Recent advances in genomics and proteomics have produced rapid changes in drug discovery, increasing the speed of the High Throughput Screening (HTS) methods by using simple assays on isolated protein targets but sacrificing depth of information and making decisions difficult as to which compounds to pursue in the pipeline. Thus, the need for smarter screening platforms is being recognized, especially platforms that emphasize Cellular Systems Biology. More sophisticated screening strategies are particularly important for discovering new molecules that target G-protein Coupled Receptors (GPCRs). A growing number of studies in vitro and in vivo have shown interactions between GPCRs that modify their pharmacological profile and trafficking, or even switch their coupling to different intracellular signaling pathways. Moreover, in the field of Neuroscience it is highly relevant to consider the cellular background of the cells (i.e. neuronal type) because of unique drug responses that occur in neurons and glia. Originus Inc. can contribute to the development of more sophisticated screening platforms for brain GPCRs that address all of the above considerations, by using a proprietary solid phase cell transfection technology termed Surface Transfection and Expression Protocol (STEP) exclusively licensed from the University of Michigan. The proposed new High-Density STEP platform will increase the efficiency and economy of the screens while enhancing the depth of information by comparing the effects of small molecules on multiple brain GPCRs simultaneously and in a neuronal background. Moreover, this new platform can be developed to monitor the activation of several signaling pathways for each individual or multiplexed brain GPCRs at the same time. Most GPCRs expressed in brain have potential significance in healthy mental function and in pharmaceutical treatment of mental dysfunction, including schizophrenia, anxiety, depression and feeding- related disorders. We will focus during Phase I of this proposal on some of the neurally relevant targets, including the melanocortin system, the orexin system, the cannabinoid system, and the dopaminergic system. The High-Density STEP platforms will be optimized for a 96-well microplate format but containing 36 individual transfection spots per well, resulting in 3,456 individual data-points per plate. Each transfection spot will express individual or """"""""physiological relevant"""""""" combinations of brain GPCRs. The first prototype will use calcium flux readout, while the second prototype will allow the simultaneous screening of four different reporter gene assays. We plan to study the activation responses of the different GPCRs in two different cellular backgrounds and compare the compound's pharmacological profiles in cell lines commonly used in HTS and neuronal cell lines. During Phase II we plan to test more GPCR's combinations based on their co-expression in neural circuits, to characterize allelic variants found in high frequency in populations, to analyze specific intracellular pathways by RNAi loss-of-function, and to move to subcellular imaging via High Content Analysis.
Although new medicines today in the research and development pipeline offer hope of reducing the human and economic costs of mental disorders, approvals of drugs with novel mechanisms of action for brain dysfunction have been recently very limited. The ultimate goal of this project is the development of """"""""smart"""""""" drug discovery platforms that allow a better understanding of the complex interrelationships of molecular pathways, cellular activities and ultimately in vivo neuronal function to enable therapies rationally aimed at complex mental diseases. ? ? ?
