Tubulin Inhibitors: Decades of controversy
The interaction of Rigosertib (ON-01910) with Tubulin has been studied since the early 2010s, with some initial reports around 2012-2014 suggesting that rigosertib may function as a microtubule destabilizer.
In the last decade, conflicting data on the effectiveness of Rigosertib have accumulated. The effectiveness of Rigosertib in vitro was achieved (or not achieved) only at elevated concentrations 10 mkM -50 mkM of the inhibitor compared to the effectiveness (nM)
in vivo experimental cell lines.
Also, special attention was paid to the purity of the obtained preparations, which contain a small amount of chemically active substances ON01500. A typical batch of clinical material is 99.9% pure and could include 0.1% of these impurities. Several storage conditions, including higher temperature, acidic pH and exposure to intense light can lead to the degradation of rigosertib into ON01500. Rigosertib purchased from Selleckchem contained approximately 5% of ON01500 as well as a few additional contaminants, while clinical grade Rigosertib obtained from Onconova Therapeutics had undetectable amounts of these contaminants.
Microscale thermophoretic analysis of ON01500 and VIN with purified tubulin. Tubulin was labeled using the Monolith NT protein labeling kit RED-NHS according to the instructions of the manufacturer. Labeled protein was incubated with increasing concentrations of rigosertib, ON01500, or VIN for 30 min and subjected to MST.
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
Determination the tubulin efficiency inhibitors by calculation methods.
However, debates about its actual mechanism of action persisted. There is some controversy surrounding the effect of rigosertib on microtubule assembly, as the concentration of rigosertib required for in vitro experiments is much higher to destabilize microtubules, compared to in vivo experiments, where a much lower concentration is required to destabilize microtubules.Some of the researchers claim that It is also important to note that many microtubuledestabilizing agents require substantially higher concentrations in vitro for robust microtubuledestabilizing activity as compared to cell culture.
Part 1.0.
The interaction between Rigosertib and Tubulin has been investigated in various studies, employing different experimental approaches and concentrations.
Below is a summary table highlighting key studies, their methodologies, rigosertib concentrations used, and the corresponding findings:
Below is a summary table highlighting key studies, their methodologies, rigosertib concentrations used, and the corresponding findings:
Part 1.2.
Part 1.2. How to put an end to the effectiveness of tubulin inhibitors?
The topic of the second video: Determining the efficiency of tubulin inhibitors using computational methods.Tubulins form various molecular formations in solution as they reach thermodynamic equilibrium. The study of the thermodynamic equilibrium of molecular formations in solution is the objective of our research. The results of the computational studies presented in this video were performed using the open AI platform on the Binomlabs website, the link is in the description under the video.
Historical overview of research results Rigosetib investigations
Clinical grade rigosertib, which is free of this impurity, does not exhibit tubulin binding activity. In vivo, cell lines that express mutant b-tubulin (TUBBL240F) were also reported to be resistant to the effects of rigosertib. However, our studies showed that both wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells
[Mechanism of action of rigosertib does not involve tubulin binding]
Stacey J. Baker, Stephen C. Cosenza, Saikrishna Athuluri-Divakar, M.V. Ramana Reddy, Rodrigo Vasquez-Del Carpio, Rinku Jain, Aneel K Aggarwal, E. Premkumar Reddy
doi: https://doi.org/10.1101/2019.12.12.874719
[Mechanism of action of rigosertib does not involve tubulin binding]
Stacey J. Baker, Stephen C. Cosenza, Saikrishna Athuluri-Divakar, M.V. Ramana Reddy, Rodrigo Vasquez-Del Carpio, Rinku Jain, Aneel K Aggarwal, E. Premkumar Reddy
doi: https://doi.org/10.1101/2019.12.12.874719
Клинический ригосертиб, свободный от этой примеси, не проявляет активности связывания тубулина. Также сообщалось, что in vivo клеточные линии, экспрессирующие мутантный b-тубулин (TUBBL240F), устойчивы к воздействию ригосертиба. Однако наши исследования показали, что как клетки дикого типа, так и клетки, экспрессирующие TUBBL240F, не могли пролиферировать в присутствии ригосертиба в концентрациях, которые летальны для клеток дикого типа.
Rigosertib, at high micromolar concentrations (>20µM), showed microtubule depolymerizing activity
Wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of clinical grade rigosertib at concentrations that are lethal to wild-type cells
Purified preparation of Rigosertib obtained from Onconova (O-RGS) shows little or no tubulin depolymerizing activity at doses up to 50µM.
