Elsevier

Biochemical Pharmacology

Volume 79, Issue 8, 15 April 2010, Pages 1118-1124
Biochemical Pharmacology

Identification of small molecule inhibitors of pyruvate kinase M2

https://doi.org/10.1016/j.bcp.2009.12.003Get rights and content

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.

Introduction

One of the first distinctions noted between cancer tissues and normal tissues was a difference in metabolism [1], [2]. Cancer cells, unlike their normal counterparts, metabolize glucose by aerobic glycolysis. This phenomenon, known as the Warburg effect, is characterized by increased glycolysis with lactate production and decreased oxidative phosphorylation [3], [4]. The propensity for tumor cells to rely on increased glucose uptake which accompanies the Warburg effect has been exploited for diagnostic purposes and forms the basis of 18F-deoxyglucose positron emission tomography (FDG-PET) as a tool for detecting and staging malignancy [2], [4]. However, although this observation was made over 90 years ago, the fundamental difference in cellular metabolism exhibited by cancer cells has not yet been exploited for therapeutic benefit [5].

Achieving specificity has been a challenge for targeting cell metabolism. All cells rely on intermediary metabolism for their survival, and glucose is a major source of energy for many cell types in humans. This raises the possibility that using a small molecule to interfere with glucose metabolism would have a detrimental effect on both cancer and normal tissues. However, cancer cells have distinct metabolic needs that are required for proliferation and there is growing evidence that the genetic alterations that drive malignancy also cause cells to become addicted to high levels of flux through specific metabolic pathways [4], [6]. The metabolism of glucose via aerobic glycolysis may be a consequence of selection for cells that reprogram glucose metabolism to meet the distinct metabolic needs of cell proliferation [4].

An important determinant of how glucose is metabolized in cells depends on the glycolytic enzyme pyruvate kinase [7], [8]. Pyruvate kinase has four isoforms in mammalian cells encoded by two separate genes [9]. The L gene is alternatively spliced to produce the L and R isoforms of pyruvate kinase, which are expressed exclusively in the liver and red blood cells, respectively [10]. The M gene is alternatively spliced by mutual exclusion of a single exon to generate either the M1 or M2 isoform of pyruvate kinase [11]. Most tissues in the adult animal express pyruvate kinase M1 (PKM1), while pyruvate kinase M2 (PKM2) is expressed during embryonic development [7], [12], [13]. Tumor cells, including those tumors derived from tissues that normally express PKL or PKR, express exclusively PKM2 [12], [13]. Knockdown of PKM2 using RNA interference significantly impairs cell growth in tissue culture, inhibition of PKM2 with peptide aptamers inhibits cell proliferation, and PKM2 expression is necessary for both aerobic glycolysis and tumor growth in vivo[7], [14]. Furthermore, PKM2 selection for tumor growth in vivo suggests that reversion to another isoform of pyruvate kinase might be incompatible with cell proliferation in the setting of an established tumor. These observations identify PKM2 as an attractive target for disruption of aerobic glycolysis in tumors.

One potential challenge for identifying a small molecule that selectively inhibits PKM2 is the similarity between PKM2 and the other isoforms of pyruvate kinase. PKM2 and PKM1 are identical in size and differ only in the 56 amino acid region encoded by the alternatively spliced exon [13]. In comparison to PKM1, PKL and PKR are less similar to PKM2, but still exhibit significant sequence conservation. The unique portion of PKM2 encoded by the alternatively spliced exon does not contribute to the active site of the enzyme, but rather allows PKM2, but not PKM1, to be allosterically activated by the upstream glycolytic intermediate, fructose-1,6-bisphosphate (FBP) [13]. The unique region of PKM2 also allows for enzymatic regulation by interaction with tyrosine-phosphorylated proteins [15]. Targeting the allosteric site of PKM2 may allow for isoform selective small molecule inhibitors of pyruvate kinase.

Here we describe a screen designed to identify inhibitors with selectivity for PKM2 over PKM1. This screen identified three classes of molecules that inhibit PKM2 with minimal effect on PKM1. These molecules can mimic some aspects of PKM2 knockdown using RNAi, including inhibition of glycolysis. These data demonstrate that selective targeting of PKM2 with a drug-like molecule is possible and suggest that efforts to target PKM2 may yield compounds suitable for targeting cancer metabolism for cancer therapy.

Section snippets

Purification of recombinant pyruvate kinase isoforms

The human cDNA for PKM2, PKM1 and PKL were cloned into pET28a with a N-terminal 6×-His tag and purified from E. coli using Ni-Agarose beads (Qiagen) as described previously [15]. Briefly, E. coli grown to an OD(600 nm) of 0.7 were induced with 0.5 mM IPTG at room temperature for 6 h. Cells were collected and lysed by freeze/thaw cycles and sonication. Lysate was passed over an Ni-NTA agarose column and pyruvate kinase eluted with 250 mM imidazole in 1 ml fractions. Fractions with high concentration

Results

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 (Fig. 1). 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

Discussion

Identification of targets that provide a sufficient therapeutic window to selectively target glucose metabolism in cancer cells has been a major hurdle to the development of drugs to exploit cancer cell metabolism. PKM2 is such a metabolic target; however its similarity to PKM1 raised questions as to whether specific targeting was feasible. Here we demonstrate that selective inhibition of PKM2 with a small molecule, at least in relation to PKM1, is possible. The unique portion of PKM2 allows

Acknowledgements

We thank the Dana-Farber/Harvard Cancer Center Cancer Drug Assays screening facility for supporting this project. We thank J. Engelman for Gefitinib and HCC827 cells, C. Rudin and D. Plas for FL5.12 cells, and Agios Pharmaceuticals for providing independently synthesized Compound 3. MGVH was a fellow supported by the Damon Runyon Cancer Research Foundation when much of this work was performed.

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