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Critical Role of Enzyme Kinetics to Improve Drug Effectiveness and Decrease Drug Side Effects


Understanding the multiple enzymes that are present in glycolysis are important in knowing how to inhibit the glucose flux to tumor cells who are heavily dependent on glucose since glycolysis is very inefficient in producing ATP. The crucial enzymes involved are hexokinase, glut transporter, and phosphofructokinase, which have crucial roles in the glycolysis process. Inhibiting enzymes is advantageous over chemotherapy as it is a lot more specific and the cancer doesn’t develop a resistance to the chemotherapy drugs. Through the use of enzyme inhibiting drugs such as 3-bromopyruvate, florentin, and others the flux of glucose can be drastically reduced causing the cancer cells to have a lack of glucose, which causes them to slowly die from a lack of a proper nutrient supply. Studies have shown that inhibiting multiple enzymes requires less inhibition of each enzyme in order to reduce the flux by 50% and this shows that the surrounding healthy living cells will be impacted less as the enzymes of these healthy cells will be inhibited less. This will give patients a lot less side effects compared to if they used chemotherapy or anticancer drugs that only inhibit one enzyme.


Sai Alapati, Grade 12, Evergreen Valley High School, CA




Critical Role of Enzyme Kinetics to Improve Drug Effectiveness and Decrease Drug Side Effe
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Critical Role of Enzyme Kinetics to Improve Drug Effectiveness and Decrease Drug Side Effects Against Cancer


Sai Alapati, Grade 12, Evergreen Valley High School, CA


ABSTRACT


Understanding the multiple enzymes that are present in glycolysis are important in knowing how to inhibit the glucose flux to tumor cells who are heavily dependent on glucose since glycolysis is very inefficient in producing ATP. The crucial enzymes involved are hexokinase, glut transporter, and phosphofructokinase, which have crucial roles in the glycolysis process. Inhibiting enzymes is advantageous over chemotherapy as it is a lot more specific and the cancer doesn’t develop a resistance to the chemotherapy drugs. Through the use of enzyme inhibiting drugs such as 3-bromopyruvate, florentin, and others the flux of glucose can be drastically reduced causing the cancer cells to have a lack of glucose, which causes them to slowly die from a lack of a proper nutrient supply. Studies have shown that inhibiting multiple enzymes requires less inhibition of each enzyme in order to reduce the flux by 50% and this shows that the surrounding healthy living cells will be impacted less as the enzymes of these healthy cells will be inhibited less. This will give patients a lot less side effects compared to if they used chemotherapy or anticancer drugs that only inhibit one enzyme.


Introduction


Many cancer treatments such as chemotherapy have been developed over the ages to fight against tumor cells however chemotherapy also kills healthy cells in the human body from the lack of specificity. This causes patients undergoing chemotherapy to feel many side effects such as nausea, vomiting, hair loss, and others. In addition, cancer cells are known to build up a resistance against chemotherapy making it very difficult to kill tumor cells in the human body and completely eradicate the cancer(). Also, cancer cells are known to have a different and faster metabolism reflective of their aggressive behavior invading the human body and replicating. This metabolism is called the “Warburg Effect” as cancer cells convert glucose into lactate by creating ATP even though oxygen is available. Taking advantage of the cancer cells’ dependency on the glucose enzymes is useful to find out how to starve cancer cells of crucial nutrients. Through enzyme kinetics and anticancer drugs that inhibit several enzymes involved in glycolysis, patients are able to receive a more effective treatment that has less side effects and improves the effectiveness of the drugs.

This research paper provides insight on how enzyme kinetics plays an important role in the fight against tumor cells while also not impacting healthy living cells. Using relationships created with data between enzyme activity and flux pathways of tumor cells, this research paper provides a solution to the problem of anticancer drugs causing harm to living cells and causing side effects. Through the use of enzyme kinetics and specific enzymes involved in glycolysis like hexokinase, glucose transporter, and phosphofructokinase, this paper determines potential methods that can be taken based on data for proper treatment against tumor cells.


