Taxane Binding Research Articles

Macrocyclic lactones and their relationship to the SNPs related to benzimidazole resistance

Haemonchus contortus is an abomasal nematode of ruminants that is widely present across the world. Its ability to cause death of infected animals and rapidly develop anthelmintic resistance makes it a dangerous pathogen. Ivermectin (IVM) and moxidectin (MOX) are macrocyclic lactones (MLs). They have been successfully used to treat parasitic nematodes over the last three decades. A genetic association between IVM selection and single nucleotide polymorphisms (SNPs) on the β-tubulin isotype 1 gene was reported in H. contortus. These SNPs result in replacing phenylalanine (F, TTC) with tyrosine (Y, TAC) at position 167 or 200 on the β-tubulin protein. Recently we reported a direct interaction of IVM with α- and β-tubulin. It had been hypothesized that the SNPs (F167Y and F200Y) may change tubulin dynamics and directly affect IVM binding. The goal of the current study was to observe the effects of SNPs (F167Y and F200Y) on tubulin polymerization and IVM binding. It was also of interest to evaluate the differences between IVM and MOX on tubulin polymerization. We conclude that the SNPs cause no difference in the polymerization of wild and mutant tubulins. Furthermore, neither of the SNPs reduced IVM binding. Varying results were obtained in the degree of polymerization of parasitic and mammalian tubulin for IVM and MOX, i.e., the extent of polymerization was greater for IVM compared with MOX, for H. contortus tubulin, and vice versa for mammalian tubulin. Molecular modeling showed that IVM and MOX docked into the taxane binding pocket of both mammalian and parasitic wild type and mutant tubulins. However the binding was stronger for mammalian tubulin as compared to parasitic tubulin.

The quest for a simple bioactive analog of paclitaxel as a potential anticancer agent.

ConspectusPaclitaxel (PTX), introduced into the clinic in 1991, has revealed itself as an effective antimicrotubule drug for treatment of a range of otherwise intractable cancers. Along with docetaxel (DTX) and in combination with other agents such as cisplatin, it has proven to be a first-line therapy. Unfortunately, PTX and DTX carry severe liabilities such as debilitating side effects, rapid onset of resistance, and rather complex molecular structures offering substantial challenges to ease of synthetic manipulation. Consequently, the past 15 years has witnessed many efforts to synthesize and test highly modified analogs based on intuitive structural similarity relationships with the PTX molecular skeleton, as well as efforts to mimic the conformational profile of the ligand observed in the macromolecular tubulin–PTX complex.Highly successful improvements in potency, up to 50-fold increases in IC50, have been achieved by constructing bridges between distal centers in PTX that imitate the conformer of the electron crystallographic binding pose. Much less successful have been numerous attempts to truncate PTX by replacing the baccatin core with simpler moieties to achieve PTX-like potencies and applying a wide range of flexible synthesis-based chemistries. Reported efforts, characterized by a fascinating array of baccatin substitutes, have failed to surpass the bioactivities of PTX in both microtubule disassembly assays and cytotoxicity measurements against a range of cell types. Most of the structures retain the main elements of the PTX C13 side chain, while seeking a smaller rigid bicycle as a baccatin replacement adorned with substituents to mimic the C2 benzoyl moiety and the oxetane ring.We surmise that past studies have been handicapped by solubility and membrane permeability issues, but primarily by the existence of an expansive taxane binding pocket and the discrepancy in molecular size between PTX and the pruned analogs. A number of these molecules offer molecular volumes 50–60% that of PTX, fewer contacts with the tubulin protein, severe mismatches with the PTX pharmacophore, lessened capacity to dispel binding site waters contributing to ΔGbind, and unanticipated binding poses. The latter is a critical drawback if molecular designs of simpler PTX structures are based on a perceived or known PTX binding conformation. We conclude that design and synthesis of a highly cytotoxic tubulin-assembly agent based on the paclitaxel pharmacophore remains an unsolved challenge, but one that can be overcome by focus on the architecture of the taxane binding site independent of the effective, but not unique, hand-in-glove match represented by the PTX–tubulin complex.

Abstract 5162: Microtubule stabilization alters tumor secretome and fibroblast activation.

