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The marine metabolite diazonamide A exerts low nanomolar cytotoxicity against a range of tumor cell lines; however, its highly complex molecular architecture undermines the therapeutic potential of the natural product. We demonstrate that truncation of heteroaromatic macrocycle in natural diazonamide A, combined with the replacement of the challenging-to-synthesize tetracyclic hemiaminal subunit by oxindole moiety leads to considerably less complex analogues with improved drug-like properties and nanomolar antiproliferative potency. The structurally simplified macrocycles are accessible in 12 steps from readily available indolin-2-one and tert-leucine with excellent diastereoselectivity (99:1 dr) in the key macrocyclization step. The most potent macrocycle acts as a tubulin assembly inhibitor and exerts similar effects on A2058 cell cycle progression and induction of apoptosis as does marketed microtubule-targeting agent vinorelbine.

Studies are described toward the synthesis of an oxazole-based analog of (−)-zampanolide (2). Construction of (−)-dactylolide analog 22was achieved via alcohol 5and acid 4through esterification and Horner–Wadsworth–Emmons (HWE)-based macrocyclization; however, attempts to attach (Z,E)-sorbamide to 22proved unsuccessful. The C(8)–C(9) double bond of the macrocycle was prone to migration into conjugation with the oxazole ring, which may generally limit the usefulness of zampanolide analogs with aromatic moieties as tetrahydropyran replacements.

Noscapine is a natural alkaloid that is used as an antitussive medicine. However, it also acts as a weak anticancer agent in certain in vivomodels through a mechanism that is largely unknown. Here, we performed structural studies and show that the cytotoxic agent 7A-O-demethoxy-amino-noscapine (7A-aminonoscapine) binds to the colchicine site of tubulin. We suggest that the 7A-methoxy group of noscapine prevents binding to tubulin due to a steric clash of the compound with the T5-loop of α-tubulin. We further propose that the anticancer activity of noscapine arises from a bioactive metabolite that binds to the colchicine site of tubulin to induce mitotic arrest through a microtubule cytoskeleton-based mechanism.

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.

Targeting microtubules is the most effective wide-spectrum pharmacological strategy in antitumoral chemotherapy, and current research focuses on reducing main drawbacks: neurotoxicity and resistance. PM534 is a novel synthetic compound derived from the Structure–Activity-Relationship study on the natural molecule PM742, isolated from the sponge of the order Lithistida, family Theonellidae, genus Discodermia(du Bocage 1869). PM534 targets the entire colchicine binding domain of tubulin, covering four of the five centers of the pharmacophore model. Its nanomolar affinity and high retention time modulate a strikingly high antitumor activity that efficiently overrides two resistance mechanisms in cells (detoxification pumps and tubulin βIII isotype overexpression). Furthermore, PM534 induces significant inhibition of tumor growth in mouse xenograft models of human non-small cell lung cancer. Our results present PM534, a highly effective new compound in the preclinical evaluation that is currently in its first human Phase I clinical trial.

The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105M–1in the case of unmodified tubulin to 1.4 ± 0.3 × 105M–1in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

FtsZ is a self-assembling GTPase that forms, below the inner membrane, the mid-cell Z-ring guiding bacterial division. FtsZ monomers polymerize head to tail forming tubulin-like dynamic protofilaments, whose organization in the Z-ring is an unresolved problem. Rather than forming a well-defined structure, FtsZ protofilaments laterally associate in vitro into polymorphic condensates typically imaged on surfaces. We describe here nanoscale self-organizing properties of FtsZ assemblies in solution that underlie Z-ring assembly, employing time-resolved x-ray scattering and cryo-electron microscopy. We find that FtsZ forms bundles made of loosely bound filaments of variable length and curvature. Individual FtsZ protofilaments further bend upon nucleotide hydrolysis, highlighted by the observation of some large circular structures with 2.5–5° curvature angles between subunits, followed by disassembly end-products consisting of highly curved oligomers and 16-subunit −220 Å diameter mini-rings, here observed by cryo-electron microscopy. Neighbor FtsZ filaments in bundles are laterally spaced 70 Å, leaving a gap in between. In contrast, close contact between filament core structures (∼50 Å spacing) is observed in straight polymers of FtsZ constructs lacking the C-terminal tail, which is known to provide a flexible tether essential for FtsZ functions in cell division. Changing the length of the intrinsically disordered C-tail linker modifies the interfilament spacing. We propose that the linker prevents dynamic FtsZ protofilaments in bundles from sticking to one another, holding them apart at a distance similar to the lateral spacing observed by electron cryotomography in several bacteria and liposomes. According to this model, weak interactions between curved polar FtsZ protofilaments through their the C-tails may facilitate the coherent treadmilling dynamics of membrane-associated FtsZ bundles in reconstituted systems, as well as the recently discovered movement of FtsZ clusters around bacterial Z-rings that is powered by GTP hydrolysis and guides correct septal cell wall synthesis and cell division.

