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The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein. Congeners possessing the monodiene with a simplified lactone had superior anticancer efficacy relative to taxol, particularly in resistant models. Specifically, one of these congeners, B2, demonstrated 1) improved pharmacologic properties, specifically increased maximum response achievable and area under the curve, and decreased EC(50); 2) a uniform dose-response profile across genetically heterogeneous cancer cell lines relative to taxol or DDM; 3) reduced propensity for senescence induction relative to DDM; 4) superior long-term activity in cancer cells versus taxol or DDM; and 5) attenuation of metastatic characteristics in treated cancer cells. To contrast the binding of B2 versus DDM in tubulin, X-ray crystallography studies revealed a shift in the position of the lactone ring associated with removal of the C2-methyl and C3-hydroxyl. Thus, B2 may be more adaptable to changes in the taxane site relative to DDM that could account for its favorable properties. In conclusion, we have identified a DDM congener with broad range anticancer efficacy that also has decreased risk of inducing chemotherapy-mediated senescence. SIGNIFICANCE STATEMENT: Here, we describe the anticancer activity of novel congeners of the tubulin-polymerizing molecule (+)-discodermolide. A lead molecule is identified that exhibits an improved dose-response profile in taxane-sensitive and taxane-resistant cancer cell models, diminished risk of chemotherapy-mediated senescence, and suppression of tumor cell invasion endpoints. X-ray crystallography studies identify subtle changes in the pose of binding to β-tubulin that could account for the improved anticancer activity. These findings support continued preclinical development of discodermolide, particularly in the chemorefractory setting.

Several next-generation taxanes have been reported to possess high potency against Taxol-resistant cancer cell lines overexpressing βIII-tubulin and/or P-glycoprotein (P-gp), both of which are involved in drug resistance. Using a photoaffinity Taxol analogue, 2-(m-azidobenzoyl)-taxol, two potent next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibited distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources that differ in β-tubulin isotype composition. They also specifically inhibited photolabeling of P-gp, and the inhibitory effect correlated well with the steady-state accumulation of [(3)H]vinblastine in a multidrug resistant (MDR) cell line, SKVLB1. Several microtubule-stabilizing agents (MSAs)-resistant cell lines from the human ovarian cancer cell line Hey were isolated, and their MDR1 and βIII-tubulin levels determined. Distinct potencies of the two taxanes against different MSA-resistant cells expressing unique levels of MDR1 and βIII-tubulin were found. Cytotoxicity assays, done in the presence of verapamil, indicated that SB-T-1214 is a substrate, although not as good as Taxol, for P-gp. The mechanisms involved in drug resistance are multifactorial, and the effectiveness of new Taxol analogues depends on the interaction between the drugs and all possible targets; in this case the two major cellular targets are β-tubulin and P-gp.

5 p.-4 fig.-1 schem., Microtubule-stabilizing agents (MSAs) are widely used in chemotherapy. Here, using X-ray crystallography we describe the detailed binding modes of two potent MSAs, (+)-discodermolide (DDM) and the DDM-paclitaxel-hybrid KS-1-199-32, in the taxane pocket of B-tubulin. Both compounds bind in a very similar hairpin conformation as previously observed in solution. However, they differentially stabilize the M-loop of B-tubulin: KS-1-199-32 induces an M-loop helical conformation that is not observed for DDM. In the context of the microtubule structure, both MSAs connect the B-tubulin helices H6 and H7 and loop S9-S10 with the M-loop, which is similar to the structural effects elicited by epothilone A, but distinct from paclitaxel. Together, our data rationalize a differential binding mechanism of DDM and KS-1-199-32 on tubulin., This work was supported by grants from the Ministerio de Economia y Competitividad (BFU2016‐75319‐R (AEI/FEDER, UE), to J.F.D), the National Cancer Institute Grant CA077263 (A.B.S, H.M.D. and S.B.H), the Breast Cancer Research Foundation (H.M.D. and S.B.H.) and the Swiss National Science Foundation (31003A_166608; to M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery” and the COST action CM1470.

Eribulin mesylate is a synthetic analog of halichondrin B known to bind tubulin and microtubules, specifically at their protein rich plus-ends, thereby dampening microtubule (MT) dynamics, arresting cells in mitosis, and inducing apoptosis. The proteins which bind to the MT plus-end are known as microtubule plus-end tracking proteins (+TIPs) and have been shown to promote MT growth and stabilization. Eribulin's plus-end binding suggests it may compete for binding sites with known +TIP proteins such as End-binding 1 (EB1). To better understand the impact of eribulin plus-end binding in regard to the proteins which normally bind there, cells expressing GFP-EB1 were treated with various concentrations of eribulin. In a concentration dependent manner, GFP-EB1 became dissociated from the MT plus-ends following drug addition. Similar results were found with immuno-stained fixed cells. Cells treated with low concentrations of eribulin also showed decreased ability to migrate, suggesting the decrease in MT dynamics may have a downstream effect. Extended exposure of eribulin to cells leads to total depolymerization of the MT array. Taken together, these data show eribulin effectively disrupts EB1 +TIP complex formation, providing mechanistic insights into the impact of eribulin on MT dynamics.

