Pyruvate kinase catalyzes the transfer of phosphate from phosphoenolpyruvate (PEP) to adenosine-5′-diphosphate (ADP), generating pyruvate and adenosine-5′-triphosphate (ATP). Pyruvate kinase activity can be measured via coupled assays to detect production of either pyruvate or ATP (). Pyruvate generation can be monitored kinetically by coupling the pyruvate kinase reaction to the lactate dehydrogenase (LDH) reaction. LDH oxidizes NADH to NAD+ while reducing pyruvate to lactate. Because the reaction catalyzed by LDH is more efficient than the PK reaction, the rate of NADH fluorescence loss corresponds to PK activity.
Schematic representation of the assays used to measure pyruvate kinase activity
To generate PKM2 enzyme suitable for inhibitor screening, N-terminal 6-His tagged human PKM2 was produced in E. coli
and purified using Ni-affinity chromatography (). Kinetic characterization of rPKM2 generated was in general agreement with published values. rPKM2 demonstrated robust activation by FBP (). Given that FBP has a slow off rate from PKM2 and does not activate according to classic Michaelis-Menton kinetics [15
], a precise Ka
was not determined. However FBP activated PKM2 in the μM range as expected from previously published studies [17
], and FBP decreased the Km
of PKM2 for PEP (). The Km
for both ADP and PEP of the rPKM2 used for screening were consistent with published values in the range of 100 μM () [17
Characterization of recombinant PKM2
To identify inhibitors of PKM2, the LDH-coupled kinetic assay was adapted to 384 well format for high-throughput screening (HTS). We modified the assay buffer conditions such that the enzyme retained activity even at room temperature for 2 hours, which is the time period needed to facilitate HTS (Supplemental Figure 1
). The screen was carried out in the presence of 125 μM FBP, which was sufficient to completely activate the enzyme at the beginning of the reaction. Reproducible results were observed between plates included in a pilot screen and the same plates included in the HTS (Supplemental Figure 2
). The Z’-factor ranged from 0.6-1.0 for all the plates screened (). Of the 107,360 compounds included in the HTS, approximately 7.4% of the compounds scored as greater than 50% inhibition (). These included many compounds which were subsequently excluded from further study because of high background fluorescent signal. 515 remaining compounds were cherry picked and retested. 41.5% scored as positive upon retesting at 30 μM compound concentration with 17.8% of those scoring positive at a compound concentration of 10 μM.
A screen for small molecule inhibitors identified compounds selective for PKM2
To identify selective inhibitors of PKM2, we next screened those compounds that demonstrated confirmed activity against PKM2 for activity against PKM1. Recombinant PKM1 was generated in bacteria and exhibited a high specific activity that was insensitive to FBP (Supplemental Figure 3A, B
). 59.8% of the confirmed hits against PKM2 at 30 μM compound concentration also demonstrated significant inhibition of PKM1 (). However, compounds were identified which exhibited selective, dose-dependent inhibition of PKM2 with less inhibition of PKM1 ().
Given the assay used to identify inhibitors was coupled to LDH, it is possible that compounds with activity against LDH could score as hits in the screen. These compounds would not be expected to score as specific inhibitors of PKM2 because LDH is used for both the PKM2 and PK-M1 assays; however, to further eliminate this possibility a luminescent end-point assay to measure ATP production was used (). Using this assay, compounds identified as selective PKM2 inhibitors were tested for the ability to inhibit PKM2- or PKM1-dependent ATP production (Supplemental Figure 3C
). The majority of compounds tested demonstrated selective PKM2 inhibition by this assay.
46% of the compounds with selective activity against PKM2 at 10 μM fell into three distinct structural classes. The most potent inhibitors in each class are shown in . Compound 1 is a thiazolidinedione. Several thiazolidinediones scored as hits in the HTS, and many thiazolidinedione-like compounds are commercially available. However we were unable to identify a more potent inhibitor of PKM2 either in terms of IC50 or % inhibition (not shown). Compound 2 is a dye derivative and was insoluble in aqueous solution even at low μM concentrations. This insolubility made calculation of a precise IC50 impossible and inhibited further characterization of Compound 2. Compound 3 demonstrated the lowest IC50 among the compounds identified from the screen and was selected for further studies.
Three distinct structural classes of small molecules were identified that exhibit selective inhibition of PKM2 over PKM1
Two other compounds related to Compound 3 also demonstrated greater than 60% inhibition of enzyme activity (Supplemental Figure 4
). Neither compound demonstrated increased potency or selectivity for PKM2 when compared with Compound 3. Several commercially available analogues of Compound 3 were also obtained and tested for activity against PKM2. All of these compounds proved to be inactive as PKM2 inhibitors.
