152044-53-6HESCAJZNRMSMJG-KKQRBIROSA-NHESCAJZNRMSMJG-KKQRBIROSA-N
Epothilone ADTXSID10332288152044-54-7QXRSDHAAWVKZLJ-PVYNADRNSA-NQXRSDHAAWVKZLJ-PVYNADRNSA-N
Epothilone BDTXSID404686233069-62-4RCINICONZNJXQF-MZXODVADSA-NRCINICONZNJXQF-MZXODVADSA-N
PaclitaxelTaxol
Abraxane
BMS 181339-01
Ebetaxel
Genaxol
Genetaxyl
Genexol
Genexol-PM
Mitotax
NSC 125973
OncoGel
Pacliex
Plaxicel
Tax-11-en-9-one, 5β,20-epoxy-1,2α,4,7β,10β,13α-hexahydroxy-, 4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine
TaxAlbin
Taxol A
Yewtaxan
DTXSID9023413Epothilone A2019-01-29T08:53:132019-01-29T08:53:13Epothilone B2019-01-29T08:53:572019-01-29T08:53:57Paclitaxel2019-01-29T09:52:452019-01-29T09:52:45Zampanolide2019-01-29T09:54:492019-01-29T09:54:49Discodermolide2019-01-29T09:55:422019-01-29T09:55:42Binding of microtubule stabilizing agents (MSA) to microtubulesBinding of MSAs to microtubulesMolecular<p>MSAs bind to polymerized tubulin. [1, 2] In several studies, MSAs were shown to inhibit cell proliferation and possess antineoplastic activity. [3-9] The taxane pocket found on the β-subunit of tubulin dimers was identified as the binding site for taxol and other MSAs like the above mentioned epothilones. [6-8, 10-20] Binding of MSAs is reversible. [6-8, 13-15]</p>
<p>- Electron microscopy of tannin-embedded tubulin crystals stabilized with taxol. Projection density maps were created from the electron microscopy data and further processed to difference maps of tubulin to visualize the binding site of taxol in polymerized tubulin [12, 16]</p>
<p>- Direct photolabeling of tubulin with radiolabelled [ 3 H]taxol [13], and [ 3 H]taxoid-derivatives to identify binding sites of taxol in tubulin [10, 11, 14, 15, 18]</p>
<p>- Mapping studies: Tubulin is photolabelled with [ 3 H]taxoid-derivatives. To identify the photoincorporation site, the labelled complex is then digested by formic acid or CNBr in combination with either clostripain or trypsin. The obtained peptide fragments are analysed via reverse phase HPLC and the radioactive peak is sequenced. [14, 15, 18]</p>
<p>- X-ray crystallography [17]</p>
<p>- NMR studies [20] and solid-state rotational echo double-resonance (REDOR) NMR [19] to determine the orientation of microtubule-bound taxol</p>
<p>- Fluorescence energy transfer (FRET) spectroscopy to determine the conformation of microtubule-bound taxol [19]</p>
<p>1. Manfredi, J.J., J. Parness, and S.B. Horwitz, Taxol binds to cellular microtubules. The Journal of Cell Biology, 1982. 94(3): p. 688-696. Confidential</p>
<p>2. Parness, J. and S. Horwitz, Taxol binds to polymerized tubulin in vitro. The Journal of Cell Biology, 1981. 91(2): p. 479-487.</p>
<p>3. Riondel, J., et al., Therapeutic response to taxol of six human tumors xenografted into nude mice. Cancer Chemotherapy and Pharmacology, 1986. 17(2): p. 137-142.</p>
<p>4. Wani, M.C., et al., Plant antitumor agents. VI. Isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. Journal of the American Chemical Society, 1971. 93(9): p. 2325-2327.</p>
<p>5. Schiff, P.B., J. Fant, and S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol. Nature, 1979. 277(5698): p. 665-667.</p>
<p>6. Bollag, D.M., et al., Epothilones, a New Class of Microtubule-stabilizing Agents with a Taxol-like Mechanism of Action. Cancer Research, 1995. 55(11): p. 2325-2333.</p>
<p>7. Hung, D.T., J. Chen, and S.L. Schreiber, (+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers, blocks taxol binding and results in mitotic arrest. Chemistry & Biology, 1996. 3(4): p. 287-293.</p>
<p>8. Kowalski, R.J., et al., The Microtubule-Stabilizing Agent Discodermolide Competitively Inhibits the Binding of Paclitaxel (Taxol) to Tubulin Polymers, Enhances Tubulin Nucleation Reactions More Potently than Paclitaxel, and Inhibits the Growth of Paclitaxel-Resistant Cells. Molecular Pharmacology, 1997. 52(4): p. 613-622.</p>
<p>9. ter Haar, E., et al., Discodermolide, A Cytotoxic Marine Agent That Stabilizes Microtubules More Potently Than Taxol. Biochemistry, 1996. 35(1): p. 243-250.</p>
<p>10. Combeau, C., et al., Predominant Labeling of. beta.-over. alpha.-Tubulin from Porcine Brain by a Photoactivatable Taxoid Derivative. Biochemistry, 1994. 33(21): p. 6676-6683. Confidential</p>
<p>11. Dasgupta, D., et al., Synthesis of a photoaffinity taxol analog and its use in labeling tubulin. Journal of medicinal chemistry, 1994. 37(18): p. 2976-2980.</p>
<p>12. Nogales, E., S.G. Wolf, and K.H. Downing, Structure of the αβ tubulin dimer by electron crystallography. Nature, 1998. 391: p. 199.</p>
<p>13. Rao, S., S.B. Horwitz, and I. Ringel, Direct Photoaffinity Labeling of Tubulin With Taxol. JNCI: Journal of the National Cancer Institute, 1992. 84(10): p. 785-788.</p>
<p>14. Rao, S., et al., 3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin. Journal of Biological Chemistry, 1994. 269(5): p. 3132-3134.</p>
<p>15. Rao, S., et al., Characterization of the Taxol Binding Site on the Microtubule 2-(m-AZIDOBENZOYL) TAXOL PHOTOLABELS A PEPTIDE (AMINO ACIDS 217-231) of β-TUBULIN. Journal of Biological Chemistry, 1995. 270(35): p. 20235-20238.</p>
