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Nan Fang Yi Ke Da Xue Xue Bao = Journal... Apr 2022To investigate the therapeutic effect of Epothilone D on traumatic optic neuropathy (TON) in rats.
OBJECTIVE
To investigate the therapeutic effect of Epothilone D on traumatic optic neuropathy (TON) in rats.
METHODS
Forty-two SD rats were randomized to receive intraperitoneal injection of 1.0 mg/kg Epothilone D or DMSO (control) every 3 days until day 28, and rat models of TON were established on the second day after the first administration. On days 3, 7, and 28, examination of flash visual evoked potentials (FVEP), immunofluorescence staining and Western blotting were performed to examine the visual pathway features, number of retinal ganglion cells (RGCs), GAP43 expression level in damaged axons, and changes of Tau and pTau-396/404 in the retina and optic nerve.
RESULTS
In Epothilone D treatment group, RGC loss rate was significantly decreased by 19.12% (=0.032) on day 3 and by 22.67% (=0.042) on day 28 as compared with the rats in the control group, but FVEP examination failed to show physiological improvement in the visual pathway on day 28 in terms of the relative latency of N2 wave (=0.236) and relative amplitude attenuation of P2-N2 wave (=0.441). The total Tau content in the retina of the treatment group was significantly increased compared with that in the control group on day 3 ( < 0.001), showing a consistent change with ptau-396/404 level. In the optic nerve axons, the total Tau level in the treatment group was significantly lower than that in the control group on day 7 (=0.002), but the changes of the total Tau and pTau-396/404 level did not show an obvious correlation. Epothilone D induced persistent expression of GAP43 in the damaged axons, detectable even on day 28 of the experiment.
CONCLUSION
Epothilone D treatment can protect against TON in rats by promoting the survival of injured RGCs, enhancing Tau content in the surviving RGCs, reducing Tau accumulation in injured axons, and stimulating sustained regeneration of axons.
Topics: Animals; Disease Models, Animal; Epothilones; Evoked Potentials, Visual; Nerve Regeneration; Optic Nerve Injuries; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells
PubMed: 35527494
DOI: 10.12122/j.issn.1673-4254.2022.04.14 -
International Journal of Molecular... Mar 2017The synthesis of two deoxygenated analogues of potent epothilones is reported in an effort to analyze the relative importance of molecular conformation and ligand-target...
The synthesis of two deoxygenated analogues of potent epothilones is reported in an effort to analyze the relative importance of molecular conformation and ligand-target interactions to biological activity. 7-deoxy-epothilone D and 7-deoxy-()-14-methoxy-epothilone D were prepared through total synthesis and shown to maintain the conformational preferences of their biologically active parent congeners through computer modeling and nuclear magnetic resonance (NMR) studies. The significant decrease in observed potency for each compound suggests that a hydrogen bond between the C7-hydroxyl group and the tubulin binding site plays a critical role in the energetics of binding in the epothilone class of polyketides.
Topics: Antineoplastic Agents; Cell Line, Tumor; Epothilones; Humans; Hydrogen Bonding; Protein Binding; Tubulin
PubMed: 28304361
DOI: 10.3390/ijms18030648 -
Microbial Cell Factories Apr 2021Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains....
BACKGROUND
Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal.
RESULTS
The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS.
CONCLUSIONS
These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.
Topics: Bacteria; Bacterial Proteins; Catalytic Domain; Polyketide Synthases; Protein Engineering; Protein Structure, Tertiary; Substrate Specificity
PubMed: 33882930
DOI: 10.1186/s12934-021-01578-3 -
Biochemistry. Biokhimiia Sep 2018Alzheimer's disease (AD) is the most common incurable neurodegenerative disorder that affects the processes of memory formation and storage. The loss of dendritic spines... (Review)
Review
Alzheimer's disease (AD) is the most common incurable neurodegenerative disorder that affects the processes of memory formation and storage. The loss of dendritic spines and alteration in their morphology in AD correlate with the extent of patient's cognitive decline. Tubulin had been believed to be restricted to dendritic shafts, until recent studies demonstrated that dynamically growing tubulin microtubules enter dendritic spines and promote their maturation. Abnormalities of tubulin cytoskeleton may contribute to the process of dendritic spine shape alteration and their subsequent loss in AD. In this review, association between tubulin cytoskeleton dynamics and dendritic spine morphology is discussed in the context of dendritic spine alterations in AD. Potential implications of these findings for the development of AD therapy are proposed.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Cytoskeleton; Dendritic Spines; Epothilones; Humans; Microtubules; Neurons; Nocodazole
PubMed: 30472945
DOI: 10.1134/S0006297918090080 -
Frontiers in Bioengineering and... 2019Epothilones are a kind of macrolides with strong cytotoxicity toward cancer cells and relatively lower side effects compared with taxol. Epothilone B derivate...
