DESCRIPTION (provided by applicant): Oxysterols are a class of endogenous cellular lipids derived from cholesterol that have been implicated in the pathophysiology of atherosclerosis, inborn errors of metabolism, inflammation and cancer. In many cases, the proteins and molecular pathways through which these enigmatic molecules exert their powerful biological effects remain unknown. We established collaboration between a synthetic organic chemist and a cell biologist to understand how oxysterols activate the Hedgehog (Hh) signaling system, a pathway that plays important roles in development, regeneration and cancer. We discovered that a specific oxysterol, 20(S)-OHC, is an allosteric activator of the 7-pass transmembrane protein Smoothened (Smo), a human oncoprotein and key drug target in oncology. This finding significantly expands the regulatory scope of oxysterols as signaling molecules, demonstrating their capacity to function as direct agonists for both a human on co-protein and a signaling receptor. Based on preliminary work, we hypothesize that endogenous 20(S)-OHC functions as a second-messenger in Hh signaling. Using a combination of mutagenesis, photo affinity labeling, and mass spectrometry, we will map the region of Smo that interacts with 20(S)-OHC to provide a biochemical portrait of this novel class of receptor-ligand interaction (Aim 1). Using quantitative mass spectrometry and a click chemistry-based imaging assay, we will ask if Hedgehog signaling can alter cellular levels or distribution of 20(S)-OHC (Aim 2). Finally, we will develop and characterize novel Hh pathway inhibitors that are inspired by oxysterol scaffolds (Aim 3). We expect three major outcomes to emerge from the successful completion of this project: (1) An answer to the question of how Smo is regulated in cells, perhaps the longest-standing mystery in the Hh pathway, (2) a biochemical understanding of how oxysterols engage and regulate 7-pass signaling receptors, and (3) the development of an integrative toolkit that can be deployed to dissect any other oxysterol- regulated cellular process. To accomplish these goals, we have recruited a team of investigators with complementary expertise in cell biology, protein biochemistry, synthetic chemistry, and mass spectrometry.

Small molecule regulation of the Hedgehog oncoprotein Smoothened GM106078-01A1, PIs: Rajat Rohatgi, Douglas Covey The Hedgehog pathway is a system for cell-cell communication that can cause birth defects and cancer when damaged. We have found that cholesterol-like molecules (called sterols) can influence this powerful signaling system. We hope to understand how sterols function in this context and to develop sterol-inspired therapeutics that can be used to manipulate this pathway in disease. Modified Leadership Plan and Timeline Signal transduction by oxysterols GM106078-01A1, PIs: Rajat Rohatgi, Douglas Covey This proposal requires a multi-PI effort because it integrates techniques from three distinct fields-- developmental signaling, synthetic chemistry, and mass spectrometry-- and thus requires a range of expertise outside the scope of a single laboratory. A partnership between two of the co-PIs, Rajat Rohatgi (Stanford) and Douglas Covey (Washington University), has produced three publications that form the basis of this proposal. Covey brings his >30-year experience in the organic synthesis of sterols and steroids chemistry to the construction of oxysterol analogs and Rohatgi brings expertise in cell biology and Hedgehog signal transduction. Alex Evers, a long-standing collaborator of Covey's, has been recruited for his unique expertise in the identification of small molecule binding sites on transmembrane proteins using photocrosslinking and mass spectrometry. The following chart summarizes the overall organizational structure of this multi-PI grant, including the specific portions of the proposal relevant to each PI. Washington University Douglas Covey All Aims Leader: Chemistry Organic Synthesis of Sterol Analogs Alex Evers Aims 1a and 2a Sample preparation and mass spectrometry to identify photolabeled Smo peptides Stanford University Rajat Rohatgi All Aims Leader: Biology Biochemical and cell biological analysis of Hedgehog signaling Leadership structure and responsibilities. Covey and Rohatgi will provide oversight of the overall program, including execution of the scientific agenda according to the timeline, data analysis, and the exploration of alterative strategies and collaborations in case they are required. In addition, they will ensure adherence to NIH and institutional rules on biosafety, chemical safety, radiological safety, ethical conduct, and data sharing. Within the scientific agenda, Covey will direct the chemistry and Rohatgi the biology. Covey will be responsible for the design and syntheses of the small molecules proposed in this grant and also perform the analysis to verify their purity and structure. These molecules will then be sent to Rohatgi for use in Smoothened binding and Hh signaling assays. Since Evers is a colleague of Covey's at Washington University, Covey will also oversee his efforts in Aims 1a and 2a. In Aims 1a and 2a, photolabeling and immunopurification of Smo will be performed in Rohatgi's lab. The sample will be then sent to Evers' lab for click conjugation to biotin, streptavidin purification, digestion, purification, mass spectrometry, and data analysis. Binding and functional studies based on this data will be performed by Rohatgi. Covey, Rohatgi and the personnel in each lab working on this project will participate in a conference call monthly to discuss experimental design, data analysis, and potential obstacles. Evers will join these calls during the phases that his lab is conducting experiments related to Aims 1 and 2. Covey and Rohatgi will also communicate by phone or email as needed about administrative and personnel issues. We note that such conference calls have been a regular part of the collaboration between Covey and Rohatgi over the past four years. All results will be made freely available for comments and suggestions (through a common Box account) to all PIs and key personnel. Rohatgi will serve as the contact PI, responsible for submission of progress reports and the central point for all communication with the NIH and between the PIs. Publications. Authorship on publications will be based on the relative scientific contributions of the PIs and the key personnel. Conflict resolution. If a conflict develops, the PIs will meet to attempt a resolution, with Covey and Rohatgi providing leadership. If this attempt fails, the disagreement will be referred to an arbitration committee consisting of one impartial senior representative each from Stanford and Washington University, and a third representative mutually agreed upon by each PI. The members of this committee will not directly be involved n the research grant. Budget. Each PI will be responsible for his portion of the budget and research administration. A larger proportion of the budget is split between Covey and Rohatgi, who are responsible for the chemistry and biology aspects of all the specific aims and will have dedicated personnel fully devoted to this project in their laboratories. The budget for Evers reflects the resources required for the specific experiments described in Aims 1a and 3a. Change in PI location. If a PI moves to a new institution, full efforts will be made to transfer the relevant portion of the grant to the new institution. If a PI can no longer fulfill his duties, a new PI will be recruited as a replacement at Stanford or Washington University. Intellectual Property. The technology transfer offices of Stanford and Washington University will coordinate the filing any patents should they become necessary. Timeline.