Defenders of Mitochondria

Alright my Biochem peeps,

Here is my 2nd review article. It is based on the target therapies of cancer treatment. In particular, this article focuses on the drug known as dichloroacetate which targets a mitochondrial enzyme known as pyruvate dehydrogenase kinase. This enzyme inhibits the action of pyruvate dehydrogenase as well as increase the metabolism of cancer cells that facilitate cancer proliferation. This article is quite interesting and thought-provoking. It will aid your understanding of the concepts of glycolysis, Krebs, ETC and oxidative phosphorylation. It is also marvelous to read the mechanism of action of cancer cells and the intricacies of their metabolism that fosters survival and proliferation. Take a read and see for yourself.

Michelakis, Evangelos D. and Gopinath Sutendra. 2013. “Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology.” Frontiers in Oncology 3(38): 1-11. doi: 10.3389/fonc.2013.00038

Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology.

This article is not a study as in the previous posting, but rather delves into the strategies utilized in cancer treatment in particular targeting more efficient, effective and less destructive methods of cancer therapy. This article deals mainly with investigating the effects of inhibition of an enzyme, pyruvate dehydrogenase kinase (PDK) as well as the applications of similar techniques that promote glucose oxidation as a viable option in metabolic targeting of neoplastic cells. The authors also explore the pre-clinical and clinical evidence of the effectiveness of these inhibitors on different cancers.

Neoplastic cells have a unique metabolic property in which there is both molecular and genetic signalling that converts glucose oxidative phosphorylation in the mitochondria of cells to glycolysis in the cytosol even under normal conditions. This results in the increase in glucose consumption as well as lactate production in cancer cells. This process is referred to as the Warburg effect after Otto Warburg who published this hypothesis in 1956. This means that cancer cells depend on glycolysis as its primary energy source as well as the source of macromolecular building blocks through glycolytic intermediates.

This is initiated by the activation of hypoxia-inducible factor 1α (HIF-1α). HIF-1α is activated in hypoxic-like environments and then stimulates the expression of glycolytic enzymes, glucose transporters and mitochondrial enzymes that increase the glycolytic pathway as well as formation of lactate. One important mitochondrial enzyme that is activated is, PDK. PDK  is considered the gatekeeper of the mitochondria since it regulates the flux of pyruvate into the mitochondria to undergo oxidative phosphorylation. PDK does this by inhibition of pyruvate dehydrogenase that performs the link reaction that decarboxylates pyruvate to Acetyl-CoA to enter the Krebs cycle. This inhibition leads to pyruvate not entering the mitochondria and therefore is converted to lactate in the cytosol by lactate dehydrogenase. Oncogenes are responsible for the maintenance of hypoxic state for cancer cells to continue to activate HIF. In cancer cells, there is also a loss of p53 gene expression that was responsible for mitochondrial DNA repair and expression cytochrome c oxidase enzymes that aid in apoptosis of the cell. Loss of p53 directly induces the expression of PDK and increases the stability and transcriptional activity of HIF. Additionally, HIF also increase pro-angiogenic and metastasis-promoting factors such as VEGF and SDF-1.

With increase in glycolytic enzymes, there is an increase in glycolysis as well as these glycolytic enzymes are utilized to aid in anti-apoptotic activities, activation of transcription in neoplastic cells, inhibition of mitochondrial function. Enzymes such include, glyceraldehyde 3-phosphate dehydrogenase and hexokinase. An isoform of hexokinase, hexokinase 2 is up regulated in neoplastic cells and then translocated to the mitochondrial transition pore, a channel responsible for the release of apoptotic factors as well as the regulation of anions the facilitate oxidative phosphorylation. Thus, hexokinase 2 facilitates resistance to apoptosis as well as increase in glycolysis in tumour cells. The glycolytic intermediates may also be funnelled to alternative pathways to produce amino acids, nucleic acids and lipids.

The mechanism of action of cancer cells lies in the alteration of the mitochondria of the cell. The mitochondria are responsible for the intrinsic apoptotic mechanisms of the cells and suppression of mitochondrial function is critical to cancer cell proliferation. One method employed by cancer cells is the alteration of the mitochondrial membrane potential of the inner mitochondrial membrane. This membrane potential is crucial to oxidative phosphorylation and electron transport chain. This membrane potential is akin to the proton motive force that is generated as hydrogen ions are pumped into the inter-membrane space of the mitochondrion during electron transport chain. This membrane potential is also critical to maintenance of intracellular calcium levels since mitochondria act like calcium sinkholes that facilitate the influx of calcium.

