Modulation of taxane binding to tubulin curved and straight conformations by systematic 3'N modification provides for improved microtubule binding, persistent cytotoxicity and in vivo potency.

The taxane class of microtubule stabilizers are some of the most effective and widely used chemotherapeutics. The anticancer activity of taxanes arises from their ability to induce tubulin assembly by selectively recognizing the curved (c-) conformation in unassembled tubulin as compared to the straight (s-) conformation in assembled tubulin. We first designed and synthesized a series of 3'N-modified taxanes bearing covalent groups. Instead of discovering covalent taxanes, we found a series of non-covalent taxanes 2, in which the 3'N side chain was found to be essential for cytotoxicity due to its role in locking tubulin in the s-conformation. A representative compound bearing an acrylamide moiety (2h) exhibited increased binding affinity to the unassembled tubulin c-conformation and less cytotoxicity than paclitaxel. Further exploration of chemical space around 2h afforded a new series 3, in which derivatives such as 3l bind more tightly to both the s- and c-conformations of tubulin compared to paclitaxel, leading to more efficient promotion of tubulin polymerization and a greater persistence of in vitro efficacy against breast cancer cells after drug washout. Although 3l also had improved in vivo potency as compared to paclitaxel, it was also associated with increased systemic toxicity that required localized, intratumoral injection to observe potent and prolonged antitumor efficacy.

Structural insight into the stabilization of microtubules by taxanes.

Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules' diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a ßM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.

Targeting the tubulin C-terminal tail by charged small molecules.

The disordered tubulin C-terminal tail (CTT), which possesses a higher degree of heterogeneity, is the target for the interaction of many proteins and cellular components. Compared to the seven well-described binding sites of microtubule-targeting agents (MTAs) that localize on the globular tubulin core, tubulin CTT is far less explored. Therefore, tubulin CTT can be regarded as a novel site for the development of MTAs with distinct biochemical and cell biological properties. Here, we designed and synthesized linear and cyclic peptides containing multiple arginines (RRR), which are complementary to multiple acidic residues in tubulin CTT. Some of them showed moderate induction and promotion of tubulin polymerization. The most potent macrocyclic compound 1f was found to bind to tubulin CTT and thus exert its bioactivity. Such RRR containing compounds represent a starting point for the discovery of tubulin CTT-targeting agents with therapeutic potential.

Discovery of 12ß-oxygenated oleanolic acid alkyl esters as potent and selective FXR modulators exhibiting hyperglycemia amelioration in vivo.

Farnesoid X receptor (FXR) ligands have been actively pursued to treat metabolic disorders, liver and bile diseases, among others. Starting from a widely occurring natural product, oleanolic acid (OA), we discovered potent and selective FXR modulator from the 12ß-oxygenated OA alkyl esters, with the assistance of molecular modeling. The representative compound 7b modulated some FXR downstream genes involved in glucose and lipid metabolism in cells, and significantly improved hyperglycemia in KKay fat mice fed with high fat diet, through the reduction of mRNA expression of gluconeogenesis genes PEPCK and G6Pase. This study provides a new series of selective FXR modulator, as well as the in vitro and in vivo evidence for their potential to improve hyperglycemia in diabetic mice through FXR antagonism.

Structure Modification of FXR Antagonistic Chalcones and Their Inhibitory Effects on NSCLC Cell Proliferation and Metastasis.

Although the farnesoid X receptor (FXR) has been regarded as a promising drug target for metabolic diseases as well as anti-inflammatory, antitumor and antiviral actions, the antagonism by FXR ligands are still underrepresented in current FXR targeted therapies. In this study, we discovered selective FXR antagonists through structure optimization from the polyoxygenated chalcone scaffold. The selective antagonist 6 p [2-methoxy-2'-hydroxy-4'-(4''-methoxy-4''-oxo-E-crotonyl) chalcone] is not only inhibitory toward non-small-cell lung cancer (NSCLC) cell proliferation in an FXR-dependent manner, but is also active in metastasis models. Taken together, this chalcone-based FXR antagonist has the potential for the targeted therapy of NSCLC in which FXR is highly expressed.

Taxanes convert regions of perturbed microtubule growth into rescue sites.

Microtubules are polymers of tubulin dimers, and conformational transitions in the microtubule lattice drive microtubule dynamic instability and affect various aspects of microtubule function. The exact nature of these transitions and their modulation by anticancer drugs such as Taxol and epothilone, which can stabilize microtubules but also perturb their growth, are poorly understood. Here, we directly visualize the action of fluorescent Taxol and epothilone derivatives and show that microtubules can transition to a state that triggers cooperative drug binding to form regions with altered lattice conformation. Such regions emerge at growing microtubule ends that are in a pre-catastrophe state, and inhibit microtubule growth and shortening. Electron microscopy and in vitro dynamics data indicate that taxane accumulation zones represent incomplete tubes that can persist, incorporate tubulin dimers and repeatedly induce microtubule rescues. Thus, taxanes modulate the material properties of microtubules by converting destabilized growing microtubule ends into regions resistant to depolymerization.

