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A diagrammatic representation of the proposed interaction of colchicine with tubulin. The A ring is shown between Cys-354 and Cys-239, while it is proposed that the tropolone ring interacts with the amino terminus of-tubulin, to account for the covalent interaction reported by Uppuluri et al. (11). The 9-Å distance shown between the two cysteine sulfur atoms is that postulated to account for cross-link formation by EBI (27), and the 3 Å between the Cys-354 sulfur atom and the C-3 oxygen atom of colchicine is the calculated length of the chloroacetyl moiety. The intracolchicine distances were obtained by manipulation of the crystal coordinates in the Cambridge Structural Data Base (reference code, COLCDH, data from Ref. 33) with the molecular modeling program Quanta Version 4.0. The unit cells of the crystal have two molecules of colchicine, and the two distances shown were nearly identical in both molecules.
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![FIG. 4. Formic acid digestion of-tubulin alkylated with [ 14 C]3CTC....](profile/Joseph-Bekisz/publication/14552440/figure/fig4/AS:482622993375235@1492077696341/Formic-acid-digestion-of-tubulin-alkylated-with-14-C3CTC-Following-the-96-h_Q320.jpg)
The colchicine analog 3-chloroacetyl-3-demthylthio-colchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37 degrees C, but not at 0 degree C, and covalently reacts with beta-tubulin at 37 degree C, but not at 0 degree C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular dis...
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Context 1
... findings with 3CTC, and to a lesser extent with 2CTC, suggest that the colchicine A ring lies in the 9 Å pocket between Cys-354 and Cys-239. Such a model (Fig. 7) is consistent with the molecular distances obtained from the crystal structure of colchicine (33) and with the 3 Å length of the chloroacetyl group. Correlating our findings with those of Uppuluri et al. (11) suggests that, following photoactivation of colchicine, the reactivity observed with the -subunit sequence 214 -241 re- sults ...
Citations
... HvTUB8 homologue proteins from other species showed high level of similarity. Previous studies showed that the Cysteine 354 of β-TUBULIN was essential for binding of colchicine (Bai et al. 1996(Bai et al. , 2000 and acrolein (Uemura et al. 2019), and mutation of this cysteine in yeast β-TUBULIN could result in cold-stable microtubules (Gupta et al. 2010). Our result demonstrated that the Cys354Tyr substitution resulted in changes of subcellular location of the protein. ...
Key message
Map-based cloning, subcellular localization, virus-induced-gene-silencing and transcriptomic analysis reveal HvTUB8 as a candidate gene with pleiotropic effects on barley spike and leaf development via ethylene and chlorophyll metabolism.
Abstract
Barley lateral spikelet morphology and grain shape play key roles in grain physical quality and yield. Several genes and QTLs for these traits have been cloned or fine mapped previously. Here, we report the phenotypic and genotypic analysis of a barley mutant with round lateral spikelet (rls) from cv. Edamai 934. rls had round lateral spikelet, short but round grain, shortened awn, thick glume and dark green leaves. Histocytologic and ultrastructural analysis revealed that the difference of grain shape of rls was caused by change of cell arrangement in glume, and the dark leaf color resulted from enlarged chloroplast. HvTUBULIN8 ( HvTUB8 ) was identified as the candidate gene for rls by combination of RNA-Seq, map-based-cloning, virus-induced-gene-silencing (VIGS) and protein subcellular location. A single G-A substitution at the third exon of HvTUB8 resulted in change of Cysteine 354 to tyrosine. Furthermore, the mutant isoform Hvtub8 could be detected in both nucleus and cytoplasm, whereas the wild-type protein was only in cytoplasm and granular organelles of wheat protoplasts. Being consistent with the rare phenotype, the “A” allele of HvTUB8 was only detected in rls , but not in a worldwide barley germplasm panel with 400 accessions. VIGS confirmed that HvTUB8 was essential to maintain spike integrity. RNA-Seq results suggested that HvTUB8 may control spike morphogenesis via ethylene homeostasis and signaling, and control leaf color through chlorophyll metabolism. Collectively, our results support HvTUB8 as a candidate gene for barley spike and leaf morphology and provide insight of a novel mechanism of it in barley development.
... HvTUB8 homologue proteins from other species showed high level of similarity, however, the Cys354Tyr located in a region that was not so conserved, whereas, the Cysteine was extremely conservative among different species, also suggesting its vital role in function maintaining. Previous studies showed that the Cysteine 354 of β-TUBULIN was essential for binding of colchicine (Bai et al. 2000;Bai et al. 1996) and acrolein (Uemura et al. 2019), and mutation of this cysteine in yeast β-TUBULIN could result in cold-stable microtubules (Gupta et al. 2010). Our result further demonstrated that the C to Y substitution resulted in changes of subcellular location of the protein. ...
