PFKFB3 is a gene that encodes the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 enzyme in humans. [5] [6] [7] It is one of 4 tissue-specific PFKFB isoenzymes identified currently (PFKFB1-4). [8]
PFKFB3 is a gene that encodes the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 enzyme in humans. [5] [6] [7] It is one of 4 tissue-specific PFKFB isoenzymes identified currently (PFKFB1-4). [8]
The PFKFB3 gene is mapped to single locus on chromosome 10 (10p15-p14). [5] [6] It spans a region of 32.5kb with an open reading frame that is 5,675bp long. It is estimated to consist of 19 exons, of which 15 are regularly expressed. [8] Alternative splicing of the variable, COOH-terminal domain has been observed, leading to 6 different isoforms termed UBI2K1 to UBI2K6 in humans. [9] Different nomenclature also recognizes two broad categories of PFKFB3 isoforms, termed ‘inducible’ and ‘ubiquitous’. [10] The inducible protein isoform, iPFK2, is named as such because its expression has been shown to be induced by hypoxic conditions.
The PFKFB3 promoter is predicted to contain multiple binding sites, including Sp-1 and AP-2 binding sites. It also contains motifs for the binding of E-box, nuclear factor-1 (NF-1), and progesterone response element. Expression of the promoter is shown to be induce by phorbol esters and cyclic-AMP-dependent protein kinase signaling. [10]
The four PFKFB isoforms share high (85%) ‘2-Kase/2-Pase core’ sequence homology, but have different properties based on variable N- and C- terminal regulatory domains and variation in residues surrounding the active sites. [11] The PFKFB3 inducible isoform has higher ‘2-Kase’ (kinase) activity than other isoforms, due to phosphorylation of Ser-460 by PKA or AMP-dependent protein kinase. [11] The high ‘2-Kase’ activity of PFKFB3 is also due to the lack of a specific Ser that is phosphorylated in the other PFKFB isoforms to decrease kinase activity. [12]
The primary protein encoded by PFKFB3, iPFK2, consists of 590 amino acids. It has a predicted molecular weight of 66.9 kDa and an isoelectric point of 8.64. [8] The crystal structure was determined in 2006: [11]
iPFK2 converts fructose-6-phosphate to fructose-2,6-bisP (F2,6BP). F2,6BP is a ‘potent’ allosteric activator of 6-phosphofructokinase-1 (PFK-1), stimulating glycolysis. Click to see image of PFFKB3 function [ permanent dead link ].
In neurons, glucose metabolism via glycolysis is usually low when compared to astrocytes. According Astrocyte-to-Neuron Lactate Shuttle Hypothesis, glucose uptake by the brain parenchyma occurs predominantly into astrocytes which subsequently release lactate for the use of neurons. [13] In neurons, glucose is mainly metabolized through the pentose–phosphate pathway (PPP), which is required for NADPH(H+) regeneration and maintenance of neuronal redox status. This neuronal metabolic switch is dictated by the PFKFB3 activity. In neurons, PFKFB3 protein abundance is negligible due to the continuous proteasomal degradation of the enzyme. [14] However, overexcitation of N-methyl-D-aspartate subtype of glutamate receptors (NMDAR), known as excitotoxicity, stabilizes PFKFB3 protein in neurons, resulting in a redirection of glucose flux from PPP to glycolysis, followed by low NADPH(H+) availability for proper GSH regeneration; this ultimately leads to oxidative stress and neuronal death. Silencing of PFKFB3 with small interfering RNA in neurons in vitro prevents the increase in ROS and apoptotic death induced by excitotoxic stimulus. [15] Pharmacological inhibition of PFKFB3 in vitro also protects neurons from apoptosis induced by NMDAR overexcitation as well as from amyloid-ß peptide-induced neurotoxicity. When used in vivo in a mouse model of ischaemic stroke, PFKFB3 inhibitor alleviates motor discoordination and brain infarct injury [16]
The Warburg effect, proposed by Otto Warbug in 1956, [17] describes the upregulation of glycolysis in most cancer cells, even in the presence of oxygen. The high rate of glycolysis is accompanied by increased lactic acid fermentation, providing additional nutrients for cancer cell growth and tumorigenesis.
PFKFB3 is associated with the Warburg effect because its activity increases the rate of glycolysis. PFKFB3 has been found to be upregulated in numerous cancers, including colon, breast, ovarian, and thyroid. [18] Reduced methylation of PFKFB3 is also found in some cancers, triggering the shift to the glycolytic pathway that supports cancerous growth. [19]
PFKFB3 expression is induced by hypoxia. [20] The promoter of PFKFB3 contains binding sites, called hypoxia response elements (HREs), that recruit the binding of hypoxia-inducible factor-1 (HIF-1). [21]
Hypoxia signaling via HIF-1α stabilization upregulates the transcription of genes that permit survival in low oxygen conditions. These genes include glycolysis enzymes, like PFKFB3, that permit ATP synthesis without oxygen, and vascular endothelial growth factor (VEGF), which promotes angiogenesis.
