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 that is benzene substituted by [4-(dimethylcarbamoyl)phenyl]sulfonyl, chloro and [(2R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoyl]amino groups at positions 1, 3 and 4, respectively. It is a potent and non-ATP-competitive inhibitor of pyruvate dehydrogenase kinase 2 (PDHK2) with IC50 of 6.4 nM and exhibits glucose-lowering activity. Also inhibits PDHK1 at higher levels (IC50 = 36.8 nM). It has a role as a hypoglycemic agent and an EC 2.7.11.2 - [pyruvate dehydrogenase (acetyl-transferring)] kinase inhibitor. It is a member of benzamides, a sulfone, a tertiary alcohol, a tertiary carboxamide, a secondary carboxamide, a member of monochlorobenzenes and an organofluorine compound.

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This is the first report of the testing of a novel PDHK inhibitor in the obese Zucker rat and provides clear evidence that a PDHK inhibitor can improve the control of blood glucose levels in an animal model with impaired glucose homoeostasis. This is in contrast to the statement by Aicher et al. [14] that small-molecule inhibitors were ineffective in animal models of diabetes (ob/ob mice and ZDF rats). Clearly our data would suggest that an inhibitor of PDHK will be an effective novel therapy for type 2 diabetes.[2]

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Background: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism.
Methods: Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested.
Results: We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells.
Conclusions: In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut).[3]

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DCA has been tested in multiple cell culture and rodent models of cancer, and PDK1 knock-down has been described to enhance the sensitivity of BRAFV600E positive melanoma to BRAF inhibitors. We tested a pan-PDK inhibitor, AZD7545, which interferes with the lypoyl binding pocket of PDKs for its capacity to inhibit the growth of BRAFV600E and NRASmut positive cells. We observed that AZD7545 suppressed growth of BRAFV600E positive cells and kinase inhibitor-resistant cells when applied in μM concentrations. Interestingly, AZD7545 had no effect on keratinocytes (HaCaT) and normal fibroblasts, cell types which constitute the cutaneous microenvironment of melanoma tumors (data not shown), indicating selective effects on BRAF/NRAS-mutated or resistant cancer cells. Finally, we show that the combination of BRAF inhibitors with PDK inhibitors is more efficient in tumor growth suppression than the single treatment suggesting that the simultaneus targeting of metabolic pathways might 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.)