Barley lateral spikelet morphology and grain shape play a key role in grain physical quality and yield. Several genes and QTLs for these traits have been cloned or fine mapped previously. Here, we report the phenotypic and genotypic analysis of a barley mutant with Round Lateral Spikelet (rls) from c.v. Edamai 934. The mutant had round lateral spikelet, short but round grain, shortened awn, thick glume and dark green leaves. Histocytologic and ultrastructural analysis revealed that the difference of grain shape of rls was caused by change of cell arrangement in glume, and the dark leaf color resulted from enlarged chloroplast. HvTUBULIN8 ( HvTUB8 ) was identified as the functional gene for rls by combination of RNA-seq, map-based cloning, virus-induced-gene-silencing (VIGS) and protein subcellular location. A single G-A substitution in exon of HvTUB8 resulted in change of Cysteine to Tyrosine. Furthermore, the mutant isoform of HvTUB8 could be detected in both nucleus and cytoplasm, whereas the wild was only in cytoplasm of wheat protoplasts. Being consistent with the rare phenotype, the “A” allele of HvTUB8 was only detected in rls , but not in a worldwide barley germplasm panel consisting about 400 accessions. VIGS confirmed that HvTUB8 was essential to maintain spike integrity. RNA-sequencing results suggest that HvTUB8 control spike morphogenesis via ethylene homeostasis and signaling, and control leaf color through chlorophyll metabolism. Collectively, our results demonstrated that HvTUB8 has pleiotropic effects on barley spike and leaf morphology and provided insight of a novel mechanism of HvTUB8 in spike and leaf development.
... For example, the β-tubulin C-terminus binds microtubule-associated protein 2 (MAP-2) or tau, whereas the α-tubulin C-terminus binds these proteins only weakly [153]. The α-tubulin C-terminus determines colchicine binding properties [154] despite the fact that the binding site is located on β-tubulin [155,156]. When colchicine is delivered directly to the brain or cerebrospinal fluid of experimental animals it causes significant cognitive impairments of learning and memory [157,158]. ...
To find out whether neuronal microtubules could translate and input the information carried by electric signals entering into the brain cortex, a detailed investigation of the local electromagnetic field structure is performed. The electric and magnetic field strengths in different neuronal compartments, including dendrites, soma, and axons, are assessed from reported electrophysiological measurements. The results show that the magnetic field is too weak to input information to microtubules and cannot support quantum Hall effect. Because the magnetic flux density of individual electric spikes is 3 orders of magnitude weaker than Earth's magnetic field, any information encoded in the magnetic signal will be suffocated by the surrounding noise. In contrast, the electric field carries biologically important information and acts upon transmembrane voltage-gated ion channels that govern the generation of neuronal action potentials. If the human mind is supported by subneuronal processing of information in the brain microtubules, then the microtubule interaction with the local electric field has to be the main source for input of sensory information. The intensity of the electric field inside the neuronal cytosol, however, is less than 500 V/m, which rules out any electric sensitivity of putative ferroelectric microtubules. Although the tubulin C-terminal tails are promising candidates for substrates that are both sensitive to electric signals and possess biologically useful intraneuronal functions, they lack physical means to affect back the electric output of neurons. Thus, voltage-gated ion channels incorporated into the neuronal plasma membranes appear to be best suited to support consciousness since they remain the only known biomolecular substrates that are capable of inputting, processing and outputting of electric signals.
... Moreover, a literature survey was carried out to enhance this theoretical design by actually reported tubulin inhibitors containing similar anchor groups. The survey revealed some interesting examples of similarity e.g. the chloroacetyl derivatives of colchicine [103,104] as well as the N-chloroacetyl oxadiazole derivative reported elsewhere [57]. Although the latter was not subjected to biological evaluation, it was instead utilized as a synthetic precursor to the corresponding 2-acetoxyacetyl, 2-azidoacetyl, 2-(dimethylamino)acetyl derivatives which were all biologically active [57]. ...