It was more recently discovered that PFKFB3 promotes cell cycle progression (cell proliferation) and suppresses apoptosis by regulating cyclin-dependent kinase 1 (Cdk-1). PFKFB3's synthesis of F2,6BP in the nucleus was found to regulate Cdk-1, whereas cytosolic PFKFB3 activates PFK-1. Nuclear PFKFB3 activates Cdk1 to phosphorylate the Thr-187 site of p27, causing decreased levels of p27. [22] [23] Reduced p27 causes protection against apoptosis and progression of cells through the G1/S phase checkpoint These findings established a significant link between PFKFB3 cancer cell survival and proliferation.
Circadian clocks dysregulation is associated with many types of cancer. [24] PFKFB3 expression exhibits circadian rhythmicity that is different between cancerous and non-cancerous cells. [25] It was specifically found that the circadian-driven transcription factor ‘CLOCK’ binds to the PFKFB3 promoter at a genuine ‘E-box’ site to increase transcription in cancer cells.
Inhibition of PFKFB3 is being analyzed as a potential anti-cancer therapy. The most notable example is clinical trial by Advanced Cancer Therapeutics (ACT) with PFK158, an improved version of 3PO, a PFKFB3 inhibitor. [29] It appears, however, that further development has been discontinued following disappointing Phase I results (see also the discussion of ACT compounds in § Small molecule inhibitors of PFKFB3). [30]
Several small-molecule inhibitors of PFKFB3 are currently in development.
For a long time a small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) was believed to be an inhibitor of PFKFB3 and used as PFKFB3 inhibitor in many scientific publications. 3PO decreases glucose uptake and increases autophagy. [31] Research is currently exploring various 3PO derivatives (i.e. PFKF15) [32] in an effort to increase their efficacy as anti-cancer therapies, but the data on 3PO derivatives being actually PFKFB3 inhibitors are also unavailable.
Recent research of one of the leading pharmaceuticals companies AstraZeneca and CRT Discovery Laboratories of world's largest independent cancer research charity Cancer Research UK showed that 3PO was inactive in a kinase PFKFB3 inhibition assay (IC50 > 100 μM). [26] The crystal structures of 3PO, as well as its analogues PFK15 and PFK158, with the PFKFB3 enzyme are also not available. The findings of AstraZeneca and Cancer Research UK regarding to 3PO remain unchallenged neither by 3PO developers since April 7, 2015.
The efficacy of two known PFKFB3 inhibitors, namely AZ67 (from AstraZeneca and CRT Discovery Laboratories [26] ), and PFK158, an improved but structurally close derivative of 3PO, were recently investigated for their ability to reduce F2,6BP production in A549 cells. Both compounds (AZ67 and PFK158) were able to reduce the cellular levels of F2,6BP in a dose-dependent manner, with IC50 of 0.51 μM and 5.90 μM, respectively. To see if the reduction of cellular F2,6BP levels was a result of direct PFKFB3 inhibition, both compounds were tried in the enzymatic cell-free assay. The study revealed that AZ67 inhibited the enzymatic activity of PFKFB3 with an IC50 of 0.018 μM, a value that is in accordance with previously published results. However, PFK158 had no effect on PFKFB3 enzymatic activity at any of the concentrations tested (up to 100 μM). Accordingly, although PFK158 is able to decrease F2,6BP and glycolytic flux, the experiments show that these effects are not due to PFKFB3 enzymatic inhibition. [16]
Together, these findings put into question the range of scientific research and publications where 3PO and its derivatives (such as PFKF158) was used as a PFKFB3 inhibitor.
In 2018 Kancera reported development and characterization of KAN0438241 (and its pro-drug KAN0438757) as a potent and highly selective PFKFB3 inhibitor and a radiosensitizer. [33]
Enhanced activity of PFKFB3 accelerates ROS production as an end product of glycolysis, and thus increases autophagy. Likewise, inhibition of PFKFB3 has been found to induce autophagy. [34] [35]
Autophagy can prolong cellular survival during low energy conditions. This finding was discovered in relation to rheumatoid arthritis. [36] It was found that RA T cell fail to upregulate autophagy, and knockout experiments placed PFKFB3 as an upstream regulator of this process.