Rigosertib purchased from Selleckchem (S-RGS), on the other hand, exhibits depolymerizing activity when used at concentrations above 25µM, reaching complete depolymerization at a concentration of 100µM (Fig. 1D), suggestive of the effect of the impurity.
Expression of the L240F beta-Tubulin mutant provides resistance to Rigosertib, suggesting that tubulin binding is critical to its cytotoxic activity. For these experiments, Jost et al. (2017) prepared lentiviral constructs that encoded empty vector or wildtype (wt) TUBB or TUBB L240F, which were individually transduced into wt K562, HeLa, or H358 cells.
Wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of clinical grade rigosertib at concentrations that are lethal to wild-type cells
Purified preparation of Rigosertib obtained from Onconova (O-RGS) shows little or no tubulin depolymerizing activity at doses up to 50µM.
Rigosertib purchased from Selleckchem (S-RGS), on the other hand, exhibits depolymerizing activity when used at concentrations above 25µM, reaching complete depolymerization at a concentration of 100µM (Fig. 1D), suggestive of the effect of the impurity.
Expression of the L240F beta-Tubulin mutant provides resistance to Rigosertib, suggesting that tubulin binding is critical to its cytotoxic activity. For these experiments, Jost et al. (2017) prepared lentiviral constructs that encoded empty vector or wildtype (wt) TUBB or TUBB L240F, which were individually transduced into wt K562, HeLa, or H358 cells.
M. Jost, Y. Chen, L.A. Gilbert, M.A. Horlbeck, L. Krenning, G. Menchon, A. Rai, M.Y. Cho, J.J. Stern, A.E. Prota, et al. [Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent] Mol. Cell, 68 (2017), pp. 210-223
[Mechanism of action of rigosertib does not involve tubulin binding]
[Mechanism of action of rigosertib does not involve tubulin binding]
С другой стороны, ригосертиб, приобретенный у Selleckchem (S-RGS), проявляет деполимеризующую активность при использовании в концентрациях выше 25 мкМ, достигая полной деполимеризации при концентрации 100 мкМ (рис. 1D), что свидетельствует о влиянии примеси.
M. Jost, Y. Chen, L.A. Gilbert, M.A. Horlbeck, L. Krenning, G. Menchon, A. Rai, M.Y. Cho, J.J. Stern, A.E. Prota, et al.
[Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent]
Mol. Cell, 68 (2017), pp. 210-223
[Mechanism of action of rigosertib does not involve tubulin binding]
[Mechanism of action of rigosertib does not involve tubulin binding]
С другой стороны, ригосертиб, приобретенный у Selleckchem (S-RGS), проявляет деполимеризующую активность при использовании в концентрациях выше 25 мкМ, достигая полной деполимеризации при концентрации 100 мкМ (рис. 1D), что свидетельствует о влиянии примеси.
M. Jost, Y. Chen, L.A. Gilbert, M.A. Horlbeck, L. Krenning, G. Menchon, A. Rai, M.Y. Cho, J.J. Stern, A.E. Prota, et al.
[Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent]
Mol. Cell, 68 (2017), pp. 210-223
Examined the binding affinity of pure ON01500, O-RGS and vincristine to purified Tubulin preparations using microscale thermophoresis (MST) (Wienken et al., 2010). The results of this study, shown in figure, demonstrate that both vincristine and ON01500 bind to tubulin with similar high affinities, which are reflected in their dissociation constants of Kd=74nM and Kd=21nM, respectively. When we examined the binding of
O-RGS using this technique, we were unable to detect binding even at a concentration of 50µM
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
O-RGS using this technique, we were unable to detect binding even at a concentration of 50µM
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
Сalculation scheme Description.
Description of graphs and diagrams
The complexity of the analysis of the effect of mutation on tubulin binding to rigosertib is that wild-type tubulins are slow to attach a rigosertib molecule, and mutant forms are even less susceptible to rigosertib.
Figure A shows a dimer of the wild-type tubulins alpha and beta, containing two small molecules of guanosine triphosphate.
Above the dimer, two calculated values are given - the stability parameter lg(cond(W)) and the dissociation constant lg[Kd].
The first parameter lg(cond(W)) is the stability parameter, characterizing the stability of the dimeric complex from the point of view of quantum mechanics. The smaller this parameter, the more stable the biochemical compound and the closer its state to thermodynamic equilibrium.
Next to the wild-type dimer, other dimers containing mutations are shown, for example, or containing small molecules and inhibitors, such as Rigosertib, marked in red in
Figures C and D.
