N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274): a potential novel PI3K inhibitor with low cardiotoxicity in mice

doi:10.21203/rs.3.rs-3990537/v1

Download PDF

Research Article

N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274): a potential novel PI3K inhibitor with low cardiotoxicity in mice

https://doi.org/10.21203/rs.3.rs-3990537/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Considering the common occurrence of the adverse cardiovascular events caused by PI3K inhibitors in clinical trials, a comprehensive assessment of the cardiotoxicity of promising PI3K inhibitor candidates for their potential clinical translation is of great importance. Based on our previous studies of the 7-azaindazole-based PI3K inhibitors, in this work, we report a potential novel PI3K inhibitor, N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274), with low cardiotoxicity in mice. FD274 exhibited minimal adverse effects in inducing cardiac dysfunction, oxidative stress and cardiac injury when compared to the positive control FD268, a bioisostere of FD274. Additionally, western blot analyses suggested that the minimal adverse effects of FD274 could be related to the preservation of the activity of the antioxidant pathway protein Nrf2. In contrast, the downregulation of Nrf2 as well as the upregulation of NADPH oxidase and the apoptosis-related proteins resulted in the cardiotoxicity of FD268. Finally, we confirmed the low cardiotoxicity of FD274 after the 20-day treatment process in the HL-60 xenograft mice model. Our results confirmed the low cardiotoxicity of FD274 which deserves further development.

7-azaindazole

PI3K inhibitor

cardiotoxicity

oxidative stress

Nrf2

Drug-induced cardiotoxicity, as one of the major reasons for the withdrawal of drugs from the market, takes the blame for up to 33% of drug failure^{[1, 2]}. Despite served as a target for cancer therapies, the PI3K pathway plays a cardioprotective role, leading to the cardiotoxic risk of PI3K inhibitors^{[3, 4]}. In fact, the clinical use of several PI3K inhibitors such as copanlisib, alpelisib, BKM120 and so forth has been reported to be hampered by cardiac-related toxicities^{[3, 5, 6]}, which suggests the importance of a comprehensive assessment of the cardiotoxicity of promising PI3K inhibitors as early as the development stage. One of the major standards for reporting cardiotoxicity induced by antitumor drugs is the drop in left ventricle ejection fraction (LVEF), which indicates a decline in the cardiac function^{[7, 8]}. As the most commonly used research animals, rodent model, particularly the mice model, is increasingly utilized in cardiovascular researches^{[9, 10]}, and echocardiography is ideally suitable for the evaluation of the cardiac function^[11–13], facilitating the assessment of potential cardiotoxic effects of antitumor drugs.

The generation of oxidative stress is one of the main mechanisms underlying the cardiotoxicity caused by antitumor drugs^[14–16]. Rather than a simple imbalance between the production and clearance of reactive oxygen species (ROS), oxidative stress is related to the dysfunction of enzymes involved in the ROS production^[17]. Nox2 (gp91phox), one of the NADPH oxidases, plays a predominant role in the accumulation of ROS in response to doxorubicin (DOX) exposure in mammalian hearts, which participates in the generation of superoxide anion and hydrogen peroxide^{[14, 17, 18]}. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major regulator of cellular responses against oxidative stress, and its down-regulation leads to a decrease in the activity of antioxidant enzyme^{[19, 20]}. The PI3K/Akt pathway is involved in the regulation of the oxidative stress response^[21–23], raising the concern whether the inhibition of the PI3K/Akt signaling pathway would increase the susceptibility of oxidative stress and the subsequent cardiotoxicity.

We have developed a series of novel PI3K inhibitors based on the structural cornerstone of 7-azaindazole^[24]. Among them, compound N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274) has been screened out for its high in-vitro and in-vivo potency against PI3Ks^[25]. From the viewpoint of the structure-cardiotoxicity relationship for the information of the design strategy of novel PI3K inhibitors, the potential cardiotoxicity of the 7-azaindazole-based PI3K inhibitors has been preliminarily examined by using zebrafish model, and FD274 exhibited the lowest cardiotoxicity among three bioisosteres under test^[26]. And the present study further aimed to perform a comprehensive assessment of the cardiotoxicity of FD274 for its potential clinical translation. Our results shown its minimal cardiotoxic effects in inducing cardiac dysfunction, oxidative stress and cardiac injury in C57BL/C mice model and H9C2 cell model, which associated with the preservation of Nrf2 antioxidant pathway. In contrast, FD268, a bioisostere of FD274 which served as the positive control, exhibited cardiotoxicity associated with the downregulation of Nrf2 as well as the upregulation of NADPH oxidase and the apoptosis-related proteins. Furthermore, after consecutive 20-day intraperitoneal (i.p.) administration of FD274, the HL-60 xenograft model mice didn’t exhibit any adverse cardiovascular effects. These studying results suggested that FD274 is a promising 7-azaindazole-based PI3K inhibitor with low cardiotoxicity that worth further development.