<p>16. Nogales, E., et al., Structure of tubulin at 6.5 Å and location of the taxol-binding site. Nature, 1995. 375(6530): p. 424-427.</p>
<p>17. Prota, A.E., et al., Molecular mechanism of action of microtubule-stabilizing anticancer agents. Science, 2013. 339(6119): p. 587-590.</p>
<p>18. Rao, S., et al., Characterization of the Taxol Binding Site on the Microtubule: IDENTIFICATION OF Arg282 IN β-TUBULIN AS THE SITE OF PHOTOINCORPORATION OF A 7-BENZOPHENONE ANALOGUE OF TAXOL. Journal of Biological Chemistry, 1999. 274(53): p. 37990-37994.</p>
<p>19. Li, Y., et al., Conformation of Microtubule-Bound Paclitaxel Determined by Fluorescence Spectroscopy and REDOR NMR. Biochemistry, 2000. 39(2): p. 281-291.</p>
<p>20. Snyder, J.P., et al., The binding conformation of Taxol in β-tubulin: A model based on electron crystallographic density. Proceedings of the National Academy of Sciences, 2001. 98(9): p. 5312-5316.</p>
2019-01-29T08:47:492019-01-29T10:01:42Disturbance in microtubule dynamic instabilityDisturbance in microtubule dynamic instabilityCellular<p>Microtubules consist of α- and β-tubulin heterodimer subunits which assemble into protofilaments. These protofilaments further form a hollow cylinder, the microtubule. Microtubules are polar structures due to the head-to-tail assembly of the tubulin heterodimers. The faster growing end of microtubules is called the plus end whereas the slower growing end is the minus end. The plus end is protected from rapid polymerisation by a GTP cap which is a ring of GTP-tubulin . However, when a new tubulin dimer is added to the plus end, GTP gets hydrolysed in the catalytic domain of α-tubulin and the tubulin subunit gets non-exchangeable. [1] Microtubules continuously undergo de- and repolymerization which is summarized as “microtubule dynamic instability”. [2] Microtubules are also stabilized by different proteins like microtubule-associated proteins (MAPs). Microtubule-related proteins often have preferential affinity for specifically modified microtubule regions, which is known as the microtubule code, including post-translational modifications like acetylation, detyrosination or polyamination. [1, 3] Furthermore, microtubules in axons exhibit specific orientation with the plus end pointing away from the soma whereas dendrites show mixed orientation of microtubules. [3] Due to the morphology of peripheral neurons possessing processes that can reach a length of more than one meter, an intact microtubule network is indispensable to ensure the supply of even the most distant parts of the neurites. [4]</p>
<p>- Nocodazole approach: Nocodazole is a microtubule-depolymerizing agent. Treatment with nocodazole over different time periods and subsequent quantification of microtubule mass by electron microscopy can give an insight into microtubule stability and the ratio of stabile and labile microtubule fractions. Labile microtubules will be degraded faster while stabile microtubules require more time for depolymerization. [5]</p>
<p>- Immunofluorescence staining for acetylated tubulin, which is a marker for stable microtubules and for tyrosinated tubulin which is a marker of labile microtubules gives insight into the composition of microtubules in cells. [3, 5]</p>
<p>- Fluorescence recovery after photobleaching: Cultured neurons are transfected to express fluorescently-labelled tubulin or injected with x-rhodamine-tubulin (tubulin with fluorescent-label). Multiple sites along a neurite are photobleached and fluorescence recovery after photobleaching was assessed at these sites as a measure for microtubule turnover. [3, 6]</p>
<p>1. Conde, C. and A. Caceres, <em>Microtubule assembly, organization and dynamics in axons and dendrites.</em> Nat Rev Neurosci, 2009. <strong>10</strong>(5): p. 319-332.</p>
<p>2. Mitchison, T. and M. Kirschner, <em>Dynamic instability of microtubule growth.</em> Nature, 1984. <strong>312</strong>(5991): p. 237-242.</p>
<p>3. Baas, P.W., et al., <em>Stability properties of neuronal microtubules.</em> Cytoskeleton (Hoboken), 2016. <strong>73</strong>(9): p. 442-60.</p>
<p>4. Griffin, J.W. and D.F. Watson, <em>Axonal transport in neurological disease.</em> Annals of Neurology, 1988. <strong>23</strong>(1): p. 3-13.</p>
<p>5. Baas, P.W. and M.M. Black, <em>Individual microtubules in the axon consist of domains that differ in both composition and stability.</em> J Cell Biol, 1990. <strong>111</strong>(2): p. 495-509.</p>
<p>6. Edson, K.J., et al., <em>FRAP analysis of the stability of the microtubule population along the neurites of chick sensory neurons.</em> Cell Motil Cytoskeleton, 1993. <strong>25</strong>(1): p. 59-72.</p>
