Exploring the cellular effects of small molecule inhibitors of Oxysterol-binding Protein

Oxysterol-binding protein (OSBP) is the founding member of a conserved protein family found among eukaryotes that functions as a lipid transporter between the ER and Golgi. OSBP is ubiquitously expressed in all tissues and is an essential host protein in the viral replication of many public health menaces, especially the Enteroviruses. Over the past decade, many broad-spectrum anti-viral small molecules have been identified as OSBP inhibitors. The anti-cancer and anti-viral natural product, OSW-1, is of interest due to its high affinity for OSBP and its ability to cause proteasomal degradation of OSBP. The work outlined in this dissertation details the unique ability of the OSW-1 compound to induce persistent OSBP repression in cells through multiple days without affecting cell viability. Even in the absence of the OSW-1 compound, the reduced OSBP levels confers a prophylactic anti-viral activity against clinical isolates of Enterovirus viruses. This long-term reduction is specific for OSBP in the proteasome, and reduction occurs through an unknown mechanism that does not involve OSBP proteolysis or transcriptional repression. Of the known OSBP small molecule inhibitors with anti-viral activity, only OSW-1 triggered the long-term repression of OSBP. The OSBP inhibitor compound T-00127-HEV2 was the only compound tested able to protect OSBP levels from the OSW-1-induced repression of the protein. Of the OSBP inhibitor compounds, only the OSW-1 compound was able to induce the prophylactic anti-viral response against two clinical isolates of Enterovirus viruses. The results produced will be beneficial in future research that seeks to define OSBP cellular regulation, especially upon OSW-1 treatment, and to potentially develop prophylactic anti-viral therapeutics through targeting OSBP

Identification and Isolation of Bioactive Natural Product Compounds Targeting Oxysterol-Binding Proteins

OSW-1, a plant-derived cholestane glycoside, has been shown to exhibit broad-spectrum antiviral activity by targeting oxysterol-binding protein (OSBP) and potent antiproliferative activity by targeting OSBP-related protein 4 (ORP4). In order to develop OSW-1 and OSW-1-derived compounds as potential antiviral or anticancer therapeutics, an understanding of OSW-1 structure-activity relationships (SAR) for binding to OSBP and ORP4 must be established. This SAR will allow for the identification of compounds with improved pharmacological properties and the development of selective OSBP- or ORP4-targeting compounds. To date, there are no reported protein structures of OSBP or ORP4, and the published research on OSW-1 only provide partial insight into the compound SAR. The major goal of this research project is to purify and identify OSW-1-related natural product compounds from Ornithogalum saundersiae bulbs. The new OSW-1-related compounds can then be subjected to biological testing, including binding to OSBP and ORP4, anticancer assays, and antiviral assays. Through the development of an efficient analytical method for the purification of natural products from Ornithogalum saundersiae, several OSW-1-related compounds, including newly discovered compounds were purified and structurally characterized. Testing the isolated OSW-1-related compounds for OSBP and ORP4 binding will provide further SAR understanding, and progressively guide the discovery and synthesis of new OSBP and ORP4 specific compounds for pre-clinical drug development as new antiviral and anticancer drugs

Design and Synthesis of OSW-1 Analogs and Other Bioactive Small Molecules for Potential Therapeutic Applications

OSW-1 is a cholestane glycoside natural product with potent anti-proliferative activity, broad spectrum anti-viral activity, and a novel mechanism of action. The OSW-1’s cellular targets are oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Recently, OSBP has been determined to execute an essential role in the proliferation of many classes of pathogenic viruses. ORP4 has been determined, also recently, to be a driver of cellular proliferation and to be a precision cancer target. The main project of this dissertation research is focused on using organic synthesis to produce new synthetic routes to access new OSW-1-derived compounds. To better understand the compound’s mechanism of action and molecular pharmacology, a set of OSW-1 probe analogs were designed and produced. To further the potential development of OSW-1-derived therapeutic compounds, a new approach for the rapid and convergent synthesis of a diverse library of OSW-1 analogs based on a new scaffold was developed. This new approach to OSW-1 analogs synthesis will allow for the future production of analogs with selective affinity for OSBP or ORP4 and improved pharmacological properties. Additionally, a set of stable-isotopically-labeled anti-cancer agents were synthesized to allow the absolute quantification of the drug inside the individual cancer cells through a new mass spectrometry technique. The last project describes the design and synthesis of a retinaldehyde’s analog with high selectivity for retinaldehydrogenase (RALDH) enzymes. This novel compound could form the basis for the development of new treatments for blindness caused by pathological myopia

A study of lipid recognition and membrane binding by the human oxysterol-binding protein (OSBP).

Recent studies have established oxysterol-binding protein (OSBP) and members of the OSBP-related protein (ORP) family as global cellular sterol sensors that participate in non-vesicular anterograde transport of monomeric sterols from the endoplasmic reticulum to other organelles such as the Golgi and the plasma membrane. By exchanging sterols for phosphoinositides, these multi-domain proteins change the bilayer composition at membrane contact sites and thus, regulate various signaling pathways. Despite the wealth of knowledge garnered from the study of fluorescent/radiolabeled ligand-protein interactions and inter-vesicular lipid transfer assays in vitro, the precise nature of the association of ORPs with organellar membranes and the factors modulating such interactions have remained largely enigmatic. The goal of my project was to characterize the behaviour of human OSBP using a label-free analytical technique called dual polarization interferometry (DPI). This technique enables surface-immobilization of phospholipid vesicles to observe and analyze the behaviour of proteins towards adsorbed bilayers. From my investigation, I found that OSBP prefers binding to membranes containing anionic phospholipids, such as phosphatidylinositol-4-phosphate (PI(4)P), over membranes made up of neutral phosphatidylcholine (PC). In the presence of PI(4)P, the wild-type protein clearly demonstrated a rapid bilayer association, followed by PI(4)P extraction and a slower dissociation, in a dosage-dependent fashion. The OSBP-related domain (ORD) mutant, OSBP-HH/AA, due to its impaired ability to extract PI(4)P, failed to dissociate from the membrane while the pleckstrin homology domain (PHD) mutant, OSBP-RR/EE, could not associate with membranes at all. The presence of sterols did not alter OSBP’s affinity for PC membranes despite a two-fold increase in protein adsorption per unit area in the presence of cholesterol in the membrane, compared to 25-hydroxycholesterol. Both cholesterol and 25-hydroxycholesterol competed with 22-NBD-cholesterol for the binding site in the ORD of OSBP, with resulting EC50 values of 15.6 ± 0.7 nM for the former and 5.0 ± 0.5 nM for the latter. OSBP also transferred ORD-bound fluorescent cholesterol to acceptor vesicles, but the rate remained unaltered upon incorporation of PI(4)P in those membranes. These results provide useful insight into the preferential association of OSBP with membranes containing specific recognizable ligands, such as sterols and PI(4)P, and help build a molecular level description of the mechanism of this protein