Arrows indicate the direction to thermodynamic equilibrium. Earlier, in experimental studies, it was suggested that the L240F mutation protects microtubules from the effects of rigosertib.
According to our calculations, this assumption has some basis, since tubulins containing this mutation are characterized by a slight decrease in stability when combined with a molecule of Rigosertib compared to wild-type compounds.
Thus, using calculation methods, we can compare the stability of dimeric formations that differ from each other in mutations, as well as in the attached inhibitors and small molecules
The complexity of the analysis of the effect of mutation on tubulin binding to rigosertib is that wild-type tubulins are slow to attach a rigosertib molecule, and mutant forms are even less susceptible to rigosertib.
Figure A shows a dimer of the wild-type tubulins alpha and beta, containing two small molecules of guanosine triphosphate.
Above the dimer, two calculated values are given - the stability parameter lg(cond(W)) and the dissociation constant lg[Kd].
The first parameter lg(cond(W)) is the stability parameter, characterizing the stability of the dimeric complex from the point of view of quantum mechanics. The smaller this parameter, the more stable the biochemical compound and the closer its state to thermodynamic equilibrium.
Next to the wild-type dimer, other dimers containing mutations are shown, for example, or containing small molecules and inhibitors, such as Rigosertib, marked in red in
Figures C and D.
Arrows indicate the direction to thermodynamic equilibrium. Earlier, in experimental studies, it was suggested that the L240F mutation protects microtubules from the effects of rigosertib.
According to our calculations, this assumption has some basis, since tubulins containing this mutation are characterized by a slight decrease in stability when combined with a molecule of Rigosertib compared to wild-type compounds.
Thus, using calculation methods, we can compare the stability of dimeric formations that differ from each other in mutations, as well as in the attached inhibitors and small molecules
Part 5: Figures a)-b)-c)
Part 6: Figures d)-e)-f)
Figure (a) stability parametr lg(cond(W))
Figure (a) shows the calculated stability lg(cond(W)) data for the wt dimers [tba-tbb] containing a mutation in the TBB protein that was not toxic when overexpressed. Note that the stability levels for the wild-type and mutant dimers are approximately the same, with the differences in stability occurring in the third decimal place. The following mutations (yellow in the graph) are found in the disease. The effect on the stability of tubulin dimers can be seen for each mutation in TBA protein.
Figures (b)-(c)
Figures (b) and (c) are symmetrical with respect to dimeric mutations, so we will consider them simultaneously. The neutral L240F mutation in the dimer, which does not disrupt tubulin assembly upon overexpression, is shown in green. The dissociation constant Kd is approximately at the same level as for the wt dimer, and the dimer with L240F mutation is characterized by a minimal change in the level of ordering compared to the wild-type dimer.
Mutations in TBA occur in the lissencephaly disease, and the calculated values are given for dimers containing one mutation in the TBA protein. We interpret the results for the “yellow” mutations as follows:
the more the calculated data for mutant dimers deviate from the calculated data obtained for wild-type dimers, the more noticeable are the differences in the formation and behavior of the assembly of mutant tubulins as a whole.
For example, if we consider the entropic contribution to the formation of a mutant dimer, then the presence of a negative value of the entropy change value may indicate the complexity of the formation and transitions in the formation of such a dimer.
Mutations in TBA occur in the lissencephaly disease, and the calculated values are given for dimers containing one mutation in the TBA protein. We interpret the results for the “yellow” mutations as follows:
the more the calculated data for mutant dimers deviate from the calculated data obtained for wild-type dimers, the more noticeable are the differences in the formation and behavior of the assembly of mutant tubulins as a whole.
For example, if we consider the entropic contribution to the formation of a mutant dimer, then the presence of a negative value of the entropy change value may indicate the complexity of the formation and transitions in the formation of such a dimer.
Figures (d)-(e)
Figures (d)-(e) contain similar results of stability calculations and entropy change measures for different types of TBA-TBB protein dimers. Now we have attached one GTP molecule to each dimer and calculated stability and entropy change measures for different types of mutations. Toxic TBB protein mutations and their corresponding calculated data are shown in blue.
TBA mutations occurring in the specified disease are shown in yellow. The effect of each mutation on dimer assembly, taking into account the addition of a GTP molecule, can be traced in two graphs of calculated values.
Toxic “blue” TBB protein mutations sharply increase the stability of dimer formations, which has a negative effect on the subsequent ability to attach subsequent tubulins.