Glycolysis and Enzymes Involved


When the rapid replication or proliferation of cancer cells occurs to make tumors in the human body, oftentimes regions are formed with low oxygen concentrations as these regions are far away from proper blood supplies. Also, the mitochondria’s aerobic processes become dysfunctional in a tumor environment with severe hypoxia (Rodrigues & Ferrez, 2020). In order for these cancer cells to keep on replicating, they must undergo an anaerobic process called glycolysis where glucose is converted into ATP for the cancer cells to have a supply of energy (Granchi & Minutolo, 2012). This process is known to be very inefficient in making ATP as anaerobic glycolysis makes around 2 ATP (Granchi & Minutolo, 2012) and in order to sustain the high proliferation rates of cancer cells, they must use a lot more glucose. Glycolysis also produces pyruvate which with a lack of oxygen is later turned into lactate and is discharged to maintain a pH balance. This would cause healthy living cells to experience acidosis causing healthy living cells to die and giving way for the tumor to grow (Granchi & Minutolo, 2012). Cancer cells therefore heavily rely on glucose to sustain their rapid tumor growth rates, this causes an outside appearance that tumors heavily rely on glycolysis. However, parts of the tumor close to blood supplies and with higher oxygen concentrations do not have to rely on such an inefficient method of generating ATP. Rather they are able to use oxidative phosphorylation (Herling et al., 2011). In order to maintain a high supply of glucose to keep the cancers with low oxygen concentrations alive and healthy, the cells will redirect the flux of glucose to pathways where cancer cells need glucose. The maintenance of this glycolytic pathway requires many enzymes that are crucial in the steps of glycolysis, such as hexokinase (HK), glut transporter (GLUT), phosphofructokinase-1 (PFK-1), and many other enzymes. These enzymes work together mainly in cancer cells to keep a steady state of glucose and this process isn’t seen in healthy living cells.




Figure 1. The metabolism of glucose through the process of glycolysis involving several important enzymes like Hexokinase (HK), Glut transporter (GLUT), and Phosphofructokinase(PFK-1). (Granchi & Minutolo, 2012)



Importance of Enzymes


Chemotherapy is a procedure that is very popular among cancer patients in order to treat tumors and cause the inhibition or retreat of tumor growth. However, anticancer drugs involved in chemotherapy have shown to not be as effective after initial uses because the cancer cells are known to develop a drug resistance making it harder to inhibit tumor growth. The drug resistance is caused by mutations of the p53 gene that causes a higher production of anti-apoptotic proteins that resist apoptosis which is when cells die (Mansoori et al., 2017). Also, tumors with cancer stem cells are known to be able to resist anticancer drugs in chemotherapy through special drug transporters known as ABCB1 and this causes chemotherapy to be completely ineffective (Mansoori et al., 2017). With this disadvantage of chemotherapy in mind, the inhibition of enzymes to cause cancer cells to starve from the lack of glucose seems to be a better option for tackling cancer. The glycolysis process has shown that cancer cells are very dependent on the aforementioned enzymes and this relationship can be used to have an effect on the cancer cells. Through this method, the growth of tumors may be inhibited and although the cancer cells could use other enzymes in the glycolysis process it will severely be limited as hexokinase, glut transporter, and phosphofructokinase are key rate limiting steps of glycolysis that have an impact on the rest of the glycolytic pathway.


Hexokinase(HK)


There are many forms or types of hexokinase such as HK-1, HK-2, HK-3, HK-4, and others but mainly HK-2 is involved in the glycolytic process and learning about this enzyme is essential for inhibiting the flux of glucose to cancer cells (Granchi & Minutolo, 2012). It plays an important role in the phosphorylation of glucose to glucose-6-phosphate which is an essential step in glycolysis (Granchi & Minutolo, 2012). HK-2 is important for tumor growth as it is known for doubling the phosphorylation of glucose which helps increase the rate of glycolysis and therefore an increase in the flux of glucose for the survival of the tumor (Granchi & Minutolo, 2012). This shows that HK-2 has a prominent role in tumor growth through its role in glycolysis and as a rate-limiting step in glycolysis.


