Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules

Abstract

Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 μmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 μmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 μmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule.

Figure 1

Structures of maytansine and the maytansine thiomethyl analogs S-methyl DM1 and S-methyl DM4.

Figure 2

Concentration-dependence for inhibition of microtubule assembly by the maytansinoids. A. Microtubule protein (3 mg/mL) was assembled in the absence or presence of maytansine (◇), S-methyl DM1 (■), and S-methyl DM4 (△) in the presence of 1 mmol/L GTP in PEM buffer at 30 °C for 45 min. The microtubules were collected by centrifugation (35000 × g, 1 h, 30 °C) and the amount of sedimented protein was determined. Maytansine, S-methyl DM1, S-methyl DM4 inhibited MT assembly with IC₅₀’s of 1 ± 0.02 μmol/L, 4 ± 0.1 μmol/L, and 1.7 ± 0.4 μmol/L, respectively. Mean and SD of two experiments. B. Electron micrographs of microtubules treated with vehicle (DMSO), S-methyl DM1, or S-methyl DM4. S-methyl DM1 showed few aggregates where as S-methyl DM4 induced extensive aggregation. Images are 100000 × magnifications.

Figure 3

Effect of maytansinoids on microtubule dynamic instability. Life history plots of changes in microtubule length at steady state in the absence and presence of 100 nmol/L compound. A, control; B, maytansine; C, S-methyl DM1; and D, S-methyl DM4.

Figure 4

Binding of maytansine (A) or S-methyl DM1 (B) to tubulin. Maytansine (0.5–20 μmol/L) or S-methyl DM1 (1–8 μmol/L) was incubated with 3 μmol/L tubulin in PEM buffer for 45 min at 30 °C. The relative intrinsic fluorescence intensity of tubulin was monitored as described in ‘Materials and Methods’. The plot indicates a dissociation constant (K_D) of 0.86 ± 0.23 μmol/L for maytansine (A) and of 0.93 ± 0.22 μmol/L for S-methyl DM1 (B). The Y–axis shows the inverse of the fractional receptor occupancy (a) of compound and the X–axis shows the inverse of free maytansinoid concentration.

Figure 5

Concentration-dependence for binding of S-methyl DM1 to microtubules. A. S-methyl DM1 binds to microtubules in a concentration-dependent manner. The graph represents mean and SEM of three independent experiments B. Scatchard plot showing binding to both the high affinity and low affinity sites on microtubules. Microtubules were assembled to steady-state (MTP, 3 mg/mL) and then incubated with [³H]-S-methyl DM1 for 1 h. Microtubules were collected by centrifugation through a glycerol/DMSO cushion. From microtubule lengths, sedimented protein, and incorporated radioactivity, the stoichiometry of the binding of S-methyl DM1 per microtubule was determined. C. Scatchard plot showing binding at low drug concentrations and indicating that there are 37 high affinity sites at microtubule ends, K_D of 0.1 ± 0.05 μmol/L.