Wei-Shuo Fang

Optimization of Taxane Binding to Microtubules: Binding Affinity Dissection and Incremental Construction of a High-Affinity Analog of Paclitaxel

The microtubule binding affinities of a series of synthetic taxanes have been measured with the aims of dissecting individual group contributions and obtaining a rationale for the design of novel compounds with the ability to overcome drug resistance. As previously observed for epothilones, the positive and negative contributions of the different substituents to the binding free energies are cumulative. By combining the most favorable substitutions we increased the binding affinity of paclitaxel 500-fold. Insight into the structural basis for this improvement was gained with molecular modeling and NMR data obtained for microtubule-bound docetaxel. Taxanes with affinities for microtubules well above their affinities for P-glycoprotein are shown not to be affected by multidrug resistance. This finding strongly indicates that optimization of the ligand-target interaction is a good strategy to overcome multidrug resistance mediated by efflux pumps.

Lx2-32c, a novel taxane and its antitumor activities in vitro and in vivo

Lx2-32c, a novel taxane derivative, is a semisynthetic analogue from cephalomannine. Its antitumor activity in vivo and in vitro was investigated in this study. Lx2-32c was cytotoxic (IC50=1.7+/-1.6nM) to various human tumor cell lines after 72h incubation. In vitro it enhanced the rate of tubulin polymerization in a dose-dependent manner and induced the bundling of microtubule in BGC-823 cells with the mode similar to that of paclitaxel. As determined by flow cytometry, after either 12 or 24h exposure, Lx2-32c caused BGC-823 cells G2/M phase arrest in a time- and dose-dependent manner. Moreover, we demonstrated that Lx2-32c had significant antitumor activity on BGC-823 (human gastric carcinoma) and A549 (human non-small cell lung carcinoma) xenograft in nude mice. These data suggest that Lx2-32c is a microtubule-stabilizing agent, which has significant antitumor activity in vitro and in vivo.

Overcoming Tumor Drug Resistance with High-Affinity Taxanes: A SAR Study of C2-Modified 7-Acyl-10-Deacetyl Cephalomannines

A series of C2‐modified 10‐deacetyl‐7‐propionyl cephalomannine derivatives was designed, prepared, and biologically evaluated. Some C2 meta‐substituted benzoate analogues showed potent activity against both drug‐sensitive and drug‐resistant tumor cells in which resistance is mediated through either P‐gp overexpression or β‐tubulin mutation mechanisms. The taxoid 15 b and related compounds are of particular interest, as they are much more cytotoxic than paclitaxel, especially against drug‐resistant tumor cells; they are able to kill both drug‐resistant and drug‐sensitive cells (low R/S ratio), and they have high affinity for β‐tubulin. Our research results led to an important hypothesis, that is, a taxane with very high binding affinity for β‐tubulin is able to counteract drug resistance, which may assist in future taxane‐based drug‐discovery efforts.

Taxachitrienes A and B, two new bicyclic taxane diterpenoids from Taxus chinensis

Abstract Two new bicyclic taxane diterpenoids, taxachitrienes A(1) and B(2), were isolated from the needles of Taxus chinensis. Their structure were elucidated on the basis of spectroscopic data. The crystal structure of 2 was also presented. It is the first time for the isolation of bicyclic diterpenoid which is proposed as the biogenetic precursor for taxanes.

Microtubule stabilising agents for cancer chemotherapy.

Background: Microtubules are very important targets for cancer chemotherapy. During the past 15 years, three structurally diverse classes of microtubule stabilising agents (MSAs), namely, taxanes, epothilones and discodermolides, have been approved as anticancer agents or subjected to clinical trials, and many novel MSAs have also been identified. Objective: This review focuses on recent advances in the use of taxanes, epothilones and discodermolides, as well as some recently described natural product based MSAs. Methods: Data were identified through the search of PubMed for research articles and reviews, the website of European patents (http://ep.espacenet.com) and US patents (www.uspto.gov) for patents up to October 2008. Only well-established MSAs are mentioned in this review. Results/conclusion: Great advances have been made in the application of MSAs in antitumour clinical practice and drug discovery. Many structure–activity relationship studies of MSAs as novel anticancer drugs have been conducted in recent years. To enhance the efficacy of MSAs as clinically useful therapeutics, numerous efforts have been made in combination chemotherapy regimens and include: i) the combination of structurally different MSAs, ii) combination of MSAs with other classes of anticancer agent with a different mechanism of action and iii) novel ways of administrating well-established MSAs.

