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. Several storage conditions, including higher temperature, acidic pH and exposure to intense light can lead to the degradation of rigosertib into ON01500.
AI platform for discovering and validating novel target combinations
Get the size distribution of biochemical compounds in solution
Identify the most stable biochemical formations
Select the temperature and salt concentration of the solution
Identify the effectiveness of the inhibitor at different levels of biochemical formation
Identify the most effective inhibitor
Identify the order of assembly of a large biochemical formation.
Input parameters for calculating the direction of biochemical reactions, finding transitional and final formations
A set of small chemical molecules and proteins that participate in the formation of biochemical structures of varying complexity
List of molecules and conditions for the system.
ON01500, Rigosertib, GTP and tubuline proteins.
ON01500, Rigosertib, GTP and tubuline proteins.
A calculation graph indicating:
Compounds with Rig are transitional,
the most stable monomeric formation
does not contain Rig
- molecules,
- biochemical formations, and
- the direction of biochemical processes.
Compounds with Rig are transitional,
the most stable monomeric formation
does not contain Rig
Comparative analysis of the monomeric compound's stability binding to GTP and Rig
According to the comparative analysis, the dimer containing GTP is the most stable compared to the dimer containing Rig according to the calculated values:
lg(cond(W)), lg[Kd]
lg(cond(W)), lg[Kd]
Comparative analysis of dimeric compounds involving GTP and Rig chemical small molecules
Compared to calculations performed for monomeric tubulins,
the difference between dimers is insignificant in binding GTP and Rig
the difference between dimers is insignificant in binding GTP and Rig
According to the numerical comparative analysis, it can be seen that GTP molecules bind preferentially to the tubulin trimer than Rig.
Comparative analysis of trimers containing GTP and Rig molecules
This diagram of biochemical processes of wild type tubulin proteins, as well as mutated type tubulin proteins, which form dimers and monomers when GTP and Rig molecules are added to the solution.
Arrows indicate the calculated values of the movement of biochemical processes within the framework of the given formations
The system is in search of molecular formations that meet the above conditions of thermodynamic equilibrium.
Arrows indicate the calculated values of the movement of biochemical processes within the framework of the given formations
The system is in search of molecular formations that meet the above conditions of thermodynamic equilibrium.
Let's add the mutation L240F, which in some sources is indicated as protecting the assembly of microtubules from the destructive effects of Rig.
Let's move on to the analysis of the most stable monomers. As can be seen from the graphs, Rig does not contribute to increasing the stability of the monomers and the monomers, in turn,
"spit out" the Rigosertib
"spit out" the Rigosertib
Conclusion. Given that Rig. has a low affinity for tubulin proteins in vitro (Rig. reduces the stability of monomeric tubulin), the protective properties of the mutation must be analyzed under conditions of low binding to tubulin.
Computational analysis demonstrated a minimal difference in affinity between the wild type and mutant tubulin protein, however, indicating that mutant tubulins have an even lower affinity for tubulin
Computational analysis demonstrated a minimal difference in affinity between the wild type and mutant tubulin protein, however, indicating that mutant tubulins have an even lower affinity for tubulin
The diagram contains the calculated data for each firm, the arrows go from the value with the maximum value to the side with the minimum values of the Kd and cond(W)) quantities.
The most stable tetramer of four tubulins was found, which does not contain additional chemical molecules.
The most stable tetramer of four tubulins was found, which does not contain additional chemical molecules.
We expand our system to the simultaneous presence of both dimers and tetramers of tubulin proteins.
Comparative numerical analysis
Rigosertib or ON01500?
Let's make biochemical graphs for tetramers, calculate thermodynamic parameters for each formation, find intermediate and final biochemical formations
Let's analyze these compounds and find the lowest stability values lg(cond(W))
The numerical difference in the stability of compounds containing Rig and oN015 is observed already in the first decimal place on a logarithmic scale.
The numerical difference in the stability of compounds containing Rig and oN015 is observed already in the first decimal place on a logarithmic scale.
Let us conduct a comparative computational analysis for tubulin tetramers containing small chemical molecules (inhibitors):
GTP, Rig, oN015000
GTP, Rig, oN015000
The trimer system contains a mutation and small chemical molecules. Each tetramer is characterized by a set of numerical values.
Arrows connect tetramers from the highest values to the lowest values.
The minimum values lg(cond(W))=4.58 correspond to tetramers containing oN015000
Arrows connect tetramers from the highest values to the lowest values.
The minimum values lg(cond(W))=4.58 correspond to tetramers containing oN015000
Now we will add the presence of mutations in the system under study.
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.
Also presented are studies of mutant tubulin forms containing a non-toxic mutation and the effect of this mutation on binding to inhibitors.
Conclusion: tubulin "spits out" the Rigosertib compound,
__________oN01500 actively interacts with tubulin
__________oN01500 actively interacts with tubulin
Historical overview of research results Rigosetib investigations.
Research summary of various tubulin experiments
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:
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]
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]
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 and repeated these studies using K562 cells that were transduced with empty vector or the TUBB L240F expression vector.
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 A.
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 A.
Studies using K562 cells. The results of this study show that both mCherry+ cells that express TUBBL240F or empty vector and mCherry- cells are inhibited by rigosertib as well as BI2536 in a concentration-dependent manner with very similar kinetics.
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
Protective functions of L240F mutation, does it?
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, was observed a slightly higher fraction of TUBB L240F expressing cells in rigosertibtreated cells. Thus, the ratio of mCherry+ cells in vehicle-treated cultures was increased from 65% to 70% in cells that express L240FTUBB. While this increase was statistically not significant, this trend was repeatedly seen in multiple experiments.
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
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.
K562 cells expressing TUBBL240F were combined with WT cells and seeded at a density of 1 × 105 cells/mL. The cells were treated with the indicated concentrations of rigosertib or BI2536, and the percentage of mCherry+ cells was determined by flow cytometry. The percentage of mCherry+ cells also increases in the presence of BI2536, indicating that this is not a rigosertib-specific phenomenon.
[A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent]
