Cysteamine (2-aminoethanethiol) dioxygenase (ADO) is non-heme iron-dependent thiol dioxygenase, initially implicated in sulphur metabolism. However, recently, ADO has also been identified as a novel oxygen-sensing enzyme in mammalian cells. It marks cysteine-initiating proteins for degradation through the Cys-branch of the N-degron pathway in an oxygen-dependent manner. During periods of low oxygen (hypoxia), ADO becomes inactive, allowing its substrates, including Regulators of G-protein Signalling (RGS), to accumulate. RGS proteins attenuate G-protein coupled receptor (GPCR) activity by accelerating GTP hydrolysis, terminating signal transduction and altering cell behaviour. Accordingly, ADO may be a promising therapeutic target for several hypoxic diseases given its role in oxygen adaptation.
To help study the oxygen sensing system, and establish a platform for drug discovery, peptide-based ligands of ADO were identified using RaPID mRNA display technology, an affinity screening strategy with a trillion-compound library. The top hits were synthesized by Solid Phase Peptide Synthesis (SPPS) and characterised by various biophysical and kinetic methods, leading to the discovery of both competitive and allosteric inhibitors of ADO. Structural studies have been initiated to determine their mode of inhibition and cell-penetrating peptides have been conjugated to the most promising hits to enhance cellular uptake. Ultimately, this will generate tools that can be used to study ADO and help understand how oxygen fluctuations influence physiology and disease.