Background
GDC-0068 (RG7440, Ipatasertib) is a novel highly selective ATP-competitive pan-Akt inhibitor that inhibits all three isoforms of Akt1, Akt2, and Akt3 at an IC50 of 5,18,8 nM, respectively [1,5].
GDC-0068 decreased cell viability, induced apoptosis, and inhibited phosphorylation of proline rich Akt substrate 40 kDa and p70 S6 kinase in a dose-dependent manner in PIK3CA-mutant breast cancer brain metastatic cell lines compared with PIK3CA-wildtype cell lines[6]. The combined treatment of GDC-0068 (RG7440) plus anti-microtubule chemotherapy, including vinorelbine, paclitaxel, eribulin, is contributed to anti-proliferative, pro-apoptotic, and anti-metastatic effect on human breast cancer cells[7]. GDC-0068 (RG7440) blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 (RG7440) resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines[2].
Preclinical Pharmacology demonstrates GDC-0068 (RG7440) exposure resulting in a dose-dependently pharmacodynamic effect and robust anti-tumor activity in a broad spectrum of human cancer cells in vitro and in vivo[5]. GDC-0068 (RG7440) therapy significantly inhibited the growth of WT tumors, however, for the PUMA / tumors, GDC-0068 (RG7440) therapy caused some suppression, but not so markedly compared with that of WT tumors. Immunohistochemistry staining shows that the expression of P-Akt reduced in both WT and PUMA / tumors after GDC-0068 (RG7440) therapy. PUMA-dependent antitumor effects of GDC-0068 (RG7440) in colon cancer[4]. As an ATP-competitive Akt inhibitor, GDC-0068 (RG7440) is a strong and safe target for Akt, which has been proved in a first-in-human phase I study[3].
参考文献:
[1]. Lin K, Lin J, et,al. An ATP-site on-off switch that restricts phosphatase accessibility of Akt. Sci Signal. 2012 May 8;5(223):ra37. doi: 10.1126/scisignal.2002618. PMID: 22569334.
[2]. Lin J, Sampath D, et,al.Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models. Clin Cancer Res. 2013 Apr 1;19(7):1760-72. doi: 10.1158/1078-0432.CCR-12-3072. Epub 2013 Jan 3. PMID: 23287563.
[3]. Saura C, Roda D, et,al. A First-in-Human Phase I Study of the ATP-Competitive AKT Inhibitor Ipatasertib Demonstrates Robust and Safe Targeting of AKT in Patients with Solid Tumors. Cancer Discov. 2017 Jan;7(1):102-113. doi: 10.1158/2159-8290.CD-16-0512. Epub 2016 Nov 21. Erratum in: Cancer Discov. 2018 Nov;8(11):1490. PMID: 27872130; PMCID: PMC5463454.
[4]. Sun L, Huang Y, et,al. Ipatasertib, a novel Akt inhibitor, induces transcription factor FoxO3a and NF-κB directly regulates PUMA-dependent apoptosis. Cell Death Dis. 2018 Sep 5;9(9):911. doi: 10.1038/s41419-018-0943-9. PMID: 30185800; PMCID: PMC6125489.
[5]. Blake JF, Xu R, et,al. Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors. J Med Chem. 2012 Sep 27;55(18):8110-27. doi: 10.1021/jm301024w. Epub 2012 Sep 18. PMID: 22934575.
[6]. Ippen FM, Grosch JK, et,al. Targeting the PI3K/Akt/mTOR pathway with the pan-Akt inhibitor GDC-0068 in PIK3CA-mutant breast cancer brain metastases. Neuro Oncol. 2019 Nov 4;21(11):1401-1411. doi: 10.1093/neuonc/noz105. PMID: 31173106; PMCID: PMC6827829.
[7]. Yan Y, Serra V, et,al.Evaluation and clinical analyses of downstream targets of the Akt inhibitor GDC-0068. Clin Cancer Res. 2013 Dec 15;19(24):6976-86. doi: 10.1158/1078-0432.CCR-13-0978. Epub 2013 Oct 18. PMID: 24141624.
GDC-0068 (RG7440, Ipatasertib) 是一种新型高选择性 ATP 竞争性泛 Akt 抑制剂，可抑制 Akt1、Akt2 和 Akt3 的所有三种亚型，IC50 分别为 5、18、8 nM [1,5].
