Am J Cancer Res 2021;11(6):2590-2617 www.ajcr.us /ISSN:2156-6976/ajcr0131974 Original Article A preclinical report of a cobimetinib-inspired novel anticancer small-molecule scaffold of isoflavones, NSC777213, for targeting PI3K/AKT/mTOR/MEK in multiple cancers Bashir Lawal1,2*, Wen-Cheng Lo3,4,5*, Ntlotlang Mokgautsi1,2, Maryam Rachmawati Sumitra1,2, Harshita Khedkar1,2, Alexander TH Wu6,7,8,9, Hsu-Shan Huang1,2,10,11 1PhD Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 11031, Taiwan; 2Graduate Institute for Cancer Biology & Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan; 3Department of Surgery, Division of Neurosurgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan; 4Department of Neurosurgery, Taipei Medical University Hospital, Taipei 11031, Taiwan; 5Taipei Neuroscience Institute, Taipei Medical University, Taipei 11031, Taiwan; 6TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei 11031, Taiwan; 7The PhD Program of Translational Medicine, College of Science and Technology, Taipei Medical University, Taipei 11031, Taiwan; 8Clinical Research Center, Taipei Medical University Hospital, Taipei Medical University, Taipei 11031, Taiwan; 9Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei 11490, Taiwan; 10School of Pharmacy, National Defense Medical Center, Taipei 11490, Taiwan; 11PhD Program in Biotechnology Research and Development, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan. *Equal contributors. Received February 17, 2021; Accepted May 14, 2021; Epub June 15, 2021; Published June 30, 2021 Abstract: The phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways are critical for normal human physiology, and any alteration in their regulation leads to several human cancers. These pathways are well interconnected and share a survival mechanism for escaping the depressant effect of antago- nists. Therefore, novel small molecules capable of targeting both pathways with minimal or no toxicity are better alternatives to current drugs, which are disadvantaged by their accompanying resistance and toxicity. In this study, we demonstrate that the PI3K/AKT/mTOR/MEK is a crucial oncoimmune signature in multiple cancers. Moreover, we describe NSC777213, a novel isoflavone core and cobimetinib-inspired small molecule, which exhibit both anti- proliferative activities against all panels of NCI60 human tumor cell lines (except COLO205 and HT29) and a selec- tive cytotoxic preference for melanoma, non-small-cell lung cancer (NSCLC), brain, renal, and ovarian cancer cell lines. Notably, for NSC777213 treatment, chemoresistant ovarian cancer cell lines, including SK-OV-3, OVCAR-3, OVCAR-4, and NCI/ADR-RES, exhibited a higher antiproliferative sensitivity (total growth inhibition (TGI) = 7.62-31.50 µM) than did the parental cell lines OVCAR-8 and IGROV1 (TGI > 100 µM). NSC777213 had a mechanistic correla- tion with clinical inhibitors of PI3K/AKT/mTOR/MEK. NSC777213 demonstrates robust binding interactions and higher affinities for AKT and mTOR than did isoflavone, and also demonstrate a higher affinity for human MEK-1 kinase than some MEK inhibitors under clinical developments. In addition, treatment of U251 and U87MG cells with NSC777213 significantly downregulated the expression levels of the total and phosphorylated forms of PI3K/ AKT/mTOR/MEK. Our study suggests that NSC777213 is a promising PI3K/AKT/mTOR/MEK inhibitor for further preclinical and clinical evaluation as a chemotherapeutic agent, particularly for the treatment of NSCLC, melanoma, and brain, renal, and ovarian cancers. Keywords: PI3K/AKT/mTOR-MEK pathways, NSC777213, NCI60 human tumor cell lines, immune infiltrations, anticancer activities, molecular docking simulations Introduction (mTOR) pathway is a crucial intracellular signal- ing pathway that plays a vital regulatory role in The phosphatidylinositol 3-kinase (PI3K)/pro- metabolism and cell cycle [1, 2]. Alterations of tein kinase B/mammalian target of rapamycin this signaling pathway is a common event that A preclinical report of a cobimetinib-inspired novel anticancer small-molecule contributes to the cell survival and prolifera- hyperactivation occurs in approximately 70% of tion, angiogenesis, tumorigenesis, and metas- human tumors, and this activation is often tasis of almost all human malignancies [3, 4]. associated with therapy resistance and overall PI3K is a heterodimer protein consisting of p85 poor prognosis [19, 21]. Ras/Raf/MEK/extra- regulatory and p110 catalytic subunits [5]. cellular regulated kinase (ERK) is a crucial sig- PI3K pathway dysregulation occurs