Promotion of tubulin assembly by poorly soluble taxol analogs

Abstract

Promotion or inhibition of tubulin assembly into microtubules is the standard in vitro assay for evaluating potential antimicrotubule agents. Many agents to be tested are poorly soluble in aqueous solution and require a cosolvent such as dimethyl sulfoxide (DMSO). However, DMSO itself can promote tubulin assembly, and its inclusion in assays for compounds that induce tubulin assembly complicates interpretation of the results. Substituting GDP for GTP in the exchangeable nucleotide binding site of tubulin produces a less active form of the protein, tubulin-GDP. Here it is shown that tubulin-GDP can be assembled into normal microtubules in DMSO concentrations up to 15% (v/v), and polymerization assays performed under these conditions can be compared with assays run under more standard conditions. Assays for measuring the effective concentration of a ligand for promotion of tubulin assembly (EC(50)), measuring the concentration for inhibition of tubulin assembly (IC(50)) by a colchicine site ligand, and measuring tubulin critical concentrations in the presence of poorly soluble taxol derivatives are illustrated.

Fig. 1

Structure of TG-185-219.

Fig. 2

Solubility of taxol as a function of DMSO concentration. The concentration of taxol soluble at different concentrations of DMSO was determined as described under Materials and Methods.

Fig. 3

Polymerization of tubulin-GDP, 15 μM (■), 13.5 μM (▽), 12 μM (▼), 10.5 μM (○) and 9 μM (●) induced by 15% DMSO. Tubulin-GDP samples in PME buffer were incubated with pre-warmed 15% DMSO at 37 °C for 53 min and the extent of assembly was monitored in terms of apparent absorption at 350 nm.

Fig. 4

The extent of polymerization of tubulin-GDP induced by taxol (●) and TG-185-219 (○). Tubulin-GDP (5 μM) in PME buffer was incubated with 40 μM taxol or TG-185-219 in the presence of (A) 4% DMSO or (B) 15% DMSO at 37 °C and the polymerization was monitored as apparent absorption at 350 nm.

Fig. 4

The extent of polymerization of tubulin-GDP induced by taxol (●) and TG-185-219 (○). Tubulin-GDP (5 μM) in PME buffer was incubated with 40 μM taxol or TG-185-219 in the presence of (A) 4% DMSO or (B) 15% DMSO at 37 °C and the polymerization was monitored as apparent absorption at 350 nm.

Fig. 5

EC₅₀ of taxol for tubulin-GDP in the presence of 15% DMSO (▲) and 4% DMSO (●). Tubulin-GDP samples (5μM) in PME buffer were incubated with different concentrations of taxol in 15% DMSO and 4% DMSO at 37 °C for 54 min. The percentage polymerization was measured in terms of the apparent absorption at 350 nm. The EC₅₀ of taxol in 4% and 15% DMSO was 4.2 ±0.54 μM and 0.54 ± 0.16 μM respectively. The values are an average of three independent determinations.

Fig. 6

Affinity of taxol for GMPCPP-microtubules in the presence of 4% DMSO (○) and 15% DMSO (●). Affinity constants were determined by competition of taxol with N-AB-PT, a fluorescent analog of taxol towards GMPCPP-microtubules, in 4% and 15% DMSO, respectively. The affinity constants of taxol were identical within experimental error (6.1 × 10⁷ M⁻¹).

Fig. 7

IC₅₀ of podophyllotoxin for tubulin-GDP in 15% DMSO (○) and tubulin-GTP in 4% DMSO (●). Tubulin-GDP samples (8μM) in PME buffer with 15% DMSO were treated with increasing concentration of podophyllotoxin and polymerized with 8 μM taxol at 37^oC. Tubulin-GTP samples (6μM) in PME buffer with 4% DMSO were treated in the same way and polymerized with 6 μM taxol under identical conditions. The percent polymerization was measured in terms of the apparent absorption at 350 nm.