Negative areas of the entropy change measure also indicate an increase in the orderliness of the mutant dimer system.
How toxic the yellow type of mutations are can be judged from a comparative analysis between two types of mutations with known properties (marked in blue) and mutations that occur in diseases
TBA mutations occurring in the specified disease are shown in yellow. The effect of each mutation on dimer assembly, taking into account the addition of a GTP molecule, can be traced in two graphs of calculated values.
Toxic “blue” TBB protein mutations sharply increase the stability of dimer formations, which has a negative effect on the subsequent ability to attach subsequent tubulins.
Negative areas of the entropy change measure also indicate an increase in the orderliness of the mutant dimer system.
How toxic the yellow type of mutations are can be judged from a comparative analysis between two types of mutations with known properties (marked in blue) and mutations that occur in diseases
Figure (f)
Fig. (f) shows the process of “flow” of all kinds of dimeric formations containing GTP molecules and rigosertib molecules (marked in red) in the direction of the most stable compounds. Let me remind you that the least value of the calculated value of lg(cond(W)) corresponds to the most stable dimer.
Dimers characterized by the greatest value of lg(cond(W)) are marked as “unstable”
Dimers characterized by the greatest value of lg(cond(W)) are marked as “unstable”
Rigosertib and L240F
Comparison of thermodynamic quantities for different variants of Rigosertib and derivative.
The stability parameter lg(cond(W)) tends to a minimum under conditions of thermodynamic equilibrium.
lg[Kd]
T(delta)H
The stability parameter lg(cond(W)) tends to a minimum under conditions of thermodynamic equilibrium.
lg[Kd]
T(delta)H
Simplified version
Comparison of thermodynamic quantities for different variants of Rigosertbi and derivative.
The stability parameter lg(cond(W)) tends to a minimum under conditions of thermodynamic equilibrium.
lg[Kd]
T(delta)H
The stability parameter lg(cond(W)) tends to a minimum under conditions of thermodynamic equilibrium.
lg[Kd]
T(delta)H
Investigation stability of tubulin dimers upon the addition of various inhibitors: Rigosertib and oN01500
Rigosertib does not provide sufficient stability of the tetramer compared to its derivative oN015
Rigosertib and oN01500
Comparison of thermodynamic quantities for different variants of Rigosertib and oN01500 derivative.
The stability parameter lg(cond(W)) tends to a minimum under conditions of thermodynamic equilibrium.
Also presented are studies of mutant tubulin forms containing a non-toxic mutation and the effect of this mutation on binding to inhibitors.
lg[Kd]
T(delta)H
The stability parameter lg(cond(W)) tends to a minimum under conditions of thermodynamic equilibrium.
Also presented are studies of mutant tubulin forms containing a non-toxic mutation and the effect of this mutation on binding to inhibitors.
lg[Kd]
T(delta)H
The arrow indicates the direction of increasing stability of the molecular complex, starting with dimers and ending with tetramers containing small inhibitors or GTP molecules.
Blue background shows toxic mutations in TBB during overexpression, yellow background shows mutations in TBA that occur in the disease.
Figures (a),(c), (e), (g)-(i) containe stability information for dimers and tetramers.
Figures (b),(d),(f) contain information about entropy change for dimers and tetramers.
Blue background shows toxic mutations in TBB during overexpression, yellow background shows mutations in TBA that occur in the disease.
Figures (a),(c), (e), (g)-(i) containe stability information for dimers and tetramers.
Figures (b),(d),(f) contain information about entropy change for dimers and tetramers.
Detailed representation of the final tetrameric formation in this study with stability values shown in log scale.
Comparison of thermodynamic quantities
The stability parameter lg(cond(W)) tends to a minimum under conditions of thermodynamic equilibrium.
lg[Kd]
T(delta)H
The stability parameter lg(cond(W)) tends to a minimum under conditions of thermodynamic equilibrium.
lg[Kd]
T(delta)H
Part 6 is devoted to thermodynamic quantities calculated for monomeric and dimeric tubulins containing small inhibitors with different affinities to tubulin proteins
The first digit represents the stability value, the second blue digit represents the logarithm of the dissociation constant, the third digit represents the change in potential energy as the difference between the energy of the mutant tree and the energy of the wild-type protein. The last digit represents the difference in the rate of change of entropy.
The first digit represents the stability value, the second blue digit represents the logarithm of the dissociation constant, the third digit represents the change in potential energy as the difference between the energy of the mutant tree and the energy of the wild-type protein. The last digit represents the difference in the rate of change of entropy.
