scholarly journals Mutations in the β-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding

Cell Cycle ◽  
2011 ◽  
Vol 10 (19) ◽  
pp. 3387-3396 ◽  
Author(s):  
Adrian Begaye ◽  
Shana Trostel ◽  
Zhiming Zhao ◽  
Richard E. Taylor ◽  
David C. Schriemer ◽  
...  
Keyword(s):  
Binding Site ◽  
Side Chain ◽  
Peloruside A ◽  
Tubulin Binding ◽  
Β Tubulin

scholarly journals Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in Chlamydomonas

2020 ◽  
Vol 133 (17) ◽  
pp. jcs249805 ◽  
Author(s):  
Julie Craft Van De Weghe ◽  
J. Aaron Harris ◽  
Tomohiro Kubo ◽  
George B. Witman ◽  
Karl F. Lechtreck
Keyword(s):  
Binding Site ◽  
Critical Concentration ◽  
Intraflagellar Transport ◽  
Microtubule Assembly ◽  
Strong Reduction ◽  
Tubulin Binding ◽  
Respective Contribution ◽  
Β Tubulin

ABSTRACTTubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP–β-tubulin by altering the IFT74N–IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP–β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP–β-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP–β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

Molecular basis for benzimidazole resistance from a novel β-tubulin binding site model

2013 ◽  
Vol 45 ◽  
pp. 26-37 ◽  
Author(s):  
Rodrigo Aguayo-Ortiz ◽  
Oscar Méndez-Lucio ◽  
Antonio Romo-Mancillas ◽  
Rafael Castillo ◽  
Lilián Yépez-Mulia ◽  
...  
Keyword(s):  
Binding Site ◽  
Molecular Basis ◽  
Site Model ◽  
Benzimidazole Resistance ◽  
Tubulin Binding ◽  
Β Tubulin

scholarly journals Diffusion rather than IFT provides most of the tubulin required for axonemal assembly

2018 ◽  
Author(s):  
J. Aaron Harris ◽  
Julie C. Van De Weghe ◽  
Tomohiro Kubo ◽  
George B. Witman ◽  
Karl F. Lechtreck
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Intraflagellar Transport ◽  
Strong Reduction ◽  
Tubulin Binding ◽  
High Concentrations ◽  
Β Tubulin

AbstractTubulin enters the cilia by diffusion and motor-based intraflagellar transport (IFT). The respective contributions of each route in providing tubulin for axonemal assembly are unknown. To attenuate IFT-based transport, we expressed modified GFP-tubulins in strains possessing IFT81 and IFT74 with altered tubulin binding sites. E-hook deficient GFP-β-tubulin normally incorporated into the axonemal microtubules; its transport frequency was reduced by ~90% in control cells and essentially abolished when expressed in a strain possessing IFT81 with an incapacitated tubulin-binding site. Despite the strong reduction in IFT, the share of E-hook deficient GFP-β-tubulin in the axoneme was only moderately reduced indicating that most axonemal tubulin (~80%) enters cilia by diffusion. While not providing the bulk of axonemal tubulin, we propose that motor-based IFT is nevertheless critical for ciliogenesis because it ensures high concentrations of tubulin near the ciliary tip promoting axonemal elongation.

scholarly journals Investigating the Specific Interactions Between Microtubule Stabilising Agents and β-Tubulin

2021 ◽  
Author(s):  
◽  
Benjamin Jones
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Binding Site ◽  
Drug Class ◽  
Efflux Pumps ◽  
Peloruside A ◽  
Mutant Cells ◽  
Β Tubulin

