1.1 GLUCOSE METABOLISM[1,2]
1.1.1 The role of glucose
Every living cell needs energy to maintain all the physiological functions that are fundamental to life. This energy, stored as ATP (i.e. Adenosine triphosphate) is produced through biochemical reactions that start from various organic precursors, the most important of which is glucose.
α-D-Glucose is an aldose reductant sugar, which can be involved in many biochemical pathways:
Complete oxidation to CO2 with ATP production.
Oxidation through the pentose phosphate pathway.
Storage as glycogen, especially in muscular and liver cells. Precursor for the synthesis of structural polymers.
The glucose that is taken with diet as simple sugar or that is obtained after the hydrolysis of disaccharides or polysaccharides, is absorbed by enterocytes through the symporter Na+/Glucose SGLT1. This protein exploits the sodium ion gradient created and kept by the
Na+/K+ ATPase pump, to transport glucose, through a secondary active transport
mechanism.
Once the glucose is inside enterocytes, a little percentage undergoes the glycolytic pathway, whereas a larger amount is extruded in the interstitial compartment through the transporter GLUT2 and from here it reaches the blood stream and all the tissues of the organism. The complete oxidation of glucose to CO2 occurs through several biochemical
processes: first of all we find glycolysis, which takes place in the cytosol and transforms one molecule of glucose in two molecules of pyruvate, with the production of two molecules of ATP and two molecules of NADH. Pyruvate is transported towards the mitochondrial matrix, where it is turned into Acetyl-CoA, which enters the citric acid cycle. Via this process Acetyl-CoA is processed to gain three molecules of NADH, one molecule of FADH2 , and one molecule of GTP. The molecules of NADH and FADH2
produced during the many phases of the oxidation of glucose, are used for the OXPHOS (Oxidative Phosphorylation) in order to obtain 32 molecules of ATP.
1.1.2 Glycolysis
Glycolysis is the metabolic process through which one molecule of glucose is converted in two molecules of pyruvate, producing two molecules of ATP and two molecules of NADH. This pathway can be divided in two subsequent phases: the first one is called preparatory phase and the second one is called payoff phase.
Fig. 2: Glycolysis: preparatory phase[1]
D-Glucose is phosphorylated in position 6 by the enzyme hexokinase, with the consumption of one molecule of ATP. This step is irreversible and in inhibited through a negative feedback mechanism when the ATP concentration on the cytosol is high. Also high concentrations of glucose-6-phosphate can block the action of hexokinase.
Glucose-6-phosphate is reversibly isomerized to fructose-6-phosphate, thank to the action of the enzyme phosphoglucose isomerase, passing through an enediol intermediate.
Fructose-6-phosphate is phosphorylated in position 1 with the consumption of an other molecule of ATP, thank to the enzyme phosphofructokinase-1, which can be inhibited through a negative feedback mechanism by high concentration of ATP and high concentration of citrate; On the other hand, this enzyme is stimulated by fructose-2,6-biphosphate.
Fructose-1,6-biphosphate is split in glyceraldehyde-3-phosphate and dihydroxyacetone phosphate through the action of the enzyme fructose-bisphosphate aldolase.
The enzyme triosephosphate isomerase converts dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. This means that from one molecule of glucose, two molecules of glyceraldehyde-3-phosphate are obtained.
Besides, a regulation mechanism that involves fructose-6-phosphate is provided in order to modulate the glucose consumption in conditions of hyperglycemia.
In fact, when the blood concentration of glucose is high, huge amounts of fructose-6-phosphate are produced until this compound saturates the enzyme phosphofructokinase-1. When this happens, fructose-6-phosphate is converted in Fructose-2,6-biphosphate by the enzyme phosphofructokinase-2. This product stimulates the production of fructose-1,6-biphosphate (and so the prosecution of glycolysis) in spite of great amounts of ATP. Moreover, fructose-2,6-biphosphate can inhibit the enzyme fructose-1,6-biphosphatase, involved in gluconeogenesis, with the purpose of preventing the increase of blood concentration of glucose.
Preparatory phase balance:
At the end of the first phase of glycolysis, the energy balance is negative, since, starting from one molecule of glucose, two molecules of glyceraldehyde-3-phosphate are obtained, with the consumption of two molecules of ATP.
Glycolysis: payoff phase[1]
Glyceraldehyde-3-phosphate is oxidised to 1,3-biphosphoglycerate by the enzyme glyceraldehyde phosphate dehydrogenase. This step leads to the production of one molecule of NADH for each molecule of glyceraldehyde-3-phosphate oxidised. 1,3-biphosphoglycerate is dephosphorylated by the enzyme phosphoglycerate
kinase. For each molecule of 1,3-biphosphoglycerate consumed, one molecule of ATP is gained.
3-Phosphoglycerate is isomerized through the action of the enzyme phosphoglycerate mutase.
2-Phosphoglycerate is hydrolyzed by an enolase, to give phosphoenolpyruvate. Phosphoenolpyruvate is dephosphorylated by pyruvate kinase (this catalysis is
stimulated by insulin) and one molecule of pyruvate and one molecule of ATP are produced.
Payoff phase balance:
At the end of this phase of glycolysis, for each molecule of glyceraldehyde-3-phosphate consumed, one molecule of NADH and two molecules of ATP are obtained.
Total energetic balance of glycolysis:
Since one molecule of glucose gives two molecules of glyceraldehyde-3-phosphate and so two molecules of pyruvate, the total energetic balance of glycolysis can be summarized as follows (Table 1): Glucose ↓ Glucose-6-phosphate ↓ - 1 ATP Preparatory phase Fructose-6-phosphate ↓ Fructose 1,6-biphosphate ↓ - 1 ATP Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate ↓ 2 Glyceraldehyde 3-phosphate ↓ 1,3-biphosphoglycerate ↓ + 2 NADH Payoff phase 3-phosphoglycerate ↓ + 2 ATP 2-phosphoglycerate ↓ phosphoenolpyruvate ↓ pyruvate + 2 ATP
TOTAL GAIN 2 ATP + 2 NADH
Table 1: Total energetic balance of glycolysis 1.1.3 Krebs cycle
The pyruvate obtained through glycolysis is transported to the mitochondrial matrix by a specific protein called MPC (i.e. Mitochondrial pyruvate carrier) which is located on the inner mitochondrial membrane. Once inside the mitochondrion, pyruvate is processed through oxidative decarboxylation, by a multienzymatic complex called pyruvate dehydrogenase, in order to form Acetyl coenzyme A, also called Acetyl-CoA.
Fig. 4: Oxidation of Pyruvate to Acetyl-CoA[1]
The pyruvate dehydrogenase complex is constituted of three different enzymes, that use thiamine pyrophosphate (TPP), lipoate, FAD, coenzyme A and NAD+ as cofactors. The
chemical transformation of pyruvate in Acetyl-CoA leads to the formation of one molecule of NADH for each molecule of pyruvate consumed.
At this particular step we have the crossing of many metabolic pathways; in fact the degradation processes of carbohydrates, fats and proteins all converge at this passage, with the generation of Acetil-CoA.