Glucose transporter (GLUT)


There are four types of glucose transporter GLUT-1, GLUT-2, GLUT-3, and GLUT-4 but GLUT-1 is known to have a crucial role in the glycolytic process because this enzyme is very active in tumor cells as there needs to be a large flux of glucose. GLUT and hexokinase are known to have 71% of control over the glycolytic flux, which goes to show how important it is for inhibiting tumor growth (Herling et al., 2011). Hypoxia or the lack of oxygen makes the glucose transporter a lot more apparent and overexpressed because of the need of more glucose by tumor cells, causing this enzyme to be a primary focus in order to inhibit tumor growth.


Enzyme Inhibiting Drugs and Flux Pathway


Table 1 shows how enzyme activity is much more present in hexokinase and phosphofructokinase as the numbers in the cancerous cells are much higher


Table 1. Shows a comparison of enzyme activities between non-cancerous cells and cancerous cells with the presence of 5mM of glucose. (Marín-Hernández et al., 2006)

EnzymesHepatocytes (non-cancer cells)AS-30D (Cancer cells of rodent origin)HeLa Cells (Cancer cells of human origin)HK0.003 ± 0.002 U(mg*protein)^-10.46 ± 0.1 U(mg*protein)^-10.02 ± 0.006 U(mg*protein)^-1PFK-10.01 ± 0.002 U(mg*protein)^-10.21 ± 0.1 U(mg*protein)^-10.09 ± 0.02 U(mg*protein)^-1



Table 2. A comparison between high availability of glucose to low availability of glucose and the impact it has on the glycolytic flux. (Marín-Hernández et al., 2006)

ConditionHepatocytes (non-cancer cells)AS-30D (Cancer cells of rodent origin)HeLa Cells (Cancer cells of human origin)

  • Glucose

2.4 ± 1.7 nmol*min^-121 ± 9 nmol*min^-132 ± 10 nmol*min^-1

  • Glucose

-0.4 ± 1 nmol*min^-1-2.2 ± 2.6 nmol*min^-17 ± 9 nmol*min^-1



3-bromopyruvate (3BP)


The HK-II inhibitor 3- Bromopyruvate (3-BP) dissociates HK-II from the mitochondrial complex, which leads to enhanced sensitization of leukemic cells to anti-leukemic drugs. From Figure 2, it can be seen how 3-bromopyruvate plays a crucial role in stopping the growth of tumors from the preclinical trials and further tests need to be done. There is a general trend that can be seen from Figure 2, which is that as the dosage increases the growth of the tumor continues to decline.












Figure 3. Chemical structure of 3-bromopyruvate. (Akins et al., 2018)



Figure 2. Relationship between increased dosage

3-bromopyruvate and inhibition of tumor growth.

(Rai et al., 2019)






Fasentin and Phloretin


Studies showed that fasentin and its analogues not only exhibit partial inhibition of the glucose transportation pathway but also break down the resistance of caspase activation, which is normally seen in malignant cells that are resistant to chemotherapy and other treatments.















Figure 4. Chemical structure of fasentin



Figure 5. Chemical structure of phloretin



Using Multiple Enzyme Inhibiting Drugs to Prevent Side Effects


From the data below, it shows that when multiple enzymes are inhibited the flux activity associated with the tumor cells is drastically reduced by approximately 50% and the enzyme activity is not inhibited as much. Compared to only inhibiting one enzyme, which also decreases the flux activity but the enzyme activity is greatly decreased. In order to inhibit enzymes, drugs have to be used and will always have an effect on healthy living cells. If multiple enzymes were to be inhibited the enzyme activity of the normal healthy living cells will slightly decrease, potentially causing minimal side effects. On the other hand, inhibiting one enzyme causes enzyme activity of both the tumor cells and healthy living cells to be severely decreased. This would cause a lot more severe side effects as enzyme activity is drastically reduced and the body isn’t able to use vital enzymes for digestion, or liver function.




