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The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme. [ABSTRACT FROM AUTHOR]

Abstract Objectives This study was aimed to develop small molecule inhibitors of the P. aeruginosa heme oxygenase (pa -HemO) as potential treatment of infections caused by P. aeruginosa. Methods New compounds were designed based on the crystal structure of pa -HemO. The binding affinities (K D) were determined using intrinsic fluorescence quenching assays. The anti-microbial effects of the new compounds was evaluated by minimal inhibitory concentration 50% (MIC 50). Results Eleven compounds were synthesized as potential pa -HemO inhibitors. New compounds demonstrated K D values ranging from 1.5 to 180 μ M, and MIC 50 values ranging from 26 to 260 μg/mL. The compounds had good affinity with HemO and promising anti-microbial effects on P. aeruginosa. Conclusions The new inhibitors described herein can inhibit the growth of P. aeruginosa via the inhibition of pa -HemO. There may be broad prospects for HemO inhibitors to treat P. aeruginosa related infections. Highlights • Inhibitors of pa-HemO were developed to suppress the binding of heme to HemO and the use of heme by P. aeruginosa. • New compounds were designed based on the x-ray crystal structure of pa-HemO. • The development of these new HemO inhibitors will help open up new avenues for the treatment of P. aeruginosa infections. [ABSTRACT FROM AUTHOR]

New therapeutic targets are required to combat multidrug resistant infections, such as the iron-regulated heme oxygenase (HemO) of Pseudomonas aeruginosa, due to links between iron and virulence and dependence on heme as an iron source during infection. Herein we report the synthesis and activity of a series of iminoguanidine-based inhibitors of HemO. Compound 23 showed a binding affinity of 5.7 μM and an MIC50 of 52.3 μg/mL against P. aeruginosa PAO1. An in cellulo activity assay was developed by coupling HemO activity to a biliverdin-IXα-dependent infrared fluorescent protein, in which compound 23 showed an EC50 of 11.3 μM. The compounds showed increased activity against clinical isolates of P. aeruginosa, further confirming the target pathway. This class of inhibitors acts by binding to an allosteric site; the novel binding site is proposed in silico and supported by saturation transfer difference (STD) NMR as well as by hydrogen exchange mass spectrometry (HXMS). [ABSTRACT FROM AUTHOR]