2019-01-29T08:48:192019-01-29T10:06:08Impaired axonial transportImpaired axonial transportCellular<p>The cytoskeleton plays an important role in neurons as it is required for the typical neuronal architecture of one long process, the axon, and several shorter dendrites. [1] Furthermore, the intact cytoskeleton is also of high importance as it is needed for processes like axonal transport. As axons lack the machinery to synthesize proteins, all necessary proteins have to be transported from the cell body to the periphery. Microtubules which are a basic element of the cytoskeleton play an important role in axonal transport and the maintenance of neurons. [2]<strong> </strong>They are highly dynamic and polarized structures with a stable minus end and a dynamic plus end. In axons, the plus end is directed away from the soma. [1] Microtubules serve as molecular tracks in neurons to ensure the transport of cargoes to different parts of the cell as well as the clearance of damaged cell organelles. The kinesins are microtubule-based molecular motors and are necessary for the anterograde transport of materials needed for maintenance of axons and synapses. [3, 4] Retrograde transport of degradation products from the axon/synapse back to the cell body is crucial for neuronal maintenance and survival as well. [5] Retrograde transport is carried out by dynein-motorproteins. [6]</p>
<p>- Vesicle motility assay: Axoplasm from squid giant axons is isolated and kept in axoplasm buffer. Preparations are analysed using a Zeiss Axiomat and organelle velocities are measured either in an automated process or by matching calibrated cursor movements to the speed of moving vesicles in agreement of two observers. [7-9]</p>
<p>- Kinesin-driven microtubule gliding assay: Slide chambers are covered with kinesins which adhere e.g. to specific antibodies on the glass slides. Rhodamine-labelled tubulin and unlabelled tubulin are mixed and assembled to microtubule structures. Microtubules are applied to the chamber and the rhodamine fluorescence is visualized to evaluate microtubule gliding. Microtubule-bodies are located and tracked to collect data on gliding velocity, trajectory curvature and microtubule length. [7, 10]</p>
<p>- Horseradish peroxidase (HRP) microinjection: HRP is injected into dorsal root ganglia neurons and visualized by 3,3’-diaminobenzidine. Microscope recordings of the neurons showing the transport of HRP are evaluated and the transport length is measured. [11]</p>
<p>- Mitochondrial trafficking: Cells are incubated with drug or DMSO solution and afterwards mitochondria are labelled with MitoTracker Green FM. Cells are kept in a live cell chamber and imaged in regular intervals. The time-lapse is used to track mitochondrial movement in neurites. [12]</p>
<p>- Axonal transport in mouse sciatic nerve: The drug is administered to mice intravenously. Mice are anesthetized and the left sciatic nerve is exposed and ligated at two points. After 24h, the ligated sciatic nerves are dissected and segments from proximal and distal sides of the ligation are collected, homogenized and analysed by Western blot. [12]</p>
<p>1. Baas, P.W., et al., <em>Stability properties of neuronal microtubules.</em> Cytoskeleton (Hoboken), 2016. <strong>73</strong>(9): p. 442-60.</p>
<p>2. Hirokawa, N., <em>Axonal transport and the cytoskeleton.</em> Current Opinion in Neurobiology, 1993. <strong>3</strong>(5): p. 724-731.</p>
<p>3. Leopold, P.L., et al., <em>Association of kinesin with characterized membrane-bounded organelles.</em> Cell Motility and the Cytoskeleton, 1992. <strong>23</strong>(1): p. 19-33.</p>
<p>4. Elluru, R.G., G.S. Bloom, and S.T. Brady, <em>Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.</em> Molecular Biology of the Cell, 1995. <strong>6</strong>(1): p. 21-40.</p>
<p>5. Delcroix, J.-D., et al., <em>Trafficking the NGF signal: implications for normal and degenerating neurons</em>, in <em>Progress in Brain Research</em>. 2004, Elsevier. p. 1-23.</p>
<p>6. Susalka, S.J. and K.K. Pfister, <em>Cytoplasmic dynein subunit heterogeneity: implications for axonal transport.</em> Journal of Neurocytology, 2000. <strong>29</strong>(11): p. 819-829.</p>
<p>7. LaPointe, N.E., et al., <em>Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy.</em> Neurotoxicology, 2013. <strong>37</strong>: p. 231-9.</p>
<p>8. Morfini, G., et al., <em>Tau binding to microtubules does not directly affect microtubule‐based vesicle motility.</em> Journal of Neuroscience Research, 2007. <strong>85</strong>(12): p. 2620-2630.</p>
<p>9. Morfini, G., et al., <em>JNK mediates pathogenic effects of polyglutamine-expanded androgen receptor on fast axonal transport.</em> Nature Neuroscience, 2006. <strong>9</strong>: p. 907.</p>
<p>10. Peck, A., et al., <em>Tau isoform‐specific modulation of kinesin‐driven microtubule gliding rates and trajectories as determined with tau‐stabilized microtubules.</em> Cytoskeleton, 2011. <strong>68</strong>(1): p. 44-55.</p>
<p>11. Theiss, C. and K. Meller, <em>Taxol impairs anterograde axonal transport of microinjected horseradish peroxidase in dorsal root ganglia neurons in vitro.</em> Cell Tissue Res, 2000. <strong>299</strong>(2): p. 213-24.</p>
<p>12. Smith, J.A., et al., <em>Structural Basis for Induction of Peripheral Neuropathy by Microtubule-Targeting Cancer Drugs.</em> Cancer Research, 2016. <strong>76</strong>(17): p. 5115-5123.</p>