<p>Microtubule stabilising agents are a class of cytotoxic compounds that cause mitotic arrest through inhibition of microtubule function. They specifically target β-tubulin subunits promoting tubulin polymerisation, which eventually leads to cell death. Members of this drug class include the cancer chemotherapeutics paclitaxel and ixabepilone. However, like many cytotoxic agents, tumour cells often develop multi-drug resistance phenotypes limiting the effectiveness of such compounds. This results from the expulsion of these drugs from cells by efflux pumps, as well as mutation of their binding site. Much effort has been focused on improving the utility of this important drug class in the ongoing fight against cancer.  The microtubule stabilising agents peloruside A and laulimalide originate from marine sponge species native to the South Pacific. They have similar pharmacological profiles to paclitaxel and ixabepilone, however with several unique properties. They are poor substrates for efflux pumps and target a different region on β-tubulin subunits, giving them the potential for treatment of resistant tumours. This represents a novel mechanism of action that may be exploited for drug development, and further characterisation of the binding site is warranted.  The aim of this study is to investigate the contribution of two amino acids of human βItubulin to the interactions with peloruside A and laulimalide. Specifically, glu127 and lys124 have been predicted by computational modelling and analogue studies to form hydrogen bonds and other associations with the two compounds. These amino acids are located on β-tubulin subunits adjacent to the main binding pocket of peloruside A and laulimalide, and represent a potential inter-protofilament interaction that does not occur with other microtubule stabilising agents. This binding mechanism has not yet been shown by crystallography and is hence based solely on in silico work, requiring biological validation.  HEK293 cells were transfected with βI-tubulin with these amino acids mutated to alanines to prevent hydrogen bond formation. Cell proliferation assays, flow cytometry, and immunoblotting were used to study the effect loss of the inter-protofilament interaction has on the bioactivity of peloruside A and laulimalide. These mutations did not significantly alter the concentration-response of cells to either drug in the cell proliferation assay. However, accumulation of cells in the G2/M phase of the cell cycle and the proportion of transfected cells showing signs of mitotic arrest significantly decreased for E127A mutant cells compared to wild type βI-tubulin transfected control cells treated with peloruside A. Furthermore, a similar reduction in cell cycle block was also seen in E127A mutant cells treated with the negative control ixabepilone, which binds to a different site on β-tubulin.  No evidence seen in this study suggests that either amino acid plays a major role in peloruside A or laulimalide target binding. However, the amino acid E127 is important for inter-protofilament associations independent of drug treatment, as its mutation appeared to reduce global stability of microtubule structures. This information requires further validation, it may be useful in the design of future analogue syntheses as development of these promising drug candidates continues.</p>

3D QSAR studies for the β-tubulin binding site of microtubule-stabilizing anticancer agents (MSAAs)

Il Farmaco ◽  
2003 ◽  
Vol 58 (9) ◽  
pp. 659-668 ◽  
Author(s):  
Laura Maccari ◽  
Fabrizio Manetti ◽  
Federico Corelli ◽  
Maurizio Botta
Keyword(s):  
Binding Site ◽  
Anticancer Agents ◽  
3D Qsar ◽  
Qsar Studies ◽  
Tubulin Binding ◽  
Β Tubulin

Structure-Activity Relationship of Taxol Inferring from Docking Taxol Analogues to Microtubule Binding Site

2009 ◽  
Vol 64 (7-8) ◽  
pp. 551-556 ◽  
Author(s):  
Fu Xiang ◽  
Jiangyan Yu ◽  
Rui Yin ◽  
Yunfeng Ma ◽  
Longjiang Yu
Keyword(s):  
Binding Site ◽  
Specific Binding ◽  
Structure Activity Relationship ◽  
Activity Relationship ◽  
Side Chain ◽  
Structural Requirements ◽  
Microtubule Binding ◽  
Structure Activity ◽  
Relationship Of ◽  
Β Tubulin

In order to find the minimal structural requirements to maintain microtubule binding, 12 taxol analogues have been docked to the taxol binding site of tubulin. By comparing the interactions of each analogue with β-tubulin, the structure-activity relationships are summarized as follow: C-2 benzoyl and taxane ring systems are the essential groups for microtubule binding, the improvements of bioactivity and bioavailability are dependent on the substituents at positions C-1, C-4, C-7, C-9, C-10, and C-14, whereas the C-13 side chain mainly provides a specific binding.

scholarly journals Investigating the Specific Interactions Between Microtubule Stabilising Agents and β-Tubulin

2021 ◽  
Author(s):  
◽  
Benjamin Jones
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Binding Site ◽  
Drug Class ◽  
Efflux Pumps ◽  
Peloruside A ◽  
Mutant Cells ◽  
Β Tubulin

