Dual blocking of PI3K and mTOR signaling by DHW‐221, a novel benzimidazole derivative, exerts antitumor activity in human non‐small cell lung cancer

Dear Editor, We previously designed and synthesized a novel benzimidazole compound, DHW-221.1 Here, we evaluated its antiNSCLC activity as a PI3K/mTOR dual-target inhibitor and comprehensively described for the first time the mechanismunderlying the PI3K/AKT/mTOR signaling pathwaymediated antitumor effects of DHW-221. Our findings indicated that the antitumor activity of DHW-221 was significantly stronger than that of NVP-BEZ235, a dual PI3K/mTOR inhibitor currently undergoing a phase II clinical trial. The PI3K/AKT/mTOR pathway is a key mediator of receptor tyrosine kinase signal transduction. The PI3K family can be divided into three categories.2 Class I PI3K and mTOR, sharing structural homology in the kinase domain,3 are considered important targets for malignancy therapy.4 Thus, the thought of blocking the PI3K/AKT/mTOR pathway by double inhibition of PI3K/mTORwas proposed.5 Herein,we systematically elucidated the anti-NSCLC effects associated with DHW-221mediated dual PI3K/mTOR inhibition and identified the mechanism underlying this effect both in vitro and in vivo. First, we identified potential targets of DHW-221. Molecular docking indicated that DHW-221 fitted well into the PI3K-ATP binding pocket and formed hydrogen bonds with Gln859 and Arg770. For mTOR, DHW-221 precisely overlapped with it and formed two more hydrogen bonds (Val2240 and Thr2245) than NVP-BEZ235 (Figure 1A). Docking results with PI3Kβ/γ/δ are shown in Figure S1. Subsequent kinase assay suggested that DHW-221 significantly inhibits the kinase activity of the main PI3K isoforms1 (IC50: PI3Kα,0.5 nM; PI3Kβ,1.9 nM; PI3Kγ,1.8 nM; PI3Kδ,0.74 nM), as well as that of mTOR (IC50: 3.9 nM) (Table S1). Cellular thermal shift assay was used to further evaluate the binding mode, the thermal stability of DHW-221 combined with PI3K/mTOR was concentration-dependently enhanced (Figure 1B).