2019-01-29T08:49:262019-01-29T10:07:16Sensory axonal peripheral neuropathySensory axonal peripheral neuropathyIndividual<p>The peripheral nervous system (PNS) connects the central nervous system with peripheral tissues and can be divided into the visceral and the somatic nervous system.The somatic nervous system consists of sensory and motor neurons. While the motor neurons control the contraction of skeletal muscles, the sensory neurons receive information from joints, muscles and skin and send it to the CNS. Motor neuron cell bodies lie in the spinal cord but cell bodies of the sensory neurons are located in the dorsal root ganglia (DRG). In contrast to the central nervous system (CNS), the PNS is not protected by the blood-brain-barrier, the skull or the spinal column and is therefore highly vulnerable to toxicants and mechanical damage. [1] However, the PNS neurons exhibits a greater ability of regeneration compared to neurons of the CNS. [2]</p>
<p>Peripheral neuropathies mostly affect sensory neurons in a length-dependent manner and therefore are characterized by a stocking-and-glove distribution of the symptoms. Sensory symptoms occurring upon taxol treatment are for example numbness and paresthesia. [3-6] Reversibility of neuropathy upon discontinuation of treatment is reported. [7]</p>
<p>- Retrospective study: Patients are interviewed after chemotherapy treatment and symptoms are assessed. [8, 9]</p>
<p>- Abnormal pin perception: Measurement of the distance in centimeters from the tip of the great toe or index finger to a level where normal pin sensation was sensed.</p>
<p>- Abnormalities in vibration and position sensation: Measurement at distal phalanx of the great toe or index finger. [9, 10]</p>
<p>- Reductions in strength of toe extensors, index finger abductors, arm abductors, hip flexors, foot dorsiflexors: Measurement by using the MRC scale and dynamometer [9, 10]</p>
<p>- Nerve conduction studies: Electrophysiological measurement of nerve conduction amplitudes and velocities in sensory neurons [9, 10]</p>
<p>- Quantitative sensory testing of vibratory threshold: Measurement of vibratory latency in seconds between the patient and the examiner in toes and fingers. [9, 10] Automated measurement using e.g. ‘Vibration 2’ (Physitemp Instruments Inc., Clifton NJ) which can be adjusted to different vibration amplitudes and records vibration units. [6]</p>
<p>- Quantitative sensory testing of thermal threshold: Using the Thermal Sensitivity Tester or NTE-2 (Physitemp Instruments Inc., Clifton NJ), the ability to discriminate small temperature differences at the index finger or the great toe can be quantified. [6, 11, 12]</p>
<p>- Sural nerve biopsy: A part of the sural nerve (some millimetres) is removed, fixed and investigated via light or electron microscopy. [13]</p>
<p>1. Benoy V. , d.Y.C., Van Den Bosch L. , Charcot-Marie-Tooth Disease and other peripheral neuropathies, in Young Perspectives for Old Diseases, M.H. G., Editor. 2015, Bentham Science Publishers. p. pp. 269-325.</p>
<p>2. Yiu, G. and Z. He, Glial inhibition of CNS axon regeneration. Nature Reviews Neuroscience, 2006. 7: p. 617.</p>
<p>3. Rowinsky, E.K., et al., Neurotoxicity of Taxol. J Natl Cancer Inst Monogr, 1993(15): p. 107-15.</p>
<p>4. Rowinsky , E.K. and R.C. Donehower Paclitaxel (Taxol). New England Journal of Medicine, 1995. 332(15): p. 1004-1014.</p>
<p>5. Donehower, R.C., et al., Phase I trial of taxol in patients with advanced cancer. Cancer treatment reports, 1987. 71(12): p. 1171-1177.</p>
<p>6. Forsyth, P.a., et al., Prospective study of paclitaxel-induced peripheral neuropathy with quantitative sensory testing. Journal of Neuro-Oncology, 1997. 35(1): p. 47-53.</p>
<p>7. Brown, T., et al., A phase I trial of taxol given by a 6-hour intravenous infusion. Journal of Clinical Oncology, 1991. 9(7): p. 1261-1267.</p>
<p>8. Pignata, S., et al., Residual neurotoxicity in ovarian cancer patients in clinical remission after first-line chemotherapy with carboplatin and paclitaxel: The Multicenter Italian Trial in Ovarian cancer (MITO-4) retrospective study. BMC Cancer, 2006. 6(1): p. 5.</p>
<p>9. Rowinsky, E.K., et al., Sequences of taxol and cisplatin: a phase I and pharmacologic study. Journal of Clinical Oncology, 1991. 9(9): p. 1692-1703.</p>
<p>10. Chaudhry, V., et al., Peripheral neuropathy from taxol and cisplatin combination chemotherapy: Clinical and electrophysiological studies. Annals of Neurology, 1994. 35(3): p. 304-311.</p>
<p>11. Arezzo, J.C., H.H. Schaumburg, and C. Laudadio, Thermal Sensitivity Tester: Device for Quantitative Assessment of Thermal Sense in Diabetic Neuropathy. Diabetes, 1986. 35(5): p. 590-592.</p>
<p>12. Wiernik, P.H., et al., Phase I Clinical and Pharmacokinetic Study of Taxol. Cancer Research, 1987. 47(9): p. 2486-2493.</p>
<p>13. Behse, F., F. Buchthal, and F. Carlsen, Nerve biopsy and conduction studies in diabetic neuropathy. Journal of Neurology, Neurosurgery, and Psychiatry, 1977. 40(11): p. 1072-1082.</p>
