PDPK1
The PDK1 agonist PS48 has the power to enhance the osteogenic capacity of dexamethasone-treated MC3T3-E1 cells. In MC3T3-E1 cells treated with dexamethasone after PS48 treatment, the protein levels of p-AKT and p-mTOR are elevated. [2]

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PS48 binding to PDK1 characterized by ITC[1]
The binding properties of PS48 and its analog compound PS08 (4) to PDK150–359 Y288G Q292A were determined by isothermal titration calorimetry (ITC) at 20 °C (Table 1 and Supplementary Fig. 2). Both compounds bound to PDK150–359 with a 1:1 stoichiometry and a binding affinity in the micromolar range (PS48, Kd = 10.3 μM; PS08, Kd = 6.2 M). The close resemblance of the compounds and their thermodynamic parameters, ΔH, ΔG and TΔS (Table 1), suggested a similar binding mechanism. In both cases, binding was both enthalpy and entropy favorable and driven by the entropy term (ΔH/ΔG = 27.1% and 25.0%, respectively; Table 1)[1].

The PDK1 agonist PS48 has the power to enhance the osteogenic capacity of dexamethasone-treated MC3T3-E1 cells. In MC3T3-E1 cells treated with dexamethasone after PS48 treatment, the protein levels of p-AKT and p-mTOR are elevated. [2]

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PS48 binding to PDK1 characterized by ITC[1]
The binding properties of PS48 and its analog compound PS08 (4) to PDK150–359 Y288G Q292A were determined by isothermal titration calorimetry (ITC) at 20 °C (Table 1 and Supplementary Fig. 2). Both compounds bound to PDK150–359 with a 1:1 stoichiometry and a binding affinity in the micromolar range (PS48, Kd = 10.3 μM; PS08, Kd = 6.2 M). The close resemblance of the compounds and their thermodynamic parameters, ΔH, ΔG and TΔS (Table 1), suggested a similar binding mechanism. In both cases, binding was both enthalpy and entropy favorable and driven by the entropy term (ΔH/ΔG = 27.1% and 25.0%, respectively; Table 1)[1].

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In MC3T3-E1 pre-osteoblast cells treated with dexamethasone, the addition of PS48 (5 µM) promoted the expression of PDK1 protein, as well as osteogenic markers Runx2, OCN, and ALP, as determined by western blotting. [2]
ALP staining and Alizarin Red staining assays showed that PS48 (5 µM) enhanced the osteogenic function of MC3T3-E1 cells that were suppressed by dexamethasone treatment. [2]
Treatment of MC3T3-E1 cells with PS48 (5 µM) alongside dexamethasone led to increased protein levels of phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR), indicating activation of the AKT/mTOR signaling pathway. [2]

In vivo, the PDK1 agonist PS48 can maintain the bone mass of mice treated with dexamethasone.[2]

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Double transgenic APPsw/PSENdE9 mice, a model for Alzheimer's disease, were used to test the oral administration of PS48, a PDK-1 agonist, on preventing the expected decline in learning and memory in the Morris Water Maze (MWM). Mice were raised on either standard (SD) or high fat (HFD) diets, dosed beginning 10 months age and tested at an advanced age of 14 months. PS48 had positive effects on learning the spatial location of a hidden platform in the TG animals, on either SD or HFD, compared to vehicle diet and WT animals. On several measures of spatial memory following successful acquisition (probe trials), the drug also proved significantly beneficial to animals on either diet. The PS48 treatment-effect size was more pronounced in the TG animals on HFD compared to on SD in several of the probe measures. HFD produced some of the intended metabolic effects of weight gain and hyperglycemia, as well as accelerating cognitive impairment in the TG animals. PS48 was found to have added value in modestly reducing body weights and improving OGTT responses in TG groups although results were not definitive. PS48 was well tolerated without obvious clinical signs or symptoms and did not itself affect longevity. These results recommend a larger preclinical study before human trial[3].

Isothermal titration calorimetry.[1]
Calorimetric titrations were performed using the VP-ITC instrument as previously described with minor modifications as detailed in the Supplementary Methods.

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Protein kinase activity tests.[1]
Protein kinase activity tests were performed essentially as previously described8,28 using T308tide as a substrate for PDK1. Further information is detailed in the Supplementary Methods.

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Probing the conformation of the ATP binding site in PDK1.[1]
The activation of PDK1 by HM-polypeptides and small compounds is due to a change in the conformation of the enzyme. We probed the conformation of the ATP binding site in PDK1 by scanning the steady state fluorescence of TNP-ATP/PDK1 in the presence or absence of P-HM-polypeptides, PIFtide or low-molecular-weight compounds. Data were obtained in a Varian Cary Eclipse spectrofluorometer (excitation λ = 479 nm; emission scanning λ = 500–600 nm; excitation slit = 10 nm; emission slit = 10 nm) at a rate of 200 nm min−1, with 150 data points per 100 nm scanning and 0.3 s averaging time. Further information is detailed in the Supplementary Methods.

