Abstract Aggregation of mutated myocilin (MYOC), a protein found in the endoplasmic reticulum (ER) of trabecular meshwork (TM) cells, disrupts the outflow of aqueous humor from the eye, causing increase in intraocular pressure (IOP), progressive retinal ganglion cell (RGC) death, and degeneration of the optic nerve, leading to primary open-angle glaucoma (POAG), characterized by irreversible vision loss. About 5% of POAG and 35 % of juvenile open-angle glaucoma (JOAG) patients exhibit glaucoma that is driven by the aggregation of mutant forms of MYOC. Our research team identified the endoplasmic reticulum (ER)-residing Hsp90 isoform, glucose- regulated protein 94 (Grp94), as necessary for the assembly of mutant myocilin aggregates. In fact, our group demonstrated that Grp94 inhibitors are effective in vivo to treat this hereditary glaucoma. Interestingly, Grp94 inhibition also prevents the IOP driven by steroids; a condition that leads to a secondary steroid-induced glaucoma (SIG). These data suggest that Grp94 inhibitors have the potential to treat any open-angle glaucoma. Current glaucoma treatments, such as beta blockers and prostaglandin analogs, only treat the symptoms of the disease and not the underlying cause. Our approach through Grp94 inhibition, represents a unique opportunity to treat mutant MYOC-driven POAG and allow persons afflicted with SIG to maintain their steroid regimens without sacrificing their vision. This application aims to secure funds for the optimization of Grp94 inhibitors to improve selectivity, modulation of physicochemical properties that are appropriate for ocular administration, and conduct pre-clinical evaluation of the optimized candidates in animal models. Preliminary work at the University of Notre Dame and the University of Florida has established that selective inhibition of Grp94 via eye drops can offer a safe and effective therapeutic option to treat hereditary forms of glaucoma by utilizing a mechanism of action that differs from currently available agents, potentially without adverse effects. In this Phase 1, Grannus will optimize the efficacy, selectivity over other Hsp90 isoforms, and the physicochemical properties of Grp94 inhibitors using a rational, structure-based approach to construct a new lead compound. Aim 1. Synthesis of rationally designed Grp94 inhibitors using the data gathered from solution of the co-crystal structures and prior structure-activity relationship studies. The working hypothesis is that introduction of conformational constraint and different functional groups into current Grp94-selective inhibitor scaffolds will increase the selectivity (over other Hsp90 isoforms), binding affinity, and drug-like properties of the designed analogs. Aim 2. Evaluate the Grp94 inhibitors prepared in Aim 1 for their effectiveness in in vitro models of myocilin aggregation. Effective compounds will undergo additional analyses for in vivo efficacy, safety and distribution measurement experiments. U...