Discovery of a series of selective and cell permeable beta-secretase (BACE1) inhibitors by fragment linking with the assistance of STD-NMR.

Two ß-secreatase (BACE1) inhibitors from natural products (cinnamic acid and flavone) were linked to furnish potent, cell permeable BACE1 inhibitors with noncompetitive mode of inhibition, with the assistance of saturated transfer difference (STD)-NMR technique. Some of these conjugates also exhibited selective BACE1 inhibition over other aspartyl proteases such as BACE-2 and renin, as well as poor cytotoxicity. Taken together, conjugates 4 represent a new series of BACE inhibitors warrants further investigation for their potential in Alzheimier's disease therapy.

Synthesis and Cytotoxicity of 7,9-O-Linked Macrocyclic C-Seco Taxoids.

A series of novel 7,9-O-linked macrocyclic taxoids together with modification at the C2 position were synthesized, and their cytotoxicities against drug-sensitive and P-glycoprotein and ßIII-tubulin overexpressed drug-resistant cancer cell lines were evaluated. It is demonstrated that C-seco taxoids conformationally constrained via carbonate containing-linked macrocyclization display increased cytotoxicity on drug-resistant tumors overexpressing both ßIII and P-gp, among which compound 22b, bearing a 2-m-methoxybenzoyl group together with a five-atom linker, was identified as the most potent. Molecular modeling suggested the improved cytotoxicity of 22b results from enhanced favorable interactions with the T7 loop region of ßIII.

Extensive Structure Modification on Luteolin-Cinnamic Acid Conjugates Leading to BACE1 Inhibitors with Optimal Pharmacological Properties.

BACE1 inhibitory conjugates derived from two natural products, luteolin (1) and p-hydroxy-cinnamic acid (2), were subjected to systematic structure modifications, including various positions in luteolin segment for conjugation, different linkers (length, bond variation), as well as various substitutions in cinnamic acid segment (various substituents on benzene, and replacement of benzene by heteroaromatics and cycloalkane). Optimal conjugates such as 7c and 7k were chosen on the basis of a series of bioassay data for further investigation.

A Series of Enthalpically Optimized Docetaxel Analogues Exhibiting Enhanced Antitumor Activity and Water Solubility.

A dual-purpose strategy aimed at enhancing the binding affinity for microtubules and improving the water solubility of docetaxel led to the design and synthesis of a series of C-2- and C-3'-modified analogues. Both aims were realized when the C-3' phenyl group present in docetaxel was replaced with a propargyl alcohol. The resulting compound, 3f, was able to overcome drug resistance in cultured P-gp-overexpressing tumor cells and showed greater activity than docetaxel against drug-resistant A2780/AD ovarian cancer xenografts in mice. In addition, the considerably lower hydrophobicity of 3f relative to both docetaxel and paclitaxel led to better aqueous solubility. A molecular model of tubulin-bound 3f revealed novel hydrogen-bonding interactions between the propargyl alcohol and the polar environment provided by the side chains of Ser236, Glu27, and Arg320.

Modification of C-seco taxoids through ring tethering and substituent replacement leading to effective agents against tumor drug resistance mediated by ßIII-Tubulin and P-glycoprotein (P-gp) overexpressions.

In our efforts to improve the efficacy of taxane-based microtubule (MT) stabilizing agents against tumor drug resistance mediated by multiple mechanisms, two clinically relevant factors were focused: i.e., P-glycoprotein and ßIII-tubulin overexpression. Based on the structure of C-seco taxoid 1 m (IDN5390) which was believed to more selectively interact with ßIII-tubulin than paclitaxel, we prepared a series of C-seco taxoids bearing various 7,9-O-linkages and/or different substituents at C2 and C3' positions. Some of them exhibited much more potent binding affinity to MTs and cytotoxicity than their C-seco parent compounds in drug resistant cells with both mechanisms. SAR analysis indicated that C2 modifications significantly enhanced MT binding but brought ambiguous influence to cytotoxicity whereas 7,9-linkage and C3' modifications enhance cytotoxicity more efficiently than improve MT binding. These observations illustrate a better translation of molecular binding effect to cellular activity by C ring closure and C3' modification than C2 modification in C-seco taxoids.

An efficient semi-synthesis of 1-hydroxyl oleanolic acid analogs.