There are seven β-tubulin isotypes present in distinct quantities in mammalian cells of different origin. Altered expression of β-tubulin isotypes has been reported in cancer cell lines resistant to microtubule stabilizing agents (MSAs) and in human tumors resistant to Taxol. To study the relative binding affinities of MSAs, tubulin from different sources, with distinct β-tubulin isotype content, were specifically photolabeled with a tritium-labeled Taxol analog, 2-(m-azidobenzoyl)taxol, alone or in the presence of MSAs. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulin from different sources. To determine the exact amount of drug that binds to different β-tubulin isotypes, bovine brain tubulin was photolabeled and the isotypes resolved by high-resolution isoelectrofocusing. All bands were analyzed by mass spectrometry following cyanogen bromide digestion, and the identity and relative quantity of each β-tubulin isotype determined. It was found that compared with other β-tubulin isotypes, βIII-tubulin bound the least amount of 2-(m-azidobenzoyl)taxol. Analysis of the sequences of β-tubulin near the Taxol binding site indicated that, in addition to the M-loop that is known to be involved in drug binding, the leucine cluster region of βIII-tubulin contains a unique residue, alanine, at 218, compared with other isotypes that contain threonine. Molecular dynamic simulations indicated that the frequency of Taxol-accommodating conformations decreased dramatically in the T218A variant, compared with other β-tubulins. Our results indicate that the difference in residue 218 in βIII-tubulin may be responsible for inhibition of drug binding to this isotype, which could influence downstream cellular events.

Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRATM), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of α-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent αβ-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used.

Suzan K. Chao 1 , Susan Band Horwitz 1 , and Hayley M. McDaid 1,2 1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461 2 Department of Medicine (Oncology) Albert Einstein College of Medicine, Bronx, NY 10461 Keywords: Senescence, 4E-BP1, mTORC1, p53, discodermolide Received: February 22, 2011; Accepted: February 22, 2011; Published: February 22, 2011; Correspondence: Susan Band Horwitz, e-mail: // Hayley McDaid, e-mail: // // Abstract Senescence is a valid tumor suppressive mechanism in cancer. Accelerated cell senescence describes the growth arrested state of cells that have been treated with anti-tumor drugs, such as doxorubicin that induce a DNA damage response. Discodermolide, a microtubule-stabilizing agent, is a potent inducer of accelerated cell senescence. Resistance to discodermolide is mediated via resistance to accelerated cell senescence, and is associated with reduced expression of the mTORC1 substrate, 4E-BP1 and increased expression of p53 [1]. Although the association of p53 with senescence induction is well-characterized, senescence reversion in the presence of high expression of p53 has not been well-documented. Furthermore, studies addressing the role of mTOR signaling in regulating senescence have been limited and recent data implicate a novel, senescence-associated role for 4E-BP1 in crosstalk with the transcription factor p53. This research perspective will address these somewhat contradictory findings and summarize recent research regarding senescence and mTORC1 signaling.

The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.

Microtubules have been identified as a suitable target for anticancer therapy, primarily based on their biological importance in coordinating chromosomal segregation at mitosis. Two main classes of microtubule-targeted agents, the taxanes and vinca alkaloids, suppress the dynamic behavior of spindle microtubules, inducing mitotic arrest and subsequent apoptotic cell death. Clinical activity of taxanes and first-generation vinca alkaloids in the treatment of solid tumors and hematologic malignancies, respectively, has prompted further research for novel analogs with improved clinical efficacy and safety. Such efforts have led to the development of vinflunine, a bifluorinated vinca alkaloid endowed with unique antitumor properties. Highlighted in this review are the key features of vinflunine that lead to effective suppression of microtubule dynamics and induction of cell death in cancer cells.

Novel second-generation taxoids with systematic modifications at the C2, C10, and C3'N positions were synthesized and their structure-activity relationships studied. A number of these taxoids exhibited exceptionally high potency against multidrug-resistant cell lines, and several taxoids exhibited virtually no difference in potency against the drug-sensitive and drug-resistant cell lines. These exceptionally potent taxoids were termed "third-generation taxoids". 19 (SB-T-1214), 14g (SB-T-121303), and 14i (SB-T-1213031) exhibited excellent activity against paclitaxel-resistant ovarian cancer cell lines with mutations in beta-tubulin as well, wherein the drug resistance is mediated by the beta-tubulin mutation. These taxoids were found to possess exceptional activity in promoting tubulin assembly, forming numerous very short microtubules similar to those formed by discodermolide. Taxoids 19 and 14g also showed excellent cytotoxicity against four pancreatic cancer cell lines, expressing three to four multidrug-resistant genes. Moreover, taxoid 19 exhibited excellent in vivo efficacy against highly drug-resistant CFPAC-1 pancreatic as well as DLD-1 human colon tumor xenografts in mice.

Purpose: To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4. Experimental Design: Human microsomes were used to determine the cytochrome P450 enzyme(s) involved in the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinetics, drug-target interactions, and pharmacodynamics of ixabepilone. Results: Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepilone dose administration to 25 mg/m2 when compared with single-agent therapy of 40 mg/m2. Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC0-∞. The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral blood mononuclear cells correlated with neutropenia. Conclusions: Ixabepilone is a good CYP3A4 substrate in vitro; however, in humans, it is likely to be cleared by multiple mechanisms. Furthermore, our results provide evidence that there is a direct relationship between ixabepilone pharmacokinetics, neutrophil counts, and microtubule bundle formation in PBMCs. Strong inhibitors of CYP3A4 should be used cautiously in the context of ixabepilone dosing.

A Taxol-resistant cell line, K20T, which does not express P-glycoprotein, was selected with Taxol from human MDA-MB-231 breast adenocarcinoma cells and maintained in the presence of 20nM Taxol. K20T cells were approximately 18-fold resistant to Taxol, displayed cross-resistance to Taxotere and the epothilones, but little cross-resistance to discodermolide. Sequence analysis of the class I beta-tubulin indicated that it harbored an A593G mutation resulting in a change from glutamate to glycine at amino acid 198, which is near the intradimer interface within the alpha/beta-tubulin heterodimer. An HA-tagged wild-type class I beta-tubulin expression vector was transfected into the K20T cells. Immunofluorescence studies demonstrated that this exogenous tubulin was incorporated into cellular microtubules and Western blot analysis indicated that the K20T transfectants predominantly expressed the exogenous wild-type class I beta-tubulin. The transfected cells were only approximately 5-fold resistant to Taxol. Our results, plus the knowledge that Glu198 is the target for other anti-tubulin agents, suggest that glutamate198 in beta-tubulin is a critical determinant for microtubule stability and Taxol resistance.

