Subrata Chakravarty

Characterization of the Taxol Binding Site on the Microtubule IDENTIFICATION OF Arg282 IN β-TUBULIN AS THE SITE OF PHOTOINCORPORATION OF A 7-BENZOPHENONE ANALOGUE OF TAXOL

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically β-tubulin. Our previous studies have indicated that [3H]3′-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of β-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132–3134) and [3H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217–233 of β-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235–20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [3H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [3H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277–293. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg282 in β-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in β-tubulin as determined by photoaffinity labeling and electron crystallography.

Antithrombotic agents: From RGD to peptide mimetics

This review covers the recent advances in the development of highly potent inhibitors of platelet aggregation as potential therapeutic drugs for thrombosis related to cardiovascular and cerebrovascular diseases. The discovery of RGD sequence-directed cell surface receptors (the integrins) has led to extensive research in the development of small RGD containing peptides and their mimetics as antithrombotic agents. These agents work by inhibiting platelet aggregation through competitive blocking of fibrinogen to the platelet surface receptor, GPIIb/IIIa. The pharmacophoric nature of the aspartic acid and arginine side chains of the RGD unit has allowed the development of strategies for rational design, largely based on assumed bioactive RGD conformations and lead optimization. Applications of such strategies, from RGD peptides to peptide hybrids and then to non-peptide mimetics, are described. Also discussed is the important issue of specificity toward GPIIb/IIIa, keeping in view that the RGD unit is a key recognition signal for a variety of cell surface receptors.

Enantiopure fluorine-containing taxoids: potent anticancer agents and versatile probes for biomedical problems

Abstract Fluorine-containing paclitaxel and docetaxel analogs are reported that are obtained through the coupling of enantiopure (3 R ,4 S )-1-acyl-β-lactams with various baccatins. Some taxoids bearing the trifluoromethyl or difluoromethyl groups at the C-3′ position possessed three orders of magnitude greater potency than either paclitaxel or doxorubicin against both sensitive and drug-resistant human breast cancer cell lines. The incorporation of fluorine in taxoids as a probe has been successfully exploited for the study of their metabolism and structure. Fluorine-containing taxoids are shown to block the metabolic pathways associated with the cytochrome P-450 class of enzymes. Structural studies using 19 F and 1 H NMR techniques on fluorine-containing taxoids in combination with molecular modeling have provided new insights into the biologically relevant conformations of taxoids in solution as well as in the microtubule-bound state.

Recent advances in the medicinal chemistry of taxoid anticancer agents

Abstract Taxol ® (paclitaxel) and Taxotere ® (docetaxel) are currently considered to be the most promising leads in cancer chemotherapy. Both paciltaxel and docetaxel exhibit significant antitumor activity against various cancers, especially breast and ovarian cancers, which have not been effectively treated by existing chemotherapeutic drugs. The anticancer activity of these drugs is ascribed to their unique mechanism of action, i.e. causing mitotic arrest in cancer cells leading to apoptosis through inhibition of the depolymerization of microtubules. Although both paclitaxel and docetaxel possess potent antitumor activity, treatment with these drugs often results in a number of undesired side effects as well as multidrug resistance (MDR). Therefore, it has become essential to develop new anticancer agents with fewer side effects, superior pharmacological properties, and improved activity against various classes of tumors. This chapter describes the accounts of our research on the chemistry of paclitaxel and taxoid anticancer agents at the biomedical interface including: (i) the development of a highly efficient method for the semisynthesis of paclitaxel and a variety of taxoids by means of the β-Lactam Synthon Method (β-LSM), (ii) the structure-activity relationship (SAR) study of taxoids for their activities against human cancer cell lines, (iii) the discovery and development of “second-generation” taxoid anticancer agents that possess exceptional activities against drug-resistant cancer cells expressing the MDR phenotype as well as solid tumors (human cancer xenografts in mice), (iv) the development of fluorine-containing taxoids as a series of the second-generation taxoid anticancer agents and as excellent probes for the identification of bioactive conformation(s) of paclitaxel and taxoids by means of 19 F NMR in solution as well as in solid state for the microtubule-taxoid complex, (v) the development of radiolabeled photoreactive analogues of paclitaxel for photoaffinity labeling and mapping of the drug-binding domain on microtubules as well as P-glycoprotein that is responsible for MDR, and (vi) an SAR study of taxoids on their activities for inducing NO and tumor necrosis factor (TNF) through macrophage activation, which may be operative as an alternative mechanism of action. Thus, this chapter covers a wide range of issues associated with these powerful taxoid anticancer agents, discussing current status and future prospects.

The synthesis and evaluation of 12,13-benzodesoxyepothilone B: A highly convergent route

Abstract The title compound retains some of the affinity for microtubule assemblies as does 12,13-desoxyepothilone B.

Design, synthesis and SAR of RGD peptide hybrids as highly efficient inhibitors of platelet aggregation

Abstract A new series of peptide hybrids is developed as highly potent and selective antagonists of the GPIIb/IIIa receptor through rational modification of the RGDX sequence. Structure-activity relationships of these peptide hybrids have disclosed the important role of the C-terminal hydrophobic moiety and the N-terminal arginine side chain surrogates. Molecular modeling study strongly suggests the significance of a γ-turn conformation to achieve exceedingly high activity and receptor specificity.