General

The synthesis of FD274 and FD268 was reported in our previous work^[24]. Adult female C57BL/6 mice (6 ~ 8 weeks old) and male BALB/C nude mice (4 ~ 5 weeks old) were respectively maintained in an animal room on a 12/12 h light/dark cycle with free access to water and food. All experiments were performed in strict accordance with the guidelines of the China Council on Animal Management. The protocols were approved by the Committee on the Ethics of Animal Experiments of Fudan University. H9C2 cell line was purchased from Procell Life Science&Technology Co., Ltd. (Wuhan, China), and were cultivated in a 5% CO₂ incubator at 37°C with high-glucose DMEM (Genombio, Zhejiang, China) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1× penicillin and streptomycin.

Drug administration

C57BL/6 mice were randomly divided into three groups (n = 3), which were control group, FD274 group and FD268 group. The control group was treated with the vehicle which is saline containing 40% PEG400 and 5% Tween-80 (v/v). The FD274 and FD268 group were respectively treated with these two compounds dissolved in vehicle to a dose of 7.5 mg/kg.

The HL-60 xenograft mice model was established as described in the previous work^[25]. Briefly, BALB/C nude male mice were subcutaneously implanted 5 × 10⁶ HL-60 cells in the right axilla region. When the tumors reached to 50 ~ 100 mm³, the mice were randomly divided into five groups (n = 3), which were the control group, FD274-5 mg/kg group, FD274-7.5 mg/kg, FD274-10 mg/kg, and the BEZ235 group (n = 3). The control group was subjected to the i.p. administration of the vehicle, saline with 40% PEG400 + 5% Tween-80, every day. The FD274 groups were subjected to the i.p. administration of FD274 suspended in the vehicle at the doses of 5 mg/kg/day, 7.5 mg/kg/day and 10 mg/kg/day, respectively. The positive group was subjected to the i.p. administration of BEZ235 (MedChemExpress LLC, Shanghai, China) suspended in the vehicle at the dose of 5 mg/kg/day. After consecutive 20-day administration, mice were euthanized, and the blood and the heart of mice in each group was prepared for the serum biochemical analysis, histopathological analysis and myocardial apoptosis determination.

Echocardiography

Mice were anesthetized by a small animal gas anesthesia machine, with 1 ~ 2% isoflurane in the room air. M-mode images were acquired by a Visual Sonic high-resolution 3100 system (FUJIFILM Visual Sonics, Canada). Ejection fraction (EF), fractional shortening (FS), left ventricular diameter (LVID), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular anterior wall end-systolic dimension (LVAWs), LVAW-diastolic dimension (LVAWd), left ventricular posterior wall end-systolic dimension (LVPWs) and LVPW-diastolic dimension (LVPWd) were recorded.

Serum biochemical analysis

The blood of mice was collected and stood overnight at 4°C, and was centrifuged at 3000 rpm for 10 min. The determinations of the levels of Glutathione peroxidase (GSH-PX, GPX), malondialdehyde (MDA), Creatine Kinase (CK), Creatine Kinase, MB form (CK-MB), lactate dehydrogenase (LDH) and LDH1 in serum were performed by using the corresponding commercial kits according to the instructions. All kits were purchased from Jiancheng Institute of Biological Engineering (Nanjing, China).

Histopathological analysis

The heart tissues were weighted by an analytical balance, and were placed in 4% Paraformaldehyde Fix Solution (Sangon Biotech, Shanghai, China). The fixed heart was embedded in paraffin, and was cut into 4-µm-thick sections for H&E staining and Masson trichrome staining. Three 400× microscopic fields from each section were captured using a fluorescence microscope (Zeiss, Oberkochen, Germany). The H&E staining for the liver, spleen, lung and kidney tissues was performed accordingly.

