PS-48

PDPK1

PDK1 激动剂 PS48 能够增强地塞米松处理的 MC3T3-E1 细胞的成骨能力。在 PS48 处理后用地塞米松处理的 MC3T3-E1 细胞中，p-AKT 和 p-mTOR 的蛋白水平升高。 [2]

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以ITC为特征的PS48与PDK1的结合[1]
在20°C下，通过等温滴定量热法（ITC）测定了PS48及其类似化合物PS08（4）与PDK150-359 Y288G Q292A的结合性能（表1和补充图2）。这两种化合物都以1:1的化学计量比与PDK150-359结合，结合亲和力在微摩尔范围内（PS48，Kd=10.3μM；PS08，Kd=6.2 M）。化合物及其热力学参数ΔH、ΔG和TΔS（表1）的密切相似性表明了类似的结合机制。在这两种情况下，结合都是焓和熵有利的，并且是由熵项驱动的（ΔH/ΔG分别为27.1%和25.0%；表1）[1]。

在体内，PDK1激动剂PS48可以维持地塞米松治疗小鼠的骨量。 [2]

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双转基因APPsw/PSENdE9小鼠是阿尔茨海默病的模型，用于测试口服PDK-1激动剂PS48预防Morris Water Maze（MWM）中学习和记忆预期下降的效果。小鼠在标准（SD）或高脂肪（HFD）饮食中饲养，从10个月大开始给药，并在14个月大时进行测试PS48对学习TG动物隐藏平台的空间位置有积极影响，无论是SD还是HFD，与载体饮食和WT动物相比。在成功获得空间记忆后的几个测量指标上（探针试验），该药物也被证明对任何一种饮食的动物都有显著益处。在几个探针测量中，与SD相比，HFD组TG动物的PS48治疗效果更为明显。HFD产生了体重增加和高血糖的一些预期代谢效应，并加速了TG动物的认知障碍PS48被发现在TG组中适度减轻体重和改善OGTT反应方面具有附加值，尽管结果尚不明确。PS48耐受良好，无明显临床体征或症状，本身不影响寿命。这些结果建议在人体试验之前进行更大规模的临床前研究[3]。

等温滴定量热法。[1]
如前所述，使用VP-ITC仪器进行量热滴定，并按照补充方法中的详细说明进行了小幅修改。

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蛋白激酶活性测试。[1]
蛋白激酶活性测试基本上如前所述8,28，使用T308tide作为PDK1的底物。更多信息详见补充方法。

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探索PDK1中ATP结合位点的构象。[1]
HM多肽和小化合物激活PDK1是由于酶构象的变化。我们通过扫描TNP-ATP/PDK1在P-HM多肽、PIFtide或低分子量化合物存在或不存在的情况下的稳态荧光，探讨了PDK1中ATP结合位点的构象。数据是在Varian Cary Eclipse荧光分光光度计（激发λ=479 nm；发射扫描λ=500-600 nm；激发狭缝=10 nm；发射狭缝=10纳米）中以200 nm min-1的速率获得的，每100 nm扫描150个数据点，平均时间为0.3秒。更多信息详见补充方法。

MC3T3-E1 细胞在含有 4 mM 甘油磷酸盐和 25 g/mL 抗坏血酸的成骨分化培养基中培养直至 70% 汇合。然后向成骨分化培养基中补充不同浓度的地塞米松（最终乙醇浓度，0.01%，vol/vol）14天。每两天更换一次培养基。添加或不添加 PS48 (5 M) 的地塞米松 107 M 培养基用于培养 MC3T3-E1 细胞。

