Background
ML-265 is widely used as an acknowledged PKM2 (pyruvate kinase muscle isoform 2) activator[1][2] ML-265 activates of PKM2 in both biochemical (AC50 = 92 nM) and cell-based assays with high selectivity over PKM1(pyruvate kinase muscle isoform 1), PKL and PKR. ML-265 was originally developed as a potential cancer therapy. Therefore, ML-265 represents an ideal lead molecule to explore the neuroprotective effects of pharmacologically activating PKM2[2]. PKM2, which is overexpressed in many cancers, is inhibited by ROS, allowing glycolytic flux to be shuttled into the oxidative PPP for NADPH generation. The small-molecule compounds, ML-265, can positively modulate PKM2, thereby decreasing glycolytic flux into the oxidative PPP and blunting NADPH biogenesis during ROS(fig.) [3].
ML-265 displayed powerful activation of recombinant human PKM2 with a maximum activation of 249 ± 14% (best-fit value ± std. error) , the half-maximum activating concentration (AC50) shows 108 ± 20 nM. To investigate the ability of ML-265 to activate PK in photoreceptors, the 661W cell line was utilized. Similar to the results observed with the recombinant enzyme, ML-265 increased PK activity in vitro (maximum activation = 515 ± 12% and AC50 = 19 ± 2 nM) [2].
To determine the intraocular pharmacokinetic profile of intravitreally administered ML-265 in rabbits. Single, intravitreal injections (50 μL) of two different doses of ML-265 were performed in rabbits. Assuming a vitreous volume of 1.15 mL, the final concentrations in the rabbit vitreous were approximately 100 μM and 1000 μM, respectively. The aqueous humor was sampled at multiple time points after the single intravitreal injection. The distribution phase has a half-life (t1/2) of 8.3 ± 0.5 hours. The elimination phase has a t1/2 = 141.6 ± 35 hours. ML-265 has a relatively long half-life in the eye following intravitreal injection and remains active at least two weeks after injection.[2].
参考文献:
[1]: Walsh MJ, Brimacombe KR, Anastasiou D, et al. ML265: A potent PKM2 activator induces tetramerization and reduces tumor formation and size in a mouse xenograft model. Probe Reports from the NIH Molecular Libraries Program [Internet]
[2] TJ Wubben, M Pawar, E Weh, et,al. Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2,circumvents photoreceptor apoptosis. Sci. Rep., 10 (2020), p. 2990, doi.org /10.1038/s41598-020-59999-w.
[3] Chakrabarti G, Gerber DE, Boothman DA. Expanding antitumor therapeutic windows by targeting cancer-specific nicotinamide adenine dinucleotide phosphate-biogenesis pathways. Clin. Pharmacol. Adv. Applications. 2015;7:57.
[4]: Xu W, Yang H, et,al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell. 2011 Jan 18;19(1):17-30. doi: 10.1016/j.ccr.2010.12.014. PMID: 21251613; PMCID: PMC3229304.
ML-265 被广泛用作公认的 PKM2（丙酮酸激酶肌肉亚型 2）激活剂[1][2] ML-265 激活 PKM2 在生物化学（AC50 = 92 nM）和基于细胞的测定中对 PKM1（丙酮酸激酶肌肉亚型 1）、PKL 和 PKR 具有高选择性。 ML-265 最初是作为一种潜在的癌症疗法而开发的。因此，ML-265 是探索药理激活 PKM2[2] 神经保护作用的理想先导分子。在许多癌症中过度表达的 PKM2 被 ROS 抑制，允许糖酵解通量穿梭到氧化 PPP 中以生成 NADPH。小分子化合物 ML-265 可以正向调节 PKM2，从而减少糖酵解通量进入氧化 PPP 并减弱 ROS 期间的 NADPH 生物合成（图）[3].
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ML-265 显示重组人 PKM2 的强大激活，最大激活为 249 ± 14%（最佳拟合值 ± 标准误差），半数最大激活浓度（AC< sub>50) 显示 108 ± 20 nM。为了研究 ML-265 在光感受器中激活 PK 的能力，使用了 661W 细胞系。与用重组酶观察到的结果相似，ML-265 增加了体外 PK 活性（最大激活 = 515 ± 12% 和 AC50 = 19 ± 2 nM）[2].
确定玻璃体内注射 ML-265 在兔体内的眼内药代动力学特征。在兔子中进行了两种不同剂量的 ML-265 的单次玻璃体内注射 (50 μL)。假设玻璃体体积为 1.15 mL，兔玻璃体中的最终浓度分别约为 100 μM 和 1000 μM。在单次玻璃体内注射后，在多个时间点对房水进行取样。分布相的半衰期 (t1/2) 为 8.3 ± 0.5 小时。消除阶段的 t1/2 = 141.6 ± 35 小时。 ML-265 在玻璃体内注射后在眼中具有相对较长的半衰期，并且在注射后至少两周仍保持活性。[2]。
Protocol
| Cell experiment [1]: |
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Cell lines
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661 W photoreceptor cell line
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Preparation Method
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For 661 W cell line experiments, media was replaced prior to the start of treatment with ML-265.
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Reaction Conditions
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Cells were incubated with10-7, 10-6, 10-5, 10-4, 10-3, 10-2 M concentrations of ML-265 for 2 hours
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Applications
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ML-265 represents an ideal lead molecule to explore the neuroprotective effects of pharmacologically activating PKM2 in photoreceptors. This in vitro data suggests the small molecule activator is able to cross the cell membrane and enhance PK activity.
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| Animal experiment [1]: |
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Animal models
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Brown-Norway mice
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Preparation Method
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Adult rats were utilized for all in vivo ML-265 studies except ocular pharmacokinetic studies. All rats were housed at room temperature with 12-hour light and 12-hour dark cycle.
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Dosage form
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Inject 2 μL of different concentrations（10-7, 10-6, 10-5, 10-4, 10-3, 10-2 M） of ML-265 slowly into the vitreous cavity，and harvest 4 hours later.
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Applications
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Intravitreal injection of ML-265 was able to activate PK in vivo up to 170 ± 26% with an AC50 = 59 ± 38 μM. Considering both the specificity of ML-265 for PKM2 and the fact that PKM2 expression is confined to the outer retina, ML-265 is most likely able to traverse the retina and the cell membranes of photoreceptors to activate PKM2 。
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参考文献:
[1]. TJ Wubben, M Pawar, E Weh, et,al. Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2,circumvents photoreceptor apoptosis. Sci. Rep., 10 (2020), p. 2990, doi.org /10.1038/s41598-020-59999-w.
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