The bioactive Taxol conformation on beta-tubulin: experimental evidence from highly active constrained analogs

Abstract

The important anticancer drug Taxol (paclitaxel) binds to tubulin in a stoichiometric ratio and promotes its assembly into microtubules. The conformation of microtubule-bound drug has been the subject of intense study, and various suggestions have been made. In this work we present experimental and theoretical evidence that Taxol adopts a T-shaped conformation when it is bound to tubulin.

Scheme 1.

Structures of Taxol (1a), docetaxel (1b), discodermolide (2), epothilone B (3), and eleutherobin (4).

Fig. 1.

Synthetic scheme for the bridged paclitaxels 13a, 13b, 14a, and 14b. The reagents and conditions for the “a” series of β-lactams were similar to those below for the “b” series. Reagents and conditions for the “b” series β-lactams follow. i, 5b, p-MeOC₆H₄NH₂, MgSO₄, CH₂Cl₂, 100%. ii, CH₃COOCH₂COCl, Et₃N, -78°C to room temperature (rt), 12 h, 85%. iii, Lipase (Amano PS), phosphate buffer, pH 7.2, CH₃CN, 24 h, 98%. iv, 1M KOH, THF, 0°C, 100%. v, TIPSCl, imidazole, DMF, 94%. vi, CAN, CH₃CN, -5°C, 62%. vii, PhCOCl, Et₃N, DMAP, CH₂Cl₂, 95%. viii, LiHMDS, THF, 0°C, CH₂—CHCOCl, 52%. ix, HF-pyridine, THF, 70%. x, CeCl₃,Ac₂O, THF, 96%. xi, Et₃SiCl, imidazole, DCM, 72%. xii, 8a, NaH, THF, 0°C to rt, 24 h. xiii, (H₂IMes)(PCy₃)(CI)₂Ru—CHPh, CH₂Cl₂, 3 h. xiv, HF-pyridine, 12 h. xv, H₂,Pd/C(10%), 35 psi, 2.5 h. xvi, 8b, NaH, THF, 0°C to rt, 24 h, 50%. xvii, HF-Pyridine, THF, 81%. xviii, (H₂IMes)(PCy₃)(CI)₂Ru—CHPh, CH₂Cl₂, 3 h, 64%. xix, HF-pyridine, 12 h, 98%. xx, H₂, Pd/C(10%), 35 psi, 2.5 h, 96%.

Scheme 2.

Structure of the fluorescent Taxol derivative N-AB-PT (15).

Fig. 3.

Competition displacement of fluorescent Taxol 15 from tubulin by Taxol (black dots) and by compound 13b (red dots). Compound 15 was maintained at 5 μM, and increasing amounts of Taxol or compound 13b were added.

Fig. 2.

T-Taxol conformation. (a) The similar and extended ring-to-ring distances between the C-2 benzoyl phenyl and the C-3′ phenyl and benzamido phenyl centers, respectively. (b) H—H separations between the C-4 acetate methyl group and the ortho and meta postions of the C-3′ phenyl ring.

Scheme 3.

Structure of bridged Taxol 16.

Fig. 4.

NAMFIS-derived T-conformations for 13b (76% red, 7% green) superimposed on the tubulin-bound T-Taxol form (blue).

Fig. 5.

T-Taxol (blue) bound to β-tubulin as described in ref. . The ortho-bridged T-form of 13b (red, both ligand and protein binding site subjected to MD relaxation similar to T-Taxol) superimposed on Taxol avoids the steric contact with Phe-270 at the bottom of the hydrophobic taxoid pocket experienced by the meta-bridged analog 16 (27).

Fig. 6.

A view of ortho-bridged 13b in which phenyl rings emanating from C-2 and C-3′ surround the imidazole of His-227 in a sandwich motif; the C-4 OAc to C-3′ phenyl bridge (yellow) avoids the latter while stabilizing the T-Taxol conformation.