Part 7.
A short video devoted to the binding of tubulin to small inhibitors, calculation of thermodynamic parameters.
This diagram illustrates the biochemical interactions of alpha and beta tubulin. These proteins form monomers and dimers, which are essential for microtubule assembly in cells. Figure A contains predicted data for monomers that can bind inhibitors, small molecules, and contain a toxic mutation that interferes with tubulin assembly. The first digit represents the stability value, the second blue digit represents the logarithm of the dissociation constant, the third digit represents the change in potential energy as the difference between the energy of the mutant protein and the energy of the wild type protein. The last digit represents the difference in the rate of change in entropy.
Tubulin proteins can also bind to small molecules and inhibitors, influencing dimer formation and affecting overall stability. The arrows indicate transitions between different states, progressing from less stable to more stable structures.
Understanding these molecular interactions is crucial for biochemistry and pharmaceutical research. Tubulin stability plays a key role in drug development, particularly in cancer therapies that target microtubules.
Tubulin proteins can also bind to small molecules and inhibitors, influencing dimer formation and affecting overall stability. The arrows indicate transitions between different states, progressing from less stable to more stable structures.
Understanding these molecular interactions is crucial for biochemistry and pharmaceutical research. Tubulin stability plays a key role in drug development, particularly in cancer therapies that target microtubules.
Сonsideration some experimental results:
cell lines, which express the mCherry marker, were combined at a 1:1 ratio with their respective parental lines and treated with rigosertib or DMSO, and the fraction of TUBB-expressing cells was measured up to 7 days after treatment as mCherry-positive cells by flow cytometry. An elevated ratio of mCherry-expressing cells after rigosertib treatment compared with DMSO was interpreted to indicate that expression of L240F mutant tubulin confers resistance.
Obtained lentiviral vectors that encode wt tubulin or the L240F beta-tubulin mutant from Dr. Weissman’s laboratory and repeated these studies using K562 cells that were transduced with empty vector or the TUBB L240F expression vector.
72 hours post-infection, the cells were mixed 1:1 with uninfected cells and treated with DMSO, increasing concentrations of rigosertib, a pan-PLK inhibitor as a control. Growth curves of the mCherry+ cells that express TUBB L240F and that of mCherry- cells (uninfected controls) is shown in Fig 3A. The results of this study show that both mCherry+ cells that express TUBBL240F or empty vector and mCherry- cells that represent parental cells are inhibited by rigosertib as well as BI2536 in a concentration-dependent manner with very similar kinetics.
72 hours post-infection, the cells were mixed 1:1 with uninfected cells and treated with DMSO, increasing concentrations of rigosertib, a pan-PLK inhibitor as a control. Growth curves of the mCherry+ cells that express TUBB L240F and that of mCherry- cells (uninfected controls) is shown in Fig 3A. The results of this study show that both mCherry+ cells that express TUBBL240F or empty vector and mCherry- cells that represent parental cells are inhibited by rigosertib as well as BI2536 in a concentration-dependent manner with very similar kinetics.
Studies using K562 cells
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
Cells treated with rigosertib at concentrations of 100 or 200nM had less that 10% viable cells on day 6 compared to their untreated controls, suggesting that expression of mutant TUBB had no effect on the growth inhibitory or apoptosis-inducing activities of rigosertib
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
examined the ratio of mCherry+ cells in the small viable fraction that remained at days 4 and 6, we observed a slightly higher fraction of TUBB L240F expressing cells in rigosertibtreated cells. Thus, the ratio of mCherry+ cells in vehicle-treated cultures was approximately 50% on days 0,4 and 6 and this ratio of mCherry+ cells increased to 65% to 70% in cells that express L240FTUBB. While this increase was statistically not significant, this trend was repeatedly seen in multiple experiments. This observation is consistent with that made by Jost et al (2017), who interpreted this population to be rigosertib-resistant cells. However, when we examined the surviving cells under the microscope, they were unusually large in size with a cellular morphology that is characteristic of senescent cells.
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
Growth curves of the mCherry+ cells that stably express TUBB L240F and vector control are shown in Fig 4B. Cell cultures expressing L240 mutant tubulin as well as empty vector expressing cells that were treated with rigosertib at concentrations of 100 or 200nM had fewer than 10% viable cells remaining at day 6. When it was examine the ratio of mCherry+ cells in this viable fraction, we observed a slightly higher fraction of TUBB L240F expressing cells in rigosertib-treated cultures.
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