The Krebs cycle (also called tricarboxylic acid cycle or citric acid cycle) consists in the oxidation of Acetyl-CoA with the formation of three molecules of NADH, one molecule of FADH2 and one molecule of ATP or GTP.
This metabolic pathway, as stated above, is a cycle, and that means that the intermediates are not consumed, but they are recovered at the end of each cycle.
Fig. 5: Krebs Cycle[1]
Acetyl-CoA is condensed with oxaloacetate by the enzyme citrate synthase, with the formation of citrate through the intermediate citryl coa.
Citrate is isomerized to isocitrate thank to the enzyme aconitase, though the intermediate cis-aconitate.
Isocitrate undergoes an oxidative decarboxylation, which transforms it in α-ketoglutarate. This reaction is catalyzed by isocitrate dehydrogenase, and provokes the release of one molecule of CO2 and the formation of one molecule of NADH.
α-ketoglutarate itself undergoes oxidative decarboxylation, generating succinyl-CoA. The reaction is catalyzed by the enzyme α-ketoglutarate dehydrogenase and produces one molecule of NADH.
Succinyl-CoA synthetase breaks the thioester bond of Succinyl-CoA, producing succinate and one molecule of ATP or GTP, starting from ADP (or GDP) and inorganic phosphate.
Succinate is oxidised to fumarate, through the action of the enzyme succinate dehydrogenase, with the production of one molecule of FADH2.
L-malate is oxidised to oxaloacetate thank to the enzyme L-malate dehydrogenase, gaining one molecule of NADH.
Oxaloacetate is condensed again with one molecule of Acetyl-CoA to start a new cycle.
Total balance of glucose metabolism: Glucose → 2 ATP + 2 NADH + 2 Pyruvate 2 (Pyruvate → Acetyl-CoA + NADH) 2 (Acetyl-CoA → 3 NADH + FADH2 + ATP)
Glucose → 4 ATP + 10 NADH + 2 FADH2
1.1.4 Oxidative phosphorylation (OXPHOS)
Oxidative Phosphorylation is the final step of the metabolic process to obtain ATP, and consists in the reduction of O2 to H2O, utilizing the electrons released by NADH and
FADH2, generated through the catabolism of glucids, lipids and amino acids. This process
occurs across the inner mitochondrial membrane, which is almost completely impermeable, with few exceptions, such as some particular molecules and electrons.
Fig. 6: Oxidative Phosphorylation[1]
OXPHOS exploits the establishment of a protonic transmembrane gradient to make multienzymatic transporters operate the chemical reactions needed to gain more ATP and restore the NAD+/NADH and FAD/FADH
2 balance. The equilibrium between the oxidised
and reduced forms of these cofactors, and in particular the restoration of the species consumed through glycolysis and Krebs cycle, allows the starting over of the glucose metabolism.
1. Complex I, also called NADH-coenzyme Q oxidoreductase, contains a flavoprotein coupled to FMN and many iron-sulfur clusters. The role of this multienzymatic complex is the catalysis of two coupled reactions: an exergonic and an endergonic reaction.
NADH + H+ + Q → NADH + QH
2 (exergonic reaction)
NADH + 5H+ + Q → NAD+ + 4H+ + QH
2 (endergonic reaction)
The first transformation consists in the electrons transfer from NADH to ubiquinone (Q) with the restoration of NAD+ and the formation of ubiquinol (QH
2).
The second one, instead, through the same redox reaction, allows the passage of four protons from the mitochondrial matrix to the mitochondrial intermembrane space.
The proton flux is unidirectional and makes the mitochondrial intermembrane space positively charged.
Ubiquinol is one of the few molecules able to diffuse through the mitochondrial inner membrane and, once it is generated, it reaches complex III.
2. Complex II, also called Succinate-Q oxidoreductase, is the same enzyme that is responsible for the reduction of succinate to fumarate in the citric acid cycle. It contains many heme groups, iron-sulfur clusters and a FAD. The FADH2 generated, transfers its
electrons to ubiquinone, transforming it in ubiquinol that, also in this case, reaches complex III.
3. Complex III, also called Q-cytochrome c oxidoreductase, catalyzes the electron transfer from ubiquinol (coming from the reduction reactions of ubiquinone that took place thank to complex I and II) to cytocrome, allowing other protons to diffuse from the mitochondrial matrix to the mitochondrial intermembrane space. Cytocrome c in its reduced form, reaches complex IV.
QH2 + 2 cytocrome c oxidised form + 2H+ → Q + 2 cytocrome c reduced form + 4H+
4. coming in its reduced form from complex III, to molecular oxygen, allowing its reduction to water and transferring protons from the mitochondrial matrix to the mitochondrial intermembrane space.
4 cytocrome c reduced form + 8H+ + O2 → 4 cytocrome c oxidised form + 4H+ + 2H2O
The electronic transfer reactions that lead to the reduction of molecular oxygen to water, are highly exergonic. The energy that is produced is mainly used to generate the protonic gradient. In particular, for each electron pair that is transferred to oxygen, as described
above, complexes I and III promote the transfer of four protons, whereas complex IV promotes the transfer of two protons, through the mitochondrial inner membrane.
When the protonic gradient is sufficiently high, the protons invert the direction of their flux, going back to the mitochondrial matrix, through a ionic channel that is associated to the enzyme ATP-synthase. So the protonic flux moves down the concentration gradient and simultaneously ATP is synthesized as follows:
ADP + Pi + nH+ → ATP + H2O + nH+
It is possible to calculate that each molecule of NADH reaching OXPHOS produces 2,5 molecules of ATP, whereas each molecule of FADH2 produces 1,5.
On this bases we can state that the total energetic balance due to the complete oxidation of one molecule of glucose, through glycolysis, Krebs cycle and OXPHOS amounts to 32 molecules of ATP.
1.1.5 Anaerobic conditions
All the metabolic processes described above refer to healthy cells, living in normoxic conditions. Glycolysis is the only pathway that is maintained also in hypoxia or anaerobic conditions.
The lack of oxygen is a state that can characterize many tissues of the organism, even in physiological conditions, such as skeletal muscle during a strenuous physical exertion; but hypoxia can be related also to many pathological statuses like cancer or ischemia.
Anerobiosis, on the other hand, can be facultative or obligate and is typical of some microorganisms, among which we can find bacteria, fungi and protozoa.
In these cases, both hypoxia or anaerobiosis, the pyruvate produced with glycolysis undergoes a different metabolic process, called fermentation, with the purpose of restoring the NAD+ consumed and allowing the starting over of another glycolytic cycle. Otherwise,
without the restoration of the optimal redox equilibrium, glycolysis would be forbidden and this status would lead to starvation and cell death.
Many types of fermentation are known, but the most important ones are alcoholic fermentation and lactic fermentation.
Alcoholic fermentation is typical of some yeast and largely exploited in the food industry; it leads to the formation of ethanol and CO2, starting from pyruvate.
Fig. 7: Alcoholic fermentation[1]
Lactic fermentation, instead, occurs not only in many bacteria species, but also in the skeleton muscle during physical exertion, in erythrocytes and in testis. It transforms pyruvate in lactate as follows:
Fig. 8: Lactic fermentation
The reaction is catalyzed by the enzyme lactate dehydrogenase (LDH) and the lactate generated is transported to liver through the blood stream. Inside the hepatocytes lactate is reconverted in glucose through gluconeogenesis.