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the alpha/beta-tubulin heterodimer, GTP bound to beta-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to beta-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both beta- and alpha-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography-electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both beta-and alpha-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimer-dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography-electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers.

Purpose: To evaluate the drug combination of discodermolide and Taxol in human ovarian cancer cells and in an in vivo model of ovarian carcinoma. Experimental Design: The combination index method was used to evaluate the interaction of Taxol and discodermolide in human ovarian SKOV-3 carcinoma cells. Data were correlated with alterations in cell cycle distribution and caspase activation. In addition, SKOV-3 xenograft-bearing mice were treated with either Taxol, discodermolide, or a combination of both drugs given concurrently to evaluate the antitumor efficacy and toxicity of this combination. The Matrigel plug assay and CD31 immunohistochemistry were done to assess antiangiogenic effects. Results: Taxol and discodermolide interact synergistically over a range of concentrations and molar ratios that cause drug-induced aneuploidy in ovarian carcinoma cells. In SKOV-3 xenograft-bearing mice, the combination is significantly superior to either single agent, and induces tumor regressions without notable toxicities. Immunohistochemical analysis of CD31 and Matrigel plug analysis show decreased vessel formation in mice treated with the combination relative to either drug alone. Conclusions: The synergistic activity of Taxol and discodermolide in cells is most potent at drug concentrations that result in drug-induced aneuploidy rather than mitotic arrest. Moreover, in an animal model of ovarian carcinoma, this is a well-tolerated combination that induces tumor regressions and suppresses angiogenesis. These data confirm the potency of this combination and support the use of concurrent low doses of Taxol and discodermolide for potential use in cancer therapeutics.

[structure: see text] The design, synthesis, and biological evaluation of a series of (+)-discodermolide molecular probes possessing photoaffinity and fluorescent appendages has been achieved. Stereoselective olefin cross-metathesis comprised a key tactic for construction of two of the molecular probes. Three photoaffinity probes were radiolabeled with tritium.

Purpose: Because induction of drug efflux transporters is one of the major underlying mechanisms of drug resistance in cancer chemotherapy, and human pregnane X receptor (hPXR) is one of the principal “xenobiotic” receptors whose activation induces transporter and drug-metabolizing enzyme gene transcription, it would be ideal to develop chemotherapy drugs that do not activate hPXR. This report describes studies undertaken to explore the characteristics of hPXR stimulation and mechanisms of drug-receptor interactions in vitro with new anti-tubulin drugs. Experimental Design: In vitro transient transcription, glutathione S-transferase pull-down assays, and mammalian one-hybrid and two-hybrid systems were used to explore drug-receptor interactions. Loss of righting reflex was used to assess effects of drugs on PXR activity in vivo. Results: The current study showed that paclitaxel, discodermolide, and an analogue of epothilone B, BMS-247550, induced CYP3A4 protein expression in HepG2 hepatoma cells. Transient transcription assays of a luciferase reporter in the presence and absence of a GAL4-steroid and xenobiotic receptor (SXR) plasmid in HepG2 cells showed that these drugs activate hPXR. This was not true for the inactive analogue of paclitaxel, baccatin III, or for an analogue of epothilone A, analogue 5, none of which stabilizes microtubules. To determine the mechanisms by which paclitaxel, discodermolide, and BMS-247550 activate hPXR, a mammalian two-hybrid assay was done using VP16SRC-1 (coactivator) and GAL4-SXR. SRC-1 preferentially augmented the effects of these drugs on hPXR. Expression of SMRT (corepressor) but not NCoR suppressed the drug-induced activation of SXR by ∼50%, indicating a selectivity in corepressor interaction with hPXR. These drugs resulted in shortened duration of loss of righting reflex in vivo, indicating drug-induced activation of PXR in mice. Conclusion: These findings suggest that activation of hPXR with selective displacement of corepressors is an important mechanism by which microtubule-stabilizing drugs induce drug-metabolizing enzymes both in vitro and in vivo.

Discodermolide is a microtubule stabilizing agent that suppresses dynamic instability and blocks cells in mitosis. Selection of A549 nonsmall cell lung carcinoma cells with increasing concentrations of discodermolide yielded a clone that proliferated in 8 nM. When these cells were exposed to any concentration greater than 8 nM, replication ceased and the cells developed a flattened, enlarged, granular morphology. Accelerated senescence was demonstrated by a functional beta-galactosidase activity at pH 6. When parental A549 cells were treated with IC50-concentrations of doxorubicin, Taxol or discodermolide, the latter two drugs quickly produced aberrant mitosis. However, discodermolide, but not Taxol, also produced a large increase in senescence-associated beta-galactosidase activity and altered levels of known senescence markers. Although some of these differences between Taxol and discodermolide were dose dependent, only discodermolide produced a doxorubicin-like induction of a senescence phenotype, including a senescence-associated beta-galactosidase activity, up-regulation of PAI-1 and p66Shc, and a strong, sustained, Erk1/2 activation. This research provides insights into the mechanism of action of discodermolide and provides the first demonstration of a microtubule stabilizing agent that inhibits tumor cell growth with a powerful induction of accelerated senescence.

