Paclitaxel

Function

Paclitaxel (also known as taxol, Figure 1) is a mitotic inhibitor used in cancer chemotherapy. It has been approved to treat ovarian, breast, and lung cancer, as well as Kaposi’s sarcoma. Paclitaxel is an antitumor drug and it plays a major role in cancer chemotherapy. to stable microtubules. Microtubules consist of polymers of tubulin which form part of the cytoskeleton and provide structure and shape to the cytoplasm of various cells. They are involved in cell division (by mitosis and meiosis) and are the major constituents of mitotic spindles. Paclitaxel partly induces cell death through disrupting mitosis by binding to and stabilizing the microtubule proteins. When paclitaxel binds to the microtubules, it essentially freezes them in place, preventing the separation of chromosomes during cell division. The stabilization is accompanied by structural modifications in the microtubules. The effects are different if assembly of mitotic apparatus is accompanied with the presence of paclitaxel, compared to when paclitaxel is added after the assembly. ^[1] See also Cancer.

History

Paclitaxel was first discovered in a US National Cancer Institute program in 1962. Monroe E. Wall and Mansukh C. Wani isolated it from the bark of a Pacific yew tree. Upon doing more research, they discovered that endophytic fungi in the bark of the tree synthesize paclitaxel. In 1977, scientists were able to confirm antitumor activity in mouse melanoma. During this year, Dr. Susan Horwitz discovered that paclitaxel was able to bind to a cell’s microtubule assembly and slow or cease cell division and growth. Ever since 1992, paclitaxel has been used for the treatment of various cancers. ^[2]

Structure

Paclitaxel has the molecular formula C₄₇H₅₁NO₁₄ and has a molecular weight of 853.92 Da, a melting point of 213°C and a boiling point of 218-222°C. is a complex diterpene having a taxane ring with a four-membered oxetane ring and an ester side chain at position C-13. Microtubules are long, hollow cylinders made up of polymerized ^[3] dimers and are approximately 24 nm in diameter. ^[4]The walls of microtubules consist of a lattice of tubulin heterodimers that are arranged head-to-tail to form protofilaments. The α- and β- tubulin dimers polymerize end-to-end and associate laterally to form a single microtubule. The two subunits are 50% identical in terms of amino acids, with each subunit having a molecular weight of 50 kDa. Tubulin polymerizes end to end, with the β- subunits of one tubulin dimer, binding to the α- subunit of the next dimer. This results in a protofilament containing one end with an α- subunit exposed, and one end with a β- subunit exposed. These ends are designated (-) and (+), respectively. The protofilaments align parallel to one another according to polarity, therefore in a microtubule, there is one end with only β- subunits (+), and the other end with only α- subunits (-). Elongation occurs at both ends, however, it is a lot more rapid at the (+) end. ^[5]

Figure 1. Paclitaxel (Modified from Wikipedia)

Reactions and Mechanism

Paclitaxel has a specific binding site on the microtubule polymer, and this makes it different and more effective than other chemotherapeutic agents. It has the ability to polymerize tubulin in the absence of cofactors, which is unusual and unique. Paclitaxel binds to cells, and in a specific and saturable manner and then blocks cells in the G1/M phase of the cell cycle by stabilizing the microtubule cytoskeleton against depolymerization. These cells are then unable to form normal mitotic apparatus, and eventually die. The mechanism of stabilization is not known, however, there has been strong research in support of the following mechanism. Paclitaxel binds into a pocket in the second globular domain of β- tubulin, facing the central hole in a microtubule. The corresponding space in α- tubulin is occupied by an eight-residue insertion in the loop between β- strands S9 and S10. Paclitaxel also makes close contact with the shorter S9-S10 loop in β-tubulin. The molecule also appears to touch the core helix and approach the loop between S7 and H9, now known as the M loop. When , the taxane ring (from the Paclitaxel) associates with the M group. It is not constrained by the dimer structure; however, it becomes strongly immobilized after the polymerization of tubulin. The amino acids that interact with the α- and β- tubulin are slightly different depending on the assembled isotypes. The amino acids that make up the include Asp226, His229, Val23, Arg369, Gly370, Thr276 and Arg278. The strong interaction allows for the binding of . The amino acids involved with the binding of Paclitaxel to the include Arg229, Leu23, Leu26, Arg320, Pro360, Tyr272, Lys370, Ala369, Ala278 and Leu217. The strong interaction allows for the binding of . The ‘stickiness’ of the M loop side of the protofilament appears to be largely responsible for the polymorphic nature of the protofilaments. Whilst the microtubules remain assembled, an observed 3-6% shortening of the ~4nm average spacing of tubulin monomers, is thought to be responsible for stabilizing the microtubule polymer and protecting it from disassembly. Chromosomes are thus unable to achieve a metaphase spindle configuration. ^[1] The is colored according to whether it is polar or nonpolar; pink representing polar, grey representing nonpolar. Before Paclitaxel binds, the binding pocket is highly nonpolar (all grey in color). Once the binding center is occupied by Paclitaxel, it shows excellent shape complementarity. The addition of some pink in the diagram illustrates that a nonpolar depression has been converted to a polar surface upon Paclitaxel binding. ^[6]