与 PIK3CA 野生型细胞系相比，GDC-0068 在 PIK3CA 突变型乳腺癌脑转移细胞系中以剂量依赖性方式降低细胞活力、诱导细胞凋亡并抑制富含脯氨酸的 Akt 底物 40 kDa 和 p70 S6 激酶的磷酸化[6]。 GDC-0068 (RG7440) 联合抗微管化疗，包括长春瑞滨、紫杉醇、艾日布林，有助于对人乳腺癌细胞产生抗增殖、促凋亡和抗转移作用[7] 。 GDC-0068 (RG7440) 在培养的人类癌细胞系和肿瘤异种移植模型中阻断 Akt 信号，下游靶标磷酸化的剂量依赖性降低证明了这一点。 GDC-0068 (RG7440) 对 Akt 活性的抑制导致细胞周期进程受阻并降低癌细胞系的活力[2]。
临床前药理学证明 GDC-0068 (RG7440) 暴露会在体外和体内的广谱人类癌细胞中产生剂量依赖性药效学效应和强大的抗肿瘤活性[5] . GDC-0068（RG7440）治疗显着抑制WT肿瘤的生长，然而，对于PUMA/肿瘤，GDC-0068（RG7440）治疗引起一些抑制，但与WT肿瘤相比没有那么显着。免疫组织化学染色显示，在 GDC-0068 (RG7440) 治疗后，P-Akt 的表达在 WT 和 PUMA/肿瘤中均降低。 GDC-0068 (RG7440) 在结肠癌中的 PUMA 依赖性抗肿瘤作用[4]。作为一种 ATP 竞争性 Akt 抑制剂，GDC-0068 (RG7440) 是 Akt 的一个强大而安全的靶点，这一点已在人体第一阶段研究中得到证实[3]。
Protocol
| Kinase experiment [1]: |
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Preparation Method
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Inhibitor(GDC-0068 (RG7440)), enzyme (9 nM Akt1 or 100 pM PKA), and substrate (100 nM Crosstide) were incubated with 5 μM ATP in assay buffe, final DMSO 2% (v/v)) for 60 min at ambient temperature in a 5 μL reaction volume. Reactions were initiated by addition of enzyme + peptide substrate to ATP solutions. IMAP binding reagent (15 μL) was added to terminate the reaction, and the stopped reactions were incubated for a minimum of 30 min at room temperature (rt).
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Applications
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GDC-0068 (RG7440) is a selective, ATP-competitive pan-Akt inhibitor that inhibits Akt1, Akt2, and Akt3 at an IC50 of 5,18,8 nM, respectively.
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| Cell experiment [2]: |
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Cell lines
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HCT116 cell
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Preparation Method
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To study how GDC-0068 (RG7440) influences tumor progression, cell viability was detected by CCK-8 in HCT116 at indicated time points after 1–20?μmol/L GDC-0068 (RG7440)treatment.
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Reaction Conditions
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1–20?μmol/L GDC-0068 (RG7440) for 0, 3, 6, 12, and 24?h
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Applications
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Cell viability decreased markedly with increasing dose or time, cell proliferation was inhibited by GDC-0068 (RG7440) in a dose- and time-dependent manner.
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| Animal experiment [3]: |
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Animal models
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Nude mice
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Preparation Method
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Nude mice were injected s.c. with HCT116 WT or PUMA?/?. Once the tumor was measurable, mice were treated daily with 30?mg/kg GDC-0068 (RG7440) by oral gavage, for 15 consecutive days.
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Dosage form
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30?mg/kg GDC-0068 (RG7440) by oral gavage, for 15 consecutive days
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Applications
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GDC-0068 (RG7440) therapy significantly inhibited the growth of WT tumors, however, for the PUMA / tumors, GDC-0068 (RG7440) therapy caused some suppression, but not so markedly compared with that of WT tumors
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参考文献:
[1]. Blake JF, Xu R, et,al. Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors. J Med Chem. 2012 Sep 27;55(18):8110-27. doi: 10.1021/jm301024w. Epub 2012 Sep 18. PMID: 22934575.
[2]. Sun L, Huang Y, et,al.Ipatasertib, a novel Akt inhibitor, induces transcription factor FoxO3a and NF-κB directly regulates PUMA-dependent apoptosis. Cell Death Dis. 2018 Sep 5;9(9):911. doi: 10.1038/s41419-018-0943-9. PMID: 30185800; PMCID: PMC6125489.
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