Herein, we report the first structural studies of HPPK from S. aureus using a combination of solution NMR and x-ray crystallographic structure determination, and the identification of a novel pterin-site inhibitor 8-mercaptoguanine byin silico ROCS screening and differential scanning fluorimetry assay. The atomic structure of SaHPPK has been determined in complex with a new pterinsite inhibitor, revealing the molecular details of inhibitor association. Binding of the inhibitor, substrate and cofactor molecules were quantified using isothermal titration calorimetry and surface plasmon resonance, while in vitro enzyme inhibition data was WZ4002 1213269-23-8 measured using a luciferase based luminescent assay. Detailed studies of ligand interactions using NMR highlight critical ligand-induced dynamic changes upon inhibitor, substrate and cofactor binding, which correlate with large entropic penalties to the binding thermodynamics of the inhibitor measured by ITC. Titration of 8-mercaptoguanine into a sample of the apo enzyme produced a range of CSPs and exchange regimes in the NMR spectra. 15 cross peaks broadened completely around the binding site and along the sheet and most others exhibited slow exchange, indicative of a Kd likely in the low mM range. In contrast, when performed in the presence of saturating ATP or AMPCPP, widespread perturbations were observed in slow exchange for all resonances, despite no change in binding affinity measured by ITC and SPR. Chemical shift perturbations clearly mapped to the respective substrate and cofactor site. This work reports the discovery, binding properties and mechanism of a novel, competitive pterin site inhibitor, presented in complex with the first crystal structure of SaHPPK. The pterin site is highly specific and restricts the chemical space available for inhibitor design to structures closely resembling the pterin scaffold. Consequently, the literature is devoid of non-pterin like HPPK inhibitors, despite mounting structural information that has been reported over the last decade. In line with the high pterin-site specificity is the high ligand efficiency of 8-mercaptoguanine. 8-Mercaptoguanine has previously been reported to have biological activity. Early studies revealed some lipolytic PF-4217903 activity while in a number of cases 8-mercaptoguanine has been shown to inhibit enzymes that normally bind purines. Antiviral activity,without significant toxicity, was also reported in an in vivo mouse model. Close analogues, such as 8-mercaptoguanosine, were also shown to induce interleukin-1 activity in macrophages. Despite these studies, no antibacterial activity has been reported previously. Interestingly, 8mercaptoguanine has been shown to bind to, but not inhibit, B. anthracis DHPS by co-crystallisation, which may open the possibility for a multi target inhibitor derived from this scaffold. In the present work, we did not observe growth inhibition in vivo by 8mercaptoguanine in E. coli cell-based assays. Given the unfavourable logP, this is likely to be due to poor membrane permeability. This may be a disadvantage for pterin-like inhibitors in general given the hydrophilic nature and restrictive chemical space of the pterin scaffold in folate pathway enzymes. Nevertheless, while insufficient transport of a set of closely related pyrimidines as potential antifolates was implicated in their poor in vivo inhibition, derivatives with an additional phenyl substituent displayed sub micromolar activity in vivo to T. brucei and L. major. The known phenethyl in vitro inhibitor of HPPK suggests that a suitably positioned phenyl group on 8-mercaptoguanine may thus be beneficial to both binding and assist cell permeability.