Summary 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 that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.

Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R. In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients.

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.

Bacteriophage endolysins include a group of new antibacterials reluctant to development of resistance. We present here the first structural study of the Cpl-7 endolysin, encoded by pneumococcal bacteriophage Cp-7. It contains an N-terminal catalytic module (CM) belonging to the GH25 family of glycosyl hydrolases and a C-terminal region encompassing three identical repeats of 42 amino acids (CW_7 repeats). These repeats are unrelated to choline-targeting motifs present in other cell wall hydrolases produced by Streptococcus pneumoniae and its bacteriophages, and are responsible for the protein attachment to the cell wall. By combining different biophysical techniques and molecular modeling, a three-dimensional model of the overall protein structure is proposed, consistent with circular dichroism and sequence-based secondary structure prediction, small angle x-ray scattering data, and Cpl-7 hydrodynamic behavior. Cpl-7 is an ϳ115-Å long molecule with two well differentiated regions, corresponding to the CM and the cell wall binding region (CWBR), arranged in a lateral disposition. The CM displays the (␤␣) 5 ␤ 3 barrel topology characteristic of the GH25 family, and the impact of sequence differences with the CM of the Cpl-1 lysozyme in substrate binding is discussed. The CWBR is organized in three tandemly assembled three-helical bundles whose dispositions remind us of a super-helical structure. Its approximate dimensions are 60 ؋ 20 ؋ 20 Å and presents a concave face that might constitute the functional region involved in bacterial surface recognition. The distribution of CW_7 repeats in the sequences deposited in the Entrez Database have been examined, and the results drastically expanded the antimicrobial potential of the Cpl-7 endolysin.Bacteria and bacteriophages produce a variety of enzymes that cleave the peptidoglycan, the cell wall hydrolases (CWHs), 3 either to lyse host cells or to re-model the cell wall during growth and division. Many of these enzymes have a modular structure and are composed of catalytic units linked to a number of other modules. The Cpl-7 lysozyme, the lytic enzyme (endolysin) encoded by the pneumococcal bacteriophage Cp-7, consists of a catalytic N-terminal domain fused to a C-terminal region containing three identical tandem repeats of 48 amino acids (the third motif has the last six residues missing), which is essential for activity and unique among CWHs encoded by the pneumococcus and its bacteriophages (1, 2). However, the same type of repeats (termed CW_7 hereafter) has been later identified in other proteins putatively associated with bacterial cell wall degradation activity (3). The catalytic module (CM) of Cpl-7 belongs to the GH25 family of glycosyl hydrolases and shows 85.6% sequence identity with the CM of the Cpl-1 lysozyme, the endolysin encoded by the pneumococcal bacteriophage Cp-1 (4). However, the cell wall binding region (CWBR) of Cpl-1, made of six tandem copies of ϳ20 amino acid residues plus a terminal tail, belongs to the family of pneumococcal choline-binding...

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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.

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.

Peloruside is a microtubule-stabilizing agent that targets the same site as laulimalide. It binds to microtubules with a 1:1 stoichiometry and with a binding affinity in the low-muM range; thereby reducing the number of microtubular protofilaments in the same way as paclitaxel. Although the binding affinity of the compound is comparable to that of the low-affinity stabilizing agent sarcodictyin, peloruside is more active in inducing microtubule assembly and is more cytotoxic to tumor cells; this suggests that the peloruside site is a more effective site for stabilizing microtubules. Acetylation of the C24 hydroxyl group results in inactive compounds. According to molecular modeling, this substitution at the C24 hydroxyl group presumably disrupts the interaction of the side chain with Arg320 in the putative binding site on alpha-tubulin. The binding epitope of peloruside on microtubules has been studied by using NMR spectroscopic techniques, and is compatible with the same binding site.