Neoplastic cells, inhibit glycogen synthase kinase in the cytosol of the cells that lead to the translocation of hexokinase 2 to the mitochondrial membrane. Hexokinase 2 then binds to the inner membrane and blocks and inhibits the voltage-dependent anion channel found in the mitochondrial transition pore that is responsible for the efflux of anions to maintain membrane potential of the mitochondria that is crucial for oxidative phosphorylation and ETC. This results in the hyperpolarization of the membrane and prevents the opening of the channel in the mitochondrial transition pore that is responsible for release of apoptotic factors such as mitochondrial reactive oxygen species, cytochrome c oxidase  as well as apoptotic inducing factor.

The suppression of mitochondrial functioning leads to the decrease in oxidative phosphorylation as well as the release of apoptotic factors. With diminishing functioning, the mitochondria of tumour cells begin to degenerate and further increase the proliferative potential of the neoplastic cell. However, this information can be utilized in targeting specific cancers especially solid tumours.

A drug known as dichloroacetate is able to penetrate most cell membranes and tissues that are not accessible to chemotherapy treatments such as the brain. This molecule is able to activate pyruvate dehydrogenase by inhibition of pyruvate dehydrogenase kinase and its isoforms. Dichloroacetate carboxylic group forms a salt bridge with Arg154 residue of the active site of pyruvate dehydrogenase kinase. Dicholoroacetate is able to decrease the membrane potential of inner mitochondria and aid in normalization of mitochondrial function in cancer cells. However, it does not affect non-cancer cells. It functions to increase apoptotic activity in the cancer cells as well as to increase the intake of pyruvate into the mitochondria and increase more ETC./oxidative phosphorylation. This decreases the production of lactate and the alternate pathways of glycolytic intermediates.

Subsequently, there is a deactivation of HIF since HIF is highly regulated by the TCA intermediate, alpha ketogluterate. Hence, an increase in TCA cycle leads to increase production of alpha ketogluterate that facilitates the inhibition of the action of HIF. This results in decrease tumour perfusion, glucose uptake, resistance to apoptosis as well as decrease production of macromolecules essential for tumour proliferation. DCA has also shown to increase p53 activity in cancer cells leading increase activation of apoptotic action by the mitochondria.

DCA is therefore very effective at selectively targeting cancer metabolism by inhibition of pyruvate dehydrogenase kinase. Clinical evidence of this has been observed in non-small cell lung carcinoma, breast carcinoma, glioblastoma, colon cancer, prostate and endometrial cancer as well as leukaemia.

However, other therapies may mimic the action of DCA and acquire similar results. Some therapies include the inhibition of lactate dehydrogenase to facilitate the channelling of pyruvate into the mitochondria to undergo oxidative phosphorylation. Similarly, decrease activation of the M2 isoform of pyruvate kinase that is expressed in neoplastic cells may prove to be another selective target for cancer treatment.

In concluding this article, the authors note that the aforementioned techniques are not a permanent solution to cancer therapy since there is no absolute action of these inhibitors on the metabolism of cancer.

My initial thoughts of this paper, was that it was long-winded and boring. However, as I continued past the introduction, the content became more interesting and captivating. The explanations and the organization of the information laid a good base level to which anyone reading was able to easily follow. It was greatly surprised at the intricacies of cancer metabolism and the very dynamic alterations made for the survival and proliferation of these cells. I knew that the mitochondria of cells were integral to the optimal functioning of the cell, nevertheless, I had no knowledge of the specific actions involved in mitochondrial functioning.

The authors aptly summarized the pathophysiology of neoplastic cells and then linked this to the action of the small DCA molecules and its efficiency in cancer treatment through selective inhibition. The importance of pyruvate dehydrogenase kinase was also elucidated. I was also intrigued by the importance of glycolysis to cancer metabolism and how effective glycolytic pathway is to the rapid growth and sustainability of the tumour.

However, it must be stated that even though DCA and the other techniques mentioned in this paper would prove highly valuable to oncology, there is no absolute solution since neoplastic cells have the ability to adapt and evolve to avoid the actions of these target drugs. There is no certainty that these therapies will show significant reduction in tumour proliferation in cancer patients and is subjected to individual differences. These therapies also have the potential to produce effects on non-cancer cells with prolonged use and high doses. Therefore, I am hesitant to believe that DCA and similar inhibitory therapies are a permanent solution to reducing the prevalence of cancer.