Modulation of Microtubule Interprotofilament Interactions by Modified Taxanes

Microtubules assembled with paclitaxel and docetaxel differ in their numbers of protofilaments, reflecting modification of the lateral association between αβ-tubulin molecules in the microtubule wall. These modifications of microtubule structure, through a not-yet-characterized mechanism, are most likely related to the changes in tubulin-tubulin interactions responsible for microtubule stabilization by these antitumor compounds. We have used a set of modified taxanes to study the structural mechanism of microtubule stabilization by these ligands. Using small-angle x-ray scattering, we have determined how modifications in the shape and size of the taxane substituents result in changes in the interprotofilament angles and in their number. The observed effects have been explained using NMR-aided docking and molecular dynamic simulations of taxane binding at the microtubule pore and luminal sites. Modeling results indicate that modification of the size of substituents at positions C7 and C10 of the taxane core influence the conformation of three key elements in microtubule lateral interactions (the M-loop, the S3 β-strand, and the H3 helix) that modulate the contacts between adjacent protofilaments. In addition, modifications of the substituents at position C2 slightly rearrange the ligand in the binding site, modifying the interaction of the C7 substituent with the M-loop.

Probing the Pore Drug Binding Site of Microtubules with Fluorescent Taxanes: Evidence of Two Binding Poses

The pore site in microtubules has been studied with the use of Hexaflutax, a fluorescent probe derived from paclitaxel. The compound is active in cells with similar effects to paclitaxel, indicating that the pore may be a target to microtubule stabilizing agents. While other taxanes bind microtubules in a monophasic way, thus indicating a single type of sites, Hexaflutax association is biphasic. Analysis of the phases indicates that two different binding sites are detected, reflecting two different modes of binding, which could arise from different arrangements of the taxane or fluorescein moieties in the pore. Association of the 4-4-20 antifluorescein monoclonal antibody-Hexaflutax complex to microtubules remains biphasic, thus indicating that the two phases observed arise from two different poses of the taxane moiety.

Synthesis and Biological Evaluation of Colchicine B‑Ring Analogues Tethered with Halogenated Benzyl Moieties

Deacetylcolchicine was reacted with substituted benzyl halides to provide a library of compounds for biological analysis. Compound 7 (3,4-difluorobenzyl-N-aminocolchicine) was shown to possess cytotoxicity in cancer cell lines in the low nanomolar range. Significantly, it showed no loss of activity in the resistant A2780AD ovarian carcinoma cell line known to overexpress the ABCB1 drug transporter and was also unaffected by overexpression of class III β-tubulin in HeLa transfected cells.

Jacaranone glycosides from Senecio scandens.

Bioassay-guided fractionation of the ethanol extract of Senecio scandens led to the isolation of four new compounds 1–4. These compounds were obtained as tautomeric mixture of α/β epimers, but their structures were confirmed unambiguously by 1D and 2D NMR spectra and LC NMR technology. 1H NMR spectra of pure 1α and 1β were furnished by HPLC NMR technology. Compounds 1–4 exhibited moderate cytotoxicities against five tumor cell lines.

Taxanes with high potency inducing tubulin assembly overcome tumoural cell resistances

We have found that four taxanes with chemical modifications at positions C10 and C13 were active against all types of taxane resistant cell lines, resistant by P-gp overexpression, by mutations in the β-tubulin binding site or by overexpression of the highly dynamic βIII-tubulin isotype. We have characterized the interaction of taxanes with high activity on chemotherapy resistant tumoural cells with microtubules, and also studied their cellular effects. The biochemical property enhanced in comparison with other taxanes is their potency at inducing tubulin assembly, despite the fact that their interactions with the microtubule binding sites (pore and luminal) are similar as studied by NMR and SAXS. A differential interaction with the S7-S9 loop (M-loop) is responsible for their enhanced assembly induction properties. The chemical changes in the structure also induce changes in the thermodynamic properties of the interaction, indicating a higher hydrophilicity and also explaining their properties on P-gp and βIII overexpressing cells and on mutant cells. The effect of the compounds on the microtubular network is different from those observed with the classical (docetaxel and paclitaxel) taxanes, inducing different bundling in cells with microtubules being very short, indicating a very fast nucleation effect and reflecting their high assembly induction power.