Design and Discovery of Novel Small Molecule Inhibitors targeting Heme Oxygenase (HemO) Dependent Iron Acquisition and Heme Signaling in Pseudomonas aeruginosa.

Abstract

The recent rise in antibiotic resistance particularly those pertaining to hospital acquired infections has highlighted the need for alternative therapeutic approaches. Alternative approaches include anti-virulence strategies that differ from current treatments targeting essential pathways in pathogens and instead target systems and factors required for virulence and infection. Gram-negative opportunistic multi-drug resistant (MDR) pathogens like Pseudomonas aeruginosa are ranked at a serious threat level by the CDC and are infamous for causing life threatening infections in immunocompromised populations such as patients with ventilator-assisted pneumonia, open surgical wounds, and cystic fibrosis. Pathogenic bacteria including P. aeruginosa, require the essential micronutrient iron for their survival and virulence. It has been reported that during acute and chronic infections P. aeruginosa preferentially utilizes heme as its iron source over siderophore mechanisms. P. aeruginosa encodes two non-redundant heme uptake systems that both utilize the iron regulated heme oxygenase enzyme (HemO) to release iron and the biliverdin (BVIX) metabolites BVIXβ and -δ. It has been shown that HemO catalytic activity is required to drive heme uptake into the cell. Furthermore, the products of extracellular heme metabolism BVIXβ and -δ function as signaling and regulatory molecules in several virulence traits. Therefore, HemO represents an ideal therapeutic target due to its dual function in limiting both iron and the heme metabolites that regulate several virulence traits. We hypothesize such a dual function strategy will also increase the barrier to resistance. The work herein applies a structure-based design and high-throughput screening approach followed by in vitro and in cell characterization of lead compounds. Further computer-aided drug design (CADD) and guided chemical synthesis optimization were employed to design and discover novel small molecule scaffolds and inhibitors of HemO. The approaches resulted in lead compounds with nanomolar binding affinity and inhibition of HemO enzyme activity both in vitro and in vivo. Additionally, I developed a new method for the production and purification of BVIXβ and -δ, in > 500-fold increased yields, to further study their role in P. aeruginosa virulence and infection.