Identification of Redox Partners and Development of a Novel Chimeric Bacterial Nitric Oxide Synthase for Structure Activity Analyses

Abstract

Background: Inhibition of bacterial nitric oxide synthase (NOS) improves the efficacy of antibiotics. Results: Development and characterization of a novel bacterial NOS chimera supports NO production in the presence of NADPH and a flavodoxin reductase. Conclusion: Structure activity relationship between NOS inhibitors and bacterial NOS can now be evaluated. Significance: Assays can be adapted for high-throughput screening to identify new bacterial NOS inhibitors.

Nitric Oxide Synthase as a Target for Methicillin-Resistant Staphylococcus aureus

“…To carry this out, we adapted a bNOS activity assay (Holden et al, 2014) to screen through a series of NOS inhibitors using a single time point approach (Fig 2). Concurrently, we measured the K S for each inhibitor using the imidazole displacement assay.…”

“…Compounds 19 and 32 were next assayed separately against purified NOS isoforms at varying concentrations (Holden et al, 2014). Even though the IC 50 s for both mNOS and bsNOS were measured by complimentary methods, both methods allowed for an excellent comparison of inhibitor potency, as IC 50 was used to calculate K i using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).…”

“…Both WT and I218V bsNOS were expressed and purified from E. coli as previously described for bsNOS (Pant et al, 2002). YumC and bBiDomain were also purified from E. coli and used for activity analysis (Holden et al, 2014). Recombinant rat nNOS and murine iNOS were expressed in E. coli and isolated as reported(Hevel et al, 1991; Roman et al, 1995).…”

“…Selectivity over nNOS may represent less of an immediate problem, since many of the polar NOS inhibitors characterized thus far are not very effective at crossing the blood-brain barrier (Silverman, 2009). Recent structure-based studies utilizing B. subtilis NOS (bsNOS) as a model system for bNOS suggest that specificity can be achieved through targeting the pterin-binding site (Holden et al, 2013; Holden et al, 2014), as the bNOS and mNOS pterin binding sites are quite different.…”

“…To quickly identify potent bNOS inhibitors we screened a diverse set of NOS inhibitors (Figure 1) using a novel chimeric enzyme recently reported for bNOS activity analysis (Holden et al, 2014). From this high-throughput analysis we were able to identify two potent and chemically distinct bNOS inhibitors.…”