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 the rate-limiting enzyme in the glycolysis pathway and is highly expressed in cancer cells. Cancer cells are highly dependent on PKM2 to meet their anabolism and energy needs, therefore, specific targeting of PKM2 has the potential to become a cancer treatment strategy. This article reports the synthesis and biological evaluation of a novel naphthoquinone derivative as a selective small molecule inhibitor of PKM2. Some target compounds, such as compound 3k, have shown stronger PKM2 inhibitory activity than the best reported PKM2 inhibitor, shikonin. The high-performance compound 3k also showed nanomolar antiproliferative activity against a range of cancer cell lines that highly express PKM2, including HCT116, HeLa and H1299, with IC50 values ranging from 0.18 to 1.56 μM. In addition, compound 3k is more cytotoxic to cancer cells than to normal cells. The discovery of a novel and highly effective small molecule inhibitor of PKM2 not only provides candidate compounds for cancer treatment, but also provides a tool for in-depth evaluation of the function of PKM2. [1]

---

This study describes the identification and characterization of a previously unreported PKM2 inhibitor. These novel naphthoquinone derivatives, as PKM2 inhibitors, exhibit stronger inhibitory activity than shikonin, with similar selectivity. These compounds lock PKM2 in a low-activity conformation, thereby forcing altered metabolism in cancer cells, reducing their metabolic flexibility compared to the normal state. Furthermore, some target compounds show higher antiproliferative activity than shikonin. However, some unexpected results emerged in enzyme activity and cytotoxicity experiments. The lack of correlation between PKM2 inhibitory activity and the in vitro cytotoxicity of the target compounds suggests that cells may activate these compounds through other mechanisms besides PKM2 inhibition. Nevertheless, PKM2 is undoubtedly the target of these synthetic naphthoquinone derivatives, as the IC50 values of some compounds inhibiting PKM2 activity are close to the nanomolar concentration range. PKM2 has been shown to be a major player not only in pyruvate kinase metabolic reprogramming but also, as a protein kinase in cancer cells, directly regulates gene expression. Upon EGFR activation, PKM2 translocates from the cytoplasm to the nucleus of cancer cells, activating β-catenin, inducing CCDN1 and c-Myc expression, and upregulating GLUT1 and lactate dehydrogenase A (LDHA). Upregulation of these glycolytic genes increases glucose consumption and lactate production, which in turn promotes tumorigenesis. Therefore, inhibiting the transcription of tumor genes and the expression of glycolytic genes co-activated by PKM2 is also a potential tumor treatment strategy. We will try to determine whether this mechanism applies to synthetic naphthoquinone derivatives. If applicable, this mechanism could also explain the structure-activity relationship differences between the enzyme activity and cytotoxicity of target compounds. Further research will also focus on the structural study of PKM2 protein complexes with small molecule inhibitors, which may help to discover more effective compounds. In any case, the results of this study lay the 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.)