argeting Myosin Light Chain Kinase (MLCK) Catalytic Domain to Treat Endothelial Barrier Dysfunction

Abstract

Specific Aims Aim 1: To develop an MLCK assay for a high-throughput screening (HTS) campaign.

Working hypothesis: MLCK truncation, fusion, and correct bacterial expression host will produce functional MLCK for an HTS campaign.

Aim 2: To screen for MLCK inhibitors using a natural product library for chemical diversity.

Working hypothesis: Natural products have elaborate scaffolds that might reveal novel mechanisms of inhibition, giving better specificity.

Aim 3: To convert hits to leads through biochemical, biophysical, and cellular analyses.

Working hypothesis: Initial hits will be confirmed, expanded, and optimized to produce lead compounds.

Methods Bacterial cell transformation, plasmid purification, PIPE cloning, small and large scale protein expression, and IMAC were used to purify recombinant MLCK. Purified samples were tested for size using gel electrophoresis. Main Results MLCK is insoluble in a pSpeed vector, and cannot be purified at large concentrations when expressed in a pGEX vector. No p-values or confidence intervals were used. Conclusions MLCK assay development for a HTS campaign cannot be completed due to low protein yield when expressed in bacteria.