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The purpose of this work was to introduce a chemical modification into the paclitaxel (Taxol) structure to reduce interactions with the product of the multidrug resistant type 1 (MDR1) gene, P-glycoprotein (Pgp), resulting in improved blood−brain barrier (BBB) permeability. Specifically, a taxane analogue, Tx-67, with a succinate group added at the C10 position of Taxol, was synthesized and identified as such a candidate. In comparison studies, Tx-67 had no apparent interactions with Pgp, as demonstrated by the lack of enhanced uptake of rhodamine 123 by brain microvessel endothelial cells (BMECs) in the presence of the agent. By contrast, Taxol exposure substantially enhanced rhodamine 123 uptake by BMECs through inhibition of Pgp. The transport across BMEC monolayers was polarized for both Tx-67 and Taxol with permeation in the apical to basolateral direction greater for Tx-67 and substantially reduced for Taxol relative to basolateral to apical permeation. Taxol and cyclosporin A treatments also did not enhance Tx-67 permeation across BMEC monolayers. In an in situ rat brain perfusion study, Tx-67 was demonstrated to permeate across the BBB at a greater rate than Taxol. These results demonstrate that the Taxol analogue Tx-67 had a reduced interaction with Pgp and, as a consequence, enhanced permeation across the blood−brain barrier in vitro and in situ.

The synthesis and biological evaluation of a novel paclitaxel photoaffinity probe is described. The synthesis involved the preparation of an azide-containing C13 side chain through a Staudinger cycloaddition followed by a lipase-mediated kinetic resolution to obtain the azetidinone in 99% ee. Coupling of the enantiopure side chain precursor to 7-TES-baccatin III and subsequent silyl ether deprotection afforded 3‘-(4-azidophenyl)-3‘-dephenylpaclitaxel, which was shown to be as active as paclitaxel in tubulin assembly and cytotoxicity assays.

We have previously described a model of paclitaxel−microtubule binding that led to the prediction that analogues of paclitaxel lacking any D ring could stabilize microtubules as well as paclitaxel if the substituent present at C4 did not have unfavorable steric interactions with the binding pocket. We report the synthesis of a 4-methyl paclitaxel analogue, compound 1, which bears this prediction out. Compound 1 is as potent as paclitaxel at microtubule stabilization in vitro; however, it has only about one-four-hundredth the cytotoxicity of paclitaxel.

The unsubstituted, 3‘-Cl, 4‘-C1, and 3‘,4‘-diCl C10 analogues of cryptophycin-24 were prepared via total synthesis and tested in vitro for cytotoxicity against MCF-7 and multi-drug-resistant MCF-7/ADR breast cancer cell lines and in a tubulin assembly assay. The ED50 values ranged from 7.2 to 15.8 μM in the tubulin assay and from 0.05 to 3.4 nM in the cell assays. The presence of a 3‘-C1 and/or 4‘-C1 substituent on the C10 phenyl ring increased cytotoxicity in the MCF-7 cell line compared to the unsubstituted phenyl ring. The most potent compound in this series possessed a 3‘-C1 substituent on the C10 phenyl ring. The 3‘-C1 analogue had ED50 values of 50 and 580 pM in the MCF-7 and MCF-7/ADR cell lines, respectively. Its activity was very similar to the parent compound cryptophycin-24. Substitution of the 4‘-MeO group in cryptophycin-24 with a 4‘-C1 moiety did not significantly affect cytotoxicity against MCF-7 and MCF-7/ADR cells compared to the parent compound. These results demonstrated that the 4‘-MeO group in cryptophycin-24 is not essential and can be replaced with 3‘-C1 or 4‘-C1 substituents.

We have developed a strategy for the purification of native microtubule motor proteins from mitotic HeLa cells and describe here the purification and characterization of human conventional kinesin and two human kinesin-related proteins, HSET and CENP-E. We found that the 120-kDa HeLa cell conventional kinesin is an active motor that induces microtubule gliding at approximately 30 microm/min at room temperature. This active form of HeLa cell kinesin does not contain light chains, although light chains were detected in other fractions. HSET, a member of the C-terminal kinesin subfamily, was also purified in native form for the first time, and the protein migrates as a single band at approximately 75 kDa. The purified HSET is an active motor that induces microtubule gliding at a rate of approximately 5 microm/min, and microtubules glide for an average of 3 microm before ceasing movement. Finally, we purified native CENP-E, a kinesin-related protein that has been implicated in chromosome congression during mitosis, and we found that this form of CENP-E does not induce microtubule gliding but is able to bind to microtubules.