An efficient route for the semi-synthesis of either 1α- or 1ß-OH epimers of 1-hydroxy-3-deoxyolean-12-en-28-oic acid (1), 6-8 steps from oleanolic acid is reported. The synthesis involves stereoselective formation of α,ß-unsaturated epoxy ketone and subsequent Wharton reaction as key steps, offering a new access to the 1-O-substituted oleanolic acid-type pentacyclic triterpenoids.

Discovery of Farnesoid X Receptor Antagonists Based on a Library of Oleanolic Acid 3-O-Esters through Diverse Substituent Design and Molecular Docking Methods.

The pentacyclic triterpene oleanolic acid (OA, 1) with known farnesoid X receptor (FXR) modulatory activity was modified at its C-3 position to find new FXR-interacting agents. A diverse substitution library of OA derivatives was constructed in silico through a 2D fingerprint similarity cluster strategy. With further docking analysis, four top-scored OA 3-O-ester derivatives were selected for synthesis. The bioassay results indicated that all four compounds 3 inhibited chenodeoxycholic acid (CDCA)-induced FXR transactivation in a concentration-dependent mode. Among them 3b and 3d are more active than the parent compound OA. A molecular simulation study was performed to attempt to explain the structure-activity relationship (SAR) and the antagonistic action. To the best of our knowledge, this is the first report on semi-synthetic pentacyclic triterpenoids with FXR-modulatory activities.

Inhibiting the proliferation, migration and invasion of triple negative breast cancer cells through anti-tumor human serum albumin nanoparticles loading aziditaxel as a novel taxane derivative.

Triple negative breast cancer (TNBC) is a severe breast cancer subtype with the high mortality rate, and still is lack of effective therapeutic means so far. Aziditaxel, a water-insoluble compound, is a novel taxane derivative with strong anti-tumor activity. In this study, we constructed an aziditaxel-loaded nano drug delivery system using human serum albumin as a carrier, and further investigated its anti-tumor effect on TNBC in vitro. An emulsion solvent evaporation method was employed to prepare aziditaxel-loaded human serum albumin nanoparticles (AT-NPs). Their physicochemical properties were characterized according to morphology, particle size, zeta potential, reconstitution stability and in vitro drug release. The in vitro anti-tumor effects of AT-NPs on TNBC were evaluated using a 4T1 murine triple negative mammary cancer cell lines as the TNBC model. The results showed that AT-NPs could be effectively taken up by 4T1 cells in a time-dependent manner. Cell Counting Kit-8 assay showed that the IC50 of AT-NPs was 0.17 µg/ml. Meanwhile, compared with AT, AT-NPs had a better ability to promote apoptosis and induce G2/M cycle arrest. On the other hand, AT-NPs had significantly inhibitory effects on the 4T1 cell adhesion, migration and invasion with the respective average inhibition ratios of 32.53%, 83.26% and 75.78%. Thus, our study revealed that AT-NPs had favorable antitumor activity in vitro and exhibited a good prospect for application in the field of TNBC therapy.

Lx2-32c-loaded polymeric micelles with small size for intravenous drug delivery and their inhibitory effect on tumor growth and metastasis in clinically associated 4T1 murine breast cancer.

Lx2-32c is a novel taxane derivative with a strong antitumor activity. In this study, we developed Lx2-32c-loaded polymeric micelles (Lx2-32c-PMs) with small size and investigated their antitumor efficacy against tumor growth and metastasis on 4T1 murine breast cancer cell line with Cremophor EL-based Lx2-32c solution as the control. In this study, copolymer monomethoxy polyethylene glycol2000-polylactide1300 was used to prepare Lx2-32c-PMs by film hydration method, and their physicochemical properties were characterized as well, according to morphology, particle size, zeta potential, in vitro drug release, and reconstitution stability. Under confocal laser scanning microscopy, it was observed that Lx2-32c-PMs could be effectively taken up by 4T1 cells in a time-dependent manner. Cell Counting Kit-8 assay showed that the IC50 of Lx2-32c-PMs was 0.3827 nM. Meanwhile, Lx2-32c-PMs had better ability to promote apoptosis and induce G2/M cycle block and polyploidy formation, compared with Lx2-32c solution. More importantly, in vivo animal studies showed that compared to Lx2-32c solution, Lx2-32c-PMs possessed better ability not only to effectively inhibit the tumor growth, but also to significantly suppress spontaneous and postoperative metastasis to distant organs in 4T1 orthotopic tumor-bearing mice. Consequently, Lx2-32c-PMs have significantly prolonged the survival lifetime of tumor-bearing mice. Thus, our study reveals that Lx2-32c-PMs had favorable antitumor activity and exhibited a good prospect for application in the field of antitumor therapy.

Synthesis and Biological Evaluations of Cytotoxic and Antiangiogenic Triterpenoids-Jacaranone Conjugates.