Epothilones are a kind of macrolides with strong cytotoxicity toward cancer cells and relatively lower side effects compared with taxol. Epothilone B derivate ixabepilone has been used for the clinical treatment of advanced breast cancer. However, the low yield of epothilones and the difficulty in the genetic manipulation of limited their wider application. Transcription activator-like effectors-Trancriptional factor (TALE-TF)-VP64 and clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-VP64 have been demonstrated as effective systems for the transcriptional improvement. In this study, a promoter for the epothilone biosynthesis cluster was obtained and the function has been verified. The TALE-TF-VP64 and CRISPR/dcas9-VP64 target P3 promoter were electroporated into strain So ce M4, and the transcriptional levels of epothilone biosynthesis-related genes were significantly upregulated. The yield of epothilone B was improved by 2.89- and 1.53-fold by the introduction of recombinant TALE-TF-VP64-P3 and dCas9-VP64-P3 elements into So ce M4, respectively. The epothilone D yield was also improved by 1.12- and 2.18-fold in recombinant dCas9-So ce M4 and TALE-VP64 strains, respectively. The transcriptional regulation mechanism of TALE-TF-VP64 and the competition mechanism with endogenous transcriptional factor were investigated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), demonstrating the combination of the P3 promoter and TALE-TF element and the competition between TALE-TF and endogenous transcriptional protein. This is the first report on the transcriptional regulation of the epothilone biosynthetic gene cluster in using the TALE-TF and dCas9-VP64 systems, and the regulatory mechanism of the TALE-TF system for epothilone biosynthesis in was also firstly revealed, thus shedding light on the metabolic engineering of to improve epothilone yields substantially and promoting the application of epothilones in the biomedical industry.
PubMed: 32039165
DOI: 10.3389/fbioe.2019.00334 -
Journal of Neurochemistry Feb 2016Exposure to binge methamphetamine (METH) can result in a permanent or transient loss of dopaminergic (DAergic) markers such as dopamine (DA), dopamine transporter, and...
Exposure to binge methamphetamine (METH) can result in a permanent or transient loss of dopaminergic (DAergic) markers such as dopamine (DA), dopamine transporter, and tyrosine hydroxylase (TH) in the striatum. We hypothesized that the METH-induced loss of striatal DAergic markers was, in part, due to a destabilization of microtubules (MTs) in the nigrostriatal DA pathway that ultimately impedes anterograde axonal transport of these markers. To test this hypothesis, adult male Sprague-Dawley rats were treated with binge METH or saline in the presence or absence of epothilone D (EpoD), a MT-stabilizing compound, and assessed 3 days after the treatments for the levels of several DAergic markers as well as for the levels of tubulins and their post-translational modifications (PMTs). Binge METH induced a loss of stable long-lived MTs within the striatum but not within the substantia nigra pars compacta (SNpc). Treatment with a low dose of EpoD increased the levels of markers of stable MTs and prevented METH-mediated deficits in several DAergic markers in the striatum. In contrast, administration of a high dose of EpoD appeared to destabilize MTs and potentiated the METH-induced deficits in several DAergic markers. The low-dose EpoD also prevented the METH-induced increase in striatal DA turnover and increased behavioral stereotypy during METH treatment. Together, these results demonstrate that MT dynamics plays a role in the development of METH-induced losses of several DAergic markers in the striatum and may mediate METH-induced degeneration of terminals in the nigrostriatal DA pathway. Our study also demonstrates that MT-stabilizing drugs such as EpoD have a potential to serve as useful therapeutic agents to restore function of DAergic nerve terminals following METH exposure when administered at low doses. Administration of binge methamphetamine (METH) negatively impacts neurotransmission in the nigrostriatal dopamine (DA) system. The effects of METH include decreasing the levels of DAergic markers in the striatum. We have determined that high-dose METH destabilizes microtubules in this pathway, which is manifested by decreased levels of acetylated (Acetyl) and detyrosinated (Detyr) α-tubulin (I). A microtubule stabilizing agent epothilone D protects striatal microtubules form the METH-induced loss of DAergic markers (II). These findings provide a new strategy for protection form METH - restoration of proper axonal transport.
Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Central Nervous System Stimulants; Corpus Striatum; Dopamine; Dopamine Plasma Membrane Transport Proteins; Epothilones; Exploratory Behavior; Glial Fibrillary Acidic Protein; Homovanillic Acid; Male; Methamphetamine; Microtubules; Motor Activity; Rats; Rats, Sprague-Dawley; Substantia Nigra; Tubulin; Tyrosine 3-Monooxygenase
PubMed: 26465779
DOI: 10.1111/jnc.13391 -
Journal of Neurochemistry Dec 2012Recent evidence underlines the crucial role of neuronal cytoskeleton in the pathophysiology of psychiatric diseases. In this line, the deletion of STOP/MAP6 (Stable...
Recent evidence underlines the crucial role of neuronal cytoskeleton in the pathophysiology of psychiatric diseases. In this line, the deletion of STOP/MAP6 (Stable Tubule Only Polypeptide), a microtubule-stabilizing protein, triggers various neurotransmission and behavioral defects, suggesting that STOP knockout (KO) mice could be a relevant experimental model for schizoaffective symptoms. To establish the predictive validity of such a mouse line, in which the brain serotonergic tone is dramatically imbalanced, the effects of a chronic fluoxetine treatment on the mood status of STOP KO mice were characterized. Moreover, we determined the impact, on mood, of a chronic treatment by epothilone D, a taxol-like microtubule-stabilizing compound that has previously been shown to improve the synaptic plasticity deficits of STOP KO mice. We demonstrated that chronic fluoxetine was either antidepressive and anxiolytic, or pro-depressive and anxiogenic, depending on the paradigm used to test treated mutant mice. Furthermore, control-treated STOP KO mice exhibited paradoxical behaviors, compared with their clear-cut basal mood status. Paradoxical fluoxetine effects and control-treated STOP KO behaviors could be because of their hyper-reactivity to acute and chronic stress. Interestingly, both epothilone D and fluoxetine chronic treatments improved the short-term memory of STOP KO mice. Such treatments did not affect the serotonin and norepinephrine transporter densities in cerebral areas of mice. Altogether, these data demonstrated that STOP KO mice could represent a useful model to study the relationship between cytoskeleton, mood, and stress, and to test innovative mood treatments, such as microtubule-stabilizing compounds.
Topics: Affect; Animals; Antineoplastic Agents; Depression; Disease Models, Animal; Epothilones; Female; Fluoxetine; Male; Memory, Short-Term; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Microtubule-Associated Proteins; Selective Serotonin Reuptake Inhibitors
PubMed: 23013328
DOI: 10.1111/jnc.12027 -
Cells Dec 2021Mitochondria move along the microtubule network and produce bioenergy in the cell. However, there is no report of a relationship between bioenergetic activity of...
Mitochondria move along the microtubule network and produce bioenergy in the cell. However, there is no report of a relationship between bioenergetic activity of mitochondria and microtubule stability in mammalian cells. This study aimed to investigate this relationship. We treated HEK293 cells with microtubule stabilizers (Taxol and Epothilone D) or a microtubule disturber (vinorelbine), and performed live-cell imaging to determine whether mitochondrial morphology and bioenergetic activity depend on the microtubule status. Treatment with microtubule stabilizers enhanced the staining intensity of microtubules, significantly increased ATP production and the spare respiratory capacity, dramatically increased mitochondrial fusion, and promoted dynamic movement of mitochondria. By contrast, bioenergetic activity of mitochondria was significantly decreased in cells treated with the microtubule disturber. Our data suggest that microtubule stability promotes mitochondrial functional activity. In conclusion, a microtubule stabilizer can possibly recover mitochondrial functional activity in cells with unstable microtubules.