PFKFB3 was identified in a kinome screen as a regulator of insulin/IGF-1. Suppression of PFKFB3 was found to decrease insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. Overexpression caused the insulin-dependent phosphorylation of Akt and Akt substrates. [37]
PFKFB3 expression increases in fat tissues during adipogenesis, but prolonged insulin exposure has been shown to decrease the expression of PFKFB3. This is thought to occur due to a negative feedback mechanism involving insulin. [38]
p38 MAPK have been found to increase PFKFB3 activity through (1) the transcriptional activation of PFKFB3 in response to stress stimuli and (2) the post-translational phosphorylation of iPFK2 at Ser-461. [39] [40]
Glycolysis is the metabolic pathway that converts glucose into pyruvate and, in most organisms, occurs in the liquid part of cells. The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH). Glycolysis is a sequence of ten reactions catalyzed by enzymes.
Gluconeogenesis (GNG) is a metabolic pathway that results in the biosynthesis of glucose from certain non-carbohydrate carbon substrates. It is a ubiquitous process, present in plants, animals, fungi, bacteria, and other microorganisms. In vertebrates, gluconeogenesis occurs mainly in the liver and, to a lesser extent, in the cortex of the kidneys. It is one of two primary mechanisms – the other being degradation of glycogen (glycogenolysis) – used by humans and many other animals to maintain blood sugar levels, avoiding low levels (hypoglycemia). In ruminants, because dietary carbohydrates tend to be metabolized by rumen organisms, gluconeogenesis occurs regardless of fasting, low-carbohydrate diets, exercise, etc. In many other animals, the process occurs during periods of fasting, starvation, low-carbohydrate diets, or intense exercise.
The enzyme fructose bisphosphatase (EC 3.1.3.11; systematic name D-fructose-1,6-bisphosphate 1-phosphohydrolase) catalyses the conversion of fructose-1,6-bisphosphate to fructose 6-phosphate in gluconeogenesis and the Calvin cycle, which are both anabolic pathways:
Tumor hypoxia is the situation where tumor cells have been deprived of oxygen. As a tumor grows, it rapidly outgrows its blood supply, leaving portions of the tumor with regions where the oxygen concentration is significantly lower than in healthy tissues. Hypoxic microenvironments in solid tumors are a result of available oxygen being consumed within 70 to 150 μm of tumor vasculature by rapidly proliferating tumor cells thus limiting the amount of oxygen available to diffuse further into the tumor tissue. In order to support continuous growth and proliferation in challenging hypoxic environments, cancer cells are found to alter their metabolism. Furthermore, hypoxia is known to change cell behavior and is associated with extracellular matrix remodeling and increased migratory and metastatic behavior.
Phosphofructokinase-1 (PFK-1) is one of the most important regulatory enzymes of glycolysis. It is an allosteric enzyme made of 4 subunits and controlled by many activators and inhibitors. PFK-1 catalyzes the important "committed" step of glycolysis, the conversion of fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP. Glycolysis is the foundation for respiration, both anaerobic and aerobic. Because phosphofructokinase (PFK) catalyzes the ATP-dependent phosphorylation to convert fructose-6-phosphate into fructose 1,6-bisphosphate and ADP, it is one of the key regulatory steps of glycolysis. PFK is able to regulate glycolysis through allosteric inhibition, and in this way, the cell can increase or decrease the rate of glycolysis in response to the cell's energy requirements. For example, a high ratio of ATP to ADP will inhibit PFK and glycolysis. The key difference between the regulation of PFK in eukaryotes and prokaryotes is that in eukaryotes PFK is activated by fructose 2,6-bisphosphate. The purpose of fructose 2,6-bisphosphate is to supersede ATP inhibition, thus allowing eukaryotes to have greater sensitivity to regulation by hormones like glucagon and insulin.
Pyruvate kinase is the enzyme involved in the last step of glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP. Pyruvate kinase was inappropriately named before it was recognized that it did not directly catalyze phosphorylation of pyruvate, which does not occur under physiological conditions. Pyruvate kinase is present in four distinct, tissue-specific isozymes in animals, each consisting of particular kinetic properties necessary to accommodate the variations in metabolic requirements of diverse tissues.
5' AMP-activated protein kinase or AMPK or 5' adenosine monophosphate-activated protein kinase is an enzyme that plays a role in cellular energy homeostasis, largely to activate glucose and fatty acid uptake and oxidation when cellular energy is low. It belongs to a highly conserved eukaryotic protein family and its orthologues are SNF1 in yeast, and SnRK1 in plants. It consists of three proteins (subunits) that together make a functional enzyme, conserved from yeast to humans. It is expressed in a number of tissues, including the liver, brain, and skeletal muscle. In response to binding AMP and ADP, the net effect of AMPK activation is stimulation of hepatic fatty acid oxidation, ketogenesis, stimulation of skeletal muscle fatty acid oxidation and glucose uptake, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipogenesis, inhibition of adipocyte lipolysis, and modulation of insulin secretion by pancreatic β-cells.
