Selective inhibition of nitric oxide synthase for multiple indications

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Project Summary This proposal is a continuation of R01 GM049725, “Selective Inhibition of Nitric Oxide Synthase for Multiple Indications”. Nitric oxide synthase (NOS) is a remarkable target, as we have found that neuronal nitric oxide synthase (nNOS) inhibitors are applicable to the potential treatment of neurodegenerative diseases (e.g., Parkinson's, Alzheimer's, cerebral palsy), bacterial infection, and melanoma. Inhibitors of nNOS block the excess NO that can cause degeneration of neurons. They also inhibit NO produced in melanocytes that leads to melanoma. Inhibitors of bacterial NOS (very similar to nNOS) can be used to protect antibiotics from bacterial degradation (i.e., resistance) and can be synergistic for bacterial growth inhibition by oxidant stress molecules, such as hydrogen peroxide. We have made outstanding progress on all three indications; two of which could not have progressed without the crystallography expertise of Thomas Poulos. In the last four years we have been able to shorten the syntheses of our inhibitors, make inhibitors that are membrane penetrable, are stabilized from metabolism, and are blood-brain barrier (BBB) penetrable with good oral bioavailability while maintaining excellent potency and nNOS selectivity. However, we have not yet gotten compounds that possess all of the desired properties. Consequently, this grant will allow us to identify compounds that have all of the desired pharmacodynamics and pharmacokinetic properties. We plan to optimize our nNOS-selective compounds for pharmacodynamics and pharmacokinetics and test in mouse models of Parkinson's and Alzheimer's diseases and in a rabbit model for cerebral palsy. We have identified several nNOS-selective compounds that inhibit the growth of bacteria, including MRSA, especially in combination with an antibiotic or hydrogen peroxide. We plan to make bacterial NOS (bNOS)-selective compounds to avoid potential side effects from inhibition of mammalian NOS. Also, there may be additional targets, other than bNOS with which our compounds interact; therefore, we will carry out protein pulldown experiments with a photoaffinity-labeled bNOS inhibitor and identify proteins to which it attaches with proteomics. This should clarify a more complete mechanism of action of bNOS inhibitors. Our best nNOS-selective inhibitors will also be tested by Dr. Sun Yang as inhibitors of a variety of melanoma cell lines and in a xenograft melanoma model for inhibition of melanoma growth and metastasis. Dr. Yang has identified a peptide carrier to target molecules to melanoma; we will conjugate active compounds to this peptide for drug delivery. We also will make a photoaffinity probe of an active inhibitor to identify targets.