Progress toward the development of an enzymatic zonulolytic reagent for the noninvasive treatment of cataracts
Sharma, Neil Christopher
Singleton, Scott F.
Doctor of Philosophy
A cataract is an opacification of the ocular lens, which is suspended behind the pupil by filaments called zonules. While cataracts are surgically treatable, limited economic resources currently hinder the treatment of this debilitating disease in the developing world. Couching of the cataractous lens by a proteolytic enzyme could provide a satisfactory and economical alternative to surgical lens removal. The HIV protease, because of its small size, could potentially be delivered non-invasively into the eye to effect release of the lens. Unfortunately, our results suggest that the wild-type protease is unable to cleave the zonules. Directed molecular evolution may provide a viable means of modifying the HIV protease to recognize and efficiently cleave the zonular fibers. Utilizing this approach, however, requires the availability of a method for screening very large protease mutant libraries. Accordingly, we have developed a novel two-tiered system for the rapid screening of site-specific protease libraries with, in some cases, as many as one billion members. The first tier is an in vivo genetic selection that links protease activity to antibiotic resistance. The activity of proteases isolated in this first tier is then verified in a more stringent second tier based on the in vitro cleavage of a recombinant fluorescent substrate bound to a solid support. This system has been adapted and optimized for screening the HIV, HCV NS3/4a, and TEV proteases, and has been demonstrated to work well in a model directed evolution experiment. With the ability to now screen large mutant libraries of the HIV protease, it should be possible in the future to isolate a mutant with cleavage activity toward the zonules. Additionally, the broad flexibility of the system for screening additional site-specific proteases should be of great assistance to the scientific community by providing a means of gathering important structure-function information and by allowing for the isolation of protease mutants with novel properties such as altered substrate specificity, stability, and/or reaction chemistry.