Overall, I think that this is a groundbreaking and lifesaving research in oncology since it will prove to reduce the mortality and morbidity of cancer. It is quite exciting to note that this method is a good step forward in finding more advance and effective treatment for a serious medical condition. Clinical evidence thus far has been positive and with more research, the methods of cancer treatment may develop into a simple cure like penicillin.

Reference

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590642/

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Parasite and Neoplastic cells Villain

Hey My Biochem peeps,

Hereunder is a review of a new study on the discovery of a new irreversible enzyme inhibitor that may be useful in the treatment of the parasitic infection, Leishmania as well as the treatment of cancers. This inhibitor targets pyruvate kinase, the last enzyme in the glycolytic pathway. Pyruvate kinase is responsible for the production of pyruvate as well as ATP. So I hope you find it as interesting as I did. Enjoy and Happy studying…….

Morgan, Hugh P., Martin J. Walsh, Elizabeth A. Blackburn, Martin A. Wear, Matthew B. Boxer, Min Shen, Henricke Veith, Iain W. McNae, Matthew W. Nowicki, Paul A. M. Michels, Douglas S. Auld, Linda A. Fothergill-Gilmore and Malcolm D. Walkinshaw. 2012. “A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase.” Biochem. J. 448: 67-72. doi:10.1042/BJ20121014.

 

A new family of covalent inhibitor block nucleotide binding to the active site of pyruvate kinase.

Pyruvate kinase is the last enzyme in the glycolytic pathway and catalyses the substrate-level phosphorylation of phosphoenolpyruvate to pyruvate and converting ADP to ATP in the process.  Human Pyruvate kinase consist of four isozymes: RPYK (Erythrocytes), LPYK (Liver), M1PYK (Muscle), M2PYK (Embryonic Tissues and Neoplastic cancer cells). Most of these isozymes are allosterically regulated by fructose 1, 6-bisphosphate meaning that increase in in fructose 1, 6-bisphosphate will lead to increase activation of pyruvate kinase. M1 isoform is constitutively active.

Leishmania mexicana is a trypanosome parasite that causes Leishmaniasis. It is transmitted by the bite of infected sand flies (Tsetse Fly). This infection results in skin sores, fever, weight loss as well as splenomegaly and hepatomegaly (Medline Plus 2012). Leishmania mexicana possesses similar pyruvate kinases as human however, trypanosome pyruvate kinase (LmPYK) is regulated by fructose 2,6-bisphosphate.

According to Morgan et al., parasitic infections are usually treated by the sulphur drug, Suramin that inhibits 7 out of the 10 glycolytic enzymes of Trypanosoma brucei. This drug works by mimicking ATP/ADP and competitively binds to the active site of pyruvate kinase to inhibit the formation of ADP. It is believed that saccharine inhibitors may have the same effect on LmPYK as suramin. This paper utilizes the saccharine derived compound to inhibit LmPYK activity and hence induce cell death in this parasite. The inhibitor is known as {4-[(1,1 dioxo-1,2-benzothiazol-3-yl)sulfonyl] benzoic acid} or DBS.

Through experimentation, this study showed that DBS was able to bind to the active site of pyruvate kinase, specifically form covalent bonding with the Lys335 residue present in the rim of the active site of pyruvate kinase. This covalent binding leads to the modification of Lys335 that prevents ADP from binding and forming ATP. This process leads to cessation of glycolysis and ATP  generation as well as pyruvate production in the parasite leading to cell death.

First, utilizing purified and characterized wild type and mutant k335R forms of LmPYK, an inhibitor assay was made in order to determine the rate of inhibition of DBS on LmPYK. The inhibitor assay contained a buffer, NADH, lactate dehydrogenase, LmPYK, Phosphoenolpyruvate and inhibitor mix of DBS. A control was created in the same manner except for replacing the inhibitor mixture with buffer solution. The inhibitor assay and control were incubated at 25oC water bath throughout the experiment. ADP was then added to the reaction mixtures and the decrease in absorbance from 340 nm was measured every 20 minutes for 300 mins. This gave the rate of inhibition by DBS.

This experiment illustrated that maximal inhibition was approximately 80% after 250 mins. However, there was never complete inhibition of 100% since after 10 hours, there was heat denaturation of LmPYK due to prolonged incubation in the water bath. The small percentage of pyruvate kinase activity was attributed to the flexibility of the side chains of Lys335. Lys335 residue in the active site has a very flexible R-group side chains that facilitate conformation to allow small access of ADP to the active site of pyruvate kinase to form ATP. However, the affinity of pyruvate kinase to ADP was greatly reduced by the covalent binding of DBS. However, Morgan et al,  posited that 80% inhibition of pyruvate kinase was sufficient to induce cell death in the parasite.