2019-01-29T08:50:082019-01-29T10:08:3714f4331e-834d-426f-a02c-9783040ba765691d7fc5-55a5-47e5-9dd6-9f61bcfa8ec2<p>It is well known that the binding of taxol and MSAs like epothilones and discodermolide to microtubules stabilizes microtubules thereby promoting polymerization and concomitantly suppressing depolymerisation. Therefore, they directly disturb microtubule dynamic instability. [1-9]</p>
<p>It is assumed that the M-Loop, which is part of the taxane pocket, undergoes conformational changes and gets more structured as a short helix is formed upon MSA binding. This structuring promotes the assembly and stabilization of microtubules as it is needed for lateral tubulin interactions. [10, 11]</p>
<p>Mutations in the β-tubulin gene were identified in patients with taxol-resistant non-small-cell lung cancer. Patients with β-tubulin mutations did not respond to taxol-treatment, whereas patients without β-tubulin mutations had complete or partial responses and survived longer. β-tubulin mutations were therefore identified as predictor of taxol-response thereby confirming β-tubulin as the binding and interaction site of taxol. [12]</p>
<p>In two taxol-resistant ovarian cancer cell lines, two point mutations were identified in the β-tubulin gene. Taxol-driven polymerization was shown to be impaired in these cells. Taxol-resistant cells did not exhibit microtubule polymerization upon taxoll treatment whereas the parental cells show increasing tubulin-polymerization with increasing doses of taxol. [13]</p>
<p>In two epothilone-resistant ovarian carcinoma cell lines two point mutations were identified in the β-tubulin gene. Epothilone- as well as taxol-driven polymerization was shown to be impaired in these cells while parental cells exhibit dose-dependent increase in tubulin polymerization upon epothilone A- and taxol-treatment. [14]</p>
<p>- Taxol known to bind along the lumen of microtubules was proven to block microtubule dynamics. Various parameters of microtubule dynamic instability were proven to be changed upon taxol treatment in a dose-dependent manner. The growth or shortening rates were shown to be decreased and the attenuation time was increased with increasing taxol concentrations. [6, 15, 16]</p>
<p>- Taxol was shown to decrease the lag-time for microtubule assembly and also the critical concentration of tubulin needed for microtubule assembly. The latter was proven by centrifugation and turbidity measurements in absence and presence of taxol using different tubulin concentrations. [1, 4, 17] The critical tubulin concentration was also proven to be decreased by discodermolide and epothilone A and B. [4, 17]</p>
<p>- Microtubules that were polymerized in the presence of taxol were shown to be resistant to cold- or CaCl<sub>2</sub>-depolymerization. [1, 2, 5] Cold- and CaCl<sub>2</sub>-depolymerization resistance could also be proven for epothilone A and B treated [2] and also for discodermolide treated [3, 5] microtubules.</p>
<p>- Tubulin polymerization assay proved that taxol-resistant cells exhibit less/impaired taxol-driven tubulin polymerization compared to parental cells. [13] The same was true for epothilone-resistant cells. [14]</p>
<p>- Taxol, discodermolide and epothilone A and B were shown to enhance <em>in vitro</em> tubulin-polymerization. [2, 3, 5] Furthermore, MSAs arrest cells in mitosis shown by quantification of cells in G1 phase or at G2-M transition, quantification of mitotic figures, overall assessment of the cell number or analysis of cyclin B1 expression. [2, 3, 5, 18] It was also shown that cells, which were treated with taxol and therefore arrested in mitosis, re-enter cell cycle after washout of taxol. [18]</p>
<p>The binding of MSAs to microtubules is extensively studies and well established. Its impact of this interaction on microtubule dynamic instability is addressed in numerous studies and the findings are largely consistent in the point of stabilization of microtubules accompanied by the disturbance of microtubule dynamic instability.</p>
<p>It has to be noted that microtubule destabilizing agents like the vinca alkaloids are known to bind to microtubules and disturb microtubule dynamic instability as well. However, vinca alkaloids differ in their mode of action as they bind to the end of microtubules and, in case of stoichiometric binding, promote depolymerization. [19]</p>
<p>1. Schiff, P.B., J. Fant, and S.B. Horwitz, <em>Promotion of microtubule assembly in vitro by taxol.</em> Nature, 1979. <strong>277</strong>(5698): p. 665-667.</p>
<p>2. Bollag, D.M., et al., <em>Epothilones, a New Class of Microtubule-stabilizing Agents with a Taxol-like Mechanism of Action.</em> Cancer Research, 1995. <strong>55</strong>(11): p. 2325-2333.</p>
<p>3. Hung, D.T., J. Chen, and S.L. Schreiber, <em>(+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers, blocks taxol binding and results in mitotic arrest.</em> Chemistry & Biology, 1996. <strong>3</strong>(4): p. 287-293.</p>