Glucokinase is an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate. Glucokinase occurs in cells in the liver and pancreas of humans and most other vertebrates. In each of these organs it plays an important role in the regulation of carbohydrate metabolism by acting as a glucose sensor, triggering shifts in metabolism or cell function in response to rising or falling levels of glucose, such as occur after a meal or when fasting. Mutations of the gene for this enzyme can cause unusual forms of diabetes or hypoglycemia.
Phosphofructokinase-2 (6-phosphofructo-2-kinase, PFK-2) or fructose bisphosphatase-2 (FBPase-2), is an enzyme indirectly responsible for regulating the rates of glycolysis and gluconeogenesis in cells. It catalyzes formation and degradation of a significant allosteric regulator, fructose-2,6-bisphosphate (Fru-2,6-P2) from substrate fructose-6-phosphate. Fru-2,6-P2 contributes to the rate-determining step of glycolysis as it activates enzyme phosphofructokinase 1 in the glycolysis pathway, and inhibits fructose-1,6-bisphosphatase 1 in gluconeogenesis. Since Fru-2,6-P2 differentially regulates glycolysis and gluconeogenesis, it can act as a key signal to switch between the opposing pathways. Because PFK-2 produces Fru-2,6-P2 in response to hormonal signaling, metabolism can be more sensitively and efficiently controlled to align with the organism's glycolytic needs. This enzyme participates in fructose and mannose metabolism. The enzyme is important in the regulation of hepatic carbohydrate metabolism and is found in greatest quantities in the liver, kidney and heart. In mammals, several genes often encode different isoforms, each of which differs in its tissue distribution and enzymatic activity. The family described here bears a resemblance to the ATP-driven phospho-fructokinases; however, they share little sequence similarity, although a few residues seem key to their interaction with fructose 6-phosphate.
Hepatocyte growth factor receptor is a protein that in humans is encoded by the MET gene. The protein possesses tyrosine kinase activity. The primary single chain precursor protein is post-translationally cleaved to produce the alpha and beta subunits, which are disulfide linked to form the mature receptor.
The mammalian target of rapamycin (mTOR), also referred to as the mechanistic target of rapamycin, and sometimes called FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1), is a kinase that in humans is encoded by the MTOR gene. mTOR is a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases.
The study of the tumor metabolism, also known as tumor metabolome describes the different characteristic metabolic changes in tumor cells. The characteristic attributes of the tumor metabolome are high glycolytic enzyme activities, the expression of the pyruvate kinase isoenzyme type M2, increased channeling of glucose carbons into synthetic processes, such as nucleic acid, amino acid and phospholipid synthesis, a high rate of pyrimidine and purine de novo synthesis, a low ratio of Adenosine triphosphate and Guanosine triphosphate to Cytidine triphosphate and Uridine triphosphate, low Adenosine monophosphate levels, high glutaminolytic capacities, release of immunosuppressive substances and dependency on methionine.
Fructose 2,6-bisphosphate, abbreviated Fru-2,6-P2, is a metabolite that allosterically affects the activity of the enzymes phosphofructokinase 1 (PFK-1) and fructose 1,6-bisphosphatase (FBPase-1) to regulate glycolysis and gluconeogenesis. Fru-2,6-P2 itself is synthesized and broken down in either direction by the integrated bifunctional enzyme phosphofructokinase 2 (PFK-2/FBPase-2), which also contains a phosphatase domain and is also known as fructose-2,6-bisphosphatase. Whether the kinase and phosphatase domains of PFK-2/FBPase-2 are active or inactive depends on the phosphorylation state of the enzyme.
Enzyme activators are molecules that bind to enzymes and increase their activity. They are the opposite of enzyme inhibitors. These molecules are often involved in the allosteric regulation of enzymes in the control of metabolism. In some cases, when a substrate binds to one catalytic subunit of an enzyme, this can trigger an increase in the substrate affinity as well as catalytic activity in the enzyme's other subunits, and thus the substrate acts as an activator.
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 is an enzyme that in humans is encoded by the PFKFB2 gene.
Serine/threonine-protein kinase ULK1 is an enzyme that in humans is encoded by the ULK1 gene.
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 is an enzyme that in humans is encoded by the PFKFB1 gene.
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 also known as PFKFB4 is an enzyme which in humans is encoded by the PFKFB4 gene.
mTORC1, also known as mammalian target of rapamycin complex 1 or mechanistic target of rapamycin complex 1, is a protein complex that functions as a nutrient/energy/redox sensor and controls protein synthesis.
The TP53-inducible glycolysis and apoptosis regulator (TIGAR) also known as fructose-2,6-bisphosphatase TIGAR is an enzyme that in humans is encoded by the C12orf5 gene.
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