Tadross lab is known for its novel drug delivery system called DART (Drugs Acutely Restricted by Tethering) that allows delivery of conventional small-molecule drugs to specific cell sub-types. It is based on a covalent interaction between HaloTag protein and a HaloTag ligand that acts as a homing device. This homing device guides drugs attached to the ligand towards neurons that express the protein. DART’s ability to be specified to a single cell type allows for a more precise investigation of potential malfunctions in the circuitry involved in neurodegenerative diseases. For example, DART has been used to restrict the binding of an inhibitor to a “go” cell type allowing for the investigation of the circuitry of Parkinson’s disease. Understanding the circuit mechanism of a drug enables the creation of more efficacious drugs for use in humans.
The overall goal of the project is to develop an orthogonal DART system, which would allow for two different drugs to be used at the same time. This system requires two unique pairs of HaloTag protein and ligand, where each ligand would only work with its specified protein. Tadross lab has a set of ligands with low affinity to the current protein. We are aiming to derive a unique variant of the high-affinity protein through directed protein mutagenesis, changing the amino acid sequence of HaloTag protein to have a more favorable binding with the second ligand. Creating a second DART protein will allow us to deliver two different drugs into two different cell types concurrently. The second DART protein will allow to manipulate brain circuits with even more precision and to develop combinatorial drugs as a treatment.
To achieve the development of the second HaloTag protein, Tadross lab developed a protein display system called GRIP (Gluing RNA to Its Protein). GRIP display creates a stable linkage between RNA and the protein it encodes. Using GRIP and a technique called Iterative Panning we can derive the best binding protein out of a trillion different protein variants. Panning for best binders can be compared to panning for gold, where more stringent filters of a pan dish extract the gold from the dirt. After each cycle, more waste is removed until only gold is left. Similarly, this is done with GRIP display, where protein variant mix is exposed to the ligand. Each cycle the weakest binders are removed, wheres the strongest binding proteins become a large portion of the overall mix. After a few cycles of panning, next-generation sequencing is used to read the resulting RNA and to determine the amino acid sequence of the protein. The goal after a few cycles is that the protein extracted is unique from the original DART and can be used in the orthogonal DART system.