Determination of myocardial apoptosis

Myocardial apoptosis was examined by using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. A TUNEL Apoptosis Detection Kit (Servicebio, China) for detecting DNA fragmentation was used for tissue sections according to the manufacturer’s instructions. The fluorescent signal was captured using a fluorescence microscope.

Determination of cell viability

The cell viability was determined using a Cell Counting Kit-8 (CCK-8) kit (Vazyme, Nanjing, China). FD274 and FD268 were firstly dissolved in DMSO (Sinopharm Chemical Reagent Co., Ltd., Beijing, China) respectively to prepare 1000× stock solution, and then were diluted with cultivation medium to the concentrations of 0.1, 0.2, 0.5, 1 and 2 µM to prepare the corresponding working solutions. H9C2 cells were planted at a density of 5000 cells/well in 96-well plates (Thermo Scientific Nunc, USA), and stood overnight in incubator. Then, cells were respectively treated with FD274 and FD268 working solutions prepared as above, along with 0.1% DMSO as control. Blank wells added equal amount of culture medium were set as background. After 24-h-treatment, 10 µL CCK-8 solution was added to all treatment, control and background wells and were placed in incubator for 2 h. The cell viability was calculated according to the absorbance read by a microplate reader (SYNERGY H1, BioTek) at 450 nm of each well by using the following formula: (OD_treatment – OD_background)/(OD_control – OD_background) ×100%.

Determination of intracellular ROS level

The 0.1, 0.2, 0.5 and 1 µM FD274 and FD268 working solutions were prepared as described above. The intracellular ROS level was determined by DCFH-DA (Beyotime Biotechnology, Shanghai, China) staining. H9C2 cells were planted at a density of 1.5 × 10⁵ cells/plate in 35mm Confocal Dishes (Biosharp, Anhui, China), and were respectively treated with 0.1, 0.2 and 0.5 µM FD274 and FD268 working solutions. After 24-h-treatment, DCFH-DA staining was performed according to the manufacturer’s instructions. The fluorescent signal was captured using a confocal laser scanning microscopy (CLSM) (Nikon, Japan). The fluorescent intensity of each group was measured by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Flow cytometry (FCM) was also performed to determine ROS level. H9C2 cells were planted at a density of 6 × 10⁵ cells/plate in 60 mm dishes (Thermo Scientific Nunc, USA), and were respectively treated with 0.1, 0.2, 0.5 and 1 µM FD274 and FD268 working solutions. After 24-h-treatment, cells were collected, and DCFH-DA staining was performed according to the manufacturer’s instructions. The positive cell ratio and mean fluorescence intensity were detected by FCM (Beckman Coulter, USA).

Determination of apoptosis level

The 0.1, 0.2, 0.5 and 1 µM FD274 and FD268 working solutions were prepared as described above. H9C2 cells were planted at a density of 6 × 10⁵ cells/plate in 60 mm dishes, and were respectively treated with 0.1, 0.2, 0.5 and 1 µM FD274 and FD268 working solutions. After 24-h-treatment, cells were collected, and Annexin V FITC/PI staining was performed according to the manufacturer’s instructions of kit (Beyotime Biotechnology, Shanghai, China). FCM was performed, and the apoptosis level in each group was measured as the ratio of the Annexin V positive and both Annexin V and PI positive cells.

Western blot

The 0.5 and 1 µM FD274 and FD268 working solutions were prepared as described above. H9C2 cells were planted at a density of 6 × 10⁵ cells/plate in 60 mm dishes, and were respectively treated with 0.5 and 1 µM FD274 and FD268 working solutions. After 24-h-treatment, cells were collected, and then proteins were obtained by using cell lysis buffer (CST; Shanghai, China) containing 1× phosphatase and protease inhibitor Cocktail (Roche; Basel, Switzerland).

Protein concentration was determined by the BCA protein assay kit (Thermo Scientific, USA). The protein samples were subjected to SDS-PAGE gel with sample volume of 30 µg. After the SDS-PAGE finished, proteins were transferred to PVDF membranes (Sigma-Aldrich, Germany). The membranes were blocked with 5% BSA in TBST for 1.5 h, and then were incubated with primary antibody for 14 ~ 16 h at 4°C. After washing for 3 times with TBST, the membrane was treated with secondary antibody (1:3000 dilution, CST, USA) at room temperature for 1 h. The target bands were visualized using an enhanced ECL detection kit by ChemiDocTM MP imaging system (BioRad, Shanghai, China).

