Design and Characterization of a Novel Intravitreal Dual-Transgene Genetic Medicine for Neovascular Retinopathies.

Purpose: Intravitreal delivery of therapeutic transgenes to the retina via engineered viral vectors can provide sustained local concentrations of therapeutic proteins and thus potentially reduce the treatment burden and improve long-term vision outcomes for patients with neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy.

Methods: We performed directed evolution in nonhuman primates (NHP) to invent an adeno-associated viral (AAV) variant (R100) with the capacity to cross vitreoretinal barriers and transduce all regions and layers of the retina following intravitreal injection. We then engineered 4D-150, an R100-based genetic medicine carrying 2 therapeutic transgenes: a codon-optimized sequence encoding aflibercept, a recombinant protein that inhibits VEGF-A, VEGF-B, and PlGF, and a microRNA sequence that inhibits expression of VEGF-C.

Site-specific and redox-controlled S-nitrosation of thioredoxin.

Protein S-nitrosation on cysteine residues has emerged as an important posttranslational modification in mammalian cells. Previous studies have suggested a primary role for thioredoxin (Trx) in controlling protein S-nitrosation reactions. Human Trx contains five conserved Cys, including two redox-active catalytic Cys (Cys32 and Cys35) and three non-active-site Cys (Cys62, Cys69, and Cys73), all of which have been reported as targets of S-nitrosation.

Functional proteomic and structural insights into molecular recognition in the nitrilase family enzymes.

Nitrilases are a large and diverse family of nonpeptidic C-N hydrolases. The mammalian genome encodes eight nitrilase enzymes, several of which remain poorly characterized. Prominent among these are nitrilase-1 (Nit1) and nitrilase-2 (Nit2), which, despite having been shown to exert effects on cell growth and possibly serving as tumor suppressor genes, are without known substrates or selective inhibitors.

Activity-based protein profiling for the functional annotation of enzymes.

Activity-based protein profiling (ABPP), the use of active site-directed chemical probes to monitor enzyme function in complex biological systems, is emerging as a powerful post-genomic technology. ABPP probes have been developed for several enzyme classes and have been used to inventory enzyme activities en masse for a range of (patho) physiological processes. By presenting specific examples, we show here that ABPP provides researchers with a distinctive set of chemical tools to embark on the assignment of functions to many of the uncharacterized enzymes that populate eukaryotic and prokaryotic proteomes.

Discovering disease-associated enzymes by proteome reactivity profiling.

Proteomics aims to identify new markers and targets for the diagnosis and treatment of human disease. To realize this goal, methods and reagents are needed to profile proteins based on their functional properties, rather than mere abundance. Here, we describe a general strategy for synthesizing and evaluating structurally diverse libraries of activity-based proteomic probes.

A bifunctional molecule that displays context-dependent cellular activity.

The cell-permeable dihydrofolate reductase inhibitor methotrexate was covalently linked to a ligand for the protein FKBP to create a bifunctional molecule called MTXSLF. The covalent tether between the two ligands was designed to be prohibitively short, so that unfavorable protein-protein interactions between DHFR and FKBP preclude formation of a trimeric complex. In vitro and in vivo experiments demonstrate that MTXSLF is an effective inhibitor of human DHFR, but that efficacy is decreased in the presence of human FKBP due to the high concentration of FKBP and its tight affinity for MTXSLF.