Glaucoma, a leading cause of blindness, is managed medically by treating the causal risk factor of increased intraocular pressure (IOP), which is typically observed prior to retina degeneration and loss of visual field. IOP is controlled in the anterior region of the eye, which contains the trabecular meshwork (TM) extracellular matrix, the anatomical pathway for drainage of aqueous humor fluid. Of the ~45 million cases of open angle glaucoma worldwide, ~3% are linked to mutations in myocilin, a protein highly expressed in the TM. Despite considerable research effort over ~20 years, little is known about the structure or function of myocilin. An improved molecular understanding of myocilin in its normal and disease states will change the paradigm for anti-glaucoma therapeutics by enabling agents that target the disease process instead of indirectly controlling IOP. Disease-associated mutations in myocilin are found throughout its sequence. In the prior grant period, we biophysically and structurally characterized the variants clustered in its C-terminal olfactomedin (mOLF) domain, lending critical new details and support for the predominant working hypothesis in which mutations localized to myoc-OLF lead to a gain of toxic function: Endoplasmic-reticulum (ER)-associated degradation is inhibited by an aberrant interaction between myocilin and the ER-resident chaperone Grp94, leading to amyloid deposits of mutant myocilin within TM cells, which are cytotoxic. The resulting accumulation of TM cell debris is thought to impede fluid outflow from the TM, causing IOP elevation. Continued structure/dysfunction studies of myocilin will not only contribute to our understanding of glaucoma and its role in the TM, but would also broaden our comprehension of the many other OLF domains, which are implicated broadly in physiology and diseases. The objectives of this proposal are to expand our molecular comprehension of structure and misfolding in myocilin-associated glaucoma as well as provide a path forward for functional studies and the discovery of small molecules that mitigate aberrant myocilin behavior. We will (1) elucidate the architecture of native full- length myocilin, which is dictated by N-terminal coiled-coils, and characterize biophysical and cellular properties of disease variants found therein, (2) clarify the interaction between myocilin and Grp94 at the molecular level, and (3) implement two high throughput assays. The expected outcomes are (1) the full scope of the misfolding disease mechanism for glaucoma-associated myocilin, (2) expansion of our knowledge of protein conformational disorders, (3) new insights into Grp94 chaperone biology, and (4) novel ligand assays based on the myoc-OLF structure and mOLF/Grp94 interaction for the identification of therapeutic small molecules.
Our long-term goal is to develop a new therapy for glaucoma, a prevalent eye disease characterized by increased intraocular pressure, neurodegeneration of the retina, and vision loss. We are focusing on myocilin, an extracellular matrix protein involved in regulating eye pressure; mutations in myocilin lead to an early-onset, inherited form of glaucoma. We will study myocilin and disease-causing mutants in terms of their structure and stability, which will guide our efforts to identify therapeutic compounds targeted to mutant myocilin that treats the underlying cause of the disease.
|Wang, Yiming; Gao, Yuan; Hill, Shannon E et al. (2018) Simulations and Experiments Delineate Amyloid Fibrilization by Peptides Derived from Glaucoma-Associated Myocilin. J Phys Chem B 122:5845-5850|
|Huard, Dustin Je; Lieberman, Raquel L (2018) Progress toward development of a proteostasis drug for myocilin-associated glaucoma. Future Med Chem 10:1391-1393|
|Patterson-Orazem, Athéna C; Hill, Shannon E; Fautsch, Michael P et al. (2018) Epitope mapping of commercial antibodies that detect myocilin. Exp Eye Res 173:109-112|
|Huard, Dustin J E; Crowley, Vincent M; Du, Yuhong et al. (2018) Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma. ACS Chem Biol 13:933-941|
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|Stothert, Andrew R; Suntharalingam, Amirthaa; Tang, Xiaolan et al. (2017) Isoform-selective Hsp90 inhibition rescues model of hereditary open-angle glaucoma. Sci Rep 7:17951|
|Hill, Shannon E; Nguyen, Elaine; Donegan, Rebecca K et al. (2017) Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin. Structure 25:1697-1707.e5|
|Crowley, Vincent M; Huard, Dustin J E; Lieberman, Raquel L et al. (2017) Second Generation Grp94-Selective Inhibitors Provide Opportunities for the Inhibition of Metastatic Cancer. Chemistry 23:15775-15782|
|Donegan, Rebecca K; Lieberman, Raquel L (2016) Discovery of Molecular Therapeutics for Glaucoma: Challenges, Successes, and Promising Directions. J Med Chem 59:788-809|
|Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E et al. (2016) Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability. Mol Cell 63:337-346|
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