<p>Microtubule stabilising agents are a class of cytotoxic compounds that cause mitotic arrest through inhibition of microtubule function. They specifically target β-tubulin subunits promoting tubulin polymerisation, which eventually leads to cell death. Members of this drug class include the cancer chemotherapeutics paclitaxel and ixabepilone. However, like many cytotoxic agents, tumour cells often develop multi-drug resistance phenotypes limiting the effectiveness of such compounds. This results from the expulsion of these drugs from cells by efflux pumps, as well as mutation of their binding site. Much effort has been focused on improving the utility of this important drug class in the ongoing fight against cancer.  The microtubule stabilising agents peloruside A and laulimalide originate from marine sponge species native to the South Pacific. They have similar pharmacological profiles to paclitaxel and ixabepilone, however with several unique properties. They are poor substrates for efflux pumps and target a different region on β-tubulin subunits, giving them the potential for treatment of resistant tumours. This represents a novel mechanism of action that may be exploited for drug development, and further characterisation of the binding site is warranted.  The aim of this study is to investigate the contribution of two amino acids of human βItubulin to the interactions with peloruside A and laulimalide. Specifically, glu127 and lys124 have been predicted by computational modelling and analogue studies to form hydrogen bonds and other associations with the two compounds. These amino acids are located on β-tubulin subunits adjacent to the main binding pocket of peloruside A and laulimalide, and represent a potential inter-protofilament interaction that does not occur with other microtubule stabilising agents. This binding mechanism has not yet been shown by crystallography and is hence based solely on in silico work, requiring biological validation.  HEK293 cells were transfected with βI-tubulin with these amino acids mutated to alanines to prevent hydrogen bond formation. Cell proliferation assays, flow cytometry, and immunoblotting were used to study the effect loss of the inter-protofilament interaction has on the bioactivity of peloruside A and laulimalide. These mutations did not significantly alter the concentration-response of cells to either drug in the cell proliferation assay. However, accumulation of cells in the G2/M phase of the cell cycle and the proportion of transfected cells showing signs of mitotic arrest significantly decreased for E127A mutant cells compared to wild type βI-tubulin transfected control cells treated with peloruside A. Furthermore, a similar reduction in cell cycle block was also seen in E127A mutant cells treated with the negative control ixabepilone, which binds to a different site on β-tubulin.  No evidence seen in this study suggests that either amino acid plays a major role in peloruside A or laulimalide target binding. However, the amino acid E127 is important for inter-protofilament associations independent of drug treatment, as its mutation appeared to reduce global stability of microtubule structures. This information requires further validation, it may be useful in the design of future analogue syntheses as development of these promising drug candidates continues.</p>

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection

2020 ◽  
Vol 16 (4) ◽  
pp. 451-459 ◽  
Author(s):  
Fortunatus C. Ezebuo ◽  
Ikemefuna C. Uzochukwu
Keyword(s):  
Molecular Dynamics ◽  
Amino Acid ◽  
Binding Energy ◽  
Binding Site ◽  
Conformational Dynamics ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Conformational Selection ◽  
Hybrid Mechanism ◽  
Dynamics Simulations

Background: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. Objective: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Methods: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it’s missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. Results: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. Conclusion: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

Crystal structure of tubulin folding cofactor A from Arabidopsis thaliana and its β-tubulin binding characterization

FEBS Letters ◽  
2010 ◽  
Vol 584 (16) ◽  
pp. 3533-3539 ◽  
Author(s):  
Lu Lu ◽  
Jie Nan ◽  
Wei Mi ◽  
Lan-Fen Li ◽  
Chun-Hong Wei ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Tubulin Binding ◽  
Β Tubulin

A Role of the Heme-7-Propionate Side Chain in Cytochrome P450cam as a Gate for Regulating the Access of Water Molecules to the Substrate-Binding Site

2009 ◽  
Vol 131 (4) ◽  
pp. 1398-1400 ◽  
Author(s):  
Takashi Hayashi ◽  
Katsuyoshi Harada ◽  
Keisuke Sakurai ◽  
Hideo Shimada ◽  
Shun Hirota
Keyword(s):  
Binding Site ◽  
Substrate Binding ◽  
Side Chain ◽  
Water Molecules ◽  
Cytochrome P450cam ◽  
Substrate Binding Site
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