PDHK2 (IC50 = 6.4 nM); PDHK1 (IC50 = 36.8 nM); pyruvate dehydrogenase kinase 2 (PDHK2)

AZD7545 (10 μM; 90 hours for BRAF V600E human melanoma cells and 120 hours for NRASmut human melanoma cells) specifically inhibits the growth of cells with BRAF and NRAS mutations as well as in inhibitor-resistant human melanoma[2].

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The PDH (pyruvate dehydrogenase) multi-enzyme complex catalyses a key regulatory step in oxidative glycolysis. Phosphorylation of the E1 subunit of the complex on serine residues results in the inactivation of enzyme activity. A family of four dedicated PDH kinase isoenzymes exists, each of which displays a distinct tissue-specific expression profile. AZD7545 is one of a series of PDH kinase inhibitors developed for the treatment of type 2 diabetes. The isoenzyme-selectivity profile of AZD7545 and related compounds is described and the consequences for their in vivo mode of action are discussed. [1]

A single dose of AZD7545 (Oral administration; 10 mg/kg once daily (08:00 h) or twice daily (08:00 and 18:00 h); for 7 days) increases the proportion of liver PDH in its active, dephosphorylated form in a dose-related manner in Wistar rats. In obese Zucker (fa/fa) rats, a single 10 mg/kg dose also significantly increases muscle PDH activity[2].

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PDH (pyruvate dehydrogenase) is a key enzyme controlling the rate of glucose oxidation, and the availability of gluconeogenic precursors. Activation of PDH in skeletal muscle and liver may increase glucose uptake and reduce glucose production. This study describes the properties of AZD7545, a novel, small-molecule inhibitor of PDHK (PDH kinase). In the presence of PDHK2, AZD7545 increased PDH activity with an EC(50) value of 5.2 nM. In rat hepatocytes, the rate of pyruvate oxidation was stimulated 2-fold (EC(50) 105 nM). A single dose of AZD7545 to Wistar rats increased the proportion of liver PDH in its active, dephosphorylated form in a dose-related manner from 24.7 to 70.3% at 30 mg/kg; and in skeletal muscle from 21.1 to 53.3%. A single dose of 10 mg/kg also significantly elevated muscle PDH activity in obese Zucker (fa/fa) rats. Obese, insulin-resistant, Zucker rats show elevated postprandial glucose levels compared with their lean counterparts (8.7 versus 6.1 mM at 12 weeks old). AZD7545 (10 mg/kg) twice daily for 7 days markedly improved the 24-h glucose profile, by eliminating the postprandial elevation in blood glucose. These results suggest that PDHK inhibitors may be beneficial agents for improving glucose control in the treatment of type 2 diabetes[2].

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PDH activation in vivo [2]
PDH activity was assessed ex vivo in tissue extracts via the linked spectrophotometric enzyme assay described by Coore et al. Activity was assessed before and after complete dephosphorylation by purified pig heart phosphatase. A single acute dose of AZD7545 to fed Wistar rats increased the percentage of active PDH in the liver in a dose-dependent manner from a basal (vehicle-dosed) level of 24.7 ± 6.2% to 70.3 ± 2.6% at the highest dose evaluated (30 mg/kg), where it is evident that maximal activation was not attained (Figure 1). PDH in gastrocnemius muscle is increased at similar doses of compound, but again full activation is not achieved at the doses tested (from 21.1 ± 1.9 to 53.3 ± 4.0%). Aicher et al. demonstrated in vivo activity in rats which had undergone a period of fasting; PDH activation was determined either indirectly via plasma lactate or by measuring PDH activity ex vivo [14]. The increases in PDH in liver and tibialis anterior muscle elicited by representative compounds were relatively modest following doses of 20 µmol/kg (approx. 8 mg/kg), but results are not expressed as a proportion of PDH in the active state and hence cannot be compared with results from our own study.

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Effect of AZD7545 in Zucker (fa/fa) rats [2]
The obese ( fa/fa) Zucker rat is a frequently used model of the insulin-resistant or prediabetic state. It exhibits impaired glucose tolerance, hyperphagia, hyperinsulinaemia and hyperlipidaemia. While not overtly hyperglycaemic, the fa/fa rat exhibits an abnormal glucose profile following feeding compared with its lean counterpart (4 h into the dark feeding phase, blood glucose levels are 8.7 mM, compared with 6.1 mM in lean animals). This is associated with a small but consistent and significantly elevated glycated haemoglobin level (3.49 versus 3.26%). At the age used in our study (12 weeks), PDH activity in the fa/fa rat was elevated compared with that in lean Zucker or Wistar rats. We have measured no difference in expression levels of PDHK2 or PDHK4 between obese and lean Zucker rats. As in Wistar rats, PDH in fed fa/fa rats can be further activated by PDHK inhibitors (for example 10 mg/kg AZD7545 increases muscle PDH from 61.0 to 97.0% active, and liver PDH from 33.5 to 72.8%). Obese Zucker rats were treated with the PDHK inhibitor AZD7545 orally for 7 days, and at the end of this period the glucose profile was monitored for 24 h (Figure 2). In control, vehicle-treated rats, blood glucose rose to a maximum of 9.45 ± 1.11 mM, whereas in rats treated with AZD7545 once daily at 08:00 h, the concentration was 6.55 ± 0.58 mM at the same time. A similar obliteration of the postprandial glucose elevation was seen after administration twice daily.