Since its approval by the FDA in 1992 for the treatment of ovarian cancer, the use of Taxol has dramatically increased. Although treatment with Taxol has led to improvement in the duration and quality of life for some cancer patients, the majority eventually develop progressive disease after initially responding to Taxol treatment. Drug resistance represents a major obstacle to improving the overall response and survival of cancer patients. This review focuses on mechanisms of Taxol resistance that occur directly at the microtubule, such as mutations, tubulin isotype selection and post-translational modifications, and also at the level of regulatory proteins. A review of tubulin structure, microtubule dynamics, the mechanism of action of Taxol and its binding site on the microtubule are included, so that the reader can evaluate Taxol resistance in context.

Differential expression of tubulin isotypes, mutations, and/or post-translational modifications in sensitive and Taxol-resistant cell lines suggests the existence of tubulin-based mechanisms of resistance. Since tubulin isotypes are defined by their C-terminal sequence, we previously described a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of tubulin diversity in human cell lines by analysis of their CNBr-released C-terminal peptides [Rao, S., Aberg, F., Nieves, E., Horwitz, S. B., and Orr, G. A. (2001) Biochemistry 40, 2096-103]. We now describe the liquid chromatography/electrospray ionization mass spectrometry analysis of native tubulins in Taxol-stabilized microtubules from parental and Taxol/epothilone-resistant human cancer cell lines. This method allows the direct determination of tubulin isotype composition, including post-translational modifications and mutations occurring throughout the entire protein. Four major isotypes, betaI-, betaIVb-, Kalpha1-, and alpha6-tubulin, were detected in two human carcinoma cell lines, A549 and HeLa. betaIII-Tubulin represented a minor species, as did alpha4-tubulin which was detected for the first time in both cell lines. The three alpha-tubulins were almost totally tyrosinated, and post-translational modifications were limited to low levels of monoglutamylation of Kalpha1-, betaI-, and betaIII-tubulin. betaII- and betaIVa-tubulins were not detected in either parental or drug-resistant cell lines, in contrast to previous RNA-based studies. Since mutations can occur in a single tubulin allele, the question as to whether the wild-type and mutant transcripts are both translated, and to what levels, is important. Heterozygous expression of Kalpha1- or betaI-tubulin mutants that introduced mass changes as small as 26 Da was readily detected in native tubulins isolated from Taxol- and epothilone-resistant cell lines.

Taxol is an antitumor drug approved by the FDA for the treatment of ovarian, breast and non-small-cell lung carcinomas. Originally isolated from the bark of the Pacific yew, Taxus brevifolia, it was the first natural product described that stabilized microtubules. In the past five years, a group of novel natural products, including the epothilones, discodermolide, eleutherobin, sarcodictyins and the laulimalides, all of which have biological activities similar to those of Taxol, has been discovered. In this review, we discuss each of these novel microtubule-stabilizing agents and the search for a common pharmacophore among them, taking into consideration recent advances in our understanding of the taxanes and tubulin.

Taxol is an anticancer agent of natural origin with significant activity against a number of human cancers including ovarian and breast carcinomas. Its cytotoxic activity has been attributed to its ability to stabilize microtubules and to promote microtubule assembly. Recently it has become clearer that Taxol has additional activities including effects in cell signaling and gene expression. We have shown previously that Taxol activates ERK 1/2 MAP-kinases and results in the formation of GRB2/SHC complexes in murine macrophage-like RAW 267.4 cells. Here we demonstrate that Taxol activates ERK 1/2 and p38 MAP-kinases in human ovarian carcinoma cells with distinct kinetics. Activation of ERK1/2 has been observed at low concentrations of Taxol (1-100 nM) within 0.5-6 h, whereas longer exposure(24 h) to nanomolar concentrations of Taxol resulted in an abrogation of the ERK1/2 phosphorylation/activation. Higher concentrations (1-10 microM) resulted in a sharp inhibition of ERK1/2 activity. p38 kinase was activated by high concentrations (1-10 microM) of Taxol within 2 h and remained active for more than 24 h. The kinetic studies showed that these effects of Taxol coincided with an inhibition of proliferation, and the onset of apoptosis. The appearance of the fragmented chromatin visualized by DAPI staining, and DNA fragments seen on an agarose gel, coincided with the decrease in ERK1/2 activation and concomitant increase of the level of active p38 MAPK. The inhibitor PD98059 abrogated ERK 1/2 activation and increased the cytotoxic effect of Taxol. An inhibitor of p38 kinase, SB203580, protected the cells partially from Taxol and, unexpectedly, activated ERK 1/2 kinases. We conclude that the alternative use of ERK1/2 and p38 MAP-kinase pathways may be necessary for the transition from proliferation state to Taxol-induced apoptosisin human ovarian carcinoma cells.

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.

Taxol (paclitaxel), a complex diterpene obtained from the Pacific yew, Taxus brevifolia , is arguably the most important new drug in cancer chemotherapy. The mechanism of cytotoxic action for paclitaxel—i.e., the stabilization of microtubules leading to mitotic arrest—is now shared by four recently identified natural products, eleutherobin, epothilones A and B, and discodermolide. Their ability to competitively inhibit [ 3 H]paclitaxel binding to microtubules strongly suggests the existence of a common binding site. Recently, we have developed nonaromatic analogues of paclitaxel that maintain high cytotoxicity and tubulin binding (e.g., nonataxel). We now propose a common pharmacophore that unites paclitaxel, nonataxel, the epothilones, eleutherobin, and discodermolide, and rationalizes the extensive structure–activity relationship data pertinent to these compounds. Insights from the common pharmacophore have enabled the development of a hybrid construct with demonstrated cytotoxic and tubulin-binding activity.

Two new taxol analogs 6 and 10 have been prepared from baccatin III (1) and taxol (7a), respectively. Like taxol, both compounds were found to promote microtubule formation and stabilization, although they were less active than taxol. Both 6 and 10 exhibited cytotoxicity against J774.2 cells; 6 was ∼60-fold less active and 10 was ∼15-fold less active.

