Representative dot plots of BrdU and propidium iodide (PI) staining or composite histograms of PI staining of MDA-MB-231, A2780 or 22Rv1 cell lines treated for 24-h with 10 nM of PM534 (blue) or vehicle (control, red) and pulsed with BrdU for 1-h. DNA content was stained with PI (2n for G1 and 4n for G2) while BrdU incorporation and staining reflects S phase.

A: HUVEC adhesion (% of adhesion compared to control) on an extracellular matrix composed of fibronectin and type I collagen quantified with Sulforhodamine B following 24 hours of incubation with PM534 or colchicine. Results are presented as the mean percentage (n=3). Dotted line indicates a 50% reduction of adhesion. B: Survival curves for HUVEC cells following the incubation with PM534 and colchicine, in which 70% of cell were alive at 10 nM of PM534.

Protein corrected transport rate (pmol/min/pmol transporter protein) of PM534 in Abcb1KO-MDCKII-MDR1-LV cells. Data are expressed as mean (n=3) and 95% CI.

Microtubules are dynamic cytoskeletal polymers whose lattice architecture regulates force generation, nucleotide hydrolysis, and recognition by motor proteins and microtubule-associated proteins (MAPs). Microtubule-stabilizing agents (MSAs), including taxanes and laulimalide/peloruside-site ligands, suppress depolymerization by binding to defined lattice sites, yet stabilization is not structurally neutral. How ligand chemistry reshapes lattice organization and function remains unresolved. Here, we address three mechanistic questions. First, do distinct ligand classes induce defined lattice states? Using X-ray fiber diffraction, we show that MSAs selectively stabilize two preferred longitudinal conformations, a compact state (~4.06 nm monomer rise) and an expanded state (~4.17 nm), while modulating lateral organization reflected in shifts in mean MT radius. These axial spacings cluster around discrete values across chemotypes, indicating stabilization of preexisting conformational minima rather than continuous distortion. Second, are these states interconvertible upon changes in ligand occupancy? Time-resolved diffraction reveals that longitudinal transitions occur within seconds of ligand addition even at substoichiometric occupancy, whereas, lateral equilibration proceeds slower, consistent with redistribution within heterogeneous protofilament organizations. Third, do such structural states alter nucleotide hydrolysis and motor/MAP behavior? Expanded lattices are associated with reduced apparent GTP hydrolysis rates under steady-state assembly conditions and altered kinesin motility, whereas compact lattices preferentially promote tau binding and distinct motor interaction profiles. Together, these findings establish longitudinal lattice conformation as a regulatory parameter and position MSAs as chemical tools that bias a dynamic structural landscape with predictable catalytic and transport consequences.

This study evaluates PM534, a novel colchicine-binding domain inhibitor, for its potential in cancer therapy. PM534 exhibited potent in vitro efficacy against a panel of 14 human cancer cell lines, including breast, ovarian, and prostate cancers, with concentration needed to reduce the growth of treated cells to half that of untreated cells values in the low nanomolar range. Both continuous (72 hours) and short-term (1-24 hours) exposure led to irreversible effects, inducing G2-M cell cycle arrest and multinucleation. Additionally, PM534 also impaired angiogenic process. It effectively inhibited HUVEC functions, including adhesion, with an IC50 of 2.3 nmol/L, markedly more potent than colchicine (IC50 = 1,800 nmol/L). At concentrations as low as 1.6 nmol/L, PM534 delayed wound closure in migration assays, completely inhibiting migration above 4 nmol/L. Additionally, PM534 abrogated invasion and disrupted capillary-like network formation at concentrations starting from 0.5 nmol/L without inducing cytotoxicity. In vivo PM534 demonstrated robust antitumor efficacy across six xenograft models, including ovarian (A2780 and ES-2), triple-negative breast (MDA-MB-231 and HCC1937), and prostate (VCaP and 22Rv1) tumors. This treatment also led to statistically significant increases in median survival times across all models, without inducing signs of systemic toxicity. Mechanistically, PM534 induced apoptosis, mitotic catastrophe, and necrosis in tumor tissues. Importantly, PM534 retained efficacy in models overexpressing multidrug resistance proteins P-glycoprotein or β-III tubulin, overcoming common resistance mechanisms that limit the effectiveness of other tubulin-binding agents. Collectively, these findings highlight PM534 as a promising antitumor agent with potent activity against diverse and treatment-resistant malignancies. A phase I clinical trial (NCT#5835609) is underway to assess the therapeutic potential of PM534 in patients with advanced solid tumors.

