 4b). This phosphorylation event serves as a marker for DNA damage induction and subsequent repair²¹. These results provide evidence that DA-143 is an efficient inhibitor of DNA-PKcs and its activity in DNA damage repair. To rule out the possibility of our observations being due to the effect of DA-143 on the related DNA repair kinase ATM which also plays an important role in facilitating the response to DNA damage, we blotted for ATM autophosphorylation at serine 1981 (or serine 1987 in mouse). Similarly to DNA-PK, ATM is autophosphorylated upon detection of DNA damage and helps to determine which DNA repair pathway is chosen. We found that ATM is indeed phosphorylated at serine 1981 3 h after doxorubicin treatment in two different immortalized cell lines we tested—both MC38 mouse colon cells and Jurkat human lymphoma T cells (Fig. 4c). However, this phosphorylation event is not attenuated by treatment with either DA-143 or NU7441 suggesting that treatment at 5 µM is not inhibiting ATM activity.

DA-143 blocks phosphorylation of DNA-PKcs substrates

The most direct method of determining if the new inhibitor is effective in a cellular context is to compare phosphorylation levels of known DNA-PKcs substrates. Activation of DNA-PKcs occurs through autophosphorylation at serine 2056. To validate that DA-143 blocks the activation and kinase activity of DNA-PKcs in cells, we analyzed phosphorylation of s2056 in Jurkat T cells following activation through the T cell receptor. We observed a stark increase in s2056 phosphorylation after about 15 min of activation. However, cells treated with either inhibitor almost completely inhibited phosphorylation of serine 2056 indicating it is capable of inhibiting the activation and kinase activity of DNA-PKcs (Fig. 5a). We also observed phosphorylation of Kap1/Trim28. Kap1 phosphorylation at serine 824 is reported to be dependent on DNA-PKcs²². Kap1 is a transcriptional mediator known to be involved in DNA damage responses as well as control the expression of genes involved in T cell receptor signaling^23,24.

Fig. 5.

DA-143 treatment blocks phosphorylation of DNA-PKcs substrates. (a) Jurkat T cells were pre-treated with DNA-PKcs inhibitors and stimulated for 10 min before harvesting and Western blotting for DNA-PKcs autophosphorylation at serine 2056 and Kap1 phosphorylation at serine 824. (b) Jurkat cells were pre-treated with inhibitors and stimulated for 2 h before harvesting and Western blotting for AKT phosphorylation at serine 473. Blots have been cropped to shown only relevant proteins to improve the clarity and conciseness of the data. All samples were run on the same gel. Black dotted line indicates where lanes were non-adjacent on the blot. Antibodies used are listed in the methods. Uncropped blots can be found in Supplemental File 2.

We observe a significant drop in the level of phosphorylation at serine 824 with NU7441 and DA-143 treatment following T cell activation. A third phosphorylation event we observed was that of serine 473 on the kinase AKT. This is thought to be a target of multiple kinases, including DNA-PKcs²⁵. We found that treatment with DA-143 also caused a drop in levels of AKT phosphorylation after stimulation in Jurkat cells (Fig. 5b). These observations both provide evidence of DA-143 inhibitor activity toward DNA-PKcs as well as contribute to the unfolding narrative that DNA-PKcs has a role to play in early T cell activation.

DA-143 alters T cell function

Our laboratory reported that DNA-PKcs is critical for the function of primary T cells¹². The DNA-PKcs inhibitors NU7441, M3814 and AZD7648 inhibited the proliferation and activity of both CD4⁺ and CD8⁺ T cells. Here, we set out to determine if DA-143 had similar effects on primary T cell function. We activated both human PBMCs and isolated mouse CD8⁺ T cells with anti-CD3/anti-CD28 antibodies and tracked proliferation using CellTrace Violet dye. While activated vehicle-treated (DMSO) T cells proliferated aptly, cells treated with NU7441 or DA-143 had a significant disruption in proliferation. At a concentration of 5 µM DA-143, T cells were unable to divide for more than 1 generation (both human and mouse). The same concentration of NU7441 also inhibited proliferation, but to a lesser extent than DA-143. (Fig. 6a,b). To test whether this effect is potentially a general effect on cells or specific to T cells, we evaluated proliferation in MC38 mouse colon cancer cells. We found that both NU7441 and DA-143 have an effect on proliferation and cell survival at this concentration. DA-143 causes a major defect in proliferation and cell survival. NU7441 treatment causes a much less exaggerated effect but still slows cell proliferation (Fig. 6e).

Fig. 6.