Abstract Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, drugs targeting MTs, like the taxanes, often fail in the metastatic setting. Therefore it is of utmost importance to fully understand the roles of MTs and MT-targeting agents in tumor biology. An important process during tumor development and metastasis involves the remodeling of the 3D microenvironment surrounding tumor cells. In order to successfully proliferate and metastasize, tumor cells need to dynamically respond and remodel their surrounding microenvironment. We hypothesized that tumor cells may stimulate residential fibroblasts in MT-dependent manner, then activated fibroblasts may play a major role in remodeling of 3D microenvironment. To test this hypothesis, we assessed the MT-dependence of metastatic MDA-MB-231 breast cancer cell activation of HMF3S human mammary fibroblasts. HMF3S fibroblasts were treated with either vehicle- or paclitaxel (PTX)-pretreated MDA-MB-231 cell conditioned media. Vehicle conditioned media activated HMF3S fibroblasts as shown by increased directional 3D motility towards a serum gradient. In contrast, PTX-pretreatment abolished the increased directional motility of the fibroblasts. Importantly, vehicle condition media from PTX-treated K20T MDA-MB-231 cells, which carry a tubulin mutation at taxane binding site, did not show reduced 3D motility of fibroblasts. Together, these results suggest that breast cancer cells produce fibroblast activation factor(s) in MT dependent manner. In order to identify the factors responsible for this MT-dependent cell-cell communication, we performed a differential proteomic analysis of the conditioned media from vehicle- and PTX-pretreated MDA-MB-231 cells. Using splitless nanoflow chromatography coupled with quadrupole time-of-flight (QTOF) mass spectrometry, we identified a total of 2124 proteins within these tumor secretomes. Using on a 2-fold change threshold, we identified 78 under-represented and 31 over-represented proteins in the secretome of PTX- versus vehicle-pretreated cells. We focused on under-represented proteins based on the hypothesis that MT stabilization would inhibit their secretion. We have identified proteins involved in cell motility, cell-cell communication, or extracellular matrix remodeling. TGF-β, CTGF, c-Met, fibronectin, and lysyl oxidase 2 were the key down-regulated proteins identified in the tumor secretome upon PTX-induced MT stabilization. Among the factors identified by mass spectrometry analyses, TGF-β has been known to play an important role in cell migration. Supporting this idea, treatment of vehicle-pretreated condition media with TGF-β blocking antibody abolished 3D migration of fibroblasts. These findings shed a light on a novel role of interphase MTs in tumor biology and may pave the way to enhance our understanding of taxane clinical efficacy. Citation Format: Andy Tran, Katsuhiro Kita, Alexandre Matov, Duane C. Hassane, Lewis M. Brown, Paraskevi Giannakakou. Microtubule stabilization alters tumor secretome and fibroblast activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5162. doi:10.1158/1538-7445.AM2013-5162

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.

Dictyostatin Flexibility Bridges Conformations in Solution and in the β-Tubulin Taxane Binding Site

Dictyostatin (DCT, 1) is a complex, flexible polyketide macrolide that demonstrates potent microtubule-polymerization activity. Both a solution structure (2a) and a possible binding mode for DCT (Conf-1) have been proposed by earlier NMR experiments. In the present study, the conformational landscape of DCT in DMSO-d(6) and methanol-d(4) was explored using extensive force-field-based conformational searches combined with geometric parameters derived from solution NMR data. The results portray a diversity of conformations for dictyostatin that illustrates the molecule's flexibility and excludes the previously suggested dominant solution conformation 2a. One conformation present in DMSO-d(6) with a 7% population (Conf-2, 0.6 kcal/mol above the global minimum at 298°) also satisfies the TR-NOESY NMR parameters of Canales et al. that characterize the taxane binding-site interaction between DCT and assembled microtubules in water. Application of several docking methods (Glide, Autodock, and RosettaLigand) has identified a low-energy binding model of the DCT/β-tubulin complex (Pose-2/Conf-2) that is gratifyingly compatible with the emerging DCT structure-activity data.

Abstract 730: Targeting colchicine binding site in tubulin for the treatment of advanced melanoma

Abstract Inhibition of tubulin dynamics has been a fruitful drug target for cancer treatment. Out the three ligand binding sites in tubulin, drugs targeting taxane binding site (e.g. taxol and epithilones) and vinca alkaloids binding site (e.g. vinblastine) are clinically very successful drugs. However, no FDA-approved cancer drugs targeting the colchicine binding site in tubulin currently exist. In our ongoing efforts to search for effective drugs for the treatment of advanced melanoma that are highly resistant to all existing cancer treatment, we have discovered a new class of compounds that are not substrates of P-glycoprotein (Pgp) and are highly efficacious against human melanoma tumors in A375 xenograft models. At relatively low doses (5∼15 mg/kg), these compounds inhibit melanoma tumor growth more effectively than DTIC (the gold standard drug for advanced melanoma) at 60 mg/kg. Extensive biological and molecular modeling studies suggest that these compounds work by competitively binding to the colchicine site in tubulin. Further optimization of these compounds hold great promise in providing an effective agent for the treatment of advanced melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 730.

