A multidisciplinary functional proteomics-aided strategy as a tool for the profiling of a novel cytotoxic thiadiazolopyrimidone

[Display omitted] •A small set of [1,3,4] thiadiazolopyrimidones has been efficiently synthesized and then decorated by Suzuki-Miyaura cross-coupling reaction, which provided rapid access to different 2-arylated thiadiazolopyrimidones.•A multi-disciplinary strategy has been used to disclose the potential biological targets of the most active thiadiazolopyrimidone, namely compound 1, using a label-free mass spectrometry based platform.•Functional proteomics, coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring, disclosed Annexin A6 as the most reliable target.•A consistent inhibition of calcium transition, cell migration and invasion of cancer cells has been demonstrated due to Annexin A6 modulation by the thiadiazolopyrimidone-based compound 1.•The first disclosed small-molecule binder targeting Annexin A6 may pave the way to the development of novel anticancer agents. In recent years, thiadiazolopyrimidine derivatives have been acknowledged for their striking poly-pharmacological framework, thus representing an interesting scaffold for the development of new therapeutic candidates. This paper examines the synthesis and the interactome characterization of a novel bioactive thiadiazolopyrimidone (compound 1), endowed with cytotoxic activity on HeLa cancer cells. In detail, starting from a small set of synthesized thiadiazolopyrimidones, a multi-disciplinary strategy has been carried out on the most bioactive one to disclose its potential biological targets by functional proteomics, using a label-free mass spectrometry based platform coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring. The identification of Annexin A6 (ANXA6) as compound 1 most reliable cellular partner paved the way to deepen the protein–ligand interaction through bio-orthogonal approaches and to prove compound 1 action on migration and invasion processes governed by ANXA6 modulation. The identification of compund 1 as the first ANXA6 protein modulator represents a relevant tool to further explore the biological role of ANXA6 in cancer, as well as to develop novel anticancer candidates.