Conversion of Non-allosteric Pyruvate Kinase Isozyme into an Allosteric Enzyme by a Single Amino Acid Substitution

Ikeda, Yoshitaka; Tanaka, Takehiko; Noguchi, T.

doi:10.1074/jbc.272.33.20495

Cited by 78 publications

(67 citation statements)

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“…The distinctive MRSA PK C domain is of particular interest for the design of selective inhibitors that act specifically against bacteria. This was suggested by the results of several previously reported studies indicating that single mutations in the effector binding sites or sulfate removal led to an altered stability of COC interfaces along with surprisingly profound effects on the rates of enzyme catalysis due to altered dynamics of the enzyme conformation (10,13,14,20,37).…”

Section: Vol 55 2011mentioning

confidence: 60%

Identification of Pyruvate Kinase in Methicillin-Resistant Staphylococcus aureus as a Novel Antimicrobial Drug Target

Zoraghi

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Axerio-Cilies

et al. 2011

Antimicrob Agents Chemother

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Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeting of the bacterial enzyme. In silico library screening combined with functional enzymatic assays identified an acyl hydrazone-based compound (IS-130) as a potent MRSA PK inhibitor (50% inhibitory concentration [IC 50 ] of 0.1 M) with >1,000-fold selectivity over human PK isoforms. Medicinal chemistry around the IS-130 scaffold identified analogs that more potently and selectively inhibited MRSA PK enzymatic activity and S. aureus growth in vitro (MIC of 1 to 5 g/ml). These novel anti-PK compounds were found to possess antistaphylococcal activity, including both MRSA and multidrug-resistant S. aureus (MDRSA) strains. These compounds also exhibited exceptional antibacterial activities against other Gram-positive genera, including enterococci and streptococci. PK lead compounds were found to be noncompetitive inhibitors and were bactericidal. In addition, mutants with significant increases in MICs were not isolated after 25 bacterial passages in culture, indicating that resistance may be slow to emerge. These findings validate the principles of network science as a powerful approach to identify novel antibacterial drug targets. They also provide a proof of principle, based upon PK in MRSA, for a research platform aimed at discovering and optimizing selective inhibitors of novel bacterial targets where human orthologs exist, as leads for anti-infective drug development.

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Section: Vol 55 2011mentioning

confidence: 60%

Identification of Pyruvate Kinase in Methicillin-Resistant Staphylococcus aureus as a Novel Antimicrobial Drug Target

Zoraghi

See

Axerio-Cilies

et al. 2011

Antimicrob Agents Chemother

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“…4 The isoemzyme specific domains are important for the interaction of the respective pyruvate kinase subunits, 29 and, although structurally very similar, 30-32 the nonallosteric M1 isoenzyme exists only as tetramer while M2-PK is an allosteric enzyme that can exist in two different conformations (a tetrameric and dimeric form). 6,30,[32][33][34] To specifically elucidate the effect of changes in the M2-PK conformation, it was important to design molecules that interact with M2-PK, but not with the closely related M1-PK isoenzyme. To do this, we employed the peptide aptamer technology 17 to select peptide sequences that specifically bind to M2-PK.…”

Section: M2-pk-binding Peptide Aptamers Discriminate Between Isoenzymesmentioning

confidence: 99%

Isotype‐specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation

Spoden

Mazurek

Morandell

et al. 2008

Intl Journal of Cancer

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Tumor cells express the glycolytic regulator pyruvate kinase subtype M2 (M2-PK), which can occur in a tetrameric form with high affinity to its substrate phosphoenolpyruvate (PEP) and a dimeric form with a low PEP affinity. The transition between both conformations contributes to the control of glycolysis and is important for tumor cell proliferation and survival. Here we targeted M2-PK by synthetic peptide aptamers, which specifically bind to M2-PK and shift the isoenzyme into its low affinity dimeric conformation. The aptamer-induced dimerization and inactivation of M2-PK led to a significant decrease in the PK mass-action ratio as well as ATP:ADP ratio in the target cells. Furthermore, the expression of M2-PK-binding peptide aptamers moderately reduced the growth of immortalized NIH3T3 cell populations by decelerating cell proliferation, but without affecting apoptotic cell death. Moreover, the M2-PK-binding peptide aptamers also reduced the proliferation rate of human U-2 OS osteosarcoma cells. In the present study, we developed the first specific inhibitors of the pyruvate kinase isoenzyme type M2 and present evidence that these inhibitors moderately decelerate tumor cell proliferation. ' 2008 Wiley-Liss, Inc.Key words: tumor metabolism; peptide aptamer; proliferation; pyruvate kinase Tumorigenesis is characterized by an increase in glycolytic enzyme activities as well as distinct changes in the glycolytic isoenzyme equipment. 1-3 A key glycolytic enzyme which is consistently altered in tumor cells is pyruvate kinase (EC 2.7.1.40), catalyzing the dephosphorylation of phosphoenolpyruvate (PEP) to pyruvate, which significantly contributes to net adenosine triphosphate (ATP) production within the glycolytic pathway. Depending on the metabolic functions of various tissues, different isoenzymes of pyruvate kinase are expressed. 4 The pyruvate kinase isoenzyme M1 (M1-PK) is characteristic for tissues with high energy turnover (muscle and brain), whereas the pyruvate kinase isoform L (L-PK) is found in tissues with gluconeogenesis, such as liver. When quiescent cells re-enter the cell cycle, as during tumorigenesis, the tissue-specific isoenzymes disappear and the pyruvate kinase isoenzyme type M2 (M2-PK) is expressed. 3,4 According to the different metabolic functions, M1-PK has the highest affinity to its substrate PEP, whereas the PEP affinity of M2-PK depends on its quaternary structure. M2-PK occurs as a tetrameric form characterized by a high affinity to its substrate PEP which is highly active at physiological PEP concentrations and as a dimeric form with low PEP affinity which is nearly inactive under physiological conditions. 3 The tetrameric but not the dimeric form of M2-PK is associated with other glycolytic enzymes as well as with lactate dehydrogenase (LDH), nucleotide diphosphate kinase and adenylate kinase, within a large complex, referred to as the glycolytic enzyme complex. 5-7 A high amount of the tetrameric form of M2-PK correlates with a relative high PK mass-action ratio ([pyruvate] AE[ATP])...