Figure 6. Cause and effect of reduced enzyme activity on the glucose flux with various enzymes. (a) GLUT + HK + HPI; (b) GLUT + HK; (c) HK or HPI; (d)GLUT; (e) PFK-1; (f) PYK and (g)TPI (Marín-Hernández et al., 2011)



Figure 7. Cause and effect of reduced enzyme activity and glycogen on glucose flux with various enzymes (a) GLUT + glycogen degradation; (b) glycogen degradation; (c) GLUT; (d) HK; (e) HPI and (f) TPI. (Marín-Hernández et al., 2011)


Conclusion


In conclusion, 3-bromopyruvate, phloretin, and fasentin can be used simultaneously in order to inhibit hexokinase and glut transporter in order to inhibit multiple enzymes. This will cause the glucose flux to decrease to 50% while the inhibition of enzymes is only slightly reduced compared to only inhibiting a single enzyme. Since it is inevitable that these enzyme inhibiting drugs will also affect the healthy living cells, having multiple enzymes be inhibited will cause the enzymes of the healthy living cells to be slightly inhibited and this will cause a lot less side effects for patients. However, more research still needs to be done with the glucose flux being reduced through specifically using 3-bromopyruvate and fasentin or phloretin together. However, enzyme inhibition has such great success in slowing down tumor growth especially with 3-bromopyruvate and in the future learning about the metabolism of cancer cells will be vital for further research on enzyme kinetics and pathways involved with the cancer’s metabolism.


References


Akins, N. S., Nielson, T. C., & Le, H. V. (2018). Inhibition of Glycolysis and Glutaminolysis: An Emerging Drug Discovery Approach to Combat Cancer. Current Topics in Medicinal Chemistry, 18(6), 494–504. https://doi.org/10.2174/1568026618666180523111351


Granchi, C., & Minutolo, F. (2012). Anticancer Agents That Counteract Tumor Glycolysis. ChemMedChem, 7(8), 1318–1350. https://doi.org/10.1002/cmdc.201200176


Herling, A., König, M., Bulik, S., & Holzhütter, H. G. (2011). Enzymatic features of the glucose metabolism in tumor cells. FEBS Journal, 278(14), 2436–2459. https://doi.org/10.1111/j.1742-4658.2011.08174.x


Mansoori, B., Mohammadi, A., Davudian, S., Shirjang, S., & Baradaran, B. (2017). The Different Mechanisms of Cancer Drug Resistance: A Brief Review. Advanced Pharmaceutical Bulletin, 7(3), 339–348. https://doi.org/10.15171/apb.2017.041


Marín-Hernández, A., Gallardo-Pérez, J. C., Rodríguez-Enríquez, S., Encalada, R., Moreno-Sánchez, R., & Saavedra, E. (2011). Modeling cancer glycolysis. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1807(6), 755–767. https://doi.org/10.1016/j.bbabio.2010.11.006


Marín-Hernández, A., Rodríguez-Enríquez, S., Vital-González, P. A., Flores-Rodríguez, F. L., Macías-Silva, M., Sosa-Garrocho, M., & Moreno-Sánchez, R. (2006). Determining and understanding the control of glycolysis in fast-growth tumor cells. FEBS Journal, 273(9), 1975–1988. https://doi.org/10.1111/j.1742-4658.2006.05214.x


Rai, Y., Yadav, P., Kumari, N., Kalra, N., & Bhatt, A. N. (2019). Hexokinase II inhibition by 3-bromopyruvate sensitizes myeloid leukemic cells K-562 to anti-leukemic drug, daunorubicin. Bioscience Reports, 39(9). https://doi.org/10.1042/bsr20190880


Rodrigues, T., & Ferraz, L. S. (2020). Therapeutic potential of targeting mitochondrial dynamics in cancer. Biochemical Pharmacology, 182, 114282. https://doi.org/10.1016/j.bcp.2020.114282


Zuo, J., Tang, J., Lu, M., Zhou, Z., Li, Y., Tian, H., Liu, E., Gao, B., Liu, T., & Shao, P. (2021). Glycolysis Rate-Limiting Enzymes: Novel Potential Regulators of Rheumatoid Arthritis Pathogenesis. Frontiers in Immunology, 12. https://doi.org/10.3389/fimmu.2021.779787

















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