<p>4. Kowalski, R.J., et al., <em>The Microtubule-Stabilizing Agent Discodermolide Competitively Inhibits the Binding of Paclitaxel (Taxol) to Tubulin Polymers, Enhances Tubulin Nucleation Reactions More Potently than Paclitaxel, and Inhibits the Growth of Paclitaxel-Resistant Cells.</em> Molecular Pharmacology, 1997. <strong>52</strong>(4): p. 613-622.</p>
<p>5. ter Haar, E., et al., <em>Discodermolide, A Cytotoxic Marine Agent That Stabilizes Microtubules More Potently Than Taxol.</em> Biochemistry, 1996. <strong>35</strong>(1): p. 243-250.</p>
<p>6. Derry, W.B., L. Wilson, and M.A. Jordan, <em>Substoichiometric Binding of Taxol Suppresses Microtubule Dynamics.</em> Biochemistry, 1995. <strong>34</strong>(7): p. 2203-2211.</p>
<p>7. Dumontet, C. and M.A. Jordan, <em>Microtubule-binding agents: a dynamic field of cancer therapeutics.</em> Nature Reviews. Drug Discovery, 2010. <strong>9</strong>(10): p. 790-803.</p>
<p>8. Jordan, M.A. and L. Wilson, <em>Microtubules as a target for anticancer drugs.</em> Nature Reviews Cancer, 2004. <strong>4</strong>: p. 253.</p>
<p>9. Carozzi, V.A., A. Canta, and A. Chiorazzi, <em>Chemotherapy-induced peripheral neuropathy: What do we know about mechanisms?</em> Neurosci Lett, 2015. <strong>596</strong>: p. 90-107.</p>
<p>10. Prota, A.E., et al., <em>Molecular mechanism of action of microtubule-stabilizing anticancer agents.</em> Science, 2013. <strong>339</strong>(6119): p. 587-590.</p>
<p>11. Snyder, J.P., et al., <em>The binding conformation of Taxol in β-tubulin: A model based on electron crystallographic density.</em> Proceedings of the National Academy of Sciences, 2001. <strong>98</strong>(9): p. 5312-5316.</p>
<p>12. Monzó, M., et al., <em>Paclitaxel Resistance in Non–Small-Cell Lung Cancer Associated With Beta-Tubulin Gene Mutations.</em> Journal of Clinical Oncology, 1999. <strong>17</strong>(6): p. 1786-1786.</p>
<p>13. Giannakakou, P., et al., <em>Paclitaxel-resistant Human Ovarian Cancer Cells Have Mutant β-Tubulins That Exhibit Impaired Paclitaxel-driven Polymerization.</em> Journal of Biological Chemistry, 1997. <strong>272</strong>(27): p. 17118-17125.</p>
<p>14. Giannakakou, P., et al., <em>A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.</em> Proceedings of the National Academy of Sciences, 2000. <strong>97</strong>(6): p. 2904-2909.</p>
<p>15. Witte, H., D. Neukirchen, and F. Bradke, <em>Microtubule stabilization specifies initial neuronal polarization.</em> The Journal of Cell Biology, 2008. <strong>180</strong>(3): p. 619-632.</p>
<p>16. Jordan, M.A., et al., <em>Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations.</em> Proceedings of the </p>
<p>National Academy of Sciences of the United States of America, 1993. <strong>90</strong>(20): p. 9552-9556.</p>
<p>17. Kowalski, R.J., P. Giannakakou, and E. Hamel, <em>Activities of the Microtubule-stabilizing Agents Epothilones A and B with Purified Tubulin and in Cells Resistant to Paclitaxel (Taxol®).</em> Journal of Biological Chemistry, 1997. <strong>272</strong>(4): p. 2534-2541.</p>
<p>18. Risinger, A.L. and S.L. Mooberry, <em>Cellular studies reveal mechanistic differences between taccalonolide A and paclitaxel.</em> Cell Cycle, 2011. <strong>10</strong>(13): p. 2162-2171.</p>
<p>19. Dumontet, C. and B.I. Sikic, <em>Mechanisms of Action of and Resistance to Antitubulin Agents: Microtubule Dynamics, Drug Transport, and Cell Death.</em> Journal of Clinical Oncology, 1999. <strong>17</strong>(3): p. 1061-1061.</p>
2019-01-29T08:50:402019-01-29T10:10:12691d7fc5-55a5-47e5-9dd6-9f61bcfa8ec2119350b6-06d2-4207-9583-04610a771c43<p>An impairment in axonal transport leads to an inadequate supply of the neuronal periphery.</p>
<p>Mutations linked to microtubules are known to cause peripheral neuropathies:</p>
<p>- Mutations in the TUBB3 coding gene are known to cause congenital fibrosis of the extraocular muscle type 3 (CFEOM3) and also lead to later-onset peripheral neuropathies. Microtubules were shown to be more stable and changes to microtubule dynamic instability were evidenced. Furthermore, Kif21a exhibits impaired interaction with microtubules thereby influencing axonal transport. [1]</p>
<p>- Patients suffering from Charcot-Marie-Tooth (CMT) neuropathy exhibit length-dependend degeneration of peripheral nerves. CMT type 2 is associated with axonal degeneration leading to reduced action potentials. CMT2F is caused by a mutation in the gene coding for heat shock protein B1 (HSPB1). [2] Heat shock proteins can stabilize or protect the structure of other proteins and specifically mutated HSPB1 was shown to bind to and stabilize microtubules and disturb microtubule dynamic instability (see below). [3]</p>
<p>- Enhanced binding to microtubules was documented for mutated HSPB1 (measured by immunoprecipitation, cosedimentation assay, K<sub>D</sub> determination (SPR)). Furthermore, mutated HSPB1 was shown to stabilize microtubules (measured by cold-induced depolymerization assay, Nocodazole approach, cell migration (scratch) assay) and thereby disrupt microtubule dynamic instability (measured by tracking of TUBB3-GFP microtubules and in HSPB1<sub> </sub>transgenic mice). [3]</p>
