(-)-Rhazinilam and the diphenylpyridazinone NSC 613241: Two compounds inducing the formation of morphologically similar tubulin spirals but binding apparently to two distinct sites on tubulin

Abstract

The most potent microtubule assembly inhibitor of newer diphenylpyridazinone derivatives examined was NSC 613241. Because NSC 613241 and (-)-rhazinilam also induce the formation of similar 2-filament spirals, these aberrant reactions were compared. Spiral formation with both compounds was enhanced by GTP and inhibited by GDP and by 15 other inhibitors of microtubule assembly. Similarly, microtubule assembly induced by paclitaxel or laulimalide is enhanced by GTP and inhibited by GDP and assembly inhibitors, but neither [(3)H]NSC 613241 nor [(3)H](-)-rhazinilam bound to microtubules or inhibited the binding of [(3)H]paclitaxel or [(3)H]peloruside A to microtubules. Differences in the pitch of aberrant polymers were found: NSC 613241-induced and (-)-rhazinilam-induced spirals had average repeats of 85 and 79-80 nm, respectively. We found no binding of [(3)H]NSC 613241 or [(3)H](-)-rhazinilam to αβ-tubulin dimer, but both compounds were incorporated into the polymers they induced in substoichiometric reactions, with as little as 0.1-0.2 mol compound/mol of tubulin, and no cross-inhibition by NSC 613241 or (-)-rhazinilam into spirals occurred. Under reaction conditions where neither compound induced spiral formation, both compounds together synergistically induced substantial spiral formation. We conclude that (-)-rhazinilam and NSC 613241 bind to different sites on tubulin that differ from binding sites for other antitubulin agents.

Keywords: (−)-Rhazinilam; 4-morpholineethanesulfonate; DPP; Diphenylpyridazinone derivatives; Glutaraldehyde-fixed microtubules; HPLC; Inhibition of tubulin assembly; MAPs; Mes; NSC 613241; Tubulin polymer of aberrant morphology; diphenylpyridazinone; gMTs; glutaraldehyde-fixed microtubules; high-performance liquid chromatography; microtubule-associated proteins.

Fig. 1

Structures of (−)-rhazinilam, a racemic rhazinilam analogue, and several DPP derivatives, including NSC 613241. IC₅₀’s for microtubule assembly for NSC 362449 and NSC 602746 were 6.2 ± 0.38 and 6.2 ±0.44 μM, respectively, obtained at the same time as the IC₅₀ of 5.8 ± 0.23 μM was obtained for NSC 362455, as described in the text. These were the most active DPP derivatives described previously [9].

Fig. 2

Aberrant tubulin assembly reactions induced by NSC 613241 (Panel A; curves 1–4) and (−)-rhazinilam (Panel B; curves 5–8). All reaction mixtures contained 10 μM tubulin, 0.6 M monosodium glutamate (pH 6.6), 2% dimethyl sulfoxide, and either 10 μM NSC 613241 or 10 μM (−)-rhazinilam, as indicated. There was no further addition to the reactions represented by curves 1 and 5. Further additions were 1.0 mM MgCl₂ only (curves 2 and 6), 50 μM GTP only (curves 3 and 7), both 1.0 mM MgCl₂ and 50 μM GTP (curves 4 and 8), and 1.0 mM MgCl₂, 50 μM GTP, and 500 μM GDP (dashed curves 4a and 8a). In addition, the arrows pointing to the baselines labeled curves 4b and 8b represent reaction mixtures containing 1.0 mM MgCl₂ and 50 μM GTP but no NSC 613241 (curve 4b) or no (−)-rhazinilam (curve 8b). Baselines at 350 nm were set with all components except NSC 613241 or (−)-rhazinilam mixed in the cuvettes. Addition of either NSC 613241 or (−)-rhazinilam, with rapid mixing into the reaction mixture, initiated the timing of the reaction. At the times indicated on the abscissa, the temperature was set at the temperatures indicated to the left of the dashed lines. Temperature rose in the cuvettes, once the temperature was set on the temperature controller at about 0.5 °C/s. It should be noted that 10 μM (−)-rhazinilam had no significant absorbance at 350 nm, whereas 10 μM NSC 613241 had absorbance of about 0.1 A₃₅₀ unit. This sudden jump when the cuvette chamber was closed could be readily distinguished from the increase in turbidity that began 30–60 s later.

Fig. 3

Electron micrographs of aberrant polymer formed with (−)-rhazinilam. Magnification is indicated by the bars in each panel. A. Lower magnification view. B. Middle magnification view. C. Higher magnification view. The arrow indicates a segment of polymer where a 2 filament substructure is clearly visible. D. Middle magnification view of polymer mildly fixed with glutaraldehyde (0.2%) before sample was applied to the grid.

Fig. 4

Electron micrographs of aberrant polymer formed with NSC 613241. Magnification is indicated by the bars in each panel. A and B. Lower magnification views. The dashed box in panel B indicates an area shown at higher magnification in panel C. C. Middle magnification view. D. Higher magnification view. Single arrows indicate areas with a 2 filament substructure. The double arrow indicates an area where there appears to be a 3 filament substructure, although this may be caused by overlapping spirals.

Fig. 5

Electron micrographs of aberrant polymer formed with vinblastine in the absence (A) and presence of GTP (B). Reaction mixtures contained 10 μM tubulin, 0.75 M monosodium glutamate, 20 μM vinblastine, and, if present, 10 μM GTP. The tubulin used in these studies was not subjected to gel filtration chromatography.

Fig. 6

Concentration effects of (−)-rhazinilam and NSC 613241 on amount of tubulin in sedimentable polymer (A) and on stoichiometry of compounds in sedimentable polymer (B). Experimental details are described in detail in the text. In brief, reaction mixtures containing the indicated concentrations of [³H](−)-rhazinilam (triangles) or [³H]NSC 613241 (circles) were incubated at 22 °C for 30 min, and the polymer was harvested by centrifugation. Protein and radiolabel content of the pellets was determined, and these data were used to calculate the values shown in the Figure.

Fig. 7

Both (−)-rhazinilam and NSC 613241 are required for aberrant assembly at 0 °C in the absence of GTP. Reaction mixtures contained 10 μM tubulin, 0.6 M monosodium glutamate (pH 6.6), 1.0 mM MgCl₂, 4% dimethyl sulfoxide, and NSC 613241 and/or (−)-rhazinilam, as indicated. A. Aberrant polymer harvested by ultracentrifugation as described in the text following a 20 min incubation at 0 °C. Symbols: circles, (−)-rhazinilam and NSC 613241 at the indicated concentrations; upright triangles, (−)-rhazinilam at the indicated concentrations; inverted triangles, NSC 613241 at the indicated concentrations. B. Formation of aberrant polymer by turbidimetry. Reactions were followed at 0 °C in a Gilford 250 spectrophotometer equipped with an electronic temperature controller. Curve 1, 15 μM (−)-rhazinilam and 15 μM NSC 613241; curve 2, 15 μM (−)-rhazinilam; curve 3, 30 μM (−)-rhazinilam; curve 4, 15 μM NSC 613241; curve 5, 30 μM NSC 613241.