Real-time proliferation assays [3]
50 × 103 cells/well of 5 melanoma cell lines were seeded in 12-well plates and 24 h later stimulated with 10 μM of AZD7545. Cellular growth was monitored in the IncuCyte ZOOM live cell microscope and images were taken in phase contrast every 3 h for a total of 90 h. Proliferation assay were carried out for three biological replicates and for each figure one representative replicate is shown.

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Long term proliferation assay [3]
The A375-iRFP cells were used to test the combination of 1 μM PLX4032 and 10 μM AZD7545 versus PLX4032 alone. 10,000 cells were seeded per well (6 well plates) and were treated with the inhibitors for 3 weeks. Medium was changed twice a week. After 3 weeks of treatment, cells were scanned and the intensity of the iRFP signal was measured using the LI-COR Odyssey instrument. The iRFP signal was quantified using the Image Studio lite version 4.0 software.

Obese male (fa/fa) Zucker rats[2];
10 mg/kg;
Oral administration; once a day (08:00 h) or Twice a day ( 08:00 and 18:00 h); for 7 days;

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Male Wistar rats (200–240 g) were dosed orally at 08:00 h with AZD7545 in suspension in 0.5% (w/w) methocel/0.1% polysorbate 80. After 2 h animals were anaesthetized with sodium pentabarbitone (60 mg/kg, intraperitoneally) and tissues excised, freeze-clamped and stored in liquid nitrogen prior to assay. Tissue extracts were prepared and PDH activity determined by the method of Coore et al. Total PDH activity (PDHt) in the extract was determined by assay following dephosphorylation by porcine heart PDP in the presence of 20 mM MgCl2/0.8 mM CaCl2. PDH activity is given as the proportion in the active (dephosphorylated) form in extracts from (
) liver and () gastrocnemius muscle. Results are compared with tissues from control.[2]

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Obese male (fa/fa) Zucker rats, housed in a 06:00 h on/18:00 h off light cycle, were dosed for 7 days with either 10 mg/kg AZD7545, given orally, at 08:00 h () or 08:00 and 18:00 h () or with vehicle (
). On day 8, glucose was measured using a hand-held glucose monitor.[2]

[1]. AZD7545 is a selective inhibitor of pyruvate dehydrogenase kinase 2. Biochem Soc Trans. 2003 Dec;31(Pt 6):1168-70.

[2]. AZD7545, a novel inhibitor of pyruvate dehydrogenase kinase 2 (PDHK2), activates pyruvate dehydrogenase in vivo and improves blood glucose control in obese (fa/fa) Zucker rats. Biochem Soc Trans. 2003 Dec;31(Pt 6):1165-7.

[3]. ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells. Mol Cancer. 2017 Jun 8;16(1):102.

AZD7545 is a sulfone compound in which the benzene ring is substituted at positions 1, 3, and 4 with [4-(dimethylcarbamoyl)phenyl]sulfonyl, chlorine, and [(2R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionyl]amino. It is a potent, non-ATP-competitive pyruvate dehydrogenase kinase 2 (PDHK2) inhibitor with an IC50 of 6.4 nM and exhibits hypoglycemic activity. Furthermore, it inhibits PDHK1 at higher concentrations (IC50 = 36.8 nM). AZD7545 can be used as a hypoglycemic agent and an EC 2.7.11.2 type pyruvate dehydrogenase (acetyltransferase) kinase inhibitor. It belongs to the benzamide, sulfone, tertiary alcohol, tertiary carboxamide, secondary carboxamide, monochlorobenzene, and organofluorine compounds.

