Inhibition of the Bacterial Heme Oxygenases from Pseudomonas aeruginosa and Neisseria meningitidis: Novel Antimicrobial Targets Lena M. Furci, # Pedro Lopes, # Suntara Eakanunkul, Shijun Zhong, Alexander D. MacKerell, Jr.,* and Angela Wilks* Department of Pharmaceutical Sciences, School of Pharmacy, UniVersity of Maryland, 20 Penn Street, Baltimore, Maryland 21201-1140 ReceiVed January 24, 2007 The final step in heme utilization and iron acquisition in many pathogens is the oxidative cleavage of heme by heme oxygenase (HO), yielding iron, biliverdin, and carbon monoxide. Thus, the essential requirement for iron suggests that HO may provide a potential therapeutic target for antimicrobial drug development. Computer-aided drug design (CADD) combined with experimental assays identified small-molecule inhibitors of the Neisseria meningitidis HO (nm-HO). CADD virtual screening applied to 800 000 compounds identified 153 for biological assay. Several of the compounds were shown to have K D values in the micromolar range for nm-HO and the Pseudomonas aeruginosa HO (pa-HO). The compounds also inhibited the growth of P. aeruginosa as well as biliverdin formation in E. coli cells overexpressing nm-HO. Thus, CADD combined with experimental analysis has been used to identify novel inhibitors of the bacterial heme oxygenases that can cross the cell membrane and specifically inhibit HO activity. Introduction The ability of bacterial pathogens to acquire iron is essential for both their survival and infectivity. In order to acquire iron, bacteria have evolved specialized systems to directly utilize iron from the host and from heme (iron protoporphyrin IX in any oxidation state) containing proteins. 1-5 In a significant number of bacterial pathogens, the final step in heme utilization is the heme oxygenase (HO) dependent oxidative cleavage of the porphyrin macrocycle to biliverdin and carbon monoxide with the release of iron. 6,7 Accordingly, as the final and critical step in iron utilization from heme, the bacterial heme oxygenases provide a unique therapeutic antimicrobial target. A variety of Gram-negative pathogens, including those that cause many endemic and life-threatening diseases such as Neisseria meningitidis 8 and Haemophilus influenzae, 9 the caus- ative agents of some forms of meningitis, and enteric pathogens such as Vibrio cholerae 10,11 and Shigella dysenteriae 12,13 have evolved sophisticated mechanisms for iron acquisition that involve direct utilization of heme-containing proteins. Neisseria meningitidis, an obligate human pathogen, is usually restricted to the nasopharynx but can invade the bloodstream and cause infections in the meninges and occasionally the synovial membranes of joints. 14 Although little is known on the bio- availability of heme on human mucosal surfaces, Neisseria spp. show phase variation in the type of iron and heme receptors expressed in response to physiological and environmental conditions. 15 This allows the bacteria to establish infections in environments where the heme availability may be extremely low through a combination of hemolysis and high-affinity outer- membrane heme receptors that internalize the heme. Heme uptake and utilization are also employed by op- portunistic pathogens such as Pseudomonas aeruginosa, which has multiple systems for iron uptake, including two Fur- regulated heme uptake operons. 16 P. aeruginosa has become an increasing cause of nosocomial infections in immune compromised patients and is the primary cause of chronic lung infections in individuals with cystic fibrosis (CF) disease. 17,18 In CF patients the virulence of P. aeruginosa is heightened by its ability to form biofilms, 19-22 leading to antibiotic resistance profiles due to the inability to effectively eliminate the infective agent from such biofilms. 21 Approximately 40% of CF patients succumb to fatal infection becasue of antibiotic-resistant P. aeruginosa infections. 17 Notably, a knockout of the heme oxygenase gene in P. aeruginosa (pigA::gen) develops heme toxicity when heme is the sole iron source, 23 suggesting that inhibiting pa-HO a in vivo will result in toxicity and ultimately decreased virulence of the pathogen. An important consideration in the design of a novel antibiotic is its specificity for the target protein. In the present case this consideration is in the context of the bacterial versus the human forms of heme oxygenase. Comparison of heme oxygenase from N. meningitidis (nm-HO) with the mammalian enzymes shows the solvent accessible surface to be significantly smaller in both nm-HO and pa-HO, ∼7.5 Å 3 , when compared to their mam- malian counterparts, which range from 43.6 to 59.7 Å 3 . 24-27 Such a structural difference suggests that low molecular weight inhibitors of nm-HO can be identified that are specific for the bacterial versus the human protein and thus may act as lead compounds in the development of potential antimicrobials. A second consideration in the development of an antibiotic is its spectrum of activity. While most of heme oxygenases have a regioselectivity for the R-meso carbon, which is released as CO to yield R-biliverdin, the iron-regulated heme oxygenase of P. aeruginosa (pa-HO) is regioselective for the δ-meso carbon. 23 However, pa-HO shares 33% identity with nm-HO while both bacterial heme oxygenases have less than 15% homology with the mammalian enzymes, increasing the prob- ability of identifying inhibitors specific for the bacterial enzymes. Thus, the potential exists for the development of novel antibiotics targeting nm-HO that may be specific for other bacterial pathogens while having the necessary specificity for bacterial over mammalian HOs. * To whom correspondence should be addressed. For A.D.M.: phone, 410-706-7442; fax, 410-706-5017; e-mail, amackere@rx.umaryland.edu. For A.W.: phone, 410-706-2537; fax, 410-706-5017; e-mail, awilks@ rx.umaryland.edu. # These authors contributed equally to this work. a Abbreviations: pa-HO, Pseudomonas aeruginosa heme oxygenase; nm- HO, Neisseria meningitidis heme oxygenase; MPA01, Pseudomonas aeruginosa PA01 strain; sGC, soluble guanylate cyclase; CYP, cytochrome P450; NOS, nitric oxide synthase; CADD, computer-aided drug design; MW, molecular weight. 3804 J. Med. Chem. 2007, 50, 3804-3813 10.1021/jm0700969 CCC: $37.00 © 2007 American Chemical Society Published on Web 07/13/2007