Bis-hydrazides 13a-h were designed and synthesized as potential tubulin inhibitors selectively targeting the colchicine site between α- and β-tubulin subunits. The newly designed ring-B substituents were assisted at their ends by ‘anchor groups’ which are expected to exert binding interaction(s) with new additional amino acid residues in the colchicine site (beyond those amino acids previously reported to interact with reference inhibitors as CA-4 and colchicine). Conformational flexibility of bis-hydrazide linker assisted these ‘extra-binding’ properties through reliving ligands’ strains in the final ligand-receptor complexes. Compound 13f displayed the most promising computational and biological study results in the series: MM/GBSA binding energy of -62.362 kcal/mol (extra-binding to Arg α:221, Thr β:353 & Lys β:254); 34% NCI-H522 cells’ death (at 10 µM), IC50= 0.073 µM (MTT assay); significant cell cycle arrest at G2/M phase; 11.6% preG1 apoptosis induction and 83.1% in vitro tubulin inhibition (at concentration = IC50). Future researchers in bis-hydrazide tubulin inhibitors are advised to consider the 2-chloro-N-(4-substituted-phenyl)acetamide derivatives as compound 13f due to extra-binding properties of their ring B.
... Additionally, the gas-phase structures of all molecules were determined using the DFT optimization with the Gaussian09 program package [52]. All calculations were carried out by the DFT method using the Becke3-Lee-Yang-Parr correlation functional (B3LYP) [53][54][55][56] with the 6-31 + G basis set, starting from the X-ray geometry of molecules. The gas-phase optimized conformations of all colchicine derivatives are, in general, in good agreement with those obtained from the X-ray single crystal investigation, however the optimized torsion angle C18-O2-C2-C3 describing the orientation of the methoxy group is significantly smaller than that provided by the X-ray analysis (Table 1 and for more details see Supplementary Table S2). ...
... Molecular orbital calculations with full geometry optimization of colchicine derivatives (6, 11, 12, 14, 15, 16, 18 and 19) were performed with the Gaussian09 program package [52]. All calculations were carried out with the DFT level using the Becke3-Lee-Yang-Parr correlation functional (B3LYP) [53][54][55][56] with the 6-31+G basis set assuming the geometry resulting from the X-ray diffraction study as the starting structure. As convergence criteria, the threshold limits of 0.00025 and 0.0012 a.u. ...
Colchicine is a well-known compound with strong antiproliferative activity that has had limited use in chemotherapy because of its toxicity. In order to create more potent anticancer agents, a series of novel colchicine derivatives have been obtained by simultaneous modification at C7 (amides and sulfonamides) and at C10 (methylamino group) positions and characterized by spectroscopic methods. All the synthesized compounds have been tested in vitro to evaluate their cytotoxicity toward A549, MCF-7, LoVo, LoVo/DX and BALB/3T3 cell lines. Additionally, the activity of the studied compounds was investigated using computational methods involving molecular docking of the colchicine derivatives to β-tubulin. The majority of the obtained derivatives exhibited higher cytotoxicity than colchicine, doxorubicin or cisplatin against tested cancer cell lines. Furthermore, molecular modeling studies of the obtained compounds revealed their possible binding modes into the colchicine binding site of tubulin.
... The binding site of colchicine on microtubules is located in the center of the tubulin dimer, right at the interface of a-and b-tubulin monomers [25][26][27][28] (Figure 4(A-C)). Binding to the colchicine site is followed by a conformational change involving an intradimer bending, where the tubulin monomers undergo twisting around the interface. ...
Cancer is a clinical situation caused by uncontrolled cell division and is responsible for a large number of deaths worldwide. Colchicine is a classical antimitotic, tubulin-binding agent (TBA) which is being explored for its antitumor activities, although its tubulin-binding ability leads to some toxicity toward normal cells proliferation. Colchicine derivatives are considered as potent antitumor compounds with less toxicity compared to colchicine. Derivatives with substituted functional groups at A-ring (methoxy), B-ring (acetamide) or C-ring (methoxy) have been synthesized via chemical and microbial routes and show modified bioactivities and altered tropolonic functionality. Earlier reports, in combination with our group’s research findings, suggest that microbial biotransformation is an efficient choice for the production of bioactive colchicine derivatives. This route has gained significant interest in the mass production of regio-specific, cost-effective, safe and eco-friendly derivatives. The present review paper critically analyzes and discusses the development and application of colchicine derivatives as a potent antitumor molecule and their production through a microbial transformation process. The information provided in this review might assist in the stimulation of new ideas regarding the development of alternative therapeutic agent(s) for cancer treatment.
... The vinca domain is present near the interchangeable GTP binding site of tubulin [plus end interface] [13,14], the taxane domain dwells in a deep hydrophobic compartment sideward between the compact protofilaments, within the lumen of the microtubule [at the minus end] [15,16,17]. However at the intra-dimer boundary between tubulin and tubulin is found the colchicine site [18,19]. ...