1.1.6 Other functions of glucose
Glucose is the most important substrate for the reactions of oxidation that lead to the generation of energy. Besides, this sugar can be processed through oxidation in the pentose phosphate pathway (PPP) in order to produce ribose, useful for nucleic acids and NADPH synthesis. NADPH is a cofactor similar to NADH, that is involved in ROS (i.e. Reactive Oxygen Species) counteracting mechanisms and more generally, it is exploited as electron donor in many redox reactions.
Fig. 9: Pentose Phosphate pathway (PPP)[1]
Two subsequent phases can be distinguished: the first phase is called oxidative, whereas the second one is called non-oxidative.
During the oxidative phase, glucose-6-phosphate is converted in ribose-5-phosphate with the production of two molecules of NADPH.
During the non-oxidative phase, ribose-5-phosphate is recycled to regenerate glucose-6-phosphate.
Another important purpose of glucose, is the synthesis of glycogen as a storage polymer, mainly in liver and skeletal muscle. The storage of glucose in this polymeric form allows the maintenance of optimal values of osmotic pressure. If an equal mass of glucose was stored as simple sugar, it would rise the osmotic pressure to levels that would be intolerable for the tissues. Glycogen, moreover, constitutes a rapidly available energetic fount, and it is particularly useful during starvation periods (especially for neurons) or during intense physical exertion; its synthesis and its hydrolysis, in fact, are regulated by hormonal messengers like insulin, glucagon and cortisol.
1.2. THE METABOLIC SWITCH OF CANCER CELLS[1,2,3,4,5]
1.2.1 The features of cancer cells
Cancer cells are characterized by various metabolic alterations, so that cancer is also defined “metabolic pathology”.
First of all they are characterized by an increase of glycolysis with a consequent high production in lactate, as cancer cells often live in hypoxic conditions and cannot rely on OXPHOS. The increase of the glycolytic rate and in general of the number of glycolytic cycles is commonly defined “Warburg effect”, from the name of the discoverer of this phenomenon.
This property is convenient for many reasons: a) The oxygen pressure can be fluctuating
With the growth of the tumor tissue, especially in solid tumors, cells can be forced to live in conditions of fluctuating oxygen pressure, because blood vessels are not always in their proximity, depending on the speed at which angiogenesis occurs.
If their metabolism was mainly based on OXPHOS, like the metabolism of healthy cells, hypoxia would be lethal for them. On the contrary, relying on lactic fermentation, in spite of its low efficiency, they can produce ATP and restore the NAD+ consumed, in every
condition.
b) Lactate has an active role in favoring tumor invasiveness, proliferation and survival.
The lactate generated starting from pyruvate, through lactic fermentation is extruded from the cell, through a specific transporter called MCT4 (Monocarboxylate transporter 4) in order to maintain cytosolic pH at physiological levels. This causes the decrease of the extracellular pH and this feature promotes the colonization by other tumor cells, as healthy cells would find too hostile this particular environment.
Lactate, besides, can activate particular membrane proteins called β-integrines, involved in tissue recognition mechanisms, tissue reparation, and cellular adhesion, stimulating the migration of cancer cells.
Lactate is also able to stimulate VEGF (i.e. Vascular Epithelium Growth Factor), that is responsible for angiogenesis, allowing growing cancer cells to receive oxygen and nourishment.
Lactate is involved also in other processes that can favor tumor growth. In fact it inhibits the immune response normally capable of identify and fight against cancer cells;
(generated by some chemotherapeutics) and it is the key for a symbiotic mechanism between normoxic and hypoxic cancer cells. More precisely, this mechanism is called lactate cell–cell shuttle system, as once lactate is pushed out in the extracellular environment, it is re-uptaken by normoxic cancer cells thank to the transporter MCT1. Here, lactate is reconverted to pyruvate through LDH-1 and enters the Krebs cycle to produce energy.
c) Glucose can be addressed to other metabolic pathways in order to produce compounds useful for the cell.
Since lactic fermentation is a less productive metabolic pathway, in cancer cells we observe an increase in glucose uptake; this phenomenon is called “Pasteur Effect” and allows the addressing of glucose towards other metabolic processes, such as the pentose phosphate pathway for the production of NADPH. This cofactor is useful for fatty acids biosynthesis, and as antioxidant protective agent against the environment in general but also against the effects of the pharmacological therapy.
In fact, in many cancer types, isoform 1 of transketolase (involved in the non-oxidative phase of PPP ) is overexpressed.
d) Glycolytic intermediates can be used for the synthesis of other molecules.
Some glycolytic intermediates are used for anabolic reactions like the synthesis of glycogen, ribose-5-phosphate, alanine, and other useful compounds.
In fact in many tumors the embrional isoform of the enzyme pyruvate kinase (PKM2) is overexpressed. This isoform can exist in two different forms: as a tetramer (with high activity) or as dimer (with low activity).
The dimeric form, as it possesses low activity, provokes an increase of the glycolytic intermediate downstream of pyruvate, allowing their transformation in amino acids, nucleic acids and lipids. Pyruvate itself can be converted in Acetyl-CoA and used for the synthesis of fatty acids and cholesterol. As a demonstration of that, in many cancer types, the enzyme FASN (Fatty acid synthase) is overexpressed.
1.2.2 Mechanisms on the basis of the metabolic switch
The fundamental cause of the metabolic change that occurs in tumor cells, lies in the fact that OXPHOS is not sufficiently exploited because of hypoxia.
In fact cancer cells mitochondria are often smaller, they lack mitochondrial cristae and possess an incomplete form of ATP-synthase.
On the consequence, aerobic glycolysis is much more relevant and, as described by Warburg, the production of lactate increases.
The factors that lead to these effects are many and they act through a chain mechanism: the activation of oncogenes (e.g. Myc) and the loss of functionality of oncosuppressors (e.g. protein p53, involved in the regulation of the cellular cycle), implicate a series of biological and biochemical interconnected alterations, that are responsible for carcinogenesis and tumor proliferation.
Fig. 10: Some molecular mechanisms on the basis of the metabolic switch of cancer cells. In Red: activated oncogenes; in green: inactivated oncosuppressors[4]
One of the central elements of the metabolic reprogramming typical of tumor cells, is the transcriptional factor HIF-1.
HIF-1 in an heterodimer constituted of two different subunits: a stable β subunit, located in the nucleus, and an α subunit that is unstable in normoxic conditions.
In particular, when the oxygen pressure is adequate, the α subunit is firstly hydroxylated by prolyl hydroxylase, then conjugated with VHL and ubiquitin and finally degradated by the proteasome. In a hypoxic environment or in case of oxidative or metabolic stress, or after the activation of oncogenes, this degradation system is inhibited and the α subunit migrates to the nucleus, where it associates with the β subunit, giving rise to the active form on HIF-1. HIF-1 activates the transcription of the glucose transporters GLUT-1 and GLUT-3, of the lactate transporter MCT4 and of many glycolytic enzymes such as hexokinase, phosphofructokinase, aldolase and more.
and of PDK1, responsible for the inactivation of the pyruvate dehydrogenase complex. On the basis of HIF-1 expression there is the mutation of two enzymes implied in the tricarboxylic acid cycle: fumarase and succinate dehydrogenase. Their alteration, in fact, causes the accumulation of fumarate and succinate, that competitively inhibit the enzyme prolyl hydroxylase, so that it cannot destroy the α subunit of HIF-1.