<p>- TUBB3 mutations were shown to change binding of KIFs to microtubules and thereby disrupt axonal transport of vesicles and mitochondria in cells derived from the peripheral nervous system. [4]</p>
<p>- Taxol, known to disrupt microtubule dynamic instability [5, 6], was proven to decrease transport of horseradish peroxidase in dorsal root ganglia neurons (and also less microtubule crosslinks were observed) Intact axonal transport can be re-gained after taxol wash-out (1 day treatment, 2 days wash-out). [7].</p>
<p>- It was shown that Taxol inhibits anterograde fast (and retrograde) axonal transport in rat sciatic nerves [8] and anterograde transport in SK-N-SH human neuroblastoma cells and mice sciatic nerves [9].</p>
<p>- Taxol, ixabepilone, vincristine and eribulin, all of which are known to suppress microtubule dynamic instability, were proven to have inhibitory effects on anterograde fast axonal transport in isolated squid axoplasm. [10]</p>
<p>- Posttranslational modifications of tubulin were shown to increase upon Taxol treatment and an increase in the stabile fraction of microtubules was observed. In the same time scale, also KIF1 accumulation was observed indicating disruption of axonal transport. [11]</p>
<p>Quantitative data illustrating a causal relationship between KE1 and the KE2 is not available.</p>
<p>Most studies in the literature only deal with the direct link of ‘known MSAs’ to KE2 ‘impaired axonal transport’. The disturbance in microtubule dynamic instability was rarely proven in studies using MSAs to investigate their effects on axonal transport. [8-10] Concentration- and/or time-dependency was investigated only in some of the studies. [7, 9-11]</p>
<p>Results of transport experiments are sometimes contradicting regarding the inhibition of retrograde axonal transport upon Taxol treatment, e.g. Smith et. al claims that only anterograde transport is inhibited [9], however, Nakata et. al found anterograde as well as retrograde transport to be inhibited [8].</p>
<p>1. Tischfield, M.A., et al., <em>Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance.</em> Cell, 2010. <strong>140</strong>(1): p. 74-87.</p>
<p>2. Ismailov, S.M., et al., <em>A new locus for autosomal dominant Charcot-Marie-Tooth disease type 2 (CMT2F) maps to chromosome 7q11-q21.</em> Eur J Hum Genet, 2001. <strong>9</strong>(8): p. 646-50.</p>
<p>3. Almeida-Souza, L., et al., <em>Small heat-shock protein HSPB1 mutants stabilize microtubules in Charcot-Marie-Tooth neuropathy.</em> J Neurosci, 2011. <strong>31</strong>(43): p. 15320-8.</p>
<p>4. Niwa, S., H. Takahashi, and N. Hirokawa, <em>beta-Tubulin mutations that cause severe neuropathies disrupt axonal transport.</em> Embo j, 2013. <strong>32</strong>(10): p. 1352-64.</p>
<p>5. Derry, W.B., L. Wilson, and M.A. Jordan, <em>Substoichiometric Binding of Taxol Suppresses Microtubule Dynamics.</em> Biochemistry, 1995. <strong>34</strong>(7): p. 2203-2211.</p>
<p>6. Witte, H., D. Neukirchen, and F. Bradke, <em>Microtubule stabilization specifies initial neuronal polarization.</em> The Journal of Cell Biology, 2008. <strong>180</strong>(3): p. 619-632.</p>
<p>7. Theiss, C. and K. Meller, <em>Taxol impairs anterograde axonal transport of microinjected horseradish peroxidase in dorsal root ganglia neurons in vitro.</em> Cell Tissue Res, 2000. <strong>299</strong>(2): p. 213-24.</p>
<p>8. Nakata, T. and H. Yorifuji, <em>Morphological evidence of the inhibitory effect of taxol on the fast axonal transport.</em> Neuroscience Research, 1999. <strong>35</strong>(2): p. 113-122.</p>
<p>9. Smith, J.A., et al., <em>Structural Basis for Induction of Peripheral Neuropathy by Microtubule-Targeting Cancer Drugs.</em> Cancer Research, 2016. <strong>76</strong>(17): p. 5115-5123.</p>
<p>10. LaPointe, N.E., et al., <em>Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy.</em> Neurotoxicology, 2013. <strong>37</strong>: p. 231-9.</p>
<p>11. Hammond, J.W., et al., <em>Posttranslational modifications of tubulin and the polarized transport of kinesin-1 in neurons.</em> Mol Biol Cell, 2010. <strong>21</strong>(4): p. 572-83.</p>
2019-01-29T08:50:572019-01-29T10:11:11119350b6-06d2-4207-9583-04610a771c4301c9081b-410c-49c2-8504-f7ec0a59fa5f<p>Defects in axonal transport are often suggested as a cause for peripheral neuropathies as the length-dependent, distal neurologic deficits observed in patients treated with taxol or other MSAs indicate an axonal loss comparable to dying-back neuropathies which are often linked to axonal transport defects. [1]</p>
<p>Mutations linked to axonal transport are known to cause Charcot-Marie-Tooth disease (peripheral neuropathy). A mutation in the gene coding for the motor protein kinesin 1B (KIF1B) was found in a CMT2A family. The transport of synaptic vesicles is mediated by KIF1B. [2]</p>
<p>Generated KIF1A mutant mice exhibited motor and sensory disturbances and transport of synaptic vesicle precursors was shown to be decreased in axons. [3]</p>