Metablastin (also called p19, stathmin, prosolin, p18, Lap18, and oncoprotein 18) is a highly conserved, cytosolic 149-amino acid polypeptide that is expressed in immature vertebrate cells and undergoes extracellular factor- and cell cycle-regulated serine phosphorylation. The protein was shown recently to destabilize microtubules in vitro (Belmont, L., and Mitchison, T. J. (1996) Cell 84, 623-631). Here we demonstrate that microinjection of recombinant metablastin induces a loss of microtubules in COS-7 cells. This effect is enhanced by serine-to-alanine mutations at several phosphorylation sites and virtually abolished by aspartate substitution at a single site, Ser-63. We also show that stoichiometric amounts of metablastin prevent assembly and promote disassembly of microtubules in vitro. Interestingly, the phosphorylation site mutations of metablastin that have dramatic differential effects in intact cells do not alter the ability of metablastin to block tubulin assembly in vitro. The data suggest that phosphorylation of metablastin controls its microtubule-destabilizing activity in vivo but that this regulation may require additional cellular factors. This control mechanism is poised to play a critical role in the dynamic reorganization of the cellular microtubule network that occurs during morphogenesis and mitosis.

BACKGROUND Paclitaxel is an antimitotic agent isolated from the Pacific yew tree. It has demonstrated antitumor activity in several cancers and is the first of a new class of antineoplastic agents containing a taxane ring system. Its levels in serum and urine have been measured previously by high performance liquid chromatography (HPLC). In this study, the authors developed two competitive radioimmunoassay methods to determine whether they could reliably be used to measure levels of paclitaxel in sera and in cerebrospinal, ascitic, and pleural fluids. METHODS A monoclonal antibody prepared against paclitaxel was employed in an immunoradiometric assay (IRMA), in which 125I-labeled antibody was used, and in a more conventional tritiated radioimmunoassay (RIA),in which 3H-paclitaxel was used. RESULTS Both radioimmunoassays detected levels of paclitaxel in sera that were comparable to those observed with HPLC. However, the IRMA was the most sensitive. Only the IRMA was able to detect low levels of paclitaxel in cerebrospinal fluid after paclitaxel infusion and in sera 3 weeks after infusion. Both the IRMA and RIA methods were able to detect paclitaxel in ascitic and pleural fluids. CONCLUSIONS Monitoring paclitaxel levels reliably in sera and other bodily fluids is possible with these radioimmunoassays and may be of value in predicting and preventing toxicity and optimizing paclitaxel treatments. Cancer 1997; 79:1022-30. © 1997 American Cancer Society.

P-glycoprotein, the multidrug resistance transporter, is phosphorylated in vivo and the major phosphorylation domain has been identified as the linker region (amino acids 629-686). The linker region is a highly charged segment of the transporter in which the negative and positive amino acid side chains are spatially segregated. Both of these charged domains contain several consensus phosphorylation sites for protein kinases. Three of the consensus phosphorylation sites for basic-directed kinases in murine mdr1b P-glycoprotein are utilized in vivo and have been identified as serines 665, 669, and 681. Mutagenesis of all the consensus basic-directed kinase phosphorylation sites in the linker region of human MDR1 P-glycoprotein did not alter the ability of the mutated transporter to confer the multidrug resistance phenotype in stably transfected cell lines. These studies would suggest that phosphorylation/dephosphorylation within the basic domain of the linker region is not directly involved in regulation of drug transporter activity. We now report that the linker region of mdr1b P-glycoprotein is also phosphorylated in vivo within the acidic domain (amino acids 631-658). These sites have been mapped using casein kinase II, a prototypic acidic-directed kinase, and a recombinant mdr1b linker region peptide (amino acids 621-687). Electrospray mass spectrometry demonstrated that casein kinase II could introduce up to five phosphates into the recombinant peptide. Two-dimensional phosphopeptide mapping indicated that all the phosphates were contained in a tryptic peptide consisting of amino acids 631-658. Phosphopeptide mapping of in vivo labeled P-glycoprotein, isolated from either J7.V1-1, a murine vinblastine-resistant cell line, or HeLa cells stably transfected with mdr1b P-glycoprotein cDNA, revealed that this tryptic peptide was phosphorylated in both proteins.

Taxol, a natural product with significant anti-tumor activity, stabilizes microtubules and arrests cells in the G2/M phase of the cell cycle. It has been reported that taxol has additional effects in cells, including an increase in tyrosine phosphorylation of proteins and activation of MAP kinase. We investigated a possible effect of taxol on tyrosine phosphorylation of Shc and on formation of the Shc/Grb-2 complex in the murine macrophage-like cell line RAW 264.7. Shc, an SH2 domain containing adaptor protein, was immunoprecipitated from lysates of taxol-treated cells with anti-phosphotyrosine antibody and its identity determined by Western blotting with anti-Shc antibody. Non-denatured Shc containing protein complexes were immunoprecipitated with anti-Shc antibody, and analysis with an anti-Grb2 antibody revealed the presence of the 24-kDa Grb2 protein. Taxol also activated Raf-1 kinase and ERK1/ERK2 MAP kinases in these cells. These results demonstrate that taxol affects tyrosine phosphorylation of Shc and this may result in the activation of the Raf-1/MAPK cascade.

Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously 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). The interaction of a second photoaffinity analogue of taxol, [3H]2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels β-tubulin and the photolabeling is competed by both taxol and unlabeled 2-(m-azidobenzoyl)taxol indicating a common binding domain. To identify the site(s) of photoincorporation, [3H]2-(m-azidobenzoyl)taxol-photolabeled β-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of β-tubulin as the major photolabeled domain.