1.2.3 Typical features of cancer and their correlation to metabolic alterations
Many cancer cells have particular properties that confer them the resistance and invasiveness typical of malignant tumors. The alterations are many and some of them can be correlated to the metabolic modifications described above.
In particular, many tumor cells are characterized by: a) Invasiveness and absence of contact inhibition b) Self-sufficiency in growth factors
c) Resistance to apoptosis
d) Ability to induce angiogenesis e) Uncontrolled proliferation
f) Ability to elude the immune response
a) Invasiveness and absence of contact inhibition
The majority of tumor cells is less adhesive to the cells belonging to their tissue of origin, than healthy cells. This implies that their detachment and migration (processes that produce metastasis) are easier. Moreover cancer cells usually do not arrest their growth because of contact inhibition, but they migrate upon the adjacent cells to continue their growth, forming a stratified tissue.
These effects are due to lactate and to the activation of HIF-1.
In particular, the lactate produced after glycolysis can actively favor migration and the formation of metastasis through the activation of β1-integrine, membrane proteins, involved in the tissue recognition and in reparation and adhesion processes. Besides, as described above, once lactate is generated, it exits the cell through MCTF4 in order to keep the cytosolic pH at physiological values. This generates extracellular acidosis that can activate cathepsins and metalloproteases, which degrade the extracellular matrix and the basement membrane.
Also HIF-1 has a crucial role in this phenomenon, as it promotes the loss of E-cadherin, which is a particular isoform necessary to the maintenance of the intercellular contact with the epithelium. E-cadherin is lost during EMT (epithelial-mesenchyme transition), a phenomenon that occurs not only in the onset of metastasis, but also during the embrional development and in the tissue reparation processes. In fact, in all these cases, the epithelial cells loose the adhesion to the other cells in order to migrate and transform in mesenchymal multipotent cells. HIF-1 can stimulate the expression of two oncogenes, called met and TWIST, which favor EMT.
b) Self-sufficiency in growth factors
Tumor cells are able to stimulate their own growth through autocrine signaling mechanisms. This happens because of alterations of the main regulators of the signaling pathways, of the second messengers or other effectors situated at different levels of the signal transduction. Besides, the natural negative modulators could be inactivated.
As far as concerning the cellular metabolism, the hyperactivation of growth provokes three main consequences:
- The growth factors usually exploit tyrosine kinase receptors, which modulate the activity of the effectors downstream, phosphorylating the tyrosine residues;
The enzyme PKM2 (specific tumor isoform of pyruvate kinase) can bind to those peptides carrying phosphorylated tyrosine residues, releasing fructose-1,6-biphosphate, that works as an allosteric activator. A partial inhibition of PKM2 follows, and so the last step of glycolysis is blocked.
This causes the accumulation of glycolytic intermediates and favors their addressing towards other anabolic reactions.
This phenomenon is very important also because it prevents an excessive production of pyruvate, promoting glycolysis.
- The hyperactivation of the PI3K-Akt system, upstream of some tyrosine kinase receptors, stimulates the expression of GLUT-1 and the translocation of GLUT-4 on the plasma membrane, increasing the flux of glucose entering the cell.
Moreover, Akt activates the glycolytic enzyme PKF2 and ATP-citrate lyase, which is involved in the fatty acids synthesis.
- The PI3K pathway leads to the downregulation of the enzyme carnitine palmitoyltransferase 1A, responsible for the esterification of long chained-fatty acids with
carnitine. This transformation occurs during the β-oxidation so that this effect increases more and more the dependence of cancer cells on glycolysis.
c) Resistance to apoptosis
Tumor cells are provided of mechanisms that allow them to evade apoptosis. This is fundamental not only for the oncogenesis process, but also as far as the resistance against the pharmacological therapy.
In particular, it is believed that cancer cells are resistant against MMP (Mitochondrial membrane permeabilization), which represents one of the decisive steps of apoptosis. This resistance can be caused by the enzyme HK that, thank to the action of Akt, is translocated to the external mitochondrial membrane, and here, inhibits the permeabilization of the membrane, acting presumably on PTPC (Permeability transition pore complex).
Also the impossibility of acting OXPHOS can confer resistance against programmed cell death. First of all, the inhibition of OXPHOS can suppress the activation of the proapoptotic proteins Bcl-2, Bax e Bak, which mediate MMP. Secondly, the absence of OXPHOS reduces the ability of some drugs to induce the generation of ROS, compromising their capability to induce apoptosis. In fact ROS (i.e. Reactive Oxygen Species) which include superoxide, hydroxyl radical, peroxide and other species, are naturally produced by the cell metabolism as byproducts, and rapidly degradated by specific mechanisms. In case of environmental stress, which can be caused by many factors such as exposition to radiations or chemotherapeutics, the production of ROS is increased. These reactive species are able to damage the DNA, oxidize polyunsaturated fatty acids, amino acids and cofactors, provoking the inactivation of certain enzymes. This leads to oxidative stress and possibly to apoptosis. Since OXPHOS is one of the most important mechanisms through which ROS are produced, its deficiency actively contributes to the resistance of cancer cells against chemotherapeutics.
d) Ability to induce angiogenesis
Both hypoxia (through the activation of HIF-1) and the hyperactivation of the signal pathways PI3K and MAP, increase the expression of VEGF, responsible for the induction of angiogenesis. Angiogenesis is really important for a growing tissue, as it allows an adequate supply of oxygen and nourishment and the elimination of waste products. This phenomenon begins with the destruction of the basement membrane, that leads to hypoxia. Hypoxia itself activates the angiogenic factors that stimulate the growth of endothelial
cells.
e) Uncontrolled proliferation
Cancer cells are characterized by the alteration or the complete loss of the proteins that can induce senescence and the arrest of the cellular growth, such as the protein p53.
The inactivation of this protein determines a decrease in the transcription of the genes that are responsible for the reparation of DNA damages and also the inhibition of cellular death caused by damages of the genetic code.
The mutations of p53 are, anyway, necessary for cancer cells survival. In fact the activation of this protein (and so the interruption of proliferation and the induction of apoptosis) is related to low values of the oxygen pressure, that are not uncommon in tumor tissues, so that an unchanged protein p53 would be incompatible with life for cancer cells.
Besides, the loss of functionality of this protein has an active role in the onset of Warburg effect, as it can activate the transcription of a particular isoform of PKF2, which inhibits glycolysis and contributes to the addressing of glucose towards the PPT.
f) Ability to elude the immune response
Tumor tissues have the ability of attract inflammatory cells that can favor cancer progression. Among them, we can find TAMs (Tumor Associated Macrophages), which facilitate angiogenesis and cellular migration, and reduce the immune response against tumor cells. Also the infiltration of TAMs in tumors is a phenomenon regulated by HIF-1 and so by hypoxia.