either naling pathway for normal human physiological through gene amplification or mutations and processes, and alteration in the regulation of induces a myriad of downstream effectors, this pathway leads to cancers [2]. MEK pro- leading to the development and progression of motes cell proliferation, cell survival, and cancers [2, 6]. Activated PI3K phosphorylates metastasis through ERK phosphorylation, phosphatidylinositol-4,5-bisphosphate to phos- which in turn translocates to the nucleus to phatidylinositol-3,4,5-triphosphate [1, 7]. Once regulate gene expression and various transcrip- phosphorylated, PI3K activates AKT, which reg- tion factors [22]. ulates several downstream molecules, includ- ing mTOR [2, 7]. Moreover, AKT may be activat- Rapamycin and its analogs have been reported ed due to mutation in the AKT PH domain and a to inhibit mTORC1 activity and have demon- loss of PTEN, a tumor suppressor gene; such strated some levels of effectiveness as a mono- activation may also be due to inositol polyphos- therapy agent [11, 23]. However, many studies phate-4-phosphatase type II, an enzyme that have reported resistance to these drugs owing negatively regulates PI3K through phosphati- to the activation of mTORC2 and PI3K signaling dylinositol-3,4,5-triphosphate dephosphoryla- pathways [24]. In addition, although mTORC1 tion [8, 9]. The activated AKT in turn phosphory- and mTORC2 inhibition can lead to the lates mTOR, which activates mTOR complex 1 decreased phosphorylation of AKT, mTOR inhib- (mTORC1) and mTOR complex 2 (mTORC2), two itors may unexpectedly upregulate the PI3K/ protein complexes with distinct structures, PDK1 pathway [9, 24]. Therefore, the concomi- functions, and substrate specificities [10, 11]. tant inhibition of both PI3K and mTOR may offer a better anticancer response compared with The mTORC1 comprises mTOR, DEPTOR, the targeting of mTOR alone. However, pan-spe- Raptor, PRAS40, and mLST8, whereas the cific PI3K/AKT/mTOR inhibitors are effective at mTORC2 comprises mTOR, DEPTOR, Proctor, high doses, and their overall success in clinical Rictor, mSin1, and mLST8 [12, 13], with mTOR trials has been limited by toxicity problems, being the catalytic subunit of both complexes. including immunosuppression, hepatic impair- The mTORC1 is the downstream component of ment, pneumonitis, hypo- and hyperglycemia, the PI3K-AKT pathway that regulates cellular mouth ulcers, constipation, cardiac toxicity, processes through signal integration from cutaneous reactions, neuropsychiatric defeats, growth factor receptors, tumor suppressors, nausea, diarrhea, and colitis [20, 25-27]. These oncoproteins, and the intracellular nutrient sta- adverse effects are limiting factors in adminis- tus [1, 14]. The mTORC1 regulates protein syn- trating the most effective dosages. Therefore, thesis and turnover through the phosphoryla- using natural products for therapeutic interven- tion of eIF4E binding protein 1 and ribosomal tion has attracted renewed interest owing of protein S6 kinase (S6K), which in turn enhanc- their ability to modulate multiple pathways and es glycolytic pathway and protein biosynthesis their nontoxic properties in contrast to conven- to initiate metabolic remodeling and regulate tional therapy [28-32]. Isoflavone derivatives translation initiation [15, 16]. Conversely, the are potent inhibitors of the PI3K/AKT/mTOR sig- mTORC2 plays a role in AKT-mediated cell sur- naling pathway [33, 34]. In addition, isoflavones vival and migration independent of mTORC1 have been reported to sensitize cancer cells to activity [17]. mTOR regulates hypoxia-inducible radiation therapy and work in synergy with con- factor 1, vascular endothelial growth factor, ventional chemotherapy in multiple cancers cyclin D1, and cell transitions from the G1 to S [33, 34]. phases, enhancing rapid cell proliferation and the growth and survival of many tumors [18]. In However, increasing evidence has suggested addition, mTOR/S6K can upregulate many pro- that the PI3K/AKT/mTOR and Raf/MEK/ERK angiogenic factors, such as AP-1, to promote pathways intersect at multiple junctions, exhib- carcinogenesis [19, 20]. Because mTOR plays it crosstalk, and contain feedback loops [8]. various roles in tumorigenic promotion, mTOR Inhibiting one pathway can result in signaling 2591 Am J Cancer Res 2021;11(6):2590-2617 A preclinical report of a cobimetinib-inspired novel anticancer small-molecule maintenance through the other pathway. The comparison set to 15% versus 85% of samples. existence of such mutual survival mechanisms Hazard ratios are