Next-generation taxanes are known to be active against taxane-resistant cell lines that overexpress P-glycoprotein (P-gp) and/or βIII-tubulin, both of which are associated with drug resistance. In this study, we examined the effect of two potent next-generation taxanes, SB-T-1214 and SB-CST-10202 (C-seco-taxane), on growth of the ovarian cancer cell line Hey and its drug-resistant daughter cell lines that overexpress different levels of P-gp and express increased levels of βIII-tubulin. We found that SB-T-1214 was ∼6-9-fold more potent than SB-CST-10202 against these drug resistant cells. Since the taxanes bind to both tubulin and P-gp, the relative binding affinity of the taxanes to these two cellular targets may influence the effectiveness of the taxanes in resistant cells. To determine the effects of these two taxanes on binding of Taxol® to P-gp and tubulin, a tritium-labeled Taxol® analog, 2-(m-azidobenzoyl)taxol (2-m-AzTax), was used. We have previously demonstrated that 2-m-AzTax photolabels a peptide (amino acids 217-231) in β-tubulin. To study the relative binding affinities of SB-T-1214 and SB-CST-10202, tubulins from bovine brain (BBT) and chicken erythrocytes (CET) were specifically photolabeled with [3H]2-m-AzTax, in the presence and absence of either Taxol®, Taxotere®, SB-T-1214 or SB-CST-10202. β-tubulin isotype content from BBT and CET is different, the former contains βI-, βII-, βIII- and βIV-tubulins, but the latter has only βVI. Taxol® had a minimal inhibitory effect (∼3%) on BBT, but a strong inhibitory effect (99%) on CET. Taxotere® appeared to have a stronger inhibitory effect than Taxol® on photolabeling (31% and 99% for BBT and CET, respectively). The inhibitory effects elicited by the two next-generation taxanes on photolabeling were distinct for β-tubulins from these two sources. SB-T-1214 and SB-CST-10202 inhibited BBT photolabeling by 38% and 32%, respectively. Interestingly, these two drugs caused a 41% and only 6% inhibition, respectively, on photolabeling of CET that does not contain βIII-tubulin. We are currently analyzing the effect of SB-T-1214 on binding of [3H]2-m-AzTax to different tubulin isotypes resolved by isoelectrofocusing. To study photolabeling of P-gp, plasma membranes from a multidrug resistant (MDR) cell line SKVLB1 that expresses very high levels of P-gp were prepared. SB-T-1214 and SB-CST-10202 inhibited photolabeling of P-gp markedly (> 80% inhibition), suggesting that these two taxanes are good substrates for P-gp. They also caused an increase in steady state [3H]2-m-AzTax accumulation in the MDR cell line SKVLB1. Since drug binding to tubulin and P-gp is the primary event that occurs in the cell following drug administration, our data on taxane binding affinity to the two targets will help in understanding the effectiveness of the next-generation taxanes in taxane-resistance. Citation Format: Chia-Ping H. Yang, Changwei Wang, Iwao Ojima, Susan Band Horwitz. The next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibit distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1229.

A series of 3′-difluorovinyl taxoids with C10 modifications, as well as those with C2 and C10 modifications, were strategically designed to block the metabolism by cytochrome P-450 3A4 enzyme and synthesized. These novel difluorovinyl taxoids were evaluated for their cytotoxicity against drug-sensitive human breast (MCF7), multidrug-resistant (MDR) human ovarian (NCI/ADR), human colon (HT-29) and human pancreatic (PANC-1) cancer cell lines. 3′-Difluorovinyl taxoids exhibit several to 16 times better activity against MCF7, HT-29 and PANC-1 cell lines and up to three orders of magnitude higher potency against NCI/ADR cell line as compared to paclitaxel. Structure–activity relationship study shows the critical importance of the C2 modifications on the activity against MDR cancer cell line, while the C10 modifications have a rather minor effect on the potency with some exceptions. The effect of the C2 modifications on potency against MCF7 cell line increases in the following order: H 3 . Among the twenty five 3′-difluorovinyl taxoids evaluated, eight taxoids exhibited less than 100 pM IC 50 values against MCF7 cell line. Difluorovinyl taxoids induced GTP-independent tubulin polymerization much faster than paclitaxel. Then, the resulting microtubules were stable to Ca 2+ -induced depolymerization, indicating strong stabilization of microtubules. Molecular modeling study indicated that a difluorovinyl taxoid binds to β-tubulin in a manner that is consistent with the REDOR-Taxol structure. The difluorovinyl group appears to mimic the isobutenyl group to some extent, but with very different electronic property, which may account for the unique activities of difluorovinyl taxoids.

Recent studies have highlighted the potential importance of posttranslational modifications of tubulin in dictating response to antitumor drugs and disease progression. These modifications include glutamylation, glycylation, phosphorylation, acetylation, and tyrosination. Some of the tubulin-modifying enzymes have been identified but the functional consequences of the posttranslational modifications remain largely unknown. In this chapter, we review the posttranslational modifications of tubulin and current knowledge of the role these alterations may play in human disease.

Two series of taxol analogs with various N -acyl groups on 3′-phenylisoserine and isoserine A-ring side chains, respectively, were prepared, and their microtubule assembly acitivities and cytotoxicities toward J774.2 cells were evaluated. Biological activities were adversely affected by the structural modifications investigated, although the 3′-phenylisoserine series was significantly more active than the isoserine series.

Taxol is a structurally novel and clinically effective antitumor drug, which, unlike other antimitotic agents, induces the assembly of tubulin into microtubules. To characterize the binding site(s) of taxol on the microtubule, taxol-based photoaffinity reagents 1 and 2 bearing photoreactive groups on the A-ring side chain were prepared and evaluated. Taxol analogue 1 exhibits better microtubule assembly activity, greater cytotoxicity toward J774.2 cells, and more specific and efficient photolabeling of the beta-subunit of tubulin than does analogue 2. Therefore, it would appear that 1 is the better candidate for further studies aimed at the characterization of the taxol binding site on the microtubule.

