Valeria La Gatta
PC19 – Valeria La Gatta
University of Modena and Reggio Emilia, Italy
valeria.lagatta@unimore.it
| Characterization of human thymidylate synthase dimer disruptor interactions by photoaffinity labeling |
| La Gatta Valeria1, Tagliazucchi Lorenzo2, Aiello Daniele1, Illuminati Davide3, Pacifico Salvatore3, Guerrini Remo3, and Costi Maria Paola1 1 Department of Life Science, University of Modena and Reggio Emilia; 2 Department of biotechnology, chemistry and pharmacy, University of Parma; 3 University of Ferrara. |
| Abstract Human thymidylate synthase (hTS) is an essential enzyme in de novo DNA biosynthesis and a validated anticancer target. hTS exists in a dynamic equilibrium between an active dimer and an inactive monomer involved in autoregulatory control of protein expression [1]. Conventional active-site inhibitors induce hTS overexpression and drug resistance [1,2]. To overcome these limitations, we developed small molecules targeting the hTS dimer interface, acting as dimer destabilizers that inhibit enzymatic activity while preserving regulatory functions [2]. To investigate ligand-protein interactions and assess selectivity, a photoaffinity labeling–mass spectrometry (PAL-MS) strategy was established. PAL employs photoreactive probes containing diazirine groups that, upon UV irradiation, generate reactive intermediates able to covalently trap proximal residues. This approach enables investigation of non-covalent and transient interactions, particularly suitable for interface-targeting ligands. The incorporation of an alkyne handle further allows bioorthogonal conjugation with reporter tags via copper-catalyzed azide–alkyne cycloaddition, enabling enrichment and identification of labeled proteins by LC–MS [3]. Among the interface-targeting compounds previously developed in our laboratory, A40 was selected as a scaffold for probe design due to its favorable solubility and synthetic accessibility. A diazirine-containing analogue was synthesized and successfully applied in PAL experiments on recombinant hTS, enabling mapping of the binding region at the dimer interface. To extend it to a cellular context, alkyne-functionalized derivatives were designed. A series of pre-probe analogues was prepared to mimic the steric and electronic properties of the final constructs. Among these, A40_3pre retained the highest inhibitory activity and was selected as precursor for the development of a fully functionalized probe suitable for cellular PAL-MS studies. This work establishes an integrated medicinal chemistry and chemical biology platform for the investigation of hTS dimer destabilizers. By combining rational probe design with PAL-MS, this approach enables characterization of ligand–protein interactions at the dimer interface and supports proteome-wide selectivity assessment, providing a foundation for next-generation anticancer agents targeting thymidylate synthase. |
| References [1] Taddia, L.; D’Arca, D.; Ferrari, S.; et al. Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance. Drug Resist. Updat. 2015, 23, 20–54. DOI: 10.1016/j.drup.2015.10.003. [2] Costantino, L.; Ferrari, S.; Santucci, M.; et al. Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth. eLife 2022, 11, e73862. DOI: 10.7554/eLife.73862. [3] Smith, E.; Collins, I. Photoaffinity labeling in target- and binding-site identification. Future Med. Chem. 2015, 7, 159–183. DOI: 10.4155/fmc.14.152. |