Abstract 7141: The novel microtubule-destabilizing compound AUS₀01 maintains unique binding to the colchicine site of tubulin and elicits reversible cellular effects relative to other anti-tubulin agents

Abstract Our previous work showed that the prenylated hydroxy-stilbene AUS₀01 exerts a strong safety profile and potent anti-proliferative responses, as assessed in 30 types of cancer cell lines, stemming from its microtubule destabilization activity. The aim of the current study was to delineate the molecular mechanism of microtubule destabilization by AUS₀01 and to provide insight into its high potency and low toxicity. Surface plasmon resonance (SPR) experiments revealed the direct interaction of AUS₀01 with tubulin with an apparent equilibrium dissociation constant, Kd, of 2. 13 × 10−5M. Size exclusion competition assays and an X-ray crystal structure of the tubulin-AUS₀01 complex at 2. 1 Å resolution establish ligand binding within the colchicine site on tubulin. Since colchicine-site ligands are well known to inhibit the curved-to-straight conformational transition of tubulin, which is an essential process for microtubule formation, these results readily explain the microtubule-destabilizing activity of the compound. Notably, while both combretastatin A4 (CA-4) and AUS₀01 are both colchicine-site stilbene ligands, CA-4 binds in the cis configuration, while AUS₀01 maintains a trans configuration leading to a substantially different pocket engagement. Comparison of the tubulin-AUS₀01 complex structure with the ones of tubulin-colchicine and tubulin-plinabulin revealed that AUS₀01 elicits different rearrangements in the T7 loop of β-tubulin versus colchicine and occupies a different zone of the pocket relative to plinabulin, further elaborating its unique binding properties. Interestingly, AUS₀01 exhibits a higher relative affinity than colchicine itself, as assessed by i) an ultrafiltration-based tubulin-ligand release assay and ii) a radioligand colchicine competition assay. However, fitting steady state SPR data for AUS₀01 binding to tubulin revealed fast association and dissociation rates and was indicative of reversible binding. The latter finding is corroborated by AUS₀01-triggered reverse biological effects including morphological alterations, cell cycle G2/M arrest, and by a decrease of cell viability upon discontinuation of the drug treatment in glioma and pancreatic cancer cells. Conversely, colchicine, CA-4 or paclitaxel administered at the same doses as those applied for AUS₀01 failed to reverse the drug-induced phenotypes upon removal resulting in sustained cytotoxicity. In conclusion, we fully characterized the unique binding mode of AUS₀01 to the colchicine site of tubulin and elucidated the reversible nature of the target engagement. AUS₀01 features a distinctive and reversible molecular interaction with tubulin which provides a plausible explanation for its favorable safety profile compared to other microtubule targeting agents. Citation Format: Herman Lelie, Yao-Chieh Chou, Alastair J. King, Zlata Boiarska, Andrea E. Prota, Michel O. Steinmetz, Marina Koutsioumpa. The novel microtubule-destabilizing compound AUS₀01 maintains unique binding to the colchicine site of tubulin and elicits reversible cellular effects relative to other anti-tubulin agents abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 7141.