MA is an orally active PG derivative with an excellent safety profile that is used primarily for the treatment of carcinomas of the breast and endometrium. We investigated the potential application of MA as an MDR-reversal agent using cell culture and human tumor xenograft models. The reversing activity of MA in vitro was compared with that of PG and VER in two human MDR cell lines, the colon carcinoma HCT-116/VM46 and the breast carcinoma MCF-7/ADR, and in a murine cell line, J774.2. At concentrations as low as 3 microM, MA was capable of partially restoring sensitivity to Act D in the HCT-116/VM46 cells and sensitivity to DOX in the MCF-7/ADR cells. Although less effective than VER, MA was about 2.5 times more potent than PG in reversing MDR at equimolar concentrations. Increased accumulation of DOX in drug-resistant cells that were treated simultaneously with MA was observed by flow cytometry. In vivo, using established human colon and breast carcinoma xenografts implanted s.c. in athymic mice, the combined therapy with MA and DOX resulted in enhanced antitumor activity relative to that of DOX alone in the MDR sublines. These results suggest that MA may be a promising clinical MDR-reversing agent.

Discodermolide is a microtubule-stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild-type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, reintroduction of a nonphosphorylatable mutant (Thr-37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32-resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation, and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence.

Epothilone B (EpoB), like Taxol, stabilizes microtubules resulting in an inhibition of microtubule dynamic instability. The drug is being evaluated in phase III clinical trials. An EpoB analog, Ixabepilone, was approved by the FDA for the treatment of taxane-resistant metastatic breast cancer. Epithelial cell adhesion antigen (EpCAM) expression is significantly higher in epithelial ovarian cancer cells compared to normal cells. The effects of EpoB and other microtubule-interacting agents on surface EpCAM expression were studied.Biochemical methods, immunofluorescence and flow cytometry were used to identify EpCAM expression on the surface of the ovarian cancer cell line, Hey, after exposure to EpoB. The relationship between EpoB-mediated surface EpCAM expression and EpoB-induced α-tubulin acetylation, a surrogate marker for stable microtubules, in Hey cells also was investigated.Nanomolar concentrations of EpoB, Taxol, discodermolide or vinblastine caused a marked increase in surface EpCAM expression in Hey cells. Alpha-tubulin acetylation was increased following treatment with Taxol, EpoB and discodermolide, but not with vinblastine, indicating that drug-enhanced surface EpCAM expression does not correlate with tubulin acetylation or stabilization. Unexpectedly, EpoB did not have a significant effect on EpCAM mRNA expression, nor did it alter the level of total cellular EpCAM in Hey cells.The results indicate that disruption of the microtubule cytoskeleton is associated with the redistribution of cell surface antigens in ovarian cancer cells. The increase in cell surface EpCAM antigen density may facilitate the antibody targeting of EpCAM-positive ovarian cancer cells.

Multi-drug-resistant cells overproduce a 130-180-kDa integral membrane phosphoglycoprotein known as P-glycoprotein which acts as an energy-dependent drug efflux pump. While P-glycoprotein has been shown to transport hydrophobic anti-tumor drugs out of multi-drug-resistant cells in tissue culture, its endogenous substrates remain unknown. This report shows that 3H-corticosterone can specifically photoaffinity label P-glycoprotein. Furthermore, corticosterone is effluxed from multi-drug-resistant cells by P-glycoprotein. These data suggest that corticosterone may be an endogenous substrate for P-glycoprotein.

Background Taxol is a potent inhibitor of the replication of eukaryotic cells and has significant antitumor activity in human malignancies. The drug induces the formation of bundles of stable microtubules and blocks cells in the mitotic phase of the cell cycle. In vitro, taxol enhances the polymerization of tubulin to microtubules that are resistant to depolymerization. Although it is evident that taxol interacts with the tubulin-microtubule system, no information has been available on the binding site for the drug on the microtubule. Purpose Our purpose was to determine if taxol binds to one or both of the tubulin subunits. Methods In the absence of a photoaffinity-labeled analogue of taxol, [3H]taxol was used directly to photolabel tubulin. A complex of microtubule protein and [3H]taxol was irradiated by ultraviolet light and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results and conclusions The radiolabeled drug preferentially binds covalently to the beta-subunit of tubulin, and the binding can be competed with unlabeled taxol. Implications This observation is the first step in a study to determine the binding site for taxol on the microtubule.

[reaction-see text] The syntheses of two epothilone analogues, 15(S)-aza-12,13-desoxyepothilone B and the epimeric 15(R)-aza-12,13-desoxyepothilone B, are described. A Mitsunobu inversion was utilized for elaboration of pre-epothilone fragments to the corresponding macrolactam. Tubulin binding and cytotoxicity profiles of these analogues are presented.

The microtubule cytoskeleton continues to be both an effective and a validated target in the therapy of cancer. Beginning with the vinca alkaloids almost 50 years ago and encouraged by the broad activity of first taxol® (paclitaxel) and then taxotere® (docetaxel) numerous investigators have identified structurally diverse compounds that interact with the tubulin/microtubule system displaying antimitotic and anticancer properties. Paclitaxel was the first of a class of agents with a binding site on the microtubule polymer that act principally by stabilizing microtubules. Other members of this class of compounds referred to as the microtubule-stabilizing agents include the epothilones, eleutherobin and discodermolide, natural products that are mechanistically similar but structurally unrelated to paclitaxel. With an emphasis on paclitaxel, the original and best-characterized member of the microtubule-stabilizing class, this chapter will review the compounds that comprise this diverse and interesting class of anti-cancer agents.