Four natural analogues of podophyllotoxin obtained from the Mexican medicinal plant Bursera fagaroides, namely, acetyl podophyllotoxin (2), 5′-desmethoxy-β-peltatin A methyl ether (3), 7′,8′-dehydro acetyl podophyllotoxin (4), and burseranin (5), have been characterized, and their interactions with tubulin have been investigated. Cytotoxic activity measurements, followed by immunofluorescence microscopy and flow cytometry studies, demonstrated that these compounds disrupt microtubule networks in cells and cause cell cycle arrest in the G2/M phase in the A549 cell line. A tubulin binding assay showed that compounds 1–4were potent assembly inhibitors, displaying binding to the colchicine site with Kbvalues ranging from 11.75 to 185.0 × 105M–1. In contrast, burseranin (5) was not able to inhibit tubulin assembly. From the structural perspective, the ligand-binding epitopes of compounds 1–3have been mapped using STD-NMR, showing that B and E rings are the major points for interaction with the protein. The obtained results indicate that the inhibition of tubulin assembly of this family of compounds is more effective when there are at least two methoxyl groups at the E ring, along with a transconfiguration of the lactone ring in the aryltetralin lignan core.

Herein, we present a phototriggered drug delivery system based on light responsive nanoparticles, which is able to release doxorubicin upon NIR light illumination. The proposed system is based on upconversion fluorescence nanoparticles of β-NaYF4:Yb,Tm@SiO2-PEG with a mean diameter of 52 ± 2.5 nm that absorb the NIR light and emit UV light. The UV radiation causes the degradation of photodegradable ortho-nitrobenzyl alcohol derivates, which are attached on one side to the surface of the nanoparticles and on the other to doxorubicin. This degradation triggers the doxorubicin release. This drug delivery system has been tested “in vitro” with HeLa cells. The results of this study demonstrated that this system caused negligible cytotoxicity when they were not illuminated with NIR light. In contrast, under NIR light illumination, the HeLa cell viability was conspicuously reduced. These results demonstrated the suitability of the proposed system to control the release of doxorubicin via an external NIR light stimulus.

The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αβ-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg2+-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulansmap to β-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating β-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.

The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/β-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/β-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/β-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubules.

A protocol based on a combination of NMR experimental data with molecular mechanics calculations and docking procedures has been employed to determine the microtubule‐bound conformation of two microtubule‐stabilizing agents, discodermolide (DDM) and dictyostatin (DCT). The data indicate that tubulin in assembled microtubules recognizes DDM through a conformational selection process, with minor changes in the molecular skeleton between the major conformer in water solution and that bound to assembled microtubules. For DCT, the deduced bound geometry presents some key conformation differences around certain torsion angles, with respect to the major conformer in solution, and still displays mobility even when bound. The bound conformer of DCT resembles that of DDM and provides very similar contacts with the receptor. Competition experiments indicate that both molecules compete with the taxane‐binding site. A model of the binding mode of DDM and DCT to tubulin is proposed.

A total, stereoselective synthesis of the naturally occurring, cytotoxic macrolide FD‐891 and of its non‐natural (Z)‐C12 isomer is described. Three fragments of the main carbon chain were stereoselectively prepared by using asymmetric aldol and allylation reactions as the key steps. The molecule was then assembled by using two Julia–Kocienski olefinations to connect the three fragments and a Yamaguchi reaction to close the macrolactone ring. Some specific biological properties (cytotoxicity, binding to tubulin) have been determined for both macrolides. The Econfiguration of the C12–C13 olefinic bond seems to be an important feature in determining the cytotoxicity but the precise biological mechanism of the latter still remains to be cleared.

GDP liganded tubulin, which is inactive in microtubule assembly, polymerizes into rings more readily than the active GTP liganded protein. The structure of double rings made of highly purified GDP-tubulin has been characterized to 3 nm resolution with synchrotron X-ray solution scattering. The scattering profile has characteristic maxima due to closely packed double rings of 38 nm mean diameter, with a 5·5 nm mean spacing between the rings, and a 4·2 nm centre-to-centre spacing between non-globular tubulin monomers within both rings. There are probably 24 and 32 monomers in the inner and outer ring, respectively, and the double ring population is more than 75% homogeneous in size. Comparison of this double ring structure to the lattice of tubulin molecules in microtubules indicates that the tubulin rings are equivalent to pairs of protofilament segments curved tangentially to the microtubule surface, with bending angle of 30° and 22·5° per tubulin αβ dimer. When the rings are modelled employing the same non-globular tubulin monomer as in microtubules, the best computer fitted scattering profiles correspond to monomer orientations equivalent to two microtubule protofilaments coiled sideways, with same or opposite polarity. Rings constitute the equilibrium assembly state of GDP-tubulin, which is tensioned inside microtubules after GTP hydrolysis, causing their functional instability. In analogy with other nucleotide binding proteins, the inactive/active structural switch of tubulin is induced by the binding of the gamma phosphate and a coordinated Mg ion. It should involve domain rearrangements which cancel the bending of the tubulin dimer in the ring structure. Copyright 1994, 1999 Academic Press