Regulation

Paclitaxel plays a key role in the regulation of tumor growth and microtubule function. Dysregulation of microtubule dynamics contributes to the development of serious diseases. Disruption of microtubules furthermore results in the induction of tumor suppressor gene p53 and inhibition of cyclin-dependent kinases and activation/inactivation of several protein kinases. It is predicted that Paclitaxel is able to induce phosphorylation of Bcl-X(L)/Bcl-2 members and thus inactivate their anti-apoptotic capacities. The down-regulation of Bcl-2 and/or the upregulation of p53 and p21-WAF-1 are one of the important modes of apoptosis induction by taxanes. ^[7] Depending on the concentration of Paclitaxel, the parts of the cell cycle that are regulated differ. At a high concentration of Paclitaxel (5-50μM), mitotic arrest at G1 or M is induced. At a low concentration of Paclitaxel (0.005-0.05 μM), apoptosis is induced at G0 and G1/S. It has recently been discovered that Parkin (an E3 ubiquitin ligase encoded by the Parkin gene) is involved in the pathogenesis of Parkinson’s disease and the development of cancer. Data has shown that Parkin upregulates and promotes the activity of Paclitaxel by binding to the outer surface of microtubules and increase the Paclitaxel-microtubule interaction. ^[8]

Kinetics

At 37°C, the binding rate constant is 3.6x10⁶ M^-1S^-1. All reactions were performed in 10mM phosphate, 1mM EGTA, 6mM MgCl2, 0.1mM GTP, pH = 6.5, and different concentrations of glycerol from 0 to 60% V/V. In order to discard the possibility that the fluorescein moiety of the fluorescent toxoids could contribute to the fast-initial binding steps of the ligands (Flutax-1 and Flutax-2), the kinetic constants of Paclitaxel association and dissociation were measured using a competition method.^[9] The kinetic constants of Paclitaxel association and dissociation from cross-linked microtubules at 37°C: Association Kinetic Constant (K+1) = 3.63x10⁶ M^-1S^-1 Dissociation Kinetic Constant (K-1) = 9.10x10^-2 M^-1s^-1 The free energy of the binding of Paclitaxel at 37°C is around -45kJ/mol. The binding reaction is endothermic, which explains why Paclitaxel induces microtubule assembly at low temperatures. ^[9]

Medical Implications

Paclitaxel has a broad activity spectrum and is clinically used to treat ovarian, breast, and lung cancer, as well as Kaposi’s Sarcoma. Cancerous tumors are characterized by cell division which is no longer controlled as it is in normal tissue. Chemotherapy is most effective at killing cells that are rapidly dividing, however, it does not know the difference between the cancerous cells and the normal cells. Paclitaxel belongs to a class of chemotherapy drugs called plant alkaloids; which are cell-cycle specific. Paclitaxel inhibits the microtubule structures within the cell. Inhibition of these structures ultimately results in cell death. ^[10] In metastatic breast cancer (MBC) specifically, weekly Paclitaxel was shown to be an effective and well-tolerated treatment for advanced breast cancer. ^[11]