Novel macrocyclic paclitaxel congeners were designed to mimic the bioactive conformation of paclitaxel. Computational analysis of the "REDOR-Taxol" structure revealed that this structure could be rigidified by connecting the C14 position of the baccatin moiety and the ortho position of C3'N-benzoyl group (C3'BzN), which are ca. 7.5 A apart, with a short linker (4-6 atoms). 7-TES-14beta-allyloxybaccatin III and (3R,4S)-1-(2-alkenylbenzoyl)-beta-lactams were selected as key components, and the Ojima-Holton coupling afforded the corresponding paclitaxel-dienes. The Ru-catalyzed ring-closing metathesis (RCM) of paclitaxel-dienes gave the designed 15- and 16-membered macrocyclic taxoids. However, the RCM reaction to form the designed 14-membered macrocyclic taxoid did not proceed as planned. Instead, the attempted RCM reaction led to the occurrence of an unprecedented novel Ru-catalyzed diene-coupling process, giving the corresponding 15-membered macrocyclic taxoid (SB-T-2054). The biological activities of the novel macrocyclic taxoids were evaluated by tumor cell growth inhibition (i.e., cytotoxicity) and tubulin-polymerization assays. Those assays revealed high sensitivity of cytotoxicity to subtle conformational changes. Among the novel macrocyclic taxoids evaluated, SB-T-2054 is the most active compound, which possesses virtually the same potency as that of paclitaxel. The result may also indicate that SB-T-2054 structure is an excellent mimic of the bioactive conformation of paclitaxel. Computational analysis for the observed structure-activity relationships is also performed and discussed.

P-glycoprotein is a 130-180-kDa integral membrane protein that is overproduced in multidrug-resistant cells. The protein appears to act as an energy-dependent drug efflux pump that has broad specificity for structurally diverse hydrophobic antitumor drugs. Many agents, such as the calcium channel blocker verapamil, reverse multidrug resistance and also interact with P-glycoprotein. The goal of this work was to determine if a common binding site participates in the transport of antitumor drugs and/or the reversal of drug resistance. This was done by comparing the peptide maps of P-glycoprotein (encoded by mdr1b) after it was labeled with a photoactive calcium channel blocker, [3H]azidopine, and a newly identified photoaffinity analog for P-glycoprotein 2-[4-(4-azido-3-[125I]iodobenzoyl) piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline [( 125I]iodoaryl azidoprazosin). [125I] Iodoaryl azidoprazosin, which classically has been used to identify the alpha 1-adrenergic receptor, bound to P-glycoprotein and was preferentially competed by vinblastine greater than actinomycin D greater than doxorubicin greater than colchicine. Peptide maps derived from P-glycoprotein labeled with [3H]azidopine or [125I]iodoaryl azidoprazosin were identical. After maximal digestion under conditions for Cleveland mapping, a single major 6-kDa fragment was obtained after digestion with V8 protease, whereas two major fragments, 6.5 and 5.5 kDa, were detected after digestion with chymotrypsin. The 6.0-kDa V8 fragment and the 6.5-kDa chymotrypsin fragment were both found when P-glycoprotein encoded by mdr1a and mdr1b was compared. Despite its specific interaction with P-glycoprotein, neither iodoaryl azidoprazosin nor prazosin markedly reversed resistance compared with verapamil or azidopine. Further, multidrug-resistant cells were 900-fold resistant to vinblastine but only 5-fold resistant to prazosin. These data demonstrate that structurally diverse reversal and/or antitumor agents are likely to have differential affinity for a small common domain of P-glycoprotein.

Two vinblastine-resistant sublines of the murine macrophage-like cell line J774.2, J7.V1-1 and J7.V3-1, overproduce unique forms of P-glycoprotein that are encoded by distinct mdr genes, mdr lb and mdr la, respectively. Degradation rates of the two P-glycoprotein isoforms were measured by immunoprecipitation of P-glycoprotein. The half-life of immunoprecipitable P-glycoprotein from J7.V1-1 cells was 16.8 ± 0.5 hours and from J7.V3-1 cells, 17.4 ± 0.5 hours. This rate was not influenced by the presence of vinblastine in the growth medium. The data indicate that P-glycoproteins derived from distinct genes have similar degradation rates.

Background: We previously demonstrated that peak microtubule bundle formation (MBF) in peripheral blood mononuclear cells (PBMCs) occurs at the end of drug infusion and correlates with drug pharmacokinetics (PK). In the current study, a new expanded evaluation of drug target effect was undertaken. Patients and methods: Patients with advanced solid malignancies were treated with ixabepilone 40 mg/m2 administered as a 1-h i.v. infusion every 3 weeks. Blood, plasma, and tumor tissue sampling was carried out to characterize pharmacodynamics and PK. Results: Forty-seven patients were treated with 141 cycles of ixabepilone. In both PBMCs (n = 27) and tumor cells (n = 9), peak MBF occurred at the end of infusion; however, at 24–72 h after drug infusion, the number of cells with MBF was significantly greater in tumor cells, relative to PBMCs. A Hill model (EC50 = 109.65 ng/ml; r2 = 0.94) was fitted, which demonstrated a relationship between percentage of PBMCs with MBF and plasma ixabepilone concentration. The percentage of PBMCs with MBF at the end of infusion also correlated with severity of neutropenia (P = 0.050). Conclusions: Plasma ixabepilone concentration and severity of neutropenia correlate with the level of MBF in PBMCs. Therefore, this technically straightforward assay should be considered as a complement to the clinical development of novel microtubule-binding agents.

Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-(3)H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, approximately 1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [3H]Taxol from microtubules, but Taxol cannot effectively displace C19-[3H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[3H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305-433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355-359, in beta-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.