BACKGROUND: The development of antiangiogenic agents arises as a more effective and selective therapeutic approach for the treatment of cancer. In addition to reduced acute toxicity, the efficacy of chemotherapy could be improved when administered in combination specific antiangiogenic with cytotoxic agents. The conjugation or hybridization of bifunctional molecules is one of the alternative rational design strategies for co-administration of anticancer drugs. OBJECTIVE AND METHODS: The goal of this work is to prepare the conjugates of an antiangiogenic triterpene, 3-oxo oleanolic acid, and structurally related triterpenoids with a cytotoxic semibenzoquinone, jacaranone. The cytotoxic, antiproliferative and antiangiogenic activities of segments and conjugates were determined. The possible targets of conjugates 6a-6h were predicted using Similarity Ensemble Approach (SEA). RESULTS: The results showed that these conjugates are more potent in both cytotoxic and antiangiogenic assays than their corresponding parent molecules, and are also selectively more active against melanoma cells B16 and metastatic B16BL6 than the two other cancer cell lines (A549 and MCF-7) tested. The predicted antiangiogenesis related targets could involve glycogen phosphorylase, neuraminidase, interferon gamma, and tubulin beta chain. CONCLUSION: The bifunctional conjugates could be useful as dual acting antitumor/antigiogenic agents.

A novel C,D-spirodioxene taxoid synthesized through an unexpected Pd-mediated ring cyclization.

A novel C,D-spirodioxene taxoid (6) was prepared from paclitaxel (1a), with the key steps including an unexpected Pd-mediated ring cyclization. The anti-tubulin activity of 6 was decreased relative to that of 1a and a previously reported C,D-spirolactone taxane (5). These observations could be rationalized on the basis of molecular modeling results. To the best of our knowledge, this is the first example indicating that 1,4-dioxenes can be synthesized from a mono-allyl vicinal diol through a Wacker-type cyclization. This strategy may be applicable to the synthesis of other C,D-spiro taxoids.

Restoration of Microtubule Interaction and Cytotoxicity in D-seco Taxanes upon Incorporation of 20-Hydroxymethyl-4-allyloxy Groups.

To probe the exact role of the oxetane D ring in both tubulin binding and cytotoxicity of taxanes, novel D-seco taxanes bearing a C4 ether substituent have been prepared from paclitaxel 1a. Among them, 20-hydroxymethyl-4-allyloxy D-seco taxane 5e is the most active in both tubulin and cytotoxicity assays. It is only slightly less potent than 1a on tubulin polymerization promotion in vitro and the most cytotoxic among all D-seco taxanes known to date. The reason for the loss and restoration of bioactivity for these D-seco taxanes is also discussed with the assistance of NMR and molecular modeling studies. From these results, we draw a conclusion that the intact D ring of taxanes is not strictly necessary for their binding to tubulin and cytotoxic effects.

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 ß-tubulin binding site or by overexpression of the highly dynamic ßIII-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 ßIII 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.

A semisynthetic taxane Yg-3-46a effectively evades P-glycoprotein and ß-III tubulin mediated tumor drug resistance in vitro.

Tumor resistance, especially that mediated by P-glycoprotein (P-gp) and ß-III tubulin, is a major obstacle to the efficacy of most microtubule-targeting anticancer drugs in clinics. A novel semisynthetic taxane, 2-debenzoyl-2-(3-azidobenzyl)-10-propionyldocetaxel (Yg-3-46a) was shown to be highly cytotoxic to breast cancer cell lines MCF-7 and MCF/ADR which overexpressed P-gp via long term culture with doxorubicin, and cervical cancer cell lines Hela and Hela/ßIII which overexpressed ßIII-tubulin via stable transfection with TUBB3 gene. siRNA transfection experiments also confirmed that Yg-3-46a can circumvent P-gp and ß-III tubulin mediated drug resistance. In addition, its cytotoxicity was lower than that of paclitaxel in the human mammary cell line HBL-100 and the human telomerase-immortalized retinal pigment epithelium cell line (hTERT-RPE1), suggesting a better safety margin for this compound in vivo. It exhibited more potent microtubule polymerization ability than paclitaxel in vitro, and also induced G2/M phase arrest in MCF-7/ADR cells. Moreover, it was found to induce apoptosis in MCF-7/ADR cells through the caspase-dependent death-receptor pathway by enhancing levels of Fas and FasL, and activating caspase-8 and 3. Yg-3-46a was found to be a poorer substrate of P-gp compared to paclitaxel, in both binding and ATPase experiments, which is likely responsible for its ability to circumvent P-gp mediated multidrug resistance (MDR). All of these results indicate that Yg-3-46a is a novel microtubule-stabilizing agent that has the potential to evade drug resistance mediated by P-gp and ß-III tubulin overexpression.