Phd in BIOCHEMISTRY

Thesis title: Development of pyridox(am)ine 5’-phosphate oxidase inhibitors as a novel antineoplastic strategy

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is an essential cofactor in amino acid metabolism, glycogen breakdown, and one-carbon transfer reactions. Mounting evidence links PLP and its biosynthetic enzymes to tumour progression, as they sustain cancer cell proliferation and metabolic plasticity. Pyridox(am)ine 5′-phosphate oxidase (PNPO), the final enzyme in PLP biosynthesis, is frequently upregulated in malignant cells; this property, together with its sensitivity PLP feedback inhibition, makes PNPO an appealing therapeutic target. Here, we report the synthesis and charachterisation of a set of effective human PNPO inhibitors identified through rational design and virtual screening. Six compounds –PLP-rhodanine (PLP-R), four PLP-hydrazones, and the acridone derivative B-2– exhibited strong inhibition, with PLP-R and PLP-isoniazid (PLP-INH) as lead molecules. Notably, the hydrazone derivatives were inspired by known hydrazide-containing drugs such as isoniazid, carbidopa, phenelzine, and hydralazine, which can form PLP adducts in vivo. This concept opens the possibility of repurposing such drugs as precursors of active PNPO inhibitors. We also report that unphosphorylated versions of two of the inhibitors (PL-INH and PL-R), which have more favourable pharmacokinetic properties according to our predictions, are converted into their PNPO-inhibiting phosphate esters by the action of pyridoxal kinase, another enzyme of the PLP biosynthesis. Preliminary experiments performed with selected compounds showed a cytotoxic effect in several cancer cell lines, particularly in acute myeloid leukaemia. These findings lay the foundation for further work, aimed at designing stronger inhibitors and defining the pathological settings where they may be most effective.