br Materials and methods br Author contributions br Acknowle

Materials and methods
Author contributions
Acknowledgments We thank members of the Skaar laboratory for critical evaluation of the manuscript. This work was supported by grants from the National Institutes of Health, R01AI069233 (EPS), and T32GM008554-21 (LJL).
Introduction Pseudomonas aeruginosa is a life-threating Gram-negative bacterium that causes infections in humans [1]. The P. aeruginosa infection can be controlled by antibiotic including β-lactams and aminoglycosides [2]. However, drug resistance has been observed and seriously increased over the past decades, which causes severe antibiotic misuse [3,4]. Iron is an essential cofactor for enzymes found within all kingdoms of life. P. aeruginosa is no exception to this rule, and therefore, it must acquire iron from their hosts in order to survive the extremely low iron environment. P. aeruginosa can obtain iron by different strategies: (i) production of Fe3+ chelating molecules and iron carriers such as pyoverdine and pyochelin, and uptake of ferric iron-carrier complexes by TonB-dependent receptors (TBDR) [5], (ii) ingestion of an exogenous iron carrier complex [6], (iii) intake of heme molecules from hemoglobin from the host [7], and (iv) RepSox synthesis and utilization of ferrous iron. Reducing Fe3+ to Fe2+ extracellularly using phenazine and Fe2+ specific uptake system (Feo system) [8]. In the host, depending on the type of infection (acute or chronic infection), P. aeruginosa adopts different iron uptake strategies to meet its own needs [9]. The majority of iron within the human body is in the form of heme [10]. P. aeruginosa has elegant systems dedicated to the acquisition of heme from host hemoproteins. P. aeruginosa can take heme from hemoglobin through both Has and Phu systems [7]. In the periplasm, the ingested heme binds to the periplasmic binding protein and gets transported into the cell via the ATP-binding cassette (ABC) transport system. In the ABC transport system, heme is first combined with the heme chaperone PhuS and then transported to the HemO that catalyses the degradation of heme to biliverdin, CO and Fe2+ [11]. Importantly, the activity of pa-HemO provides critical driving force for the flux of heme into cells. Therefore, if HemO is inhibited, P. aeruginosa loses its ability to take up heme as an iron source [12]. The pa-HemO has a unique heme-binding active site. The solvent accessible surface of pa-HemO (∼7.5 Å3) is much smaller than the mammalian enzyme HO1 (43.6–59.7 Å3) [[13], [14], [15], [16]]. Moreover, different than the binding mode in HO1 and other known heme oxygenases, heme binds in the active site of pa-HemO with a significantly rotated (∼100°) orientation as a result of the unique amino acid network present in the active site [[13], [14], [15], [16]]. These structural differences provide foundations for selective inhibition of the pa-HemO over other heme oxygenases such as HO1 [17]. In this study, we developed competitive inhibitors of the pa-HemO to suppress the binding of heme to HemO and inhibit the use of heme by P. aeruginosa. New compounds were designed based on the x-ray crystal structure of pa-HemO [18]. The development of these new HemO inhibitors will help open up new avenues for the treatment of P. aeruginosa infections.
Material and methods
Results and discussion Eleven compounds were tested as potential pa-HemO inhibitors. The KD values of the majority of compounds ranged from 8 to 90 μM (Table 1). Among these compounds, the compound 7c indicated the best KD value (1.5 ± 0.8 μM) and compound 7d has the largest KD value (180 ± 37 μM). Overall, the results show that compounds bind to the active site with modest affinity, suggesting that the compounds can be suitable developing inhibitors for pa-HemO. Compared with the control group without DMSO, only the testing group containing 1% DMSO did not show obvious effects on the growth of P. aeruginosa (P > 0.05). Other testing groups with higher concentrations of DMSO significantly inhibited the growth of the bacteria. The OD600 measurement curve is shown in Fig. 2. Therefore, in the subsequent MIC50 assay, we kept the DMSO concentration at 1%. The research data of DMSO toxicity to P.aeruginosa growth are detailed in supplementary material.