Javier Rodríguez-Salarichs

Zampanolide, a Potent New Microtubule-Stabilizing Agent, Covalently Reacts with the Taxane Luminal Site in Tubulin α,β-Heterodimers and Microtubules

Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and suggest that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.

Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach.

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.

TRAPPII regulates exocytic Golgi exit by mediating nucleotide exchange on the Ypt31 ortholog RabERAB11

Significance Ypt1 and Ypt31/32 RAB GTPases regulate traffic across the Golgi. Both are activated by TRAPP, an oligomeric GEF. Three TRAPP versions share the same core subunits. TRAPPI and TRAPPIII activate Ypt1. The third, TRAPPII, composed of TRAPPI plus specific subunits, appears to act specifically on Ypt31, but this role has been disputed. By combining the resolving power of fungal genetics with biochemical assays, we establish that the physiological target of TRAPPII is RabE, the Aspergillus Ypt31 ortholog. However, our data suggest that TRAPPII contains independent binding sites for RabE and RabO (Ypt1), possibly explaining its relative lack of discrimination in vitro. TRAPPII arrives at Golgi cisternae preceding their dissipation into carriers, determining the Golgi–to–post-Golgi transition through RabE recruitment. The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabORAB1 resides in the Golgi, RabERAB11 localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabERAB11, but not RabORAB1, immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabERAB11 in vivo. hypA1ts rapidly shifts RabERAB11, but not RabORAB1, to the cytosol, consistent with HypATrs120 being specifically required for RabERAB11 activation. Missense mutations rescuing hypA1ts at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabORAB1 did not, establishing that the essential role of TRAPPII is facilitating RabERAB11 nucleotide exchange. In vitro, TRAPPII purified with HypATrs120-S-tag accelerates nucleotide exchange on RabERAB11 and, paradoxically, to a lesser yet substantial extent, on RabORAB1. Evidence obtained by exploiting hypA1-mediated destabilization of HypATrs120/HypCTrs130/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabORAB1, which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypATrs120 colocalizes with RabERAB11, arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi–to–post-Golgi transition.

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.

New Interfacial Microtubule Inhibitors of Marine Origin, PM050489/PM060184, with Potent Antitumor Activity and a Distinct Mechanism

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 Mg(2+)-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map 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.

Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches.

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.

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 β-tubulin binding site or by overexpression of the highly dynamic βIII-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 βIII 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.

Free Energy Profile and Kinetics Studies of Paclitaxel Internalization from the Outer to the Inner Wall of Microtubules.

Several pieces of experimental evidence led us to hypothesize that the mechanism of action of paclitaxel (Taxol) could involve a two-steps binding process, with paclitaxel first binding within the outer wall of microtubules and then moving into the inner binding site. In this work, we first used multiply targeted molecular dynamics (MTMD) for steering paclitaxel from the outer toward the inner binding site. This rough trajectory was then submitted to a refinement procedure in the path collective variables space. Paclitaxel binding energy was monitored along the refined pathway, highlighting the relevance of residues belonging to the H6-H7 and the M- loops. Computational results were supported by kinetics studies performed on fluorescent paclitaxel derivatives.

Zampanolide Binding to Tubulin Indicates Cross-Talk of Taxane Site with Colchicine and Nucleotide Sites

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 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

Synthesis, Biological Profiling and Determination of the Tubulin-Bound Conformation of 12-Aza-Epothilones (Azathilones)

12-Aza-epothilones (azathilones) incorporating quinoline side chains and bearing different N12-substituents have been synthesized via highly efficient RCM-based macrocyclizations. Quinoline-based azathilones with the side chain N-atom in the meta-position to the C15 atom in the macrocycle are highly potent inhibitors of cancer cell growth in vitro. In contrast, shifting the quinoline nitrogen to the position para to C15 leads to a ca. 1000-fold loss in potency. Likewise, the desaturation of the C9-C10 bond in the macrocycle to an E double bond produces a substantial reduction in antiproliferative activity. This is in stark contrast to the effect exerted by the same modification in the natural epothilone macrocycle. The conformation of a representative azathilone bound to α/β-tubulin heterodimers was determined based on TR-NOE measurements and a model for the posture of the compound in its binding site on β-tubulin was deduced through a combination of STD measurements and CORCEMA-ST calculations. The tubulin-bound, bioactive conformation of azathilones was found to be overall similar to that of epothilones A and B.