Neurodegenerative disorders (such as Alzheimer?s, Parkinson?s, Huntington?s diseases, and others) result in severe neurological, cognitive, and motor deficits. Neuronal nitric oxide synthase (nNOS) inhibition is emerging as a promising strategy to treat neurodegenerative disorders, but the development of nNOS inhibitors is often hindered by poor pharmacokinetic (PK) properties. In the past few years, we have developed several classes of highly potent and selective inhibitors of nNOS, which exhibit improved membrane permeability, elevated metabolic stability, or promising blood-brain barrier penetration in animals. However, we have yet to identify compounds that encompass all of these desired properties, and further optimization of these nNOS inhibitors will be needed. Our current R35 award allows us to identify promising nNOS inhibitors and characterize drug- like properties in a limited capacity. We have expertise and capability to design and synthesize 40-50 novel nNOS inhibitors every year. However, only 5-6 compounds can be evaluated in comprehensive in vitro studies of absorption, distribution, metabolism, excretion and toxicity (ADMET), and only 2-3 compounds are advanced in PK in vivo studies using external resources. To facilitate our discovery of nNOS inhibitors with desired CNS drug properties, we have tried to carry out some of these ADMET/PK studies using commercially available materials and internal resources, while the following quantitative analyses were impeded by the lack of reliable and continuous LC-MS/MS support. Therefore, through this proposal, we aim to describe how the requested LC- MS/MS system will be valuable and supportive of our current R35 award to improve the druglike properties of novel nNOS inhibitors with higher efficiency and productivity. The plan of research encompasses the following aims:
Specific Aims Aim 1 Installation of a new triple-quadrupole mass spectrometer coupled with high-performance liquid chromatography to be completed within three months. A routine training/maintenance policy will be established, assisted by technicians from the vendor.
Aim 2 To establish an ADMET/PK routine screening platform within three months using internal resources. Cellular and animal experiments will be carried out at the core facilities of Northwestern University with appropriate training and approvals. Final quantitative analyses will be carried out using the new LC-MS/MS system.
Aim 3 To evaluate 20 new analogues through different in vitro ADMET studies every year to identify nNOS inhibitors with improved druglike parameters for CNS applications. Three to five inhibitors that are chosen from in vitro assessment will then be advanced to brain/plasma PK studies.
Neuronal nitric oxide synthase (nNOS) is a promising enzyme target; the development of nNOS inhibitors holds promise for the treatment of neurodegenerative diseases, which is a highly desirable unmet medical need. In the last few years, we have discovered highly potent and selective nNOS inhibitors aided by in silico predictions and co-crystallization of our inhibitors with human nNOS. Inhibitor potency is irrelevant if the inhibitor is unable to cross the blood-brain barrier and reach nNOS in vivo. The goal of this project is to facilitate druglike property screening and help to discover nNOS inhibitors with improved CNS drug properties.