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“…PKM1 constitutively forms stable tetramers (the active form of PK) and is expressed in normal adult tissues that require high levels of energy, such as the heart, brain, and skeletal muscle. In contrast, PKM2 exists in either tetramers or dimers with less activity and metabolic intermediate fructose-1,6-bisphosphate (FBP) can allosterically activate PKM2 by promoting the formation of tetramer from dimer (1,2). PKM2 is selectively expressed in normal proliferating cells as well as tumor cells, indicating an indispensable role in cancer development.…”

mentioning

confidence: 99%

A Novel Role for Pyruvate Kinase M2 as a Corepressor for P53 during the DNA Damage Response in Human Tumor Cells

Wang

et al. 2016

Journal of Biological Chemistry

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Edited by Patrick SungThe pyruvate kinase (PK) is a rate-limiting glycolytic enzyme catalyzing the dephosphorylation of phosphoenolpyruvate to pyruvate, yielding one molecule of ATP. The M2 isoform of PK (PKM2) is predominantly expressed in normal proliferating cells and tumors, and both metabolic and non-metabolic activities for the enzyme in promoting tumor cell proliferation have been identified. However, the exact roles of PKM2 in tumor initiation, growth and maintenance are not yet fully understood. Using immunoprecipitation-coupled LC-MS/MS in MCF7 cells exposed to DNA-damaging agent, we report that the nuclear PKM2 interacts directly with P53 protein, a critical safeguard for genome stability. Specifically, PKM2 inhibits P53-dependent transactivation of the P21 gene by preventing P53 binding to the P21 promoter, leading to a nonstop G 1 phase. As a result, PKM2 expression provides a growth advantage for tumor cells in the presence of a DNA damage stimulus. In addition, PKM2 interferes with phosphorylation of P53 at serine 15, known to stimulate P53 activity by the ATM serine/threonine kinase. These findings reveal a new role for PKM2 in modulating the DNA damage response and illustrate a novel mechanism of PKM2 participating in tumorigenesis.Pyruvate kinase (PK), 4 a rate-limiting enzyme of glycolysis, catalyzes the transfer of phosphate from phosphoenolpyruvate (PEP) to ADP, yielding one molecule of ATP and pyruvate. The PK consists of four isoforms, the PKLR gene-encoded PKL and PKR isoforms and the PKM gene-encoded M1 (PKM1) and M2 (PKM2) isoforms, which express in different types of mammalian cells and tissues. PKM1 constitutively forms stable tetramers (the active form of PK) and is expressed in normal adult tissues that require high levels of energy, such as the heart, brain, and skeletal muscle. In contrast, PKM2 exists in either tetramers or dimers with less activity and metabolic intermediate fructose-1,6-bisphosphate (FBP) can allosterically activate PKM2 by promoting the formation of tetramer from dimer (1, 2). PKM2 is selectively expressed in normal proliferating cells as well as tumor cells, indicating an indispensable role in cancer development.There are two known mechanisms by which PKM2 favors tumor cell growth, one dependent on and one independent of glycolysis. As a glycolytic enzyme, PKM2 in tumor cells shifts glucose metabolism from oxidative phosphorylation to glycolysis under normoxic conditions, a phenomenon termed the Warburg effect or aerobic glycolysis (3). On the other hand, non-metabolic function of PKM2, which is mainly associated with the nuclear PKM2, has been identified as a major contributor during tumorigenesis. For example, nuclear PKM2 displays kinase activity in phosphorylating a number of protein substrates, including histone H3 (4, 5), signal transducer and activator of transcription 3 (stat3) (6, 7), Bub3 (8), and myosin light chain 2 (MLC2) (9), for which PKM2 uses the high-energy phosphate from PEP but not ATP as a phosphate donor. The PKM2-induced phosphorylation...

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Conversion of Non-allosteric Pyruvate Kinase Isozyme into an Allosteric Enzyme by a Single Amino Acid Substitution

Cited by 78 publications

References 35 publications

Identification of Pyruvate Kinase in Methicillin-Resistant Staphylococcus aureus as a Novel Antimicrobial Drug Target

Identification of Pyruvate Kinase in Methicillin-Resistant Staphylococcus aureus as a Novel Antimicrobial Drug Target

Isotype‐specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation

A Novel Role for Pyruvate Kinase M2 as a Corepressor for P53 during the DNA Damage Response in Human Tumor Cells

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