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This is the first report of testing a novel PDHK inhibitor in obese Zucker rats and provides clear evidence that PDHK inhibitors can improve glycemic control in animal models of impaired glucose homeostasis. This is contrary to the claims of Aicher et al. [14], who argued that small molecule inhibitors were ineffective in diabetic animal models (ob/ob mice and ZDF rats). Clearly, our data suggest that PDHK inhibitors could become an effective new therapy for type 2 diabetes. [2]

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Background: Most patients with BRAFV600E-positive melanoma respond well to combination therapy with BRAF kinase inhibitors and MEK inhibitors. However, some patients are inherently resistant, and most eventually develop resistance to treatment. There is an urgent need to find new therapeutic targets for patients who do not respond well to BRAF and MEK inhibitors. Cellular metabolism is a promising new target: mutant BRAFV600E has been shown to affect cellular metabolism. Methods: We performed time-course experiments and a series of Western blot analyses on a co-incubation group of BRAFV600E and BRAFWT/NRASmut human melanoma cells with BRAF and MEK1 kinase inhibitors. We used siRNA to investigate related metabolic factors. Reactive oxygen species (ROS) were detected using confocal microscopy, and the inhibitor AZD7545, targeting pyruvate dehydrogenase kinase (PDK), was tested. Results: We found that inhibition of the RAS/RAF/MEK/ERK pathway induced phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in both BRAFV600E and BRAFWT/NRASmut mutant cells. Inhibition of BRAF, MEK1, and siRNA knockdown of ERK1/2 all mediated PDH phosphorylation. siRNA-mediated PDK knockdown or the use of DCA (a pan-PDK inhibitor) eliminated PDH-E1α phosphorylation. BRAF inhibitor treatment also induced ROS upregulation, accompanied by the induction of PDH phosphorylation. MitoQ's inhibition of ROS suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediates PDK activation. Interestingly, inhibition of PDK1 using AZD7545 specifically inhibited the growth of BRAF mutant and BRAF inhibitor-resistant melanoma cells. Conclusion: In BRAFV600E and BRAFWT/NRASmut melanoma cells, inhibition of the RAS/RAF/MEK/ERK pathway increased ROS production, subsequently activating PDK, which in turn phosphorylates and inactivates PDH. As a possible remedy, inhibition of the tricarboxylic acid cycle leads to reduced oxidative metabolism and decreased ROS levels. We found that inhibition of PDK using AZD7545 inhibited the growth of BRAF mutant and BRAF inhibitor-resistant melanoma cells. Therefore, small molecule PDK inhibitors like AZD7545 may be a potential combination therapy for patients with melanomas that have activating mutations in the RAS/RAF/MEK/ERK pathway (50% BRAF mutation, 25% NRAS mutation, 11.9% NF1 mutation). [3] DCA has been tested in various cell cultures and rodent cancer models, and studies have shown that PDK1 knockdown can enhance the sensitivity of BRAFV600E-positive melanomas to BRAF inhibitors. We tested a pan-PDK inhibitor, AZD7545, which inhibits the growth of BRAFV600E and NRAS-mutant positive cells by interfering with the acyl-binding pocket of PDK. We observed that AZD7545 at a concentration of μM inhibited the growth of BRAFV600E-positive cells and kinase inhibitor-resistant cells. Interestingly, AZD7545 had no effect on keratinocytes (HaCaT) and normal fibroblasts (cell types that make up the skin microenvironment of melanoma tumors) (data not shown), suggesting that it has a selective effect on BRAF/NRAS mutant or drug-resistant cancer cells. Finally, we found that the combination of BRAF inhibitors and PDK inhibitors was more effective in inhibiting tumor growth than monotherapy, suggesting that simultaneous targeting of metabolic pathways may indeed be beneficial for melanoma patients. [3]

C19H18CLF3N2O5S

478.87

478.057

C, 47.66; H, 3.79; Cl, 7.40; F, 11.90; N, 5.85; O, 16.71; S, 6.69

252017-04-2

16741245

White to off-white solid powder

1.5±0.1 g/cm3

683.1±55.0 °C at 760 mmHg

366.9±31.5 °C

0.0±2.2 mmHg at 25°C

1.573

2.66

2

8

5

31

778

1

ClC1C([H])=C(C([H])=C([H])C=1N([H])C([C@](C([H])([H])[H])(C(F)(F)F)O[H])=O)S(C1C([H])=C([H])C(C(N(C([H])([H])[H])C([H])([H])[H])=O)=C([H])C=1[H])(=O)=O

DTDZLJHKVNTQGZ-GOSISDBHSA-N

InChI=1S/C19H18ClF3N2O5S/c1-18(28,19(21,22)23)17(27)24-15-9-8-13(10-14(15)20)31(29,30)12-6-4-11(5-7-12)16(26)25(2)3/h4-10,28H,1-3H3,(H,24,27)/t18-/m1/s1

4-[3-chloro-4-[[(2R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoyl]amino]phenyl]sulfonyl-N,N-dimethylbenzamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.22 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 (5.22 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 (5.22 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.)