Statistical analysis

Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using Graphpad Prism 8.3.0 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance among three or more groups were determined by one-way ANOVA, and Dunnett was performed to correct for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

The minimal cardiotoxicity of FD274 to C57BL/6 mice

The body weight change of mice on days 0 ~ 4 after respectively single i.p. administration of FD274 and positive control FD268 was recorded to investigate their cardiotoxicity to mice. As shown in Fig. 1b, the body weight of the FD274 group and the control group increased steadily, while that of the FD268 group exhibited a sharp decrease immediately after the administration but followed by a slow recovery. After the mice were euthanized, their hearts were weighed and the heart/body weight ratios were calculated. As shown in Fig. 1c ~ d, there was no significant difference in heart weight and heart/body weight ratio between the control group and the FD274 group, while a significant decrease in heart weight and heart/body weight ratio was observed in FD268 group compared to the control group (P < 0.0001). The results above suggested the minimal cardiotoxicity of FD274.

The minimal toxic effects of FD274 in inducing cardiac dysfunction

M mode echocardiography was performed to examine cardiac function parameters of mice after the i.p. administration of compounds under test (Fig. 1e ~ k). The FD274 group shown no significant changes in LVIDs, LVIDd, LVESV, LVEDV, EF and FS, which suggested its well cardiac function. By contrast, the FD268 group shown significant increase in LVIDs, LVIDd, LVESV and LVEDV, and decrease in the EF and FS, which suggested its cardiac dysfunction. Additionally, neither FD274 nor FD268 influenced the left ventricular wall dimension (Fig. S1). All these results suggested the minimal toxic effects of FD274 in inducing cardiac dysfunction.

The minimal toxic effects of FD274 in inducing oxidative stress in mice serum

The MDA and the GSH-PX levels in the mice serum after the i.p. administration of compounds under test were examined to investigate their effects on oxidative stress (Fig. 2a ~ b). FD274 did not cause abnormalities in the levels of MDA and GSH-PX. By contrast, FD268 caused a significant increase in MDA level (P < 0.01), which suggested high lipid peroxidation level, and a significant decrease in the activity of glutathione oxidase (P < 0.05), which suggested the impaired ability to remove peroxides. All these results suggested the minimal toxic effects of FD274 in inducing oxidative stress in mice serum.

The minimal toxic effects of FD274 in inducing cardiac injury

To visualize myocardial histological alterations, the H&E and Masson trichrome staining of cardiac tissue sections were performed. As shown in Fig. 2c, FD274 didn’t influence the cardiomyocyte morphology, while the FD268 group exhibited extensive cytoplasmic vacuolization as well as the deposition of blue collagen, suggesting the myocardial injury induced by FD268. Additionally, neither FD274 nor FD268 induced cardiac apoptosis (Fig. S2). All these results suggested the minimal toxic effects of FD274 in inducing cardiac injury.

Additionally, the toxic effects of FD274 and FD268 on other organs were determined. As shown in Fig. S3, the nephridial tissue section in the FD268 group exhibited glomerulus atrophy, tubular vacuolization and the epithelial cells detachment, and the hepatic tissue section in the FD268 group exhibited inflammatory infiltration in the central veins of hepatic lobules. No obvious toxic effect was observed in organs in the FD274 group, suggesting its minimal adverse effect.

The minimal toxicity of FD274 to H9C2 cells

H9C2 cell line was served to investigate the mechanism of the cardiotoxicity of 7-azaindazole-based PI3K inhibitors, and the cell viability of H9C2 cells after respectively treated with various concentrations of FD274 and FD268 was firstly determined. As shown in Fig. 3a ~ b, over the concentration ranges tested, the cell viability of FD274 group remained higher than 90%. By contrast, the cell viability decreased to 88.43 ± 12.52% when treated with 0.5 µM FD268; after the treatment with 1 µM FD268, the cell viability decreased to 51.62 ± 2.13%, which is significantly different to the control group (P < 0.0001). The results above suggested the minimal toxicity of FD274 to H9C2 cells.

