Suppression of Microtubule Dynamics by Binding of Cemadotin to Tubulin:  Possible Mechanism for Its Antitumor Action

Abstract

Cemadotin (LU103793) (NSC D-669356) is a water-soluble synthetic analogue of dolastatin 15 that inhibits cell proliferation in vitro and the growth of human tumor xenografts. Cemadotin is in phase II clinical trials as a promising cancer chemotherapeutic agent. The drug blocks cells at mitosis. Its primary mode of action has been unclear but is believed to involve an action on microtubules. We have found that cemadotin binds to tubulin and strongly suppresses microtubule dynamics. Scatchard analysis of cemadotin binding to tubulin indicated that there are two affinity classes of cemadotin-binding sites with Kd values of 19.4 microM and 136 microM. Cemadotin did not inhibit the binding of vinblastine to tubulin, and, conversely, vinblastine did not inhibit the binding of cemadotin to tubulin. By quantitative video microscopy of individual microtubules, we found that cemadotin strongly suppressed dynamic instability of microtubules assembled to steady state using bovine brain tubulin devoid of microtubule-associated proteins. It reduced the rate and extent of growing and shortening, increased the rescue frequency, and increased the percentage of time the microtubules spent in an attenuated or paused state, neither growing nor shortening detectably. At the lowest effective cemadotin concentrations, dynamics were suppressed in the absence of significant microtubule depolymerization. The results suggest that cemadotin exerts its antitumor activity by suppressing spindle microtubule dynamics through a distinct molecular mechanism by binding at a novel site in tubulin.

Diazonamide A and a Synthetic Structural Analog: Disruptive Effects on Mitosis and Cellular Microtubules and Analysis of Their Interactions with Tubulin

“…In contrast, it is difficult to demonstrate the binding of [ 3 H]dolastatin 15 to tubulin; such binding is only demonstrable by HummelDreyer chromatography. Analogously, Jordan et al (1998) examined the binding to tubulin of an analog of dolastatin 15, All reaction mixtures contained 10 M (1.0 mg/ml) tubulin, 0.4 mM GTP, varying concentrations of peptides, and 4% (v/v) dimethyl sulfoxide. Polymerization was followed for 20 min at 30°C.…”

Identification of Tubulin as the Molecular Target of Proapoptotic Pyrrolo-1,5-benzoxazepines

“…The results from the competitive binding experiments revealed that PBOX-6 did not inhibit binding of either colchicine or vinblastine to tubulin, indicating that PBOX-6 has its molpharm.aspetjournals.org own, novel, binding site upon tubulin. Other antimicrotubule agents, such as estramustine (Panda et al, 1997), arsenic trioxide (Ling et al, 2002), naphthopyran (Dell, 1998), and cemadotin (Jordan et al, 1998), have also been shown to bind to as-yet-uncharacterized novel sites on tubulin.…”

A Phase I Study of the Dolastatin-15 Analogue Tasidotin (ILX651) Administered Intravenously Daily for 5 Consecutive Days Every 3 Weeks in Patients with Advanced Solid Tumors

“…This metabolite also prevents microtubule assembly in microtubule polymerization assays, but exhibits 8-fold less cytotoxicity than cemadotin in vitro (9). Tasidotin was rapidly cleared from plasma with an effective half-life of <45 minutes in most cases, whereas ILX651-C-carboxylate concentrations peaked at 5 hours and had a half-life of f10 hours.…”

“…Cemadotin, a dolastatin-15 pentapeptide, at concentrations as low as 10 to 100 nmol/L, suppressed the rate of tubulin growth more than shortening, a process that differentiated dolastatin-15 analogues from other tubulin-acting agents like Vinca alkaloids (which suppress the rate of growth and shortening simultaneously) and taxanes (which preferentially suppress the rate of tubulin growth; ref. 9). However, clinical trial results with cemadotin were disappointing due to rapid metabolic conversion and dose-limiting cardiovascular toxicity, including hypertension, angina, and myocardial infarction (10 -13).…”