Paclitaxel (PTX) is a widely used microtubule (MT) stabilizer whose clinical utility is limited by peripheral neuropathy, likely due to drug-induced structural perturbations of neuronal MTs. Here, we aimed to discern how taxane derivatives modify MT lattice architecture and how structural states regulate motor proteins and MAP dynamics. To decouple MT stabilization from adverse structural changes, we designed, synthesized, and characterized several PTX analogues. Our compound 1b retains PTX-like stabilizing activity in vitro and in cells while preserving a native-like MT lattice. This structural separation allowed direct interrogation of MT structure-function relationship in cells. PTX-induced lattice expansion disrupted dynein-mediated retrograde transport, altered kinesin-1 motility and suppressed dynamic Tau exchange. In contrast, 1b preserved more physiological Tau dynamics. These findings reveal that MT stabilization and lattice modulation are separable properties and establish drug-imposed MT states as regulators of intracellular transport and MAP behavior.

Cell lines used and key characteristics

A: Changes of body weights (as mean %) of mice bearing different xenografts and treated with PM534 (blue) or placebo (gray) on days 0, 7 and 14. Data are expressed as mean±SD (n≥6/group). B: Clinical chemistry parameters obtained on Day 21 in non-tumored female nu/nu mice (n=10) after the i.v. administration of PM534 (at 5 mg/kg; days 0, 7 and 14) or placebo. C: Changes (folds) in clinical chemistry parameters of PM534-treated mice (vs. placebo: median values between parenthesis).

African swine fever virus (ASFV) belongs to the family of Asfarviridae, part of the group of nucleocytoplasmic large DNA viruses (NCLDV). Little is known about the internalization of ASFV in the host cell and the fusion membrane events that take place at early stages of the infection. Poxviruses, also members of the NCLDV and represented by vaccinia virus (VACV), are large, enveloped, double-stranded DNA viruses. Poxviruses were considered unique in having an elaborate entry-fusion complex (EFC) composed of 11 highly conserved proteins integrated into the membrane of mature virions. Recent advances in methodological techniques have again revealed several connections between VACV EFC proteins. In this study, we explored the possibility of an analogous ASFV EFC by identifying ten candidate proteins exhibiting structural similarities with VACV EFC proteins. This could reveal key functions of these ASFV proteins, drawing attention to shared features between the two virus families, suggesting the potential existence of an ASFV entry-fusion complex.

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.

We have prepared a series of analogs of the complex marine macrolide (–)-zampanolide, which incorporate a dioxane-, oxathiane-, or oxathiane-dioxide ring in place of the natural tetrahydropyran moiety and we have determined their microtubule-binding affinity and antiproliferative activity against human cancer cells. The synthesis of these analogs was based on a convergent strategy with a HWE-based macrocyclization and a stereoselective aza-aldol reaction as key steps. The microtubule-binding affinity and cellular potency of the dioxane- and oxathiane-based analogs with a natural (Z,E)-sorbamide-based side chain were essentially indistinguishable from those of natural (–)-zampanolide; changing the configuration of the sorbamide unit from Z,E to E,E resulted in a slight loss in activity. In contrast, the presence of an oxathiane-dioxide ring caused a steep decrease in microtubule-binding and a significant loss in growth inhibitory activity. In addition, a substantial loss in potency was observed against a multidrug-resistant, P-glycoprotein-overexpressing cell line, while no such effect was found for the dioxane- or oxathiane-based analogs. A high-resolution X-ray crystal structure of the complex between beta-tubulin and dioxane-zampanolide was obtained, which showed that this compound, like natural (–)-zampanolide, induces helical structuring of the M-loop.

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.