Additionally, we knocked down PI3K in HCC827 cells ( Figure 1C) and compared how DHW-221, NVP-BEZ235, and wortmannin (a PI3K inhibitor) treatments affected AKT and mTOR protein phosphorylation levels. No significant difference in p-AKT levels was found with any of the treatments ( Figure 1D), suggesting that DHW-221 likely targeted PI3K. Conversely, compared with wortmannin, p-mTOR expression was significantly decreased after DHW-221 and NVP-BEZ235 intervention, implying that DHW-221 may target mTOR. Finally, western blot and immunofluorescence analysis confirmed the effect of DHW-221 on the expression of PI3K/AKT/mTOR pathway-related proteins both in vitro and in vivo ( Figure 1E-G). Combined, these results suggested that DHW-221 inhibited PI3K/AKT/mTOR pathway activation through targeting PI3K/mTOR. The effects of DHW-221 on notch pathway are provided in Result S1.
To investigate the effect of DHW-221 in vitro, SRB assay was performed. DHW-221 exhibited excellent inhibitory activity concentration-and time-dependently ( Figure 2B) without affecting the normal cells ( Figure S3), and IC 50 is shown in Table S2. Additionally, the long-term efficacy of DHW-221 was evaluated and confirmed a better antiproliferation role than NVP-BEZ235 ( Figure 2C-E). Figure 2F investigates the concentration-dependently inhibition of DHW-221 on protein synthesis in cells. HCC827 xenograft models in both subcutaneous and orthotopic were used to further evaluate its antitumor potency in vivo. As illuminated in Figure 2G-P, DHW-221 apparently inhibited tumor growth without affecting body weight ( Figure 2J, N) or exerting toxic effects ( Figure 2K), except in the spleen. Interestingly, aminotransferase and creatinine levels were not affected by DHW-221 treatment, whereas the alanine aminotransferase concentration was increased at high dose, indicating that DHW-221 exerted only mild toxicity ( Figure 2L). H&E staining also demonstrated that no abnormal pathological changes were present in the F I G U R E 1 DHW-221 is a PI3K/mTOR dual-target inhibitor and inhibits the PI3K/AKT/mTOR signaling pathway. (A) Predicted binding modes for DHW-221 (blue stick) with PI3Kα (PDB code: 4JPS) and mTOR (PDB code: 4JT6). Hydrogen bonds are shown as green dashed lines. Key residues for DHW-221 and BEZ235 interaction are highlighted. (B) A cellular thermal shift assay (CETSA) and an isothermal dose-response fingerprinting (ITDRF)cetsa were used to evaluate the thermal stability of DHW-221 bound to PI3Kα/β or mTOR. (C, D) The expression of PI3K, p-AKT, and p-mTOR in transfected NSCLC cells was assessed by western blot. (E, G) Western blot was also used to evaluate the effects of DHW-221 on PI3K/AKT/mTOR pathway-related proteins in non-small cell lung cancer (NSCLC) cells and tissues (n = 6). (F) Immunofluorescence images of p-AKT protein expression following DHW-221 treatment. Anti-rabbit p-AKT (Ser473) antibody was used for labeling and the nuclei were stained with DAPI; scale bar, 50 µm. Mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 versus the control (model). One-way analysis of variance followed by Tukey's post hoc multiple-comparisons test visceral organs ( Figure 2M). Moreover, DHW-221 treatment could ameliorate the burden of orthotopic carcinoma to nude mice ( Figure 2O, P). These findings indicated that DHW-221 exhibited significant anti-NSCLC activity with low toxicity and few side effects both in vitro and in vivo. Meanwhile, we investigated the pharmacokinetic of DHW-221 in mice, and the parameters are provided in Table S3.
With an oral bioavailability of up to 67.5%, the overall PK parameters of DHW-221 appear promising and encourage its further evaluation for antitumor study.
The PI3K/AKT/mTOR pathway is strongly linked to the regulation of cell cycle progression and apoptosis during cell proliferation. 6,7 In Figure 3A, NSCLC cells were arrested at G0/G1 phase by DHW-221 concentrationdependently. Meanwhile, the expression of endogenous cyclins in G0/G1 phase was detected by western blot, confirming that DHW-221 regulated the cell cycle (Figure 3B). Apoptosis is crucial for anticancer drugs to kill cancer cells. 7 First, Hoechst 33342 and Annexin V/PI staining verified that DHW-221 concentration-dependently  quantitative results is shown on the right. Mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 versus the control. One-way analysis of variance followed by Tukey's post hoc multiple-comparisons test induced apoptosis ( Figure 3C, D). Moreover, transmission electron microscopic analysis of cellular ultrastructure also suggested that cells treated with DHW-221 had typical apoptotic characteristics ( Figure 3E). Additionally, JC-1 staining showed that the mitochondrial membrane potential was concentration-dependently decreased, further confirming that DHW-221 induced cell apoptosis via the mitochondrial pathway ( Figure 3F). Finally, the proapoptotic effects of DHW-221 were confirmed by western blot analysis of apoptosis-related protein expression both in vitro and in vivo ( Figure 3G-I).
Activated PI3K/AKT/mTOR signaling participates in tumor metastasis, invasion, 8 and angiogenesis. 9 In Figure 4A and B, DHW-221 exerted a greater capacity in curtailing the migration and invasion of NSCLC cells. Meanwhile, western blot confirmed that DHW-221 downregulated the expression of invasion-related factor MMP-2 ( Figure 4C). Notably, migration and differentiation (tube formation) of endothelial cells are necessary for early angiogenesis. 10 DHW-221 markedly inhibited the metastatic ( Figure 4D, E) and invasive ( Figure 4E) potential of HUVEC and concentration-and time-dependently suppressed the tube formation ( Figure 4F). Additionally, DHW-221 concentration-dependently suppressed microvessel sprouting in the rat aortic ring ex vivo ( Figure 4G) and reduced chorioallantoic membrane neovascularization in vivo ( Figure 4H). Matrigel plug assay also suggested that DHW-221 could significantly inhibit angiogenesis in vivo ( Figure 4I). VEGFA and HIF-1α both play key roles in tumor angiogenesis; here, DHW-221 could concentration-dependently inhibited the expression of these two proteins ( Figure 4J, K). Together, these data indicate that DHW-221 can reduce tumor angiogenesis by inhibiting the PI3K/HIF-1α/VEGF signaling pathway.
In conclusion, DHW-221 was identified as being a dual PI3K/mTOR inhibitor, and displayed excellent anti-NSCLC activity, including the inhibition of cell proliferation, migration, invasion, and angiogenesis, by blocking the PI3K/AKT/mTOR signaling pathway both in vitro and in vivo. Additionally, DHW-221 was found to promote cell cycle arrest and mediate apoptosis through the mitochondrial pathway. Combined, these findings indicate that DHW-221 has potential as a novel anticancer drug targeting PI3K/mTOR in NSCLC to inhibit tumor initiation and progression.

A C K N O W L E D G M E N T S
This research was supported by Liaoning Natural Fund Guidance Plan (Number: 2019-ZD-0446).

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.

E T H I C S A P P R O VA L A N D C O N S E N T T O PA R T I C I PAT E
All animal experiments were conducted in accordance with the guidelines of the internationally recognized guidelines for animal studies: reporting in vivo experiments. This study was approved by the Ethics Committee for Animal Experiments of Shenyang Pharmaceutical University.

A U T H O R C O N T R I B U T I O N S
Prof. Q.C. Z. and Dr. H.W. D. for supervision and funding acquisition; X.C. Q. for conceptualization, methodology, validation, formal analysis, writing-original draft, writingreview and editing, and project administration; M.Y. L. and Y.T. W. for investigation and resources; others for editing and proofread. All authors read and approved the final manuscript.

D ATA A N D M AT E R I A L S AVA I L A B I L I T Y
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. Used research material is available by request from the corresponding author or from commercial sources when applicable.