The minimal toxic effects of FD274 in inducing ROS over-production in H9C2 cells

DCFH-DA staining was performed to visualize the ROS level of H9C2 cells after respectively treated with various concentrations of FD274 and FD268. As shown by CLSM images (Fig. 3c ~ f), no obvious ROS positive signal was observed in H9C2 cells after treated with FD274 over the concentration ranges tested; while after the treatment with 0.5 µM FD268, obvious ROS fluorescence signal was observed in H9C2 cells, and the ROS fluorescence intensity was significantly higher than that in the control group (P < 0.01). Similarly, the FCM results further confirmed the low ROS positive cell ratio and mean ROS fluorescence intensity in H9C2 cells after treated with FD274 over the concentration ranges tested (Fig. 3g ~ i). By contrast, 0.2, 0.5 and 1 µM FD268 induced the significant increase in both the ROS positive cell ratio and mean ROS fluorescence intensity compared with the control group (Fig. 3g, j and k). All these results suggested the minimal toxic effects of FD274 in inducing ROS over-production in H9C2 cells.

The minimal toxic effects of FD274 in inducing apoptosis in H9C2 cells

The apoptosis level of H9C2 cells after respectively treated with various concentrations of FD274 and FD268 was visualized by Annexin V FITC/PI staining. As shown in Fig. 4a ~ c, FD274 didn’t induce the apoptosis level over the concentration ranges tested; while after treated with 1 µM FD268, significant increase in the apoptosis level in H9C2 cells compared with the control group was observed (P < 0.001). All these results suggested the minimal toxic effects of FD274 in inducing apoptosis in H9C2 cells.

The investigation of the mechanism of the cardiotoxicity of 7-azaindazole-based PI3K inhibitors

The expression of the oxidative stress- and apoptosis-related proteins in H9C2 cells after respectively treated with 0.5 µM, 1 µM FD274 and FD268 were determined by western blot analysis. As shown in Fig. 4d ~ h, both FD274 and FD268 induced the up-regulation of NADPH oxidase gp91-phox. FD274 didn’t influence the expression of apoptosis-related proteins Bax and cleaved caspase-3 or the antioxidant pathway protein Nrf2; while FD268 induced the up-regulation of Bax and cleaved caspase-3, and down-regulated the expression of Nrf2. These results implied that the inhibition of Nrf2 and the activation of gp91-phox resulted in the oxidative stress and the following cell apoptosis in the FD268 group; while the preservation of the activity of Nrf2 resulted in the alleviation of the oxidative stress and the cell apoptosis in the FD274 group. These results further confirmed the minimal cardiotoxicity of FD274.

The low cardiotoxicity of FD274 after the consecutive 20-day treatment process in the HL-60 xenograft mice model

In our previous work, FD274 exhibited high in-vivo antitumor efficacy in HL-60 xenograft mice model, which was comparable to BEZ235 of the same dose^[25]. And the cardiotoxicity of FD274 after the same treatment process in the HL-60 xenograft mice model was further confirmed. As shown in Fig. 5, FD274 over the dose ranges tested didn’t influence the heart/body weight ratio, neither influenced the serum biochemical indexes including MDA, GSH-PX and myocardial enzymes (CK, CK-MB, LDH and LDH1). Pathological examination suggested that no myocardial fibrosis or apoptosis occurred in all groups. All these results confirmed the low cardiotoxicity of FD274 after the treatment process.

Small molecules targeting PI3K provides a promising strategy for cancer therapy. Our group has conducted a series of work on the development and research of PI3K inhibitors. Enlighted by the binding mode of GSK2126458 with PI3Kγ protein, we developed a novel scaffold that conjugating pyridinesulfonamide motif with 7-azaindole/azaindazole groups^[24]. This 7-azaindole/azaindazole scaffold exhibited ideal binding mode and inhibitory activity to PI3K as well as potent antiproliferative effects against a panel of human tumor cells, which can serve for the development of both pan-PI3K and isoform-specific PI3K inhibitors. FD223 is the first star molecule of this class, which was reported as a potent and selective PI3Kδ inhibitor for the treatment of acute myeloid leukemia (AML)^[27], yet its low inhibitory activity to the downstream of PI3K left the scope of further optimization. Several pan-PI3K inhibitor candidates were then developed. As the representative of them, FD268 was proved to inhibit PI3K/Akt/mTOR signaling pathway to exhibit high antiproliferative effects against AML cells^[28]. Shortly afterwards, FD274, a bioisostere of FD268, was discovered as an equipotent dual inhibitor of PI3K and mTOR kinases with superior in-vitro and in-vivo antitumor activities^[25]. Along with the prosperous development of lead compounds, the common occurrence of the cardiotoxicity of PI3K inhibitors in clinical trials raised our concern about the structure-cardiotoxicity relationship of these 7-azaindazole-based PI3K inhibitor which can lead to the development of PI3K inhibitors with high antitumor efficacy and low cardiotoxicity. Based on FD269, FD268 and FD274, three bioisosteric analogues that give comparable inhibitory activity against PI3Ks, we confirmed the structure-dependent reduction in the cardiotoxicities of from FD269 to FD268 and to FD274 in zebrafish model, which suggested the bioisosteric approach as a useful strategy to design novel PI3K inhibitors with low cardiotoxicity risk (Fig. 6)^[26]. On the basis of these results, the present study further provides comprehension of the cardiotoxicity of FD274 for its potential clinical translation. Our results validated the minimal adverse effects of FD274 in inducing cardiac dysfunction, oxidative stress and cardiac injury in C57BL/6 mice model and H9C2 cell model. Additionally, after the consecutive 20-day treatment process conducted on HL-60 xenograft mice model, FD274 exhibited low cardiotoxicity. In summary of our studies, FD274 is a promising antitumor drug candidate with both high antitumor efficacy and low cardiotoxicity, which deserves further efforts on improving its drug-ability as an orally bioavailable antitumor drug.

