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Tubulin is a heterodimeric protein composed of α- and β-subunits, which polymerize to form the cell’s microtubules. The latter are key components in mitotic spindle formation and essential targets in anticancer therapy. Compounds such as paclitaxel, tubulysins, dolastatins and synthetic analogues of these latter compounds, including cemadotin, exert their cytotoxic effects by disrupting microtubule dynamics. Previously, we reported the production and anticancer activity of a library of cemadotin analogues featuring a C-terminal tertiary amide functionalized with a variety of N-substituents, thus resulting in compounds occurring as a mixture of amide rotamers. Here we describe a comprehensive NMR and conformational study that provides new insights into the effect of the conformational equilibrium on the binding mode of the novel cemadotin analogues to the tubulin target. The conformational behavior of the isomer equilibrium of cemadotin’s terminal amide bond was investigated by TOCSY and ROESY NMR experiments, which allowed the identification and quantification of individual rotamer populations. A slow interconversion between the s-cis and s-trans amide rotamers was observed under standard NMR conditions (25 °C), indicating a significant energy barrier and conformational rigidity. Molecular docking and saturation transfer difference (STD) NMR experiments were performed with a representative analogue and tubulin to assess the binding mode. The results revealed that the s-trans rotamer is the predominant conformer in solution and exhibits a more favorable interaction with tubulin compared to the s-cis isomer, thus helping to understand the conformational requirements for an improved tubulin binding and the inhibition of the polymerization process.

A central process contributing to the phenotype of aging is cellular senescence. We recently identified the FOXO4 – p53 axis as pivotal in maintaining the viability of senescent cells, and that senescent cells can be targeted selectively with the senolytic peptide FOXO4-DRI. Here, we solve the solution NMR structural models of the p53 transactivation domain in complex with the FOXO4 forkhead domain and in complex with FOXO4-DRI. Strikingly, we find that the disordered FOXO4-DRI binds to the disordered p53 TAD2 and forms a transiently folded complex. In this complex, both, the FOXO4-derived region and the cationic cell permeability peptide contribute to the interaction. Furthermore, we show that p53 phosphorylation enhances the affinity for both FOXO4 and FOXO4-DRI. Summarizing we provide a detailed characterization of the interaction of p53 with FOXO4 and FOXO4-DRI which is the basis for development of p53 inhibitors to treat diseases linked to cellular senescence such as cancers. Cellular senescence drives aging, with the p53–FOXO4 axis sustaining senescent cell viability. This study reveals structural insights into p53 binding to FOXO4/FOXO4-DRI, informing the development of p53-targeted senolytics for age related diseases.

Cell-to-cell communication in microbial systems is known for its vital role in cellular signalling and gene expression. A specific form termed Quorum Sensing (QS) has received considerable attention since its discovery in the marine symbiont Aliivibrio fischeri. QS-controlled microbial functions are associated with bacterial virulence, pathogenicity, host-microbe interactions, and biofilm development. Interference in these signalling systems can modulate microbial virulence and pathogenicity, and microbial infection caused by drug-resistant pathogens. Plant-derived phytochemicals are considered a promising candidate, with coumarins emerging as significant plant-derived signalling molecules shaping microbiome dynamics and pathogen behaviours from a broad spectrum of ecosystems. Here we explored the role of natural and synthetic coumarin compounds in the control of signalling and virulence traits in Pseudomonas aeruginosa and other priority bacterial pathogens, including the fungal opportunist Aspergillus fumigatus. We uncovered an important 'hydroxylation-bias' favouring coumarin, umbelliferone (7-OH), and 6-hydroxy-coumarin (6-OH) in the specific competitive inhibition of the Pseudomonas Quinolone Signal (PQS), associated with reduced activity of a PqsR translational fusion and suppression of pyocyanin production. Conversely, while esculetin (6,7-OH) was most effective at Acyl Homoserine Lactone (AHL) QS biosensor inhibition, it did not affect PQS production. Anti-biofilm activity of coumarins against P. aeruginosa was independent of initial attachment but linked to changes in exopolysaccharide production. As the very real threat posed by antimicrobial resistance persists, these data support a role for phytochemicals such as coumarins in delivering an ecological solution to dysbiosis in the host-microbe interaction.

Intricate communication networks and sensing systems underpin the complexity of microbe-host interactions, enabling spatiotemporal control of optimised microbe-host consortia in a diverse range of ecosystems. A central component of these complex interactomes has been the signalling events that enable recognition of host and microbe, whether that niche be clinical or environmental, human or plant. Coumarins have emerged as significant plant derived signalling molecules shaping microbiome dynamics and pathogen behaviours from a broad spectrum of ecosystems. Here we explored the role of natural and synthetic coumarin compounds in signal interference and control of pathogenesis in bacterial and fungal pathogens, uncovering an important ‘hydroxylation-motif’ in the specific inhibition of two Pseudomonas aeruginosa interspecies and interkingdom communication molecules. Characterisation of the anti-biofilm activity of coumarins revealed changes in exopolysaccharide production independent of the initial attachment phenotype. Molecular modelling provides an insight into the receptor binding dynamics of three closely related natural coumarins, suggesting an intricate and highly specific host-microbe interaction at the species level. As the very real threat of antimicrobial resistance continues to shadow our horizons, phytochemicals such as coumarins have potential to deliver an ecological solution to dysbiosis in the host-microbe interaction. Natural ecosystems rely on homeostatic interactions between the kingdoms of life to ensure sustainable and balanced communities can persist. Plant-derived coumarins have recently emerged as playing an important role in shaping microbial communities, presenting a remarkable chemical diversity that can influence the behaviour of bacteria and fungi. At the same time, one of the major challenges to human health continues to be the spread of antimicrobial resistance and the parallel absence of a concerted effort to source and produce new antibiotics at industrial scale. Therefore, new approaches to the control of infection are required, and an ecosystem-level lens may offer one such innovative intervention. Coumarins have the potential to neutralise the very mechanisms used by bacteria and fungi to cause infection leading to morbidity and mortality in hosts ranging from plant to animals. Here we present a mechanistic insight into how effective these molecules can be in targeting keystone pathogens termed the ESKAPEEs and their fungal counterparts.