Drug formulation and delivery[3]
In preliminary toxicity and tolerability studies, normal adult C7/Bl6 mice were dosed for 2 weeks, one cohort by oral gavage and another intravenous. PS48 was solubilized in 100% DMSO, then diluted in corn oil to a final DMSO of 5%. Daily gavage of drug (1 mg/Kg and 50 mg/Kg) amounted to 12.5 microliter DMSO per 50 gm mouse. In the other assessment, mice were injected through the tail vein with PS48, titrating to a max dose of 20 mg/Kg. Daily weights and general activity were recorded and necropsy included liver and muscle enzyme levels.[3]
PS48was dosed orally for the clinical study of cognition in 2xTG mice beginning age week 44. Drug delivered via SD and HFD proceeded in two ways, in order to insure the full daily drug dose was ingested while providing for equivalent total kcal intake. In the mice on SD, the drug was administered in a cookie dough treat (or supplement) that was preferred over SD and always finished, so that consumption could be easily tracked. Mice were also housed individually during dough dosing to monitor complete ingestion. Thus, 1/2 of daily food consumption (∼3 g) was dough/drug and 1/2 SD food pellets. After mixing the drug/vehicle-corn oil emulsion (50 microliter) into the dough (1/20 v/v), it was partitioned into silicone candy molds (1.3 cc/well) and frozen until use within 1 week (each morsel ∼1.5 gm). The HFD, found by us to be preferred by the animals, even over cookie dough, had a softer consistency to begin with and could therefore be simply ground and mixed with compound (or Vehicle) emulsified in corn oil/5% DMSO. It was then reformed into 5-gram pellets and dried to be suitable for the hopper. The average daily food intake in the HFD-V group was 3.2 g and in the HFD-PS48 was 3.3 g.[3]
Mice were dosed 50 mg PS48/Kg/day. Based on a mean adult transgenic (2xTG) mouse weight of 46 g, the daily dose of PS48 was 2.3 mg. Based on a mean WT mouse weight of 41 g, the daily dose was 2.0 mg. No further adjustments based on weight differences between the groups occurring over time were made. The duration of drug therapy from outset through the end of cognitive testing was just over 4 months.

[1]. Nat Chem Biol. 2009 Oct;5(10):758-64.

[2]. Front Endocrinol (Lausanne). 2020 Jan 28;10:922.

[3]. Behav Brain Res. 2023 Feb 13:438:114183.

Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.[1]

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Long-term and high dose glucocorticoid treatment can cause decreased viability and function of osteoblasts, which leads to osteoporosis and osteonecrosis. In this study, we investigated the role and mechanism of action of HIF-1α in glucocorticoid-induced osteogenic inhibition in MC3T3-E1 cells. Our results showed that HIF-1α protein expression was reduced when MC3T3-E1 cells were exposed to dexamethasone (Dex) at varying concentrations ranging from 10-9 to 10-6 M. PDK1 expression was also decreased in MC3T3-E1 cells after dexamethasone treatment. MC3T3-E1 cells when treated with the glucocorticoid receptor antagonist RU486 along with dexamethasone showed enhanced HIF-1α expression. In addition, upregulated expression of HIF-1α was capable of promoting the osteogenic ability of MC3T3-E1 cells and PDK1 expression. However, the HIF-1α antagonist 2-methoxyestradiol (2-ME) had a reverse effect in MC3T3-E1 cells exposed to dexamethasone. Furthermore, the PDK1 antagonist dichloroacetate could repress the osteogenic ability of MC3T3-E1 cells, although HIF-1α was upregulated when transduced with adenovirus-HIF-1α construct. The PDK1 agonist PS48 was able to promote the osteogenic ability of MC3T3-E1 cells treated with dexamethasone. Importantly, the protein levels of p-AKT and p-mTOR were increased in MC3T3-E1 cells treated with dexamethasone after PS48 treatment. in vivo, the PDK1 agonist PS48 could maintain the bone mass of mice treated with dexamethasone. This study provides a new understanding of the mechanism of glucocorticoid-induced osteoporosis.[2]

C17H15CLO2

286.75

286.076

C, 71.21; H, 5.27; Cl, 12.36; O, 11.16

1180676-32-7

44141940

White to off-white solid powder

1.2±0.1 g/cm3

444.1±24.0 °C at 760 mmHg

222.4±22.9 °C

0.0±1.1 mmHg at 25°C

1.609

5.59

37.3

1

2

5

20

337

0

O=C(O)/C=C(C1=CC=CC=C1)/CCC2=CC=C(Cl)C=C2

LLJYFDRQFPQGNY-QINSGFPZSA-N

InChI=1S/C17H15ClO2/c18-16-10-7-13(8-11-16)6-9-15(12-17(19)20)14-4-2-1-3-5-14/h1-5,7-8,10-12H,6,9H2,(H,19,20)/b15-12-

(Z)-5-(4-chlorophenyl)-3-phenylpent-2-enoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~57 mg/mL (~198.8 mM)
Ethanol: ~57 mg/mL (~198.8 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.72 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (8.72 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (8.72 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。