PI3K/Akt pathway plays protective roles against oxidative stress and inflammatory injury^{[21, 22, 29, 30]}. Our previous research indicated that 7-azaindazole-based PI3K inhibitors FD269 and FD268 up-regulate noxo1b and evoke oxidative stress injury^[26]. The present study further investigated the effects of 7-azaindazole-based PI3K inhibitors in inducing oxidative stress injury on rodent model. Endogenous antioxidants glutathione oxidase can remove peroxides such as ROS so that helps to prevent acute myocardial injury^[14], and MDA is the main product of lipid peroxidation^[31]. Our results shown that the FD268 group exhibited the decrease in the glutathione oxidase activity and the increase in MDA level, suggested the oxidative stress in the serum; while FD274 didn’t cause the abnormality in these oxidative stress-related parameters in mice serum. Nox2 is the major NADPH oxidase that quiescently located in the cell membrane, working as enzymes and transferring electrons from the cytosol into various intracellular and extracellular compartments^{[14, 17]}. DOX can activate Nox2 and inhibit Nrf2 antioxidant pathway, which leads to the generation of superoxide anion and hydrogen peroxide and the following oxidative stress^[18]. Our results shown that FD268 down-regulated the Nrf2 antioxidant pathway and up-regulated Nox2 (gp91-phox), so that induced oxidative stress injury. By contrast, FD274, although up-regulated Nox2, didn’t influence the activity of Nrf2, so that avoided the generation of oxidative stress and the following cardiac injury. Our future studies will further focus on the mechanism of oxidative stress lying behind the cardiotoxicity induced by 7-azaindazole-based PI3K inhibitors.

A comprehensive assessment of the cardiotoxicity of PI3K inhibitors as early as the development stage is of great importance. The results in this study shown that the candidate FD274 exhibited minimal adverse effects in inducing cardiac dysfunction, oxidative stress and cardiac injury in in-vivo and in-vitro models after single-dose administration, and low cardiotoxicity in HL-60 xenograft mice model after the consecutive 20-day treatment process. Furthermore, we revealed that the low cardiotoxicity of FD274 may be ascribed to the preservation of the activity of Nrf2 antioxidant pathway. In summary, FD274, with high antitumor efficacy, exhibited low cardiotoxicity, worth further development as a promising antitumor drug.

Author Contribution

TW, YG, MZ, YC, YY, YL and YZ: planning of experiments, TW and YG: experiments, YC, CY, YY, YL, JX, TC, LL and YZ: space supply, reagents, technicians, and laboratory support, TW: manuscript writing, TW, YY, YL and YZ: manuscript edition. All authors reviewed the manuscript

Acknowledgements

We gratefully acknowledge the financial support from National Natural Science Foundation of China (22275037), International Science & Technology Innovation Collaboration Project of Shanghai (22520713700), the Commission for Science and Technology of Shanghai (20ZR1404800).