Tubulin/microtubule assembly and disassembly is characterized as one of the chief processes during cell growth and division. Hence drugs those perturb these process are considered to be effective in killing fast multiplying cancer cells. There is a collection of natural compounds which disturb microtubule/tubulin dis/assemblage and there have been a lot of efforts concerted in the marine realm too, to surveying such killer molecules. Close to half the natural compounds shooting out from marine invertebrates are generally with no traceable definite mechanisms of action though may be tough anti-cancerous hits at nanogram levels, hence fatefully those discoveries conclude therein without a capacity of translation from laboratory to pharmacy. Astoundingly at least 50% of natural compounds which have definite mechanisms of action causing disorders in tubulin/microtubule kinetics have an isolation history from sponges belonging to the Phylum: Porifera. Poriferans have always been a wonder worker to treat cancers with a choice of, yet precise targets on cancerous tissues. There is a specific order: Dictyoceratida within this Phylum which has contributed to yielding at least 50% of effective compounds possessing this unique mechanism of action mentioned above. However, not much notice is driven to Dictyoceratidans alongside the order: Demospongiae thus dictating the need to know its select microtubule/tubulin irritants since the unearthing of avarol in the year 1974 till date. Hence this review selectively pinpoints all the compounds, noteworthy derivatives and analogs stemming from order: Dictyoceratida focusing on the past, present and future.
... Additionally the gas-phase structures of molecules 2 and 6 using the DFT optimization were performed with the Gaussian09 program package [44] . All calculations were carried out by the DFT method using the Becke3-Lee–Yang–Parr correlation functional (B3LYP) [45][46][47][48]with the 6-31+G basis set, starting from the X-ray geometry of molecules. The gas-phase optimized conformations of 2 and 6 molecules are, in general, in good agreement with those obtained from the X-ray single crystal investigation (Table S2 and Fig. S21). ...
In order to create more potent anticancer agents, a series of five structurally different derivatives of Colchicine have been synthesised. These compounds were characterised spectroscopically and structurally and their antiproliferative activity against four human tumour cell lines (HL-60, HL-60/vinc, LoVo, LoVo/DX) was evaluated. Additionally the activity of the studied compounds was calculated using computational methods involving molecular docking of the Colchicine derivatives to β-tubulin. The experimental and computational results are in very good agreement indicating that the antimitotic activity of Colchicine derivatives can be readily predicted using computational modeling methods.
... Moreover, this compound forms the strongest hydrogen bond with O2A of GTP coenzyme in α-tubulin subunit with the bond length of 2.04 Å. Also, compound 4 is able to connect to α-tubulin subunit unlike colchicine that can only be connected to βtubulin subunit [33]. Collectively, it appeared from the above analysis that compound 4 was the most potent inhibitor in this compound series. ...
Apoptosis is a naturally occurring process by which a cell is directed to programmed cell death. Chemotherapy drugs affect the cancer cells by the apoptotic induction. During the present study, a series of 4H-chromene-3-carbonitrile was synthesized by one-pot method as the inducers of apoptosis. Cytotoxic effects of six compounds of 4H-chromene-3-carbonitrile were evaluated against five tumor cell lines, with the help of colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Compound 4 showed significant cytotoxic activity and was selected for conjugation with the synthesized gold nanoparticles by aspartic acid. Also, we evaluated apoptosis induction capacity of the selected compound with the help of fluorescent dyes and DNA fragmentation. The result showed that the conjugated and non-conjugated forms of compound 4 were effective in inducing apoptosis and conjugated one had more efficiency and reduced the effective dose. Also, molecular modeling experiments involving compound 4 and colchicine binding site of tubulin dimer showed several strong hydrogen bonds and hydrophobic interactions to many important amino acid residues and GTP.
... The taxane site resides in a deep hydrophobic pocket at the lateral interface between adjacent protofilaments, within the lumen of the microtubule [90][91][92][93]. The colchicine site is located at the intra-dimer interface between -tubulin andtubulin [94][95][96]. In addition to these three well characterized drug-binding sites, there is another binding site on -tubulin that is occupied by laulimalide (23), a microtubule-stabilizing drug isolated from the marine sponge Cacospongia mycofijiensis; however, the exact location of this binding site remains elusive [97,98]. ...
Plants are an important source of synthetic and herbal agents used in several pharmaceutical industries. Some of the prominent plant-derived compounds have played a major role in the development of several clinically useful anti-cancer agents such as vinblastine, vincristine, topotecan and paclitaxel (taxol). Himalayan plants, grown in high altitude are the rich source of various secondary metabolites such as anthraquinones, flavonoids, tannins, alkaloids, etc. This review article covers the active constituents isolated from several anticancerous plants of Himalaya and their effect against several anticancer cell lines.