Histological and genetic markers for non-small-cell lung cancer: Customizing treatment based on individual tumor biology

To describe how molecular and genetic markers influence the response to therapy in patients with advanced non-small-cell lung cancer (NSCLC). Cisplatin resistance has been associated with high rates of expression of endonucleases, a family of DNA repair proteins that includes ERCC1 and BRCA1. Gemcitabine is thought to produce its antitumor effects by several mechanisms, including inhibition of ribonucleotide conversion to deoxyribonucleotide through the inactivation of ribonucleotide reductase. One type of gemcitabine resistance occurs when the inactivation of ribonucleotide reductase decreases, which correlates with increased expression of the M1 subunit of ribonucleotide reductase. Taxanes produce their anti-tumor effects by binding to and stabilizing intracellular microtubules, which are necessary for DNA replication and cell division. High expression of beta-tubulin III, a microtubule subunit with low taxane binding affinity, appears to confer resistance to taxane therapy. High expression of thymidylate synthase, an enzyme that is important in purine synthesis and DNA replication, has been associated with decreased response to uracil antimetabolites, and may also be an important prognostic factor in patients receiving pemetrexed. The epidermal growth factor receptor (EGFR) is a cell- surface receptor with an intracellular tyrosine kinase domain that is thought to modulate numerous cellular functions that contribute to tumorigenicity, tumor invasiveness, and resistance to therapy. Recent clinical trials have demonstrated that treatment response to small-molecule and monoclonal antibody inhibitors of EGFR is greatest for patients who have a higher EGFR gene copy number. Several genetic, molecular, and histological markers may affect treatment response in patients with NSCLC. Evaluating patients for such markers is important not only for treatment efficacy, but also to improve safety and tolerability by avoiding exposure to treatments that are unlikely to produce significant benefits. Nearly all of the available information regarding the predictive value of these markers has been derived from retrospective studies. Prospective clinical trials are important to validate marker evaluation methodology and the prospective utility of biomarkers in clinical decision making.

Distinct Pose of Discodermolide in Taxol Binding Pocket Drives a Complementary Mode of Microtubule Stabilization

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.

Differences in Paclitaxel and Docetaxel Interactions with Tubulin Detected by Mutagenesis of Yeast Tubulin

Differences in Paclitaxel and Docetaxel Interactions with Tubulin Detected by Mutagenesis of Yeast Tubulin

Structural studies of taxol analogues for drug discovery

Background: The major mechanism of action of antitumor taxanes, including two clinically useful antitumor agents, paclitaxel and docetaxel, lies in their interactions with β-tubulin in microtubule polymers, leading to cell cycle arrest and apoptosis. However, owing to the technical limitations, the molecular interactions of ligands with the residues in taxane binding sites of tubulin as well as the conformations adopted by taxanes on binding are still not fully understood. Objective: This review focuses on the exploration of paclitaxel's interactions with tubulin, and the impact of such efforts on the drug discovery for new taxanes and microtubule stabilizing agents (MSAs). Methods: Data were identified through the search of Chemical s and PubMed databases for research articles and reviews up to April 2008. Conclusion: Based on a collection of information gathered from crystallography, 1D and 2D NMR spectroscopy (NOESY, ROESY, REDOR), and structural–activity relationship (SAR) by chemical synthesis and pharmacological assays, ‘opened’ or T-shape conformations have been predicted to be the biologically active ones in recent years, and confirmed by further SAR studies. Some more potent analogues than paclitaxel or simplified compounds with similar potencies to that of paclitaxel have been discovered. Structural studies of taxol analogues will continue to make great contributions to the rational design of taxanes and novel prototype MSAs for drug discovery.

Farnesyltransferase Inhibitors Reverse Taxane Resistance

The combination of farnesyltransferase inhibitors (FTIs) and taxanes has been shown to result in potent antiproliferative and antimitotic synergy. Recent phase I and II clinical trials have shown that this combination shows clinical activity in taxane-refractory or taxane-resistant cancer patients. To understand the mechanism behind these clinical observations, we used a cancer cell model of paclitaxel resistance and showed that the FTI/taxane combination retains potent antiproliferative, antimitotic, and proapoptotic activity against the paclitaxel-resistant cells, at doses where each drug alone has little or no activity. To probe the mechanistic basis of these observations, paclitaxel activity was monitored in living cells using the fluorescently conjugated paclitaxel, Flutax-2. We observed that all FTIs tested increase the amount of microtubule-bound Flutax-2 and the number of microtubules labeled with Flutax-2 in both paclitaxel-resistant and paclitaxel-sensitive cells. Importantly, we observed a consequential increase in microtubule stability and tubulin acetylation with the combination of the two drugs, even in paclitaxel-resistant cells, confirming that the enhanced taxane binding in the presence of FTI affects microtubule function. Furthermore, this mechanism is dependent on the function of the tubulin deacetylase, HDAC6, because in cells overexpressing a catalytically inactive HDAC6, FTIs are incapable of enhancing Flutax-2-microtubule binding. Similar results were obtained by using an FTI devoid of farnesyltransferase inhibitory activity, indicating that functional inhibition of farnesyltransferase is also required. Overall, these studies provide a new insight into the functional relationship between HDAC6, farnesyltransferase, and microtubules, and support clinical data showing that the FTI/taxane combination is effective in taxane-refractory patients.