Apart from members of the ABC superfamily, organic anion transporters such as Oat1, first identified by the PI's lab, may well be the most important xenobiotic and multi-specific metabolite transporters in the body. Oats are believed to be critical for regulating the distribution of drugs, toxins and metabolites between the CSF, blood, urine and other body fluids. In this RESUBMISSION application, we propose to employ in silico, in vitro, structural biological and in vivo methods to provide a comprehensive picture of substrate-transporter interactions and to validate this using assays that are relatively unique to the PI's group. Thus the group has multiple in silico, in vitro and in vivo approaches working, and our collaborator (Dr. Chang) is one of the premier structural biologists in the field of drug transport So far a full circuit of in silico-in vitro-in vivo analysis of any of the non-ABC mammalian drug transporters has not been performed, and we argue that we are in an ideal position to do this. We plan to use ligand and transporter-based computational modeling, with which we have extensive experience, to identify the molecular determinants which target substrates for uptake and handling via Oat1 (SA1, to be done with collaborators at the San Diego Supercomputer). Virtual screening of chemical structural libraries using pharmacophores will then be done, followed by wet lab validation of compounds. We will also crystallize and determine the high-resolution x-ray structure of Oat1 in collaboration with Dr. Chang (involving both his laboratory and TransportPDB, a component of the NIH Protein Structural Initiative) to gain key insights into the structural requirements for its substrate binding and transport (SA2). In SA3, we propose to further validate our analysis of substrate-transporter interactions (from SA1 and SA2) by testing predictions of inhibition of Oat1 transport by in vivo pharmacokinetic analysis (in comparison with the OAT1 knockout in which we have previously shown both aberrant systemic metabolism as well as defective drug and toxin handling). The results of these studies could also have broad implications for understanding the role of Oat1 in whole body and tissue-specific metabolism (quite apart from potentially leading to inhibitors that prolong drug half-life). We have tried to address all the prior criticisms, focusing on clarifying the Significance, Innovation and Approach, and we have provided preliminary data for the crystallization aim. We have also published many relevant computational-wet lab papers in 2010-2011. In one of these papers, we describe the use of pharmacophore modeling and virtual screening to identify a novel high-affinity inhibitor that was preliminarily validated in a transport assay; this high-affinity inhibior may also aid in obtaining an Oat1 substrate-bound structure. Perhaps unique for this field, the PI has brought together world-class experts (all at UCSD or Scripps) whose labs regularly use the proposed methods. Also discussed are alternative computational and wet lab approaches in case the primary approach is less successful than anticipated.
The elimination of a number of common drugs, toxins and physiological metabolites depends upon transporters in the kidney. How these substrates and the transporter interact in the process of elimination is not known. Here we seek to investigate this, as well as identify specific inhibitors of one of these transporters.
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