We have prepared a series of partially reduced or demethylated analogs of the natural microtubule stabilizer (−)-zampanolide and we have assessed their antiproliferative activity, their microtubule-binding affinity and their effects on the cellular microtubule network and on cell cycle progression. For reasons of synthetic efficiency, these analogs were derived from 13-desmethylene-(-)-zampanolide, which we had previously shown to be an equally potent cancer cell growth inhibitor as the natural product. The synthesis of all compounds was based on a unified strategy that included final formation of the macrobicyclic core by an intramolecular HWE reaction and a stereoselective aza-aldol reaction to establish the C(20) stereocenter as the key steps. For the 5-desmethyl macrocycle, ring-closure relied on macrolactonization; however, elaboration of the macrocyclic aldehyde into the corresponding zampanolide analog was unsuccessful.All structural modifications investigated led to reduced cellular activity and lower microtubule-binding affinity compared to the parent 13-desmethylene-(–)-zampanolide, which may be ascribed to increased conformational flexibility due to the formal reduction of double bonds or the removal of the C(17)-methyl group. Notwithstanding this general trend, the cellular potency of 2,3-dihydro-13-desmethylene zampanolide as the most potent analog identified remained within a 9-fold range of that of 13-desmethylene-(–)-zampanolide (for 5 out of 6 cell lines). Notably, while the formal reduction of the C=C double bond of the enone system that is required for the covalent attachment of (−)-zampanolide to beta-tubulin caused a drop in antiproliferative activity of several hundred fold, the compound does bind to microtubules and shows the typical cellular hallmarks of a microtubule-stabilizing agent.

Background: Microtubule targeting agents have demonstrated to be very effective antitumoral drugs. The development of novel anti-tubulin agents with more efficient mechanisms of action presents several challenges due to their poor solubility, troublesome synthesis or purification, and toxicities. In this work, we present the novel anti-tubulin agent PM534, a synthetic small molecule that shows efficient antitumoral and antiangiogenic properties in vitro and in vivo. Methods and Results: PM534 exhibits a potent antitumor activity in vitro with a mean GI50 value in the low nanomolar range in several different human cancer cell lines. Washout in vitro experiments show irreversible cellular effects after 1h in contact with the compound, compelling the cells to apoptotic cell death likely due to PM534 induced disorganization of the tubulin cytoskeleton. PM534 inhibits migration and invasion of tumor cells in vitro, and arrests the cell cycle in the G2/M phase, forcing them to apoptosis. In addition, PM534 presents potent antiangiogenic effects in vitro, robustly inhibiting endothelial cells proliferation and migration, and the formation of angiotubes. The in vivo antitumor activity of PM534 was characterized in human-derived tumors xenografted in athymic nu/nu mice, namely breast (MDA-MB-231), and pancreas (Mia-Paca-2). PM534 was intravenously administered once per week for three consecutive weeks at 5.0 mg/kg. In vivo PM534-induced antitumor activity (vs. placebo) was seen in MDA-MB-231 (T/C, 0.3% on Day 28; TV, p<0.0001) and Mia-Paca-2 (21.3% on Day 21; TV, p<0.0001). Also, PM534 treatment increased with statistical significance (vs. placebo) the median survival time of mice bearing MDA-MB-231 (82 vs. 33 days; p<0.0001) and Mia-Paca-2 (51 vs. 30 days; p=0.0008). Of note, complete tumor remissions were observed in 2/10 (lasting 11 days and 161 days, each), and 10/10 (lasting 45 days in 9/10 and 140 days in 1/10) PM534-treated animals bearing Mia-Paca-2 and MDA-MB-231 and tumors, respectively. Conclusions: Based on in vitro activity against different human tumor cell lines, in vivo activity in xenografted human tumors, as well as on its anti-angiogenic properties, the safety, pharmacology and preliminary antitumor activity of PM534 will be evaluated in a first-in-human clinical trial to be conducted in patients with advanced solid tumors. Citation Format: Marta Martínez Diez, María José Muñoz-Alonso, Gema Santamaría Nuñez, María José Guillén, María Ángela Oliva, Eva Maria Garrido-Martin, Pablo Avilés, J. Fernando Díaz, Carmen Cuevas. The novel antitubulin agent PM534 exhibits potent antitumoral and antiangiogenic properties in vitro and in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6243.

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 βM-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.

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.