Analogues of Taxol (paclitaxel) with the side chain conformationally restricted by insertion of a carbon linker between the 2‘-carbon and the ortho-position of the 3‘-phenyl ring were synthesized. Biological evaluation of these new taxoids showed that activity was dependent on the length of the linker and the configuration at C2‘ and C3‘. Two analogues in the homo series, 9a and 24a, showed tubulin binding and cytotoxicity comparable to that of Taxol. NAMFIS (NMR analysis of molecular flexibility in solution) deconvolution of the averaged 2-D NMR spectra for 9a yields seven conformations. Within the latter set, the hydrophobically collapsed “nonpolar” and “polar” classes are represented by one conformation each with predicted populations of 12−15%. The five remaining conformers, however, are extended, two of which correspond to the T-conformation (47% of the total population). The latter superimpose well with the recently proposed T-Taxol binding conformer in β-tubulin. The results provide evidence for the existence of two previously unrecognized structural features that support Taxol-like activity:  (1) a reduced torsion angle between C2‘ and C3‘ and (2) an orthogonal arrangement of the mean plane through C1‘, C2‘ and the 2‘-hydroxyl and the 3‘-phenyl plane, the latter ring bisected by the former plane. By contrast, epimerization at 2‘,3‘ and homologation of the tether to CH2−CH2 were both detrimental for activity. The decreased activity of these analogues is apparently due to configurational and steric factors, respectively.

Previous work using n.m.r. spectroscopy to investigate the binding between formyltetrahydrofolate synthetase and its ligands was done using the catalytically active tetrameric form of the enzyme. By removal of specific monovalent cations the tetramer dissociates to four identical, catalytically inactive monomers, which are capable of binding nucleotides with affinities similar to those obtained with the tetramer. In the studies reported here, we examined the interaction of metal-nucleotide, formate and monovalent cations with the monomer using n.m.r. relaxation measurements. We were able to demonstrate that formate binds to the monomer. The spin-lattice relaxation rate (1/T1) of the formate carbon in the monomer. M2+.ADP. formate complex is enhanced when Mg2+ is replaced by Mn2+. By assuming that the exchange of formate is not rate-limiting and that tau c of the monomer is the same as that of the tetramer, the distance between the Mn2+ and the formate carbon was calculated and found to be similar in the monomer and tetramer complexes. The spin-lattice relaxation rates of [13C]trimethylammonium ion (an inactive monovalent cation), [13C]methylammonium and [15N]ammonium ions (both active monovalent cations), were measured in the presence of tetramer, MnADP and formate. The relaxation rates of methylammonium and ammonium ions were enhanced under these conditions whereas the relaxation rate of trimethylammonium ion was not. The results indicate that the active monovalent cations bind near the MnADP binding site. A distance from the Mn2+ to the ammonium nitrogen of between 0.5 and 0.6 nm was calculated.

The synthesis and biological evaluation of the two known phenolic metabolites of paclitaxel are described. The C3‘-phenolic metabolite 2 of paclitaxel was prepared from 7-(triethylsilyl)baccatin III (8) and enantioenriched N-benzoyl-2-azetidinone 7. The C2-phenolic metabolite 3 was synthesized from paclitaxel (1a) via selective C2 debenzoylation and reacylation.

The action of a synthetic antitubulin agent, nocodazole, on tubulin self-assembly, preformed microtubules, fast axoplasmic transport and axonal ultrastructure was studied. Nocodazole completely blocks the self-assembly of tubulin containing or lacking associated proteins. This inhibition is readily reversible. At similar concentrations, nocodazole also depolymerizes preformed microtubules in vitro. Fast axoplasmic transport in cat vagus nerve is disrupted by bathing the nerve fivers in a medium containing 10 micrometer nocodazole for periods of 2.5 hr or longer. A 4-hr exposure results in extensive inhibition. The nocodazole action on fast axoplasmic transport appears to be reversible but with a high degree of variability. After 2.5 hr of exposure to 10 micrometers nocodazole, there is a slight decrease in axonal microtubules and an increase in 10 nm neurofilaments. A 4 hr exposure results in a marked reduction in microtubules. These results suggest that nocodazole has a mode of action similar to other antimicrotubule agents.