To confirm that Compound 3 and not a contaminant present in the library used for HTS was responsible for PKM2 inhibition, Compound 3 was obtained from two independent sources and retested using the LDH-coupled kinetic assay. Compound 3 from both sources demonstrated inhibition of PKM2 with an IC50
in the 50 μM range (, not shown). While this value was slightly higher than that obtained from the screening compound, the selectivity of Compound 3 for PKM2 over PKM1 was confirmed (). At least some of the variability in activity could be accounted for by assay configuration as the concentration of PKM2 influences maximal enzyme activity presumably via affecting amounts of highly active tetramer [14
]. Compound 3 was also tested for an ability to inhibit PKL. Compound 3 was found to inhibit PKL with similar potency to PKM2 (). Analogous to Compound 3, Compound 1 was also found to inhibit PKL to a similar degree as PKM2 (Supplemental Figure 5
Compound 3 inhibits PKM2 and PKL activity
To test the effects of PKM2 inhibition on cancer cells, Compound 3 was added to H1299 human lung cancer cells. H1299 cells are known to express PKM2 as their only pyruvate kinase isoform [7
]. Consistent with an IC50
of 10-50 μM, high μM concentrations of Compound 3 were required to impact cellular glucose utilization or cell proliferation. Overnight treatment of H1299 cells with 100 μM of Compound 3 resulted in a 18.5% (+/− 3.5% (SEM)) decrease in glycolysis. For comparison, a 57.7% (+/− 5.5% (SEM)) decrease in glycolysis is observed upon RNA interference mediated knockdown of PKM2 to levels that result in inhibition of cell proliferation [7
]. Consequently, greater than 100 μM of Compound 3 is needed for cytotoxicity ().
Compound 3 inhibits PKM2 and is toxic to cells in culture
To determine if pyruvate kinase inhibition was observed in cells at the concentrations needed for cytotoxicity, the activity of pyruvate kinase in cell lysates was measured following treatment with 250 μM Compound 3 or vehicle control. 250 μM Compound 3 was the lowest concentration where a significant decrease in viability was observed after two days (). No difference in viability was apparent after 6 hours of drug treatment; however lysates from Compound 3 treated cells had significantly less pyruvate kinase activity (). In addition, treatment of cells with Compound 3 prevented the ability of exogenous FBP to activate pyruvate kinase in the lysates. These data are consistent with Compound 3 inhibiting pyruvate kinase activity in cells at concentrations where cytotoxicity is seen.
Despite the correlation between PKM2 inhibition in lysates and cytotoxicity, it is possible that the toxicity results from an off target effect of Compound 3. To evaluate this possibility, we generated PKM1 expressing cell by expressing the highly conserved mouse versions of PKM1 or PKM2, and then subjecting those cells to shRNA-mediated knockdown of the endogenous pyruvate kinase (Supplemental Figure 6
]). PKM1 has a higher IC50 for Compound 3 than PKM2, so PKM1-expressing cells should be less susceptible to toxicity mediated by on-target pyruvate kinase inhibition. Thirty-six hour exposure to Compound 3 resulted in increased cell death in the PKM2-expressing cells compared with the PKM1-expressing cells (). These data show that inhibition of PKM2 activity is responsible for at least a portion of the cytotoxicity caused by Compound 3, and suggest that a window for selective inhibition of PKM2 can be achieved in cells.
Growth factor signaling has been shown to increase the rate of glucose utilization, and cell death following growth factor withdrawal correlates with decreases in glycolysis [16
]. To test if inhibition of glycolysis can sensitize cells to growth factor withdrawal, we used the FL5.12 hematopoetic cell line, which is dependent on the growth factor IL-3 for survival. Addition of Compound 3 to FL5.12 cells increased the amount of cell death observed at 20 hours following growth factor withdrawal (). To test if PKM2 inhibition can increase cell death following inhibition of growth factor signaling in cancer cells, we utilized HCC827 lung cancer cells that carry a mutation in EGFR and are sensitive to the EGFR tyrosine kinase inhibitor Gefitinib [18
]. Similar to results observed in FL5.12 cells, cell death from Compound 3 was increased in the presence of Gefitinib (). These data suggest that PKM2 inhibition might have a role in cancer therapy even in the absence of full inhibition of glycolysis.