Mutations in the olfactomedin domain of myocilin (myoc-OLF) are the strongest link to inherited primary open angle glaucoma. In this recently-identified protein misfolding disorder, aggregation-prone disease variants of myocilin hasten glaucoma-associated elevation of intraocular pressure, leading to vision loss. In spite of its well-documented pathogenic role, myocilin remains a domain of unknown structure or function. Here we report the first small-molecule ligands that bind to the native state of myoc-OLF. To discover these molecules, we designed a general label-free, mix-and-measure, high throughput chemical assay for restabilization (CARS), which is likely readily adaptable to discover ligands for other proteins. Of the 14 hit molecules identified from screening myoc-OLF against the Sigma-Aldrich Library of Pharmacologically Active Compounds using CARS, surface plasmon resonance binding studies reveal three are stoichiometric ligand scaffolds with low micromolar affinity. Two compounds, GW5074 and apigenin, inhibit myoc-OLF amyloid formation in vitro. Structure-activity-relationship-based soluble derivatives reduce aggregation in vitro as well as enhance secretion of full-length mutant myocilin in a cell culture model. Our compounds set the stage for a new chemical probe approach to clarify the biological function of wild-type myocilin, and represent lead therapeutic compounds for diminishing intracellular sequestration of toxic mutant myocilin.

Deposition of disease-specific proteins or protein fragments as fibrillar aggregates is the molecular hallmark of both neuropathic and systemic human diseases, including Alzheimer's disease, type II diabetes or rheumatoid arthritis. While the cross-b sheet architecture of mature fibrils is firmly established, insights into the structural features of intermediates formed transiently fibril assembly has been hampered by their intrinsic metastability. We show here that correlated thioflavin T (ThT) fluorescence and light scattering measurements and infrared spectroscopy can be used to monitor the structural evolution of amyloid intermediates of lysozyme emerging along an oligomer-free vs. oligomeric assembly pathway. While ThT fluorescence responded to all generations of transient intermediates, it did so with very different efficacies for the two different assembly pathways. Correlating ThT responses against light scattering indicated that the observed differences in ThT responses results from intrinsic differences (binding affinity, quantum yield) of ThT interactions with intermediates and mature fibrils in different pathways. Infrared spectroscopy confirmed that both assembly pathways result in the formation of cross-beta sheet structures early on. At the same time, there were reproducible spectral differences across these two pathways. All these pathway specific features emerged upon formation of the earliest intermediates and persisted up to late-stage fibrils. We confirmed that neither the existence of these two pathways nor their specific structural features were affected by the hydrolysis of lysozyme that occurs at highly acidic growth conditions. These observations imply that intermediates along different assembly pathways are not just morphologically but also structurally distinct and that these pathway-specific structural motifs become established during the very earliest aggregation events.