We have examined the dilution-induced in vitro disassembly kinetics of bovine brain microtubules, initially at steady state, using a wider range of dilutions (2-100-fold) than previously employed. In contrast to earlier results, as well as to the simple nucleation-condensation model for microtubule formation, the initial rate of dimer loss from microtubule ends was not a linear function of the initial concentration of unpolymerized tubulin. Over a 2-20-fold dilution range, plots of the initial rate of dimer loss versus the initial unpolymerized tubulin concentration were approximately linear. However, at greater dilutions, rates of microtubule depolymerization increased nonlinearly. For example, between a 10-fold dilution and a 100-fold dilution, the initial rate of dimer loss for microtubule-associated protein-containing microtubules increased by 300%, rather than a maximum of 11% expected on the basis of a linear rate plot. The nonlinear response was observed for dimer loss from opposite microtubule ends separately and with microtubules containing and lacking associated proteins. Qualitatively similar results were obtained using a wide range of experimental protocols, from which we can reasonably exclude methodological artifact as a basis for the data. We can also reasonably exclude the dissociation of the high molecular weight microtubule-associated proteins 1 and 2 from the microtubules as an explanation for the nonlinearity of the rate plots. The nonlinearity of the rate plots indicates that kinetic constants obtained under nonsteady state conditions of extreme microtubule dilution may not describe the steady state condition accurately.

Addition of 2-200 microM vinblastine to microtubules at steady state in vitro causes the microtubules to depolymerize, with the formation of protofilament spirals and other aggregated forms of microtubule protein. The presence of such spirals and protein aggregates, which are difficult to separate from microtubules, has complicated attempts to measure the binding of vinblastine to microtubules. We have found that stabilizing bovine brain microtubules in vitro with dimethyl sulfoxide, taxol, or a combination of dimethyl sulfoxide and taxol prevents or greatly retards the formation of protofilamentous spirals, thus permitting us to measure the binding of vinblastine to intact microtubules. Reciprocal plots of binding data indicate the presence of 1.4-1.7 vinblastine binding sites/mol of tubulin in the microtubule, with a Ka of approximately 3-4 x 10(3) M-1. The Ka value obtained is within 1 order of magnitude of the apparent intrinsic binding constant for the binding of vinblastine to tubulin dimers. The results support the idea that depolymerization of microtubules by intermediate and high concentrations of vinblastine occurs by stoichiometric binding of vinblastine to tubulin along the microtubule surface.

Tubulin from bovine brain was polymerized in vitro using a variety of assembly conditions. Many of the formed microtubules were shown to contain 14 wall protofilaments. The number of microtubules containing 14 protofilaments increased with consecutive repetitions of cold-dissociation followed by reassembly in vitro.

Triosephosphate isomerase was purified to homogeneity as judged by analytical gel electrophoresis from clostridium sp. strain 69, clostridium pasteurianum, and C. thermosaccharolyticum, which grow optimally at 18, 37, and 55 C, respectively. Comparative studies on these purified proteins showed that they had the same molecular weight (53,000) and subunit molecular weight (26,500). They were equally susceptible to the active site-directed inhibitor, glycidol phosphate. However, their temperature and pH optima, as well as their stabilities to heat, urea, and sodium dodecyl sulfate, differed. The proteins also had different mobilities in acrylamide gel electrophoresis. This difference in ionic character was also reflected in the elution behavior of the enzymes from hydroxyapatite and in the isoelectric points determined by isoelectric focusing in acrylamide gel. The amino acid composition of these proteins showed that the thermophilic enzyme contains a greater amount of proline than the other enzymes. The ratio of acidic amino acids to basic amino acids was 1.79, 1.38, and 1.66 for the thermophilic mesophilic and psychrophilic enzymes, respectively. This is consistent with the relative isoelectric point values of these three enzymes.

Epi-C3-cryptophycin-24, epi-C3-m-chlorobenzyl-cryptophycin-24, and the corresponding styrenes were synthesized and tested in vitro against the MCF-7 and multidrug-resistant MCF-7/ADR breast cancer cell lines and in an in vitro tubulin assembly assay. The results demonstrate that the S configuration at the C3 stereocenter is not required to induce potent cytotoxicity and the m-Cl substituent present on the C10 side chain did not induce any large change in activity.