MC3T3-E1 cells are cultured in an osteogenic differentiation medium containing 4 mM glycerophosphate and 25 g/mL ascorbic acid until 70% confluency. The osteogenic differentiation medium is then supplemented for 14 days with dexamethasone (final ethanol concentration, 0.01%, vol/vol) at various concentrations. Every two days, the culture medium is changed. Dexamethasone 107 M culture medium supplemented with or without PS48 (5 M) is used to cultivate MC3T3-E1 cells.

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Cell Culture & Treatment: The mouse pre-osteoblast cell line MC3T3-E1 (subclone 4) was cultured in α-MEM supplemented with fetal bovine serum. For osteogenic differentiation experiments, cells were cultured in differentiation medium containing glycerophosphate and ascorbic acid. PS48 was used at a final concentration of 5 µM in the culture medium, alongside dexamethasone (10^-7 M) where indicated. [2]
Western Blotting: Total protein was isolated from MC3T3-E1 cells using RIPA lysis buffer. Protein samples were separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific primary antibodies against PDK1, Runx2, OCN, ALP, phospho-AKT, total-AKT, phospho-mTOR, total-mTOR, and GAPDH (as a loading control). After incubation with HRP-conjugated secondary antibodies, target proteins were visualized using a chemiluminescent substrate kit. [2]
ALP Staining: After osteogenic induction, MC3T3-E1 cells were washed, fixed, and stained for Alkaline Phosphatase (ALP) activity using a BCIP/NBT reagent kit according to the manufacturer's instructions. Stained cells were photographed. [2]
Alizarin Red Staining: To assess mineralization, fixed MC3T3-E1 cells were stained with an Alizarin Red working solution for 5 minutes, then washed and photographed. [2]

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 can transmit a large number of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by inducing conformational changes in target or interacting proteins. Previous studies have described an allosteric site that mediates phosphorylation-dependent activation of AGC kinase. AGC kinase PDK1 is activated by binding to a phosphorylation motif on its substrate. This paper reports the crystal structure of PDK1 bound to a rationally designed low molecular weight activator and describes the conformational changes induced by the small molecule compound in crystals and solutions using fluorescence detection and deuterium exchange experiments. Our results show that the binding of the compound induces local changes at the target site (PIF binding pocket) and allosteric changes at the ATP binding site and activation loop. In summary, we elucidate the molecular details of the allosteric changes that trigger PDK1 activation by a small molecule compound through the mimicking of phosphorylation-dependent conformational changes. [1] Long-term high-dose glucocorticoid therapy can lead to decreased osteoblast viability and function, which in turn can lead to osteoporosis and osteonecrosis. This study investigated the role and mechanism of HIF-1α in glucocorticoid-induced osteogenic inhibition in MC3T3-E1 cells. Results showed that HIF-1α protein expression decreased when MC3T3-E1 cells were exposed to dexamethasone (Dex) at concentrations ranging from 10⁻⁹ to 10⁻⁶ M. Dexamethasone treatment also decreased PDK1 expression in MC3T3-E1 cells. Combined treatment with the glucocorticoid receptor antagonist RU486 and dexamethasone enhanced HIF-1α expression in MC3T3-E1 cells. Furthermore, upregulation of HIF-1α expression promoted osteogenic capacity and PDK1 expression in MC3T3-E1 cells. However, the HIF-1α antagonist 2-methoxyestradiol (2-ME) had the opposite effect in dexamethasone-treated MC3T3-E1 cells. Furthermore, although HIF-1α expression was upregulated after transduction of the adenovirus-HIF-1α construct, the PDK1 antagonist dichloroacetic acid still inhibited osteogenic capacity of MC3T3-E1 cells. The PDK1 agonist PS48 promoted osteogenic capacity of dexamethasone-treated MC3T3-E1 cells. Importantly, after treatment with PS48, the protein levels of p-AKT and p-mTOR in dexamethasone-treated MC3T3-E1 cells were increased. In vivo experiments showed that the PDK1 agonist PS48 maintained bone mass in dexamethasone-treated mice. This study provides new insights into the mechanism of glucocorticoid-induced osteoporosis. [2]

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This study used PS48 as a pharmacological tool to activate PDK1. It was able to antagonize the inhibition of dexamethasone-induced osteogenic function of pre-osteoblasts by activating the PDK1/AKT/mTOR signaling pathway. [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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.72 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.72 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.72 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)