This proposal ultimately seeks to develop the foundation for a virtual and wet lab high throughput screening system for drugs delivered to the central nervous system (CNS), including those useful for CNS tumors, CNS HIV infection and neuropsychiatric disorders. The goal is to identify highly specific substrates for two olfactory- expressed organic anion transporters (Oats) discovered by us, Oat6 and Oat1, thereby allowing for intranasal delivery of drugs to the CNS. Oats are considered to be the rate-limiting steps in the movement across key epithelial tissue-environment interfaces (eg. kidney, liver) of many common drugs (eg. antibiotics, antivirals, chemotherapeutics, NSAIDs, antihypertensives, diuretics). We were the first to identify the prototype, Oat1, as well as related transporters, and have recently published the Oat1 and Oat3 knockouts, which have defective organic anion transport in kidney and choroid plexus. Because of the expression of Oat6 and Oat1 at the so- called nose-brain barrier and specific substrate preferences, the pathway is attractive for a side-by-side in silico and wet lab strategy (and in vivo validation) for identification of CNS-active drugs and probes. It is hypothesized that the Oat6 and Oat1 transporters provide a unique transport mechanism for the nasal administration of drugs to the CNS, and that a side-by- side in silico (SA1) and wet lab (SA2) approach will prove synergistic and lead more rapidly to high throughput virtual and wet lab screening of a huge number of compounds to identify Oat-specific sets. These compounds may then either function as potential leads to consider for CNS-acting drugs that can be delivered nasally or, perhaps more likely, serve as a range of consensus structures for Oat6 and Oat1-mediated transport that can be considered for the design of new drugs or the derivativization of existing drugs with ineffective CNS delivery. The goal of SA1 (40-45% effort) is in silico modeling of the interaction of Oat6/Oat1 and its substrates using steered molecular dynamics in order to begin to build the basis for virtual screening. SA2 (40- 45% effort) will initially continue wet lab low-medium throughput (Xenopus oocyte transport) screening for potential Oat6 and Oat1-specific substrates and develop a high throughput assay employing stably transfected Oat1 and Oat6-expressing cell lines, which could then be used to screen chemical libraries. SA3 (10-20% effort) involves initial ex vivo and in vivo """"""""proof of concept"""""""" experiments. The proposed work will provide a paradigm for this type of screening of drugs and compounds that are transported not only across the olfactory epithelium but also the choroid plexus and brain capillary endothelium (including compounds that can be tagged for imaging of tumors and brain metabolism). We have begun preliminary experiments with NIH Roadmap funded facilities (Scripps) with the long-term goal of developing a synergistic high throughput virtual and wet lab screening strategy. All the expertise for completion of the aims exists within the PI's group. While the emphasis is on screening for CNS active drugs, it is to be emphasized that the proposal is also highly relevant to drug design for elimination via the kidney and liver. This proposal seeks to develop the basis for a virtual and wet lab high throughput screening system for drugs delivered to the central nervous system, including those useful for CNS tumors, CNS HIV infection and neuropsychiatric disorders. The goal is to identify highly specific substrates for the nasally expressed organic anion transporters, Oat1 and Oat6, thereby allowing for efficient intranasal delivery and transport of drugs to the CNS. ? ? ?