Next, a preparation of an inhibitor-modified LmPYK was made and crystallized in order to identify the structure of the LmPYK-DBS complex. The crystals formed were anisotropic and demonstrated covalent modification of Lys335 residues at the active sites of LmPYK. This modification was located at the adenine-binding site that leads to the blocking of ADP/ATP from binding. The saccharine group of DBS was covalently bonded to the Lys335 residue at the rim of the cleft of the active site. It was also found that the binding conformation of LmPYK-DBS complex mimics that of the drug Suramin.

Finally, using the mutant K335R, the modification of the Lys335 residue on LmPYK was confirmed. This mutant form of LmPYK was similar in action and kinetics of the wild type, however, it possessed a different Lysine residue at its active site and therefore when DBS  was added, there was no inhibition of mutant K335R. This illustrated that DBS is only able to form covalent bonding and irreversible inhibition of pyruvate kinase if Lys335 is present.

The selectivity of DBS for Lys335 was also investigated using rabbit lactate dehydrogenase and firefly luciferase. These are usually coupling enzymes with pyruvate kinase in the respective animals. However, it was found that even though these enzymes share similar characteristics with pyruvate kinase active sites such as lysine residue in the same position of the active site (LDH) or binds to the hydroxyl group of ribose sugar from NAD (Luciferase), DBS was unable to inhibit the action of these enzymes. Hence, DBS is very selective towards Lys335 residues in the active site of pyruvate kinase.

In concluding, Morgan et al., linked the fact that due to the similarity between LmPYK  and certain human PYK isoforms in terms of Lys335 residues at the rim of the active site, DBS would be an effective inhibitor to both human and trypanosome pyruvate kinases. This is especially important for humans since neoplastic tumour cells as well as erythrocyte pyruvate kinases possess these similarities  and thus DBS would provide a good target drug treatment for cancer.

My thoughts on this paper. Firstly, I was elated that it was not very long and the information was presented in a very organized, easily comprehensible manner. Methods, objectives, rationale and findings were clearly outlined and the language was not too technical so that it was difficult to follow the concepts of the paper. The topic was a very stimulating and informative since I am very interested in diseases and novel therapies to stem the prevalence. Leishmaniasis is especially critical since we are susceptible to this parasitic infection given our tropical geography.

Another interesting note, is the fact that isoforms of human pyruvate kinase such as those found in our red blood cells and cancer cells, are similar to those found in this parasite. This nugget of information may prove valuable in the formulation and production of novel cancer therapies. It has been established that neoplastic cells rely on aerobic glycolysis to generate energy and macromolecular building blocks for growth and metastasis of tumours. Therefore, targeting pyruvate kinase is essential to fighting cancers.

The selectivity of DBS, is an added bonus in drug therapies containing this saccharine since it would be able to only target pyruvate kinase that possesses the Lys335residue at the rim of the active site where ADP/ATP binding usually occurs. Thus, DBS will be able to act on specific cells while leaving the normal non-cancer or non-parasitic cells unaffected.

However, this study was the first to demonstrate the effectiveness of DBS and therefore the findings have not been established. There must be more research into DBS and other analogues to determine whether this phenomenon is a solid avenue to follow in terms of therapy. Little is known about the actual effects of DBS on the human cells since this experiment was done by enzyme testing and no live tissue analysis was performed. Therefore, even though DBS seems like a promising compound that may enhance the efficiency and efficacy of target drugs, there has to be more in-depth research into the compound and the implications of its use.

Overall, this paper was very intriguing and interesting. It brought together concepts of glycolysis, irreversible enzyme inhibition and illustrated how the knowledge of biochemical processes may be applied to medical uses. The research is very poignant since parasitic diseases as well as neoplastic ailments are predominant in modern life. This fosters a greater demand for more efficient and effective methods of treatment for disease. The finding of DBS inhibition on the Lys335 residue offers hope and positive connections to increase the efficacy of future medical interventions.

Thus, I believe that this was an enjoyable and educational reading and I am excited to see and hear about the developments in medicine and zoonoses that have arisen out of this novel study. I recommend that you take a read, the reference is listed at the top of the posting. This topic offers a good outlet to link your understanding of the key concepts of glycolysis and enzyme specificity to developing useful things for society.

Until next posting……..

Reference

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3498827/