As described above, also lactate is fundamental for the inhibition of the immune response, as it can generate an acidic extracellular environment that inhibits the activity of NK lymphocytes and the production of cytokines.
1.3. BACKGROUND ON THE PHARMACOLOGICAL TARGETS TO COUNTERACT TUMOR METABOLISM [2,4,6,7]
1.3.1 Why counteracting cancer cell metabolism
As described above, the typical cancer tissue metabolic alterations, are related to other physiological characteristics of tumors.
Therefore, counteracting their metabolism, we should obtain a decrease or an arrest of proliferation, the induction of apoptosis, and a decrease in the development of metastasis. These effects can be exploited both as chemotherapeutic, and as preventive or anti-relapse treatment.
Many pharmacological approaches have been studied in order to oppose to cancer metabolism; the most promising targets can be summarized in three categories:
Membrane transporters involved in glycolysis Pyruvate dehydrogenase kinase (PDK)
Glycolytic enzymes
1.3.2 Membrane transporters involved in glycolysis
The membrane transport proteins, involved in glycolysis can be divided in two main groups: glucose transporters (GLUT) and monocarboxylate transporters (MCT).
GLUTs are responsible for glucose uptake, so inhibiting their functionality would give a decrease in available glucose levels and consequently a decrease of glycolysis. This leads to cell death for starvation.
MCTs, on the other hand, are responsible for the lactate cell-cell shuttle mechanisms. Their block would destroy the symbiosis based on lactate, between hypoxic cells (that produce lactate through fermentation of pyruvate) and normoxic cells (that uptake lactate and transform it back to pyruvate, that enters the Krebs cycle). The selective inhibition of tumor GLUTs is hard and the drugs that possess this feature are really few. An example is represented by natural flavonoids, which decrease the expression of GLUTs, causing an anticancer effect. Nevertheless, this result cannot be totally attributed to GLUTs inhibitions, as these compounds possess also other contributing activities. For example, flavonoids, can reduce the expression of VEGR and modulate the expression of the proapoptotic genes Bax e Bcl-2.
Another chance to act on GLUTs consists in the conjugation of chemotherapeutics with glucose or other sugars, in order to increase their uptake in tumor cells and insure a selectivity towards rapidly proliferating cells.
Of course this approach does not reduce transporters functionality, but exploits it to promote the action of antineoplastic drugs.
As far as the inhibition of MCTs, the isoforms involved involved in the carbohydrate metabolism are 4 and 1. Isoform 4 has got low affinity for lactate and it is responsible for its efflux, whereas isoform 1 possesses medium affinity and is responsible for its uptake in normoxic tumor cells.
Their inhibition provokes the acidification of the cytosol, that leads to growth arrest , cytotoxicity and a decrease in the development of metastasis.
1.3.3 Pyruvate dehydrogenase kinase (PDK)
The pyruvate dehydrogenase multienzymatic complex (PDH), responsible for the transformation of pyruvate to Acetyl-CoA, is regulated through the transfer of phosphate groups, by the enzymes pyruvate dehydrogenase kinase and pyruvate dehydrogenase dephosphatase. The phosphorylation determines the inhibition of PDH, whereas the dephosphorylation activates it.
Inhibiting PDK, Krebs cycle is compromised and a metabolism entirely based on glycolysis is favored.
Moreover, the impossibility of conducting OXPHOS, decreases the production of ROS, and this condition confers higher survival abilities and resistance against chemotherapeutics, to cancer cells.
In tumors PDK isoforms 1 and 3 are overexpressed, because of the action of HIF-1.
High levels of this enzyme are related to aggressive phenotypes and poor prognosis. Therefore, the silencing or the inhibition of this protein would lead to positive effects, such as the induction of apoptosis, an increase in ROS production and the stabilization in the growth of metastasis.
1.3.4 Glycolytic enzymes
The inhibition of the glycolytic enzymes is another efficient method to counteract tumor metabolism, as it leads necessarily, to the accumulation of intermediates and to the impossibility of producing ATP through this metabolic pathway.
The most promising targets are the proteins involved in the first or last steps of glycolysis, such as hexokinase or lactate dehydrogenase (LDH). Of course in the attempt of interfering with tumor glycolysis we can expect side effects on healthy cells, especially on those tissues that use glucose as almost exclusive fuel, such as brain, retina and testis. Since many tissues express particular isoforms of the glycolytic enzymes, which differ
from the isoforms found in cancer cells, a certain selectivity is theoretically possible. However, the substrates of the glycolytic enzymes are small, polar and generally negatively charged, so the active sites have little space and carry cationic residues. This causes a series of inconveniences in the design of new potential inhibitors, such as slight cell penetration and off-target interference.
1.4. LACTATE DEHYDROGENASE (LDH)[8,9,10,11]
1.4.1 Structure and catalytic activity
The enzyme lactate dehydrogenase (LDH) belongs to the family of the oxidoreductases and it is responsible for the transformation of pyruvate, generated through glycolysis, to lactate and vice versa, using NAD+ as cofactor.
Fig. 11: Tetrameric structure of human LDH[8]
Six distinct isoforms of human LDH are known by now: five of them are tetramers constituted by the association of two different subunits called H and M, whereas the sixth isoform is constituted by another subunit, called X.
The H subunit takes its name from heart, as it is highly expressed in the cardiac tissue (but also in spleen, kidneys, brain and erythrocytes) and it is encoded by the gene ldha.
The M subunit takes its name from muscle (but it can be found in liver too) and it is encoded by the gene ldhb.
These two subunits associate to form two homotetramers (LDH-1 and LDH-5) and two heterotetramers (LDH-2 and LDH-3).
LDH-2 is made of three H subunits and one M subunit (H3M);
LDH-3 is made of two H subunits and two M subunits (H2M2);
LDH-4 is made of one H subunit and three M subunits (HM3);
LDH-5, also called LDHA, is made of four M subunits (M4);
The ability to catalyze the reduction of pyruvate to lactate, increases with the increase of number of M subunits, so it is particularly high for LDH-5 and particularly low for LDH-1, which, in fact, catalyzes more easily the reverse reaction.
As a matter of fact, LDH-5 and LDH-1 have interdependent roles in the lactate cell-cell shuttle mechanism.
On the contrary, the X subunit is found in testis and composes the isoform LDH-C4, which possesses an important role in male fertility, as it is implied in the process of capacitation. LDH-1 and LDH-5 share an analogous conformation; we can identify two domains: the first one, formed by the amino acid residues 20-162 and 248-266, disposed in a Rossman fold (this conformation is typical of nucleotide-binding proteins), and the second one, formed by the residues 163-247 and 267-331, which represents the substrate-binding domain.
At the interface between the two domains, we can find a loop in which pyruvate settles.
Fig. 12: Amino acid residues in the active site of LDH[8]
As far as the catalytic activity, at the beginning we observe the binding of NADH to His195, which is in a fissure of the central β-sheet that constitutes the Rossman fold. Here we can find also Ile250 that increases the lipophilicity of the site, favoring the binding with NADH and penalizing the binding with NAD+.
After that, the pyruvate binds to the active site, interacting with His195 that acts as a proton exchanger and allows the optimal orientation of the substrate.
