Identification of small molecule inhibitors of pyruvate kinase M2

Abstract

A common feature of tumors arising from diverse tissue types is a reliance on aerobic glycolysis for glucose metabolism. This metabolic difference between cancer cells and normal cells could be exploited for therapeutic benefit in patients. Cancer cells universally express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2), and previous work has demonstrated that PKM2 expression is necessary for aerobic glycolysis and cell proliferation in vivo. Because most normal tissues express an isoform of pyruvate kinase other than PKM2, selective targeting of PKM2 provides an opportunity to target cell metabolism for cancer therapy. PKM2 has an identical catalytic site as the related M1 splice variant (PKM1). However, isoform selective inhibition is possible as PKM2 contains a unique region for allosteric regulation. We have screened a library of greater than 1,00,000 small molecules to identify such inhibitors. The inhibitors identified for PKM2 fell primarily into three distinct structural classes. The most potent PKM2 inhibitor resulted in decreased glycolysis and increased cell death following loss of growth factor signaling. At least part of this effect was due to on-target PKM2 inhibition as less cell death was observed in cells engineered to express PKM1. These data suggest that isoform selective inhibition of PKM2 with small molecules is feasible and support the hypothesis that inhibition of glucose metabolism in cancer cells is a viable strategy to treat human malignancy.

Figure 1. Schematic representation of the assays used to measure pyruvate kinase activity

The pyruvate kinase (PK) reaction can be coupled to the faster lactate dehydrogenase (LDH) reaction, and pyruvate kinase activity followed by measuring a decrease in NADH fluorescence. Alternatively, ATP produced by the pyruvate kinase reaction can be measured by luminescence produced during the ATP-dependent firefly luciferase reaction.

Figure 2. Characterization of recombinant PKM2

A. PKM2 was expressed and purified from E. coli. A coomassie stained SDS-PAGE gel of the purified PKM2 fractions used for subsequent assays is shown. B. Activity of PKM2 protein was assessed by following loss of NADH fluorescence in an LDH coupled assay. PKM2 enzyme activity in the absence or presence of 50 μM FBP is shown. C. Lineweaver-Burk plot of enzyme velocity measured for PKM2 at various concentrations of PEP in the presence or absence of 50 μM FBP as shown. D. Enzyme velocity is plotted at various concentrations of ADP as shown. E. Enzyme velocity is plotted at various concentrations of PEP as shown.

Figure 3. A screen for small molecule inhibitors identified compounds selective for PKM2

A. The Z’-factor for each plate in the HTS is plotted as shown. B. The effect of two representative small molecules identified in the HTS as PKM2 selective inhibitors on PKM2 and PKM1 activity using the LDH coupled assay is shown. The data presented is from Compound 1 and Compound 2 as labeled (see Figure 4). Differences in NADH scale shown for PKM1 and PKM2 assays reflect optimization of the assay to compensate for inherent differences in specific activity of the two enzymes.

Figure 4. Three distinct structural classes of small molecules were identified that exhibit selective inhibition of PKM2 over PKM1

The structure of the most potent compound as judged by both IC₅₀ and % inhibition in each class is shown. The IC₅₀ against PKM2 as well as the percent inhibition of both PKM2 and PKM1 at 30 μM is shown for each compound. The standard error of the mean from three independent determinations of percent inhibition is shown in parentheses. The IC₅₀ for compound 2 was not determined (n.d.).

Figure 5. Compound 3 inhibits PKM2 and PKL activity

A. Independently obtained Compound 3 was re-tested for inhibition of PKM2 activity in the LDH-coupled assay at various concentrations as shown. B. The inhibition of PKM2, PKM1 and PKL activity by 50 μM of Compound 3 is shown.

Figure 6. Compound 3 inhibits PKM2 and is toxic to cells in culture

A. Viability of H1299 cells was assessed by propidium iodide exclusion using flow cytometry after two days of treatment with Compound 3 at the indicated concentration (solid diamonds) or DMSO control (open square) as shown. The Compound 3 used for in vivo experiments was independently purchased and not obtained from the screening library. B. H1299 cells were treated for six hours with DMSO (Ctrl) or 250 μM Compound 3 prior to lysis. The pyruvate kinase activity in the lysates was assessed by following loss of NADH fluorescence in the LDH coupled assay in the presence or absence of exogenously added FBP (125 μM) as shown. C. H1299 cells engineered to express either PKM1 or PKM2 were treated with DMSO (Ctrl) or 250 μM Compound 3 for 36 hours and viability assessed by propidium idodide exclusion and flow cytometry. The increased cell death observed in PKM2-expressing cells compared with PKM1-expressing cells in the presence of Compound 3 was statistically significant by Student’s t-test (p < 0.05). D. Viability of IL-3-dependent FL5.12 cells was assessed by propidium iodide exclusion using flow cytometry. Viability was assessed following treatment with Compound 3 at the indicated concentration in the presence or absence of IL-3 for 20 hours as shown. The effect of vehicle only (DMSO) is plotted as an open square on the Y axis. E. Viability of HCC827 cells was assessed by propidium iodide exclusion using flow cytometry. Viability was assessed after 2 days of treatment with Compound 3 at the indicated concentration in the presence or absence of 1 μM of the EGFR inhibitor Gefitinib (EGFRi) as shown.