Pyruvate kinase M2 (PKM2) (IC50 = 2.95 μM)

PKM2-IN-1 (compound 3k) is an inhibitor of pyruvate kinase M2 (PKM2) having an IC50 of 2.95±0.53 μM. The majority of the investigated compounds exhibited some degree of PKM2 inhibition, according to the results, and certain compounds, including compound 3k and compound 6d, which are PKM2-IN-1, demonstrated more potent activity than the positive control shikonin. Examples of substances that exhibit dose-dependent inhibitory effects on PKM2 are PKM2-IN-1 and 6d. Conversely, these compounds have inhibitory effects as modest as shikonin on PKM1 and PKL. The results of the testing indicated that 3a, PKM2-IN-1, and 3r were the most effective compounds against HCT116 and HeLa cells, with IC50 values of 0.39 to 0.41 μM, 0.18 to 0.29 μM, and 0.18 to 0.38 μM, respectively [1].

PKM2 activity assay [1]
Pyruvate kinase activity was measured with a fluorescent pyruvate kinase-lactate dehydrogenase coupled assay as described previously. All compounds were tested in a kinetic mode by coupling the generation of pyruvate by pyruvate kinase to the depletion of NADH through lactate dehydrogenase. For PKM2, 40 μL of buffer (50 mM Tris–HCl, pH 7.5, 10 mM KCl, 5 mM MgCl2), 1 μL of compound and 5 μL of enzyme solution were dispensed into Corning black solid 96-well plates and incubated for 15 min. 55 μL of substrate mix (final concentration, 0.5 mM PEP, 4.0 mM ADP, 0.12 mM NADH, 0.25 mM FBP and 1 unit LDH) was then added, and the plates were placed in a FlexStation 3, followed by determination of NADH fluorescence at 30 s exposure intervals for 3–6 min. Data was collected on the FlexStation 3.

Cell viability experiments [1]
Cell viability was detected with the MTS assa according to the manufacturer's instructions. Briefly, 5000 cells in per well were plated in 96-well plates. After incubated for 12 h, the cells were treated with different concentration of tested compound or DMSO (as negative control) for 48 h. Then 20 μL MTS was added in per well and incubated at 37 °C for 3 h. Absorbance of each well was determined by a microplate reader (Flexstation 3) at a 490 nm wavelength. The IC50 values were calculated using Prism Graphpad software of the triplicate experiment.

[1]. Discovery of novel naphthoquinone derivatives as inhibitors of the tumor cell specific M2 isoform of pyruvate kinase. Eur J Med Chem. 2017 Sep 29;138:343-352.

Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme of the glycolytic pathway which is highly expressed in cancer cells. Cancer cells rely heavily on PKM2 for anabolic and energy requirements, and specific targeting of PKM2 therefore has potential as strategy for cancer therapy. Here, we report the synthesis and biologic evaluation of novel naphthoquinone derivatives as selective small molecule inhibitors of PKM2. Some target compounds, such as compound 3k, displayed more potent PKM2 inhibitory activity than the reported optimal PKM2 inhibitor shikonin. The well performing compound 3k also showed nanomolar antiproliferative activity toward a series of cancer cell lines with high expression of PKM2 including HCT116, Hela and H1299 with IC50 values ranging from 0.18 to 1.56 μM. Moreover, compound 3k exhibited more cytotoxicity on cancer cells than normal cells. The identification of novel potent small molecule inhibitors of PKM2 not only offers candidate compounds for cancer therapy, but also provides a tool with which to evaluate the function of PKM2 in depth. [1]

---

In this study we have described the identification and characterization of a previously undescribed chemical class of PKM2 inhibitors. These novel naphthoquinone derivatives act as PKM2 inhibitors, and show high inhibitory responses that are more potent than shikonin and have selectivity similar to shikonin. These compounds lock PKM2 into a low activity conformation which forces alteration of cancer cell metabolism that is less metabolically flexible than metabolism in the normal state. Moreover, some of these target compounds show higher antiproliferative effects than shikonin. However, there are unexpected results in the enzyme and cytotoxicity experiments. The absence of correlation between PKM2 inhibitory activity and in vitro cytotoxicity of the target compounds suggests that the cells are probably using other mechanisms to activate these compounds, except in the case of PKM2 inhibitory activity. However, there is no doubt that PKM2 is a target of these synthesized naphthoquinone derivatives, through the IC50 of some compounds which inhibit PKM2 activity is closed to nanomolar concentration range.
PKM2 has been identified as a major contributor not only of metabolic reprogramming as for pyruvate kinases but also in direct regulation of gene expression as a protein kinase in cancer cells. Upon EGFR activation, PKM2 translocates from the cytoplasm into the nucleus of cancer cells where it activates β-catenin to induce CCDN1 and c-Myc expression and upregulate GLUT1 and lactate dehydrogenase A (LDHA). Upregulation of these glycolysis genes increases glucose consumption and lactate production, and subsequently promotes tumorigenesis. Therefore, inhibition of PKM2-coactivated tumor gene transcription and glycolysis gene expression are also potential tumor treatment strategies. An attempt will be made to determine whether the mechanism works for the synthesized naphthoquinone derivatives. If so, the mechanism also can explain the SAR discrepancy of enzyme activity and cytotoxicity of target compounds.
Further investigation will also focus on structural studies of PKM2 protein and small molecule inhibitor complex, which may lead to identification of compounds with higher effectiveness. In any case, the findings presented here establish a foundation for the development of PKM2-targeted anticancer therapies.[1]

C18H19NO2S2

345.478962182999

345.085

C, 62.58; H, 5.54; N, 4.05; O, 9.26; S, 18.56

94164-88-2

131698387

Light yellow to yellow solid powder

3.5

0

4

3

23

549

0

O=C1C(C)=C(CSC(N2CCCCC2)=S)C(=O)C2C1=CC=CC=2

STAFOGVMELKGRI-UHFFFAOYSA-N

InChI=1S/C18H19NO2S2/c1-12-15(11-23-18(22)19-9-5-2-6-10-19)17(21)14-8-4-3-7-13(14)16(12)20/h3-4,7-8H,2,5-6,9-11H2,1H3

(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl piperidine-1-carbodithioate

PKM2-IN-1; PKM2 IN-1; 94164-88-2; PKM2-IN-1; PKM2 inhibitor(compound 3k); PKM2 inhibitor; (3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl piperidine-1-carbodithioate; CHEMBL4128703; (3-methyl-1,4-dioxonaphthalen-2-yl)methyl piperidine-1-carbodithioate; PKM2-IN 1

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 : ~10 mg/mL (~28.95 mM)

Solubility in Formulation 1: 8 mg/mL (23.16 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 80.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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.

---

Solubility in Formulation 2: ≥ 8 mg/mL (23.16 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 80.0 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.

---

View More

Solubility in Formulation 3: ≥ 8 mg/mL (23.16 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 80.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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