Arg109 forms a hydrogen-bond that polarizes the carbonyl group, inducing the closing of the loop. In this way the enter of solvent inside the active site is prevented and the redox reaction in facilitated.
The most evident difference between LDH-1 and LDH-5 is the net superficial charge, that modifies the pKa value of His195, and so differentiates the kinetic activities of the two isoforms. In fact LDH-5 possesses a higher value of Km for pyruvate than LDH-1, which means that LDH-5 requires higher concentrations of pyruvate to reach the maximum activity. At these high concentrations, LDH-1 is, instead, inhibited.
1.4.2 Role of LDH in human cells
LDH is required for the oxidation of pyruvate to lactate and vice versa, in many organisms. In healthy human cells LDH is necessary for the maintenance of the equilibrium NAD+/NADH and, in particular, LDH-5 is involved in the rapid regeneration
of NAD+ in the skeletal muscle under physical exertion (i.e. in hypoxic conditions),
granting the possibility to continue glycolysis and to obtain energy rapidly; on the contrary, in brain, during an intense mental activity, after a first glycolytic phase, the concentration of pyruvate increases and reaches the levels that inhibit LDH-1; in this way the Krebs cycle is favored and so the metabolism stays aerobic.
LDH is a glycolytic enzyme and so it is located in the cytosol, but it can also be found in peroxisomes, mitochondria and nucleus, where it performs particular functions.
In peroxisomes, LDH-5 is predominant and in absence of pyruvate, reconverts the NADH generated through the long-chained fatty acids oxidation. Lactate and pyruvate are exchanged through MCT located on the peroxisomes membrane in order to maintain the optimal balance.
In mitochondria, LDH allows the reconversion of lactate to pyruvate, letting it enter the tricarboxylic acid cycle or the gluconeogenesis. It can happen either in the same cell that produced lactate or in adjacent cells, exploiting the cell-cell shuttle mechanism.
Finally, in the nucleus, LDH binds to single stranded DNA, together with glyceraldehyde-3-phosphate dehydrogenase; their combined action keeps the optimal NAD+/NADH
1.4.3 Lactate dehydrogenase as pharmacological target
The inhibition of LDH can be a valid therapeutic strategy for anticancer therapy, as it would lead to cytotoxic effects for hypoxic cells and to a decrease in metastasis development.
In particular, the lack of functionality of LDH in hypoxic cells, would cause the arrest of glycolysis and the accumulation of the intermediates, so that the production of ATP would be impossible. The extracellular pH, usually low because of the high amounts of lactate extruded from cancer cells, would increase, and this condition would not be optimal for cellular invasiveness and migration.
Besides, the absence of lactate would prevent the symbiotic mechanism of lactate cell-cell shuttle between normoxic and hypoxic cells.
Fig. 13: Lactate cell-cell shuttle
All these effects result in cytotoxicity for cancer cells, but should provoke only minimal side effects on healthy cells, as the congenital deficiency of LDH subunits is compatible with life.
Many clinical cases have been studied and patients lacking of the M subunit of LDH show muscular rigidity and myoglobinuria, exclusively after a strenuous physical exertion. Patients lacking of the H subunit, instead, do not show any relevant negative consequence. In particular, the isoform 1 of LDH is the main responsible for the production of ATP in erythrocytes and in spite of that, individuals lacking of subunit H do not suffer from anemia but in some cases can be affected from slight hemolysis.
On the contrary, the congenital deficiency of other glycolytic enzymes leads to anemia due to repeated hemolysis episodes; in fact, since erythrocytes do not possess mitochondria, they cannot exploit the usual aerobic metabolism (i.e. Krebs cycle and OXPHOS).
The fact that the lack of LDH do not cause serious erythrocyte damages, on the opposite to what happens with the lack of other glycolytic enzymes, can be explained with the presence of other oxidative systems independent from LDH, which reconvert NADH to NAD+. An example is represented by α-glycerophosphate dehydrogenase in the skeletal
muscle.
This enzyme, during intense physical exertion (and so in hypoxic conditions) converts glucose to glycerol and α-glycerophosphate, restoring part of the amount of NAD+
consumed through the first phase of glycolysis. However, this process provokes the complete arrest of glycolysis and so the block of the ATP synthesis. The consequence is the disruption of the plasma membrane and the release of the cytosol proteins.
Glucose
- 2 NAD+
Glyceraldehyde-3-phosphate +
Dihydroxyacetone-phosphate -glycerophosphate dehydrogenase Glycerol-3-phosphate +
NAD+
Pyruvate
Fig. 14: Alternative systems that can restore the redox equilibrium
In the erythrocytes the levels of α-glycerophosphate dehydrogenase are low but, in case of congenital deficiency of the H subunit of LDH, a further oxidative mechanism is activated, in order to restore the NAD+ needed. The oxidation is led through the action of
NADH-methemoglobin reductase, which reconvert NADH to NAD+ without affecting the
carbohydrates metabolism.
The only side effects that should be observed in case of LDH inhibition, would concern rapid proliferating cells, like those belonging to tissues that undergo reparation processes and immune cells during the activation of the immune response.
The studies conducted by now reveal that the isoform 1 has a contradictory and not fully understood role in carcinogenesis. On the other hand, the isoform 5 seems to be a fundamental factor for tumor growth and invasiveness.
The following table (Table 2) summarizes the main cellular features, related to the activity of the isoforms 1 and 5 of LDH.
LDH-1 Consequence
Reduced expression Induction of carcinogenesis in some type of cancer Increase of the invasiveness in some type of cancer High expression
Poor prognosis
Increase of cellular signal through the tyrosine kinase receptors activation
LDH-5
Silencing
Inhibition of cellular growth Inhibition of migration
Decrease of the activation of tyrosine kinase receptors Increase of the production of ROS
Decrease of the pharmacological resistance against some chemotherapeutics
Table 2: cellular features related to the activity of the isoforms 1 and 5 of LDH. All the factors reported above, suggest that the inhibition of LDH should be selective against the isoform 5, as its silencing showed univocal effects that counteract the progress of cancer. Moreover, it should not be ignored that the selective inhibition of the isoform 5 does not unbalance the proliferation and the metabolism of healthy cells.
Obviously the main pathology for which LDH inhibitors are meant, is cancer, but actually this pharmaceutical approach can be used also against other diseases. In fact, LDH is also a fundamental enzyme for the survival of Plasmodium falciparum, the etiologic agent of malaria, during its erythrocytic stage, as human parasite. The transmission of malaria from an infected mosquito to a human being, begins with the injection of Plasmodium sporozoites in blood. Through the blood stream they reach the liver, and multiply and differentiate in schizonts, containing merozoites, inside the hepatocytes. The merozoites generated are then released into the blood, from which they are able to invade erythrocytes. This is called the erythrocytic phase of the Plasmodium life cycle, during which these asexual blood parasites proliferate at high rates and therefore need large amounts of energy. Although the genome contains the genes that codify for the tricarboxylic acid enzymes, it is demonstrated that during the erythrocytic phase, the Plasmodium is characterized by a glycolytic phenotype, through which it oxidizes glucose to pyruvate, which is then transformed into lactate by a particular isoform of LDH, called pfLDH. The tricarboxylic acid cycle seems to be branched and completely uncoupled from glycolysis, in order to produce Succinyl-CoA as starting material for the synthesis of
haeme. The most important differences between human and pf LDH are the amino acid sequence, and the ability to use ADAP (i.e. 3-acetylpyridine NAD) as coenzyme, instead of NAD. Both the human and the pf isoforms of LDH can use ADAP, but the latter can utilize it at a higher rate.
