Microtubule targeting agents: from biophysics to proteomics

Abstract

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug-tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule-tubulin equilibrium and tubulin-drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton.

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

“…These ligands have different binding sites on tubulin and slightly different affinities. As discussed above, the affinity of L1 for tubulin dimer is reported to be in the 0.5×10 6 to 3×10 6 M -1 range, whereas for L9 the affinity is slightly lower, 0.1×10 6 to 0.5×10 6 M -1 [5,32]. Shown in Figure 3a is a representative ESI mass spectrum acquired for a solution of tubulin (14 μM) with L1 (14 μM) and L9 (14 μM); shown in Figure 3b-f are CID mass spectra measured in the Trap region at collision energies ranging from 25 to 120 V. Signals corresponding to both protonated L1 (m/z 400.1) and L9 (m/z 825.4) are evident in the CID spectra, and the L1 to L9 abundance ratios reach a constant value at Trap voltages ≥80 (Figure 4a).…”

“…Shown in Figure S7a and b (Supplementary Information) are CID mass Abundance ratios of L1 to L9 ions (green bars) released by CID (in Trap) from tubulin ions, produced by ESI performed in positive ion mode on an aqueous ammonium acetate solution (100 mM, pH 6.8) containing 0.05% DMSO (v/v) and tubulin (14 μM) with L1 and L9 at equimolar concentrations (2, 7, and 14 μM). The average value of the abundance ratio determined from these three concentrations is shown, as well as the expected value based on the reported affinities (Lit., shown in blue) [5,32] spectra acquired at Trap and Transfer voltages of 100 and 1 V, respectively ( Figure S7a, Supplementary Information), and 1 and 100 V, respectively ( Figure S7b, Supplementary Information). In both cases, abundant protonated L1 is detected.…”

“…T he αβ-tubulin heterodimers are the building blocks of microtubules (MTs) [1], which are long, hollow, cylindrical protein polymers and dynamic structural components of the eukaryotic cytoskeleton [2][3][4][5]. Both the α and β subunits exist as different isotypes in many organisms, with dissimilar relative amounts, e.g., there are six main isoforms of both α (αI-VI) and β (βI-VI) subunits in mammals, differing from each other in their amino acid sequences and encoded by different genes [6,7].…”

Tubulin-interactive stilbene derivatives as anticancer agents

“…It exerted aerobic metabolic stability and displayed the efficiency of hypoxiarelated release of CA-4 [44]. Investigations are ongoing into formulations that improve the bioavailability of MIAs, including nanoparticles, which are suggested to provide effective and selective delivery of cytotoxic agents to cancer cells [45]. Based on the results of preclinical studies, CA-4 derivatives were suggested to be effective for retinal neovascularization and choroidal neovascularization [28][29][30]46].…”

“…A set of novel colchicine derivatives with selective affinity to the colchicine domain of the αβIII isoform was synthesized. Their cytotoxic action correlated with the expression levels of specific tubulin isotypes [45,111]. The discovery of novel tubulin-binding agents that could selectively bind specific isoforms will be the aim of further research with the use of molecular modeling.…”