DA-143 prevents proper T cell activation. DNA-PKcs inhibitors and proliferation: (a) Isolated human PBMCs were stained with CellTrace Violet, then treated with 5 µM inhibitor or vehicle and stimulated with anti-CD3/anti-CD28 and cultured for 5 days. Proliferation was assessed by flow cytometry gating on CD3 + cells. (b) Isolated mouse CD8 + cells were stained with CellTrace Violet, then treated with 5 µM inhibitor or vehicle and stimulated with anti-CD3/anti-CD28 and cultured for 3 days. DNA-PKcs inhibitors and cytokine expression: (c) IL2 expression in human PBMCs after 48 h and (d) IL2 expression in mouse CD4 + T cells after 24 h with DNA-PKcs inhibitors (10 µM) or vehicle control was measure by ELISA from cell culture media. Error bars = s.d. of the mean of three replicates. * indicates p < 0.05 by Student’s T test (1-tailed). **Indicates p < 0.5 by T test (2 tailed). (e) An immortalized cell line, MC38, was assessed for proliferation using CellTrace Violet after treatment with 5 µM inhibitors for 2 days.

The cytokine Interleukin-2 (IL2) produced by T cells is vital for the expansion and activity of numerous immune cell populations. Our previous studies indicate that DNA-PKcs is critical for the proper expression of IL2 following T cell activation as inhibitors NU7441, M3814 and AZD7648 significantly reduced IL2 production in T cells. DA-143 had similar effects reducing IL2 production in primary human T cells as well as mouse CD4⁺ T cells following activation with anti-CD3/anti-CD28 antibodies. As observed with proliferation, DA-143 at lower concentrations had a stronger inhibitory effect on IL2 production than NU7441. These results further confirm the necessary role of DNA-PKcs in T cell activity and highlight the potential use of this new inhibitor in the treatment of T cell mediated diseases and disorders. (Fig. 6c,d).

Discussion

As the role of DNA-PKcs extends beyond DNA repair, encompassing functions in specific cell types like neurons and immune cells, the application of DNA-PKcs inhibitors for a broad spectrum of diseases is garnering significant interest. The exploration of these inhibitors' clinical utility is often hampered by their poor aqueous solubility. In our research, we introduce the DNA-PKcs inhibitor DA-143, which we developed to enhance water solubility. Beginning with the structure of NU7441, we incorporated an ionizable group to create a new compound that retains the inhibitory capabilities of NU7441 yet demonstrates a five-fold increase in aqueous solubility. This property is crucial for intravenous administration, affecting the maximum concentration at which a drug can be administered without the risk of precipitation in aqueous, minimally organic solvent-containing therapeutic formulations. In addition, DA-143 displays high selectivity for DNA-PKcs with an IC₅₀ of 2.5 nM, significantly lower than the reported 14 nM for NU7441. This suggests DA-143 can be used at lower concentrations compared to NU7441 or other DNA-PKcs inhibitors that have poor solubility.

In our comparative studies, DA-143 matched NU7441 in inhibitory efficacy in kinase assays and exhibited significant effectiveness in cell culture models. We assessed the impact of DA-143 on the DNA repair function of DNA-PKcs by examining apoptosis and DNA damage markers post-doxorubicin treatment. The results showed that DA-143 enhances the sensitivity of cancer cells to doxorubicin, indicated by increased apoptosis rates, likely due to persistent DNA damage as suggested by elevated H2AX phosphorylation levels. Moreover, DA-143 also demonstrated an ability to inhibit T cell activity, reducing proliferation and IL2 cytokine production. These findings position DA-143 as a promising candidate for further preclinical and clinical evaluations, offering insights into DNA-PKcs inhibitors' roles not only in cancer therapy but also in managing T cell-mediated conditions, including autoimmune disorders and transplant rejection.

Author contributions

Z.W. performed cell-based assays and wrote the paper. B.A. and D.A. developed DA-143. MH assisted in experimental design. R.R., S.A., M.F., A.C.A., and A.B. performed experiments. L.B. assisted in experimental design and data analysis. B.F. and M.S.B. assisted in experimental design and data analysis, wrote the paper and provided financial support. All authors reviewed the manuscript.

Funding

This work was supported by Arkansas Children’s Research Institute, the University of Arkansas for Medical Sciences Department of Surgery, the National Institutes of General Medical Sciences (P20GM109005), a UAMS College of Pharmacy Seed grant, and a 2023 UAMS College of Pharmacy Summer Research Fellowship.

Data availability

The data used and/or analyzed during the current study are included in this article and are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-70858-w.