Fig. 15: Life cycle of Plasmodium falciparum[11]
Another possible application of LDH inhibitors is as immunosuppressant, as glycolysis increases in lymphocytes during an excessive immune response.
1.4.4 Background on the most important inhibitors of Lactate Dehydrogenase
The most important inhibitors of LDH studied until now can be classified in eight categories on the basis of their chemical structure:
a) Substrate-like derivatives b) Polyphenols
c) Galloflavin d) Azoles
e) Quinolines and 1,4-dihydro-4-quinolones f) “Bifunctional” inhibitors
g) Dihidropyrimidines h) N-hydroxyindoles
a) Substrate-like derivatives HO HO O O O OH
Pyruvate Lactic acid
HO NH2 O O HO OH O O HO O OH O OH O O n
Oxamic acid Oxalic acid
Tartronic acid Cyclic polylactates
Fig. 16: Substrate-like inhibitors
Among the most known molecules belonging to this category, we find oxamic acid, which represents a bioisostere of pyruvate. Since its structural analogy with the natural substrate, oxamic acid is a competitive inhibitor of human and pf LDH and it turned out to be non-toxic on lab animals, nevertheless, it presents some inconveniences: first of all it is not a specific inhibitor of LDH, but it can inhibit also other enzymes such as AAT (i.e. Aspartate aminotransferase, an enzyme involved in the malate-aspartate shuttle mechanism) with a smaller value of Ki; secondly, oxamic acid hardly penetrates through biological membranes because of its high polarity and, finally, its potency as inhibitor is low.
Many modifications of the basic structure of oxamic acid have been studied (in particular substitutions on the nitrogen atom) none of which has led to a selective inhibitor for a specific human LDH isoform, and in some cases a certain preference for pfLDH has been obtained.
Another compound member of this group, is oxalic acid, which inhibits both isoform 1 and 5 of human LDH, competing with lactate, thank to their structural analogy.
Also tartronic acid can block glycolysis, but it is not a potent inhibitor, and above all it is not selective towards LDH, because it can inhibit MDH (i.e. Malate dehydrogenase) too. The last molecules which are part of this category are cyclic polylactates. They are able to
inhibit LDH in a non-competitive manner, but they are not selective as they can also inhibit PK; this mechanism contributes to their antiglycolytic effect.
b) Polyphenols
The most known compound of this group is gossypol, a non-selective inhibitor which competes with NADH and for this reason it can also bind to other NADH-dependent dehydrogenases (e.g. glyceraldheyde-3-phosphate dehydrogenase, a glycolytic enzyme). As far as the inhibition of LDH, gossypol is active preferentially on the human isoforms 1 and 5, but it can also block the human isoform C4 and pfLDH, affecting male fertility, and provoking an antimalarial effect.
Fig. 17: Gossypol
Gossypol exists as two atropisomers, generated by the rotation around the 2-2' bond. It is demonstrated that the R-(-) isomer is the most potent one.
The most important disadvantage of gossypol is its unspecific toxicity. In fact, its structure can chelate metal ions, the aldehyde groups can generate highly reactive Schiff bases binding to amino acids, and the cathecol hydroxyls can undergo oxidative metabolism to form toxic quinone structures. Because of this, gossypol interacts with many cellular structures, interfering with various physiological functions such as ion transport, membrane properties and many others.
In the attempt to keep the biological activity and removing the toxicity, many analogues have been designed. They lack the aldehyde groups or both the aldehyde groups and the cathecol rings in order to reduce the toxicity observed.
Essentially, gossypol derivatives can be grouped in three structural categories: the 2,3-dihydroxy-1-naphthoic acids, the cyclic derivatives and the naphthoic acids.
OH OH HO HO OH HO 2 2' OHC iPr iPr CHO
The 2,3-dihydroxy-1-naphthoic acids inhibit the pf isoform and the human 1,5 and C4
isoforms of LDH, although they are generally less active on LDH-1 than on the other isoforms. The most selective inhibitor for human LDH-5 is the compound FX11.
COOH HO
HO
CH2Ph
Fig. 19: Compound FX11
FX11 decreases both ATP and lactate production, increasing oxidative stress which leads to cell death. Moreover, hypoxia can enhance FX11 activity, making this molecule particularly suitable for anticancer therapies. However this response can be provided also from off-target effects, so that it is still unclear how much of the effect is due to the LDH inhibition. It is not neglectable to mention that the cathecol portion of FX11 makes it highly reactive and so not appropriate as a drug.
The cyclic derivatives of gossypol are lactone structures, the most selective of which (relatively to human LDH-5) is an iminolactone which, unfortunately, has got low potency.
O HN HO HO iPr Fig. 20: Gossyliciminolactone
As far as the naphthoic acid derivatives, they are naphthalene rings substituted with carboxylic acids, sulfonic acids or hydroxyl groups. They settle in the active site binding to both the pyruvate and the cofactor sites, but they are not potent inhibitors of LDH.
HO HO OH R' COOH R'' HO HO O iPr X COOH HOOC R' R''
Fig. 18: On the left: 2,3-dihydroxy-1-naphthoic acids. In the center: cyclic derivatives. On the right: Naphthoic acids
c) Galloflavin O O O O OH OH HO HO Fig. 21: Galloflavin
Galloflavin has been discovered though virtual screening based on the crystal structure of the muscular subunit of human LDH. It can inhibit subunits 1 and 5 of human LDH in a non-competitive way causing the block of glycolysis, a decrease in lactate production and ATP levels, and the activation of a stress response mediated by ROS, without lethal effects on healthy cells. Furthermore, galloflavin can interfere with the interaction between LDH and ssRNA, exploiting the NADH binding site of the enzyme, thus preventing the introduction of uridine in the nucleic acid. This brings to the complete block of RNA synthesis, and this effect contributes to the therapeutic action of galloflavin.
d) Azoles Z X Y HO COOH Fig. 22: Azole inhibitors
Azole compounds endowed with LDH inhibition properties, block human LDH-1 and pfLDH trough a mixed competition with NADH and pyruvate and a complete competition with lactate. After various modification of the chemical structures, the proximity of an hydroxyl group to a carboxylic acid has been determined as part of the pharmacophore pattern. In fact, the hydroxyl group can form an H-bond with the amino acid residues Leu140, His195 and Arg109, whereas the azole heterocycle arranges parallel to the nicotinamide ring of NAD+.
