Abstract
Purpose :
Exfoliation glaucoma (XFG) is a common form of secondary glaucoma caused by the accumulation of insoluble fibrillary deposits in the anterior segment of the eye. XFG accounts for approximately 25% of glaucoma in the United States and worldwide and there is currently no pharmacological treatment for this disease. In XFG, aberrant synthesis and abnormal aggregation of LOXL-1 (lysyl oxidase-like 1), an enzyme that catalyzes the cross-linking of elastin and collagen, contributes to the formation of fibrillary deposits. Recently, it was reported that dysfunction of autophagy likely plays a role in XFG pathology. Our goal was to identify small molecules that modulate the secretion, facilitate degradation of misfolded LOXL1 or inhibit the activity of LOXL-1 and as such, may serve as lead molecules for developing drugs for XFG.
Methods :
Full-length LOXL-1 constructs representing the healthy and diseased haplotype of the enzyme were expressed in CHO cells. The ability of autophagy modulator compounds to reduce the secretion and aggregation of LOXL-1 was assessed in cell-based assays using Western blot and other analyses of the culture media and cell lysates. A LOXL-1 form that contains the catalytic domain without the pro-region was also expressed in CHO cells and purified from the culture media. The purified enzyme was used for inhibitor screening on our proprietary compound library.
Results :
The purified LOXL-1 artificially depleted of the pro-region demonstrated significant enzymatic activity in our fluorimetric assay. Using this protein preparation, small molecule inhibitors of LOXL-1 were identified. The selectivity of the identified inhibitors was assessed by screening for inhibitory activity on LOXL2. Analysis of culture media and cell lysates from cell lines expressing full-length mutant LOXL1 identified autophagy regulator molecules that can induce LOXL1 degradation significantly reducing the amount of high molecular weight aggregates and LOXL1 secretion.
Conclusions :
Our results provide potential lead molecules from two distinct functional classes (LOXL1 inhibitors and modulators of autophagy) for the development of XFG treatment.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.