Diseases

Microtubules form important cytoskeletal structures that play important roles in establishing and maintaining neuronal polarity, transporting cargo and scaffolding signaling molecules to form signaling hubs. Reduced microtubule stability leading to the malfunction of microtubules has been linked to several neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS), and tauopathies like Progressive Supranuclear Palsy. Hyperstable microtubules, as seen in Hereditary Spastic Paraplegia (HPS), also leads to neurodegeneration. Therefore, the ratio of stable and dynamic microtubules is likely to be important for neuronal function and perturbation in microtubule dynamics might contribute to disease progression. ^[12]

References

1. ↑ ^{1.0 1.1} Amos LA, Lowe J. How Taxol stabilises microtubule structure. Chem Biol. 1999 Mar;6(3):R65-9. doi: 10.1016/s1074-5521(99)89002-4. PMID:10074470 doi:http://dx.doi.org/10.1016/s1074-5521(99)89002-4
2. ↑ “Horwitz, Susan. “Taxol® (NSC 125973).” National Cancer Institute, dtp.cancer.gov/timeline/flash/success_stories/s2_taxol.htm.
3. ↑ doi: https://dx.doi.org/10.2210/pdb5SYF/pdb
4. ↑ Arnal I, Wade RH. How does taxol stabilize microtubules? Curr Biol. 1995 Aug 1;5(8):900-8. doi: 10.1016/s0960-9822(95)00180-1. PMID:7583148 doi:http://dx.doi.org/10.1016/s0960-9822(95)00180-1
5. ↑ Manka SW, Moores CA. Microtubule structure by cryo-EM: snapshots of dynamic instability. Essays Biochem. 2018 Dec 7;62(6):737-751. doi: 10.1042/EBC20180031. Print 2018, Dec 7. PMID:30315096 doi:http://dx.doi.org/10.1042/EBC20180031
6. ↑ Snyder JP, Nettles JH, Cornett B, Downing KH, Nogales E. The binding conformation of Taxol in beta-tubulin: a model based on electron crystallographic density. Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5312-6. doi: 10.1073/pnas.051309398. , Epub 2001 Apr 17. PMID:11309480 doi:http://dx.doi.org/10.1073/pnas.051309398
7. ↑ Ganansia-Leymarie V, Bischoff P, Bergerat JP, Holl V. Signal transduction pathways of taxanes-induced apoptosis. Curr Med Chem Anticancer Agents. 2003 Jul;3(4):291-306. PMID:12769774
8. ↑ Wang H, Liu B, Zhang C, Peng G, Liu M, Li D, Gu F, Chen Q, Dong JT, Fu L, Zhou J. Parkin regulates paclitaxel sensitivity in breast cancer via a microtubule-dependent mechanism. J Pathol. 2009 May;218(1):76-85. doi: 10.1002/path.2512. PMID:19214989 doi:http://dx.doi.org/10.1002/path.2512
9. ↑ ^{9.0 9.1} Diaz JF, Barasoain I, Andreu JM. Fast kinetics of Taxol binding to microtubules. Effects of solution variables and microtubule-associated proteins. J Biol Chem. 2003 Mar 7;278(10):8407-19. doi: 10.1074/jbc.M211163200. Epub 2002, Dec 20. PMID:12496245 doi:http://dx.doi.org/10.1074/jbc.M211163200
10. ↑ Cleveland Clinic Cancer. “Taxol.” Dexamethasone - Drug Information - Chemocare, chemocare.com/chemotherapy/drug-info/Taxol.aspx.
11. ↑ Kellokumpu-Lehtinen P, Tuunanen T, Asola R, Elomaa L, Heikkinen M, Kokko R, Jarvenpaa R, Lehtinen I, Maiche A, Kaleva-Kerola J, Huusko M, Moykkynen K, Ala-Luhtala T. Weekly paclitaxel--an effective treatment for advanced breast cancer. Anticancer Res. 2013 Jun;33(6):2623-7. PMID:23749918
12. ↑ Dubey J, Ratnakaran N, Koushika SP. Neurodegeneration and microtubule dynamics: death by a thousand cuts. Front Cell Neurosci. 2015 Sep 9;9:343. doi: 10.3389/fncel.2015.00343. eCollection, 2015. PMID:26441521 doi:http://dx.doi.org/10.3389/fncel.2015.00343

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