Furthermore, azoles containing a sulfur atom are better LDH-1 inhibitors of those containing oxygen atoms, whereas the thiadiazole derivative showed to be an inhibitor of LDH-5; nevertheless these compounds are not selective for LDH and can interact with other targets.
e) Quinolines and 1,4-dihydro-4-quinolones
Fig 23: Quinolines and 1,4-dihydro-4-quinolones inhibitors[10]
4-hydroxyquinoline-2-carboxylic acids, 4-hydroxyquinoline-3-carboxylic acids and 1,4,-dihydro-4-quinolone-3-carboxylic acids were studied as inhibitors of several dehydrogenases involved in the glucose metabolism of cancer cell. The quinolines bearing the carboxylic acid in position 3 possess more activity then those carrying the COOH group in position 2 and, when substituted in the positions 5,6 and 8 with small groups (such as amino groups or methoxy group), the selectivity towards LDH over other dehydrogenases increases.
The p-nitrophenoxyalkyl-substituted dihidroquinoline turned out to be the best LDH inhibitor, although its selectivity is low. The replacement of the nitro group with an amino group increases the selectivity but decreases the potency.
Fig. 25: Quinoline-based compound[10]
The quinolines carrying an amide or sulfonamide group in position 3 and an aniline in position 4, with a carboxylic acid on the phenyl ring showed to be good inhibitors. In particular, the aniline and the amide or sulfonamide groups resemble the nicotinamide portion of NADH, whereas the carboxylic group resembles the substrate.
N O COOH MeO O NO2 Fig. 24: p-nitrophenoxyalkyl derivative
f)“Bifunctional” inhibitors
This kind of inhibitors contain a NADH-like portion and a pyruvate resembling group, connected by a linker. These compounds are thought to interact both with the cofactor and the substrate binding sites since they are close to each other. It was demonstrated that the cofactor interacts with the active site through its extremities: the nicotinamide and the
adenosine rings, which are 20 Å distant. The substrate, instead, lies parallel to the nicotinamide ring.
One of the simplest compounds of this series is the glycolic acid-NADH conjugate.
This compound showed to inhibit lactate production but, above all, it has the capability to inhibit the oxidation of lactate to pyruvate. Moreover it decreases the production of ROS but, unfortunately it has got low cell permeability.
Through fragment-based approach it was possible to design different classes of bifunctional inhibitors, which can be classified in four classes: bis(indolyl)maleimide derivatives, 3,4-dihydroxyphenylmethylene-rhodanine derivatives, malonate inhibitors and 6-(3-fluorophenyl)nicotinic acid derivatives. The first class is characterized by a bis(indolyl)maleimide group, which resembles the adenosine ring of NADH, and a carboxylic acid group (on the variable portion), connected by an alkyl or aryl-alkyl chain containing a triazole ring.
HN N NH O O (CH2)n N N N (CH2)n COOH Fig. 27: bis(indolyl)maleimidederivatives
The terminal carboxylic portion is fundamental for the activity thank to its ability to form strong polar interactions in the substrate-binding site.
As far as the 3,4-dihydroxyphenylmethylene-rhodanine derivatives, they have been discovered through the identification of a common ligand mimic for many
Fig. 26: glycolic acid-NADH conjugate[10]
oxidoreductases. The most potent inhibitor is a 3,2-dichlorobenzylamide derivative which shows selectivity for LDH over other oxidoreductases, however the selectivity for the isoform 5 has not been studied.
The malonate inhibitors have been discovered through fragment-based design. They possess a benzothiazole ring which binds to the hydrophobic pocket where the adenosine ring of NADH lies. Malonate, instead, is meant to mimic the pyruvate. The linker is an S-propyl portion, or a methyl group.
These derivatives show low activity in cell-based assays, perhaps because of their low cell permeability, due to the malonate portion. For this reason it has been thought to convert the free carboxylic groups with methyl ester groups, and this type of derivatives showed to be more permeable and to have antiproliferative activity.
Through fragment-based design, it was possible to obtain the last class of inhibitors too. The 6-(3-fluorophenyl)nicotinic acids possess two nicotinic acid functions connected by a polyhydroxy-alkyl chain. linker F N R2 MeO Cl HN O S N R1
Fig. 29: 6-(3-fluorophenyl)nicotinic acid derivatives H N HN S N R' O O linker OH HO O O HO HO N S O S R
Fig. 28: On the left: 3,4-dihydroxyphenylmethylene-rhodanine inhibitors. On the right: malonate inhibitors
The 6-(3-fluorophenyl)nicotinic acid binds to the cofactor site, in the nicotinamide pocket, whereas the linker and the second nicotinic portion lie in the adenosine site. The stereochemistry of the hydroxyl groups on the linker is fundamental for the activity, as they form many hydrogen-bonds with the enzyme. When the substituents R1 and R2 are
carboxylic acids the permeability decreases; when only one carboxylic function is kept the permeability increases, but the activity reduces; a COOH group is fundamental to interact with the substrate-binding site, in fact the dimethyl ester derivative resulted inactive. g) Dihidropyrimidines
These structures have been discovered through high throughput enzymatic screening and the most important molecules belonging to this category carry a 2-thio-6-oxo-1,6-dihidropyrimdine.
They are selective for LDH and need the presence of the cofactor in the active site for an optimal inhibition. Structural modifications showed that the sulfonamide, the cyano and the p-chloro groups are fundamental for the activity. The addition of more halogens in position R3 gives an improvement in potency, whereas R1 can be a methyl or an ethyl group
without significant consequences.
Dihidropyrimdines showed a certain selectivity for the isoform 5 of human LDH through the interaction with amino acid residues, placed near the catalytic region. These chemical groups undergo conformational adaptation due to the enzymatic activity, but they are not directly involved in the catalytic process. Anyway these derivatives did not show decreasing of lactate production, perhaps because of their low permeability.
h) N-hydroxyindoles
N-hydroxyindole derivatives have been designed ex novo considering the chemical characteristics of the active site of the enzyme. They contain a central indole portion substituted with a hydroxyl group on the nitrogen atom and a carboxylic acid in position 2, which constitute the pharmacophore pattern described above.
HN N R3 R2 CN O S H N R1 O S H2N O O
Fig. 31: N-hydroxyindole derivatives[10]
In particular, they interact with the polar cationic cavity usually occupied by the substrate and the cofactor, though several bonds: COOH forms a salt bridge with Arg 169, and an hydrogen-bond with Thr 248, whereas the N-hydroxy group forms an hydrogen-bond with Thr 248 and His 193. The aryl portion in position 6, disposes itself inside a lipophilic pocket. The substitution with a chlorine atom or a biphenyl group in position 6 increases the activity. In particular it has been demonstrated that the presence of aromatic rings in position 6 is usually beneficial for the activity and the additional presence of another phenyl ring in position 5 maintain the functionality. On the contrary, 1,2,3-triazole based compounds show a lower potency.
N-hydroxyindoles inhibit LDH competing both with pyruvate and NADH, possessing a good specificity for isoform 5 of the human enzyme. They cause a decrease lactate production and cell growth also in hypoxic conditions.
i) Compounds interfering with LDH expression
Another possible strategy to counteract the effects generated by LDH is to interfere with its expression. Many natural compounds, such as catechins present in green tea and in plants, are able to decrease the expression of LDH, through the dissociation of protein Hsp90 from the α subunit of HIF-1, in order to promote its degradation in the proteasome.
The reduction of the expression of LDH leads to tumor cell death without significant side effects.