MacDonald and Robertson. (2009). Toxicity Testing in the 21st Century: A View from the Pharmaceutical Industry. Toxicological Sciences, 110 (1), 40-46.
Ziegler, Häusermann, Kirchner and Polonchuk. (2021). Cardiac Safety of Kinase Inhibitors – Improving Understanding and Prediction of Liabilities in Drug Discovery Using Human Stem Cell-Derived Models. 8.
Sadasivan, Zhabyeyev, Labib, White, Paterson and Oudit. (2020). Cardiovascular toxicity of PI3Kα inhibitors. Clinical Science, 134 (19), 2595-2622.
Mayer and Arteaga. (2016). The PI3K/AKT Pathway as a Target for Cancer Treatment. 67 (1), 11-28.
Curigliano and Shah. (2019). Safety and Tolerability of Phosphatidylinositol-3-Kinase (PI3K) Inhibitors in Oncology. Drug Safety, 42 (2), 247-262.
Yang, Meoli, Moslehi and Roden. (2018). Inhibition of the <em>α</em>-Subunit of Phosphoinositide 3-Kinase in Heart Increases Late Sodium Current and Is Arrhythmogenic. 365 (3), 460-466.
Lenneman and Sawyer. (2016). Cardio-Oncology An Update on Cardiotoxicity of Cancer-Related Treatment. Circulation Research, 118 (6), 1008-1020.
Morelli, Bongiovanni, Da Pra, Miano, Sacchi, Lauriola and D'Uva. (2022). Cardiotoxicity of Anticancer Drugs: Molecular Mechanisms and Strategies for Cardioprotection. Frontiers in Cardiovascular Medicine, 9.
Scherrer-Crosbie and Thibault. (2008). Echocardiography in translational research: Of mice and men. Journal of the American Society of Echocardiography, 21 (10), 1083-1092.
Baumans. (2004). Use of animals in experimental research: an ethical dilemma? Gene Therapy, 11, S64-S66.
Elnakish, Hassanain and Janssen. (2012). Vascular Remodeling-Associated Hypertension Leads to Left Ventricular Hypertrophy and Contractile Dysfunction in Profilin-1 Transgenic Mice. Journal of Cardiovascular Pharmacology, 60 (6), 544-552.
Moon, Park, Kang, Lee, Cheong, Lim, Ihm, Seung, Jaffer, Narula, Chang and Hong. (2012). Noninvasive Assessment of Myocardial Inflammation by Cardiovascular Magnetic Resonance in a Rat Model of Experimental Autoimmune Myocarditis. Circulation, 125 (21), 2603-2612.
Fischer, Dechend, Gapelyuk, Shagdarsuren, Gruner, Gruner, Gratze, Qadri, Wellner, Fiebeler, Dietz, Luft, Muller and Schirdewan. (2007). Angiotensin II-induced sudden arrhythmic death and electrical remodeling. American Journal of Physiology-Heart and Circulatory Physiology, 293 (2), H1242-H1253.
Kong, Guo, Song, Zhang, Yuan, Teng, Yan and Tang. (2022). Underlying the Mechanisms of Doxorubicin-Induced Acute Cardiotoxicity: Oxidative Stress and Cell Death. International Journal of Biological Sciences, 18 (2), 760-770.
Bouitbir, Panajatovic and Kraehenbuehl. (2022). Mitochondrial Toxicity Associated with Imatinib and Sorafenib in Isolated Rat Heart Fibers and the Cardiomyoblast H9c2 Cell Line. International Journal of Molecular Sciences, 23 (4), 2282.
Angsutararux, Luanpitpong and Issaragrisil. (2015). Chemotherapy-Induced Cardiotoxicity: Overview of the Roles of Oxidative Stress. Oxidative Medicine and Cellular Longevity, 2015, 795602.
Schramm, Matusik, Osmenda and Guzik. (2012). Targeting NADPH oxidases in vascular pharmacology. Vascular Pharmacology, 56 (5-6), 216-231.
Zhao, McLaughlin, Robinson, Harvey, Hookham, Shah, McDermott and Grieve. (2010). Nox2 NADPH Oxidase Promotes Pathologic Cardiac Remodeling Associated with Doxorubicin Chemotherapy. Cancer Research, 70 (22), 9287-9297.
Li, Zhang, Li, Li, Bao, Yang, Wang, Wei, Chen and Jin. (2019). Role of Nrf2 in the antioxidation and oxidative stress induced developmental toxicity of honokiol in zebrafish. Toxicology and Applied Pharmacology, 373, 48-61.
Hu, Liu, Wang, Hu and Li. (2019). miR-200a Attenuated Doxorubicin-Induced Cardiotoxicity through Upregulation of Nrf2 in Mice. Oxidative Medicine and Cellular Longevity, 2019.
