Computationally Guided Design of Thermostable mutants of Neurotensin receptor1

Beckman Research Institute/City Of Hope, Duarte CA

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Abstract

DESCRIPTION (provided by applicant): Neurotensin receptors belong to the superfamily of peptide activated G protein-coupled receptors (GPCRs) and they are important drug targets to diseases such as schizophrenia and even cancer. The complexity in drug design for NTS1 and for many peptide activated GPCRs partly stems from the paucity of structural information, which could provide vital information for rational drug design. The barriers to pursuing structural and biophysical studies of membrane bound neurotensin receptors are due primarily to three challenges that are: 1.obtaining sufficient levels of expressed mammalian receptors, 2. functionally reconstituting them into membrane mimetics and 3. most importantly, stabilizing a particular inactive or active state conformation due to the conformational flexibility of GPCRs. While steps (1) and (2) are surmountable, step (3) the conformational flexibility in GPCRs, severely hampers the protein purification process for further crystallization or any other biophysical studies of GPCRs. Therefore meeting challenge (3) is critical in solving the macromolecular structure and ligand binding to GPCRs and requires a confluence of predictive computational models accompanied by experimental techniques. We propose to develop a computational method "LiticonDesign" that would enable design of thermally stable mutants of rat neurotensin receptor 1 (NTS1) (class A GPCRs in general). LiticonDesign will be based on the "Liticon", a predictive method that allows accurate modeling of various functional ligand stabilized conformational states of GPCRs. The predicted mutants of NTS1 will be expressed and tested for thermal stability and derive sufficient quantities of the pure functional receptor for structural studies. The results of this proposed work will provide a breakthrough method to obtain stable and functional pure protein of NTS1 which could be extended to other class A GPCRs. The proposed interdisciplinary collaboration is between Dr. Nagarajan Vaidehi (PI) at City of Hope, Dr. Reinhard Grisshammer NINDS/NIH and Dr. Christopher Tate (Univ. of Cambridge UK). Dr. Vaidehi has extensive experience in developing modeling techniques for GPCRs, while Dr. Tate is an expert in deriving thermostable mutants of GPCRs. Dr. Grisshammer is an expert on neurotensin receptors and their pharmacology. The budget is allocated only for Dr. Vaidehi, since Dr. Grisshammer is funded by the intramural funding in NINDS and Dr. Tate has sufficient funding for this work. The proposed work is ongoing in his laboratory. PUBLIC HEALTH RELEVANCE: The neurotensin receptor1, NTS1 plays an important role in the pathophysiology of schizophrenia and drug abuse. The peptide neurotensin, acts as a growth factor on cancer cells and NTS1 antagonists such as SR48692 have been suggested to prove useful as inhibitors of NT-induced cancer growth. The proposed interdisciplinary research will develop computational methods to design thermally stable mutants of NTS1 that would enable purification of sufficient quantities of stable and functional NTS1 in detergents. The resulting mutants will be experimentally made and tested for thermal stability in detergents. The results of this study will pave way to further biophysical and structural studies of NTS1, which is sparse at best.