Background Hydrogen sulfide (H₂S) is now recognised as an endogenous gasotransmitter that regulates vascular, metabolic and inflammatory processes. One of its principal biosynthetic enzymes is 3-mercaptopyruvate sulfurtransferase (3MST), which generates H₂S and reactive persulfides in both mitochondria and cytosol. Dysregulation of the 3MST-H₂S pathway has been implicated in cancer, metabolic disease and redox signalling. The discovery of selective inhibitors of 3MST is therefore essential to enable pharmacological dissection of H₂S biology, in a manner analogous to the use of nitric oxide synthase inhibitors in defining the physiological roles of nitric oxide. Although the first selective 3MST inhibitors were reported in 2017, progress has been limited by the small number of available chemical tools and by issues of selectivity, reactivity and assay interference, which continue to complicate interpretation of biological data. Aim The aim of this thesis was to design, synthesise and biologically evaluate a focused library of novel 3MST inhibitors. A structure-guided approach was employed, in which commercially available chemical fragments were screened in silico against the 3MST active site using molecular docking to prioritise candidates with favourable predicted binding and synthetic tractability. Selected fragments were incorporated into a pyrimidinone-based scaffold to generate a series of mercaptoacetyl and mercaptoacetamide derivatives. The resulting compounds were synthesised and evaluated using a fluorescence-based biochemical assay that measures enzymatic H₂S production. Comparative analysis with known inhibitors and reference compounds was used to assess inhibitory potency and selectivity. Results A total of 20 compounds were synthesised and evaluated for 3MST inhibition. A previous fluorescence-based screen in our laboratory had identified CDS004892 as a 3MST inhibitor, with an average IC₅₀ of 7.92 μM, and this compound was used as a lead scaffold for the present study. In parallel, screening of commercially available compounds identified two additional 3MST inhibitors, NCC-00043864 and NCC-00065766, with IC₅₀ values of 4.5 μM and 9.7 μM, respectively. From the newly synthesised series, four compounds, MNA1, MNA13, MNA20 and MNA23, displayed inhibitory activity with potencies comparable to those reported for the best literature 3MST inhibitors, which typically exhibit IC₅₀ values in the range of 1.7 to 6.3 μM. Notably, MNA1 and MNA23 also showed selectivity profiles comparable to reference compounds, indicating preferential inhibition of 3MST over related enzymes. Conclusion These findings demonstrate that rational, structure-guided design combined with biochemical screening can yield novel, potent and selective 3MST inhibitors. The identification of both newly synthesised and commercially available inhibitors expands the currently limited repertoire of chemical tools available to investigate the 3MST–H₂S pathway. In particular, the discovery of selective inhibitors within the MNA series provides valuable starting points for the development of improved pharmacological tools. The compounds developed here provide a platform for further optimisation and biological evaluation, particularly in cell-based assays and mitochondria-specific contexts.
Maha Almehaize (Thu,) studied this question.