Xu, Wang, Jin, Zhu, Lin, Xie and Wang. (2017). Perillyl alcohol protects human renal tubular epithelial cells from hypoxia/reoxygenation injury via inhibition of ROS, endoplasmic reticulum stress and activation of PI3K/Akt/eNOS pathway. Biomedicine & Pharmacotherapy, 95, 662-669.
Song, Xu, Liu, Li, Wang, Shan, Wainer, Hu, Zhang, Woo and Xiao. (2021). Heterodimerization With 5-HT(2B)R Is Indispensable for β(2)AR-Mediated Cardioprotection. Circulation Research, 128 (2), 262-277.
Ye, Cai, Xia, Shi, Jin, Chen, Xia, Xia, Papadimos, Xu, Liu and Wang. (2021). Apelin-13 Reverses Bupivacaine-Induced Cardiotoxicity via the Adenosine Monophosphate-Activated Protein Kinase Pathway. Anesthesia and Analgesia, 133 (4), 1048-1059.
Yang, Zhang, Wang, Yang, Liu, Deng, Jia, Ling, Meng and Zhou. (2017). Discovery of a Novel Series of 7-Azaindole Scaffold Derivatives as PI3K Inhibitors with Potent Activity. Acs Medicinal Chemistry Letters, 8 (8), 875-880.
Yang, Chen, Wu, Gao, Liu, Yang, Ling, Jia, Deng, Wang and Zhou. (2023). Discovery of N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274) as a highly potent PI3K/mTOR dual inhibitor for the treatment of acute myeloid leukemia. European Journal of Medicinal Chemistry, 258, 115543.
Wu, Chen, Yang, Lu, Geng, Guo, Pi, Ling, Xu, Cai, Lu and Zhou. (2023). Systematical Evaluation of the Structure-Cardiotoxicity Relationship of 7-Azaindazole-based PI3K Inhibitors Designed by Bioisosteric Approach. Cardiovascular Toxicology, 23 (11), 364-376.
Yang, Xu, Li, Chen, Wu, Hong, Lu, Jia, Yang, Liu, Deng, Chen, Li, Ling and Zhou. (2021). Bioisosteric replacements of the indole moiety for the development of a potent and selective PI3Kδ inhibitor: Design, synthesis and biological evaluation. European Journal of Medicinal Chemistry, 223, 113661.
Chen, Wu, Yang, Lu, Chen, Deng, Jia, Yang, Liu, Wang, Ling, Lu and Zhou. (2022). A pyridinesulfonamide derivative FD268 suppresses cell proliferation and induces apoptosis via inhibiting PI3K pathway in acute myeloid leukemia. Plos One, 17 (11).
Uchiyama, Engelman, Maulik and Das. (2004). Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning. Circulation, 109 (24), 3042-3049.
Arslan, Lai, Smeets, Akeroyd, Choo, Aguor, Timmers, van Rijen, Doevendans, Pasterkamp, Lim and de Kleijn. (2013). Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Research, 10 (3), 301-312.
Rochette, Guenancia, Gudjoncik, Hachet, Zeller, Cottin and Vergely. (2015). Anthracyclines/trastuzumab: new aspects of cardiotoxicity and molecular mechanisms. Trends in Pharmacological Sciences, 36 (6), 326-348.

No competing interests reported.

20240215CTSI.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274): a potential novel PI3K inhibitor with low cardiotoxicity in mice

Status:

Version 1

Abstract

Figures

Introduction

Materials & methods

General

Drug administration

Echocardiography

Serum biochemical analysis

Histopathological analysis

Determination of myocardial apoptosis

Determination of cell viability

Determination of intracellular ROS level

Determination of apoptosis level

Western blot

Statistical analysis

Results

The minimal cardiotoxicity of FD274 to C57BL/6 mice

The minimal toxic effects of FD274 in inducing cardiac dysfunction

The minimal toxic effects of FD274 in inducing oxidative stress in mice serum

The minimal toxic effects of FD274 in inducing cardiac injury

The minimal toxicity of FD274 to H9C2 cells

The minimal toxic effects of FD274 in inducing ROS over-production in H9C2 cells

The minimal toxic effects of FD274 in inducing apoptosis in H9C2 cells

The investigation of the mechanism of the cardiotoxicity of 7-azaindazole-based PI3K inhibitors

Discussion

Conclusion

Declarations

Author Contribution

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1