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.

Work with the elements of nature: Our building block, maytansinol, represented as a constellation of stars, is the main protagonist of the story. A ray of light coming out from it demonstrates how different moieties can be attached to it, opening new horizons. As maytansinoids show a destabilising effect on microtubules, we represent the latter by trees. The tree trunk is a microtubule, from which the tubulins are disassembling into the leaves. The TubInTrain logo in the corner represents the H2020-MSCA-ITN-2019 network that made this interdisciplinary investigation possible. More information can be found in the Research Article by S. Pieraccini, D. Passarella and co-workers. (DOI: 10.1002/chem.202203431). Created with BioRender.com.

Background: Microtubule targeting compounds are a successful class of anticancer agents in the clinic. Although highly potent, the currently approved antitumor agents targeting tubulin present some drawbacks such as the development of acquired resistances, which remain an obstacle for an effective prolonged anticancer treatment. In this work, we present a novel microtubule destabilizing agent with an optimized interaction for the colchicine site and a novel mechanism of action which confers it a high affinity binding for tubulin. Methods and Results: We have determined the X-ray crystallographic structure of the complex T2R-TTL with PM534 at a resolution of 2.45 Å. We unequivocally found PM534 ligand density at the intra-dimer interface between α and β tubulin of both tubulin dimers (the colchicine domain) within the complex. The colchicine domain of tubulin can be divided into three zones: a central pocket and two accessories. Whereas other colchicine site-ligands only bind to two of the zones, we found that PM534 binds extensively along the three zones, making multiple contacts with β-tubulin N terminal domain, central hinge helix, and the intermediate domain. PM534 interaction did not affect the overall curved conformation of tubulin and its mechanism of action consists on precluding the curve-to-straight conformational transition required for tubulin to assemble intro microtubules and thus, to perform its cellular functions. In competition assays we found that the compound displaces a bona fide high-affinity colchicine-probe with a binding affinity of 5.1±0.3 × 107 M−1 at 25̊C. Moreover, PM534 shows potent antineoplastic activity in vitro, with GI50 values in the low nanomolar range, observed in different human tumor cancer cell lines. An in vivo Proof of the Concept (PoC) was performed in athymic nu/nu mice bearing H460 (NSCLC) tumors that were treated (intravenous, on days 0, 7, 14) with different doses (ranging from 0.75 mg/kg to 2.5 mg/kg) of PM534. Results demonstrated strong, dose-related PM534-induced antitumor activity in this in vivo model. Conclusions: The mechanism of action of PM534 involves an optimized interaction with the colchicine site, which is reflected in its high affinity for the substrate, being this the ultimate reason for the observed cellular activity. Additionally, PM534 presents a potent antitumor activity in vitro that was translated into a clear positive in vivo PoC that resulted in strong antitumor effect. Citation Format: Maria Ángela Oliva, Beatriz Álvarez-Bernad, Daniel Lucena-Agell, Marta Martínez Diez, María José Guillén, Gema Santamaría Nuñez, María José Muñoz-Alonso, Eva M. Garrido-Martin, Pablo Avilés, Carmen Cuevas, J. Fernando Díaz. PM534 is a novel microtubule-destabilizing agent with high affinity and potent antineoplastic properties. [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 6239.

Invited for the cover of this issue are the groups of Professors Passarella and Pieraccini at the University of Milan, in collaboration with some of the members of TubInTrain consortium. The image depicts work with the elements of nature, in particular the destabilising effect of maytansinol (the constellation) on microtubules (the trees). Read the full text of the article at 10.1002/chem.202203431.

Maytansinol is a valuable precursor for the preparation of maytansine derivatives (known as maytansinoids). Inspired by the intriguing structure of the macrocycle and the success in targeted cancer therapy of the derivatives, we explored the maytansinol acylation reaction. As a result, we were able to obtain a series of derivatives with novel modifications of the maytansine scaffold. We characterized these molecules by docking studies, by a comprehensive biochemical evaluation, and by determination of their crystal structures in complex with tubulin. The results shed further light on the intriguing chemical behavior of maytansinoids and confirm the relevance of this peculiar scaffold in the scenario of tubulin binders.

Taxanes are microtubule-stabilizing agents used in the treatment of many solid tumors, but they often involve side effects affecting the peripheral nervous system. It has been proposed that this could be related to structural modifications on the filament upon drug binding. Alternatively, laulimalide and peloruside bind to a different site also inducing stabilization, but they have not been exploited in clinics. Here, we use a combination of the parental natural compounds and derived analogs to unravel the stabilization mechanism through this site. These drugs settle lateral interactions without engaging the M loop, which is part of the key and lock involved in the inter-protofilament contacts. Importantly, these drugs can modulate the angle between protofilaments, producing microtubules of different diameters. Among the compounds studied, we have found some showing low cytotoxicity and able to induce stabilization without compromising microtubule native structure. This opens the window of new applications for microtubule-stabilizing agents beyond cancer treatment.

Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians.

Maytansinoids are a successful class of natural and semisynthetic tubulin binders, known for their potent cytotoxic activity. Their wider application as cytotoxins and chemical probes to study tubulin dynamics has been held back by the complexity of natural product chemistry. Here we report the synthesis of long-chain derivatives and maytansinoid conjugates. We confirmed that bulky substituents do not impact their high activity or the scaffold's binding mode. These encouraging results open new avenues for the design of new maytansine-based probes.