Abstract 6239: PM534 is a novel microtubule-destabilizing agent with high affinity and potent antineoplastic properties
Authors: Oliva Maria Ángela , Álvarez-Bernad Beatriz , Lucena-Agell Daniel , Diez Marta Martínez , Guillén María José , Nuñez Gema Santamaría , Muñoz-Alonso María José , Garrido-Martin Eva M. , Avilés Pablo , Cuevas Carmen , Díaz J. Fernando 
Published: April 2023

Abstract
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.