Topics: Adenosine Triphosphate; Cell Proliferation; Cell Respiration; Cell Shape; Cell Survival; Gene Expression Regulation; HEK293 Cells; Humans; Membrane Potential, Mitochondrial; Microtubules; Mitochondria; Oxygen Consumption; Signal Transduction; TOR Serine-Threonine Kinases
PubMed: 34944107
DOI: 10.3390/cells10123600 -
Frontiers in Cellular Neuroscience 2018Microtubule dynamics underpin a plethora of roles involved in the intricate development, structure, function, and maintenance of the central nervous system. Within the...
Microtubule dynamics underpin a plethora of roles involved in the intricate development, structure, function, and maintenance of the central nervous system. Within the injured brain, microtubules are vulnerable to misalignment and dissolution in neurons and have been implicated in injury-induced glial responses and adaptive neuroplasticity in the aftermath of injury. Unfortunately, there is a current lack of therapeutic options for treating traumatic brain injury (TBI). Thus, using a clinically relevant model of mild TBI, lateral fluid percussion injury (FPI) in adult male Thy1-YFPH mice, we investigated the potential therapeutic effects of the brain-penetrant microtubule-stabilizing agent, epothilone D. At 7 days following a single mild lateral FPI the ipsilateral hemisphere was characterized by mild astroglial activation and a stereotypical and widespread pattern of axonal damage in the internal and external capsule white matter tracts. These alterations occurred in the absence of other overt signs of trauma: there were no alterations in cortical thickness or in the number of cortical projection neurons, axons or dendrites expressing YFP. Interestingly, a single low dose of epothilone D administered immediately following FPI (and sham-operation) caused significant alterations in the dendritic spines of layer 5 cortical projection neurons, while the astroglial response and axonal pathology were unaffected. Specifically, spine length was significantly decreased, whereas the density of mushroom spines was significantly increased following epothilone D treatment. Together, these findings have implications for the use of microtubule stabilizing agents in manipulating injury-induced synaptic plasticity and indicate that further study into the viability of microtubule stabilization as a therapeutic strategy in combating TBI is warranted.
PubMed: 30104961
DOI: 10.3389/fncel.2018.00223 -
NeuroImage Aug 2014The MAP6 (microtubule-associated protein 6) KO mouse is a microtubule-deficient model of schizophrenia that exhibits severe behavioral disorders that are associated with...
The MAP6 (microtubule-associated protein 6) KO mouse is a microtubule-deficient model of schizophrenia that exhibits severe behavioral disorders that are associated with synaptic plasticity anomalies. These defects are alleviated not only by neuroleptics, which are the gold standard molecules for the treatment of schizophrenia, but also by Epothilone D (Epo D), which is a microtubule-stabilizing molecule. To compare the neuronal transport between MAP6 KO and wild-type mice and to measure the effect of Epo D treatment on neuronal transport in KO mice, MnCl2 was injected in the primary somatosensory cortex. Then, using manganese-enhanced magnetic resonance imaging (MEMRI), we followed the propagation of Mn(2+) through axonal tracts and brain regions that are connected to the somatosensory cortex. In MAP6 KO mice, the measure of the MRI relative signal intensity over 24h revealed that the Mn(2+) transport rate was affected with a stronger effect on long-range and polysynaptic connections than in short-range and monosynaptic tracts. The chronic treatment of MAP6 KO mice with Epo D strongly increased Mn(2+) propagation within both mono- and polysynaptic connections. Our results clearly indicate an in vivo deficit in neuronal Mn(2+) transport in KO MAP6 mice, which might be due to both axonal transport defects and synaptic transmission impairments. Epo D treatment alleviated the axonal transport defects, and this improvement most likely contributes to the positive effect of Epo D on behavioral defects in KO MAP6 mice.
Topics: Animals; Contrast Media; Disease Models, Animal; Epothilones; Humans; Magnetic Resonance Imaging; Manganese; Mice; Mice, Inbred C57BL; Mice, Knockout; Microtubule-Associated Proteins; Schizophrenia; Somatosensory Cortex; Synaptic Transmission; Treatment Outcome; Tubulin Modulators
PubMed: 24704457
DOI: 10.1016/j.neuroimage.2014.03.071