<p>- Mutated KIF1B was shown to lose motor activity and did not move towards the plus end of microtubules. [2] A loss of function in motor proteins leads to disruption of transport along microtubules and therefore also of axonal transport. As this mutation of KIF1B was shown to be linked to CMT2A [2], a peripheral neuropathy, a direct link between axonal transport defects and peripheral neuropathy can be created.</p>
<p>- Transport of the synaptic vesicle precursor synaptotagmin was shown to be decreased in mice with a knockout of the KIF1A motor protein. These mice also exhibited axonal degeneration and showed motor and sensory disturbances. [3]</p>
<p>- Neuropathy was induced in cats by administration of acrylamide. Cats exhibited neuropathic symptoms like foot drop and also axonal degeneration was documented. Axonal transport was proven to be disturbed in acrylamide-neuropathic cats. [4]</p>
<p>- Several mutations in the neurofilament light (NFL) gene have been shown to cause Charcot-Marie-Tooth disease, a peripheral neuropathy. Neuronal cultures of NFL mutants exhibited reduced axonal transport. Furthermore, mutant NFL protein overexpression lead to degeneration of neurites. This observation points to a potential mechanism of neuropathy development in CMT patients with NFL mutations. [5]</p>
<p>The KIF1B mutation, which was identified in a CMT2A family and proven to disturb axonal transport, was only found in this family and has never been confirmed. [2, 6]</p>
<p>KIF1A knockout mice were born alive but died within 24h after birth. All measurements of sensory and motor function, axonal degeneration and axonal transport were performed within these 24h. [3]</p>
<p>Neuropathy was induced in cats by administration of either acrylamide or triorthocresyl phosphate (TOCP). Neuropathic symptoms like axonal degeneration were detected in both acrylamide- as well as TOCP-induced neuropathic cats but axonal transport of proteins was shown to be disturbed only in acrylamide-induced neuropathic cats. [4]</p>
<p>Furthermore, only limited human <em>in vivo</em> evidence is available for KE1 and its relationship to the AO.</p>
<p>1. Rowinsky , E.K. and R.C. Donehower <em>Paclitaxel (Taxol).</em> New England Journal of Medicine, 1995. <strong>332</strong>(15): p. 1004-1014.</p>
<p>2. Zhao, C., et al., <em>Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta.</em> Cell, 2001. <strong>105</strong>(5): p. 587-97.</p>
<p>3. Yonekawa, Y., et al., <em>Defect in Synaptic Vesicle Precursor Transport and Neuronal Cell Death in KIF1A Motor Protein–deficient Mice.</em> The Journal of Cell Biology, 1998. <strong>141</strong>(2): p. 431-441.</p>
<p>4. Pleasure, D.E., K.C. Mishler, and W.K. Engel, <em>Axonal Transport of Proteins in Experimental Neuropathies.</em> Science, 1969. <strong>166</strong>(3904): p. 524-525.</p>
<p>5. Pérez-Ollé, R., et al., <em>Mutations in the neurofilament light gene linked to Charcot-Marie-Tooth disease cause defects in transport.</em> Journal of Neurochemistry, 2005. <strong>93</strong>(4): p. 861-874.</p>
<p>6. Pareyson, D., et al., <em>Mitochondrial dynamics and inherited peripheral nerve diseases.</em> Neuroscience Letters, 2015. <strong>596</strong>(Supplement C): p. 66-77.</p>
2019-01-29T08:51:082019-01-29T10:12:04Microtubule interacting drugs lead to peripheral neuropathyMicrotubule interacting drugs lead to peripheral neuropathy<p>Anna Katharina Holzer, Stefan Schildknecht</p>
Under development: Not open for comment. Do not cite<p>Peripheral neuropathy is regularly observed as a side-effect in microtubule-targeted chemotherapies. The length of their axons, together with the absence of protection by a blood brain barrier, renders peripheral neurons particularly vulnerable to disturbances in microtubule dynamics.</p>
<p>The present AOP was developed to summarize mechanistic data that support the link between the interaction of “microtubule stabilizing agents” with microtubules (MIE) and sensory axonal peripheral neuropathy (AO), via disturbances in microtubule dynamic instability (KE1) and an impaired axonal transport (KE2).</p>
<p>The present AOP allowed the identification of the following knowledge gaps in the literature: (i) experimental support for the mechanistic link of microtubule interacting drugs with the onset of sensory axonal peripheral neuropathy is generally limited by laborious and circumstantial access to peripheral neurons in vivo and by limited availability of human in vitro models of peripheral neurons; (ii) it is necessary to distinguish the biological consequences of microtubule-stabilizing drugs on the one side, and microtubule-destabilizing compounds on the other; (iii) the link of clinical symptoms to the action of microtubule stabilizers is complicated by incomplete toxicokinetic data, by the time offset between drug treatment and clinical symptoms, and by partial reversibility of the AO following washout of the microtubule-interacting drugs.</p>
<p>The present AOP was developed to contribute to cross systems testing as well as to experimental studies covering the testing of National Toxicology Program compounds.</p>
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