Author Archives: Bryan Rego

Creation of an Orthogonal DART System

Bryan Rego

Mentors: Victoria Goldenshtein, Michael Tadross, Ph.D.

Department of Neurobiology

DART is a novel drug delivery system based on covalent interaction between the HaloTag ligand and the HaloTag receptor that acts as a homing device to enable the delivery of pharmaceutics to specific cell types. Currently, DART can only deliver one drug to one cell type at a time, we are trying to develop an orthogonal DART system for multiplex drug delivery. To achieve this we will use GRIP, a protein display system, and principles of directed mutagenesis to create a library with a large number of protein variants and subject them to selective binding pressure. Next-Generation Sequencing is used to identify the top DNA sequences. To measure the level of binding, cells were plated with a HaloTag vector and a fluorescent dye, and the resulting signal was quantified. Next, we will measure the binding affinity of the top sequences and the cross-reactivity with the original system in neurons. If successful, the dart system would offer potential a deeper understanding of the brain circuits.

ELP Drug Delivery

As the world is returning to normal after the pandemic new methods to potentially improve injection of drugs into the body have increased in value. When a drug, like a vaccine, is injected into a body it is put in a muscle, so it is able to slowly go into the blood stream. The allows for the slower delivery of drugs and can also lower level of potential risk of being toxic to the patient. This allows for a safer vaccine and reduces potential side effects.

Camila is working on a group of molecules called elastin-like polypeptides. These molecules are naturally occurring and can be found in beans. These are a large amount of elastin repeated. This structure allows for some interesting properties. It is able to be a liquid at room temperature but once it is injected into the body it is able to form a solid block in the tissue. This allows for the drugs that in the ELP to diffuse into the blood stream even slower reducing any potential allergic reaction.  One potential issue with ELP drug delivery is that ELP themselves could be a potential allergen. The because of the ELP structure it has to potential for itself to become an allergen, but this has yet to be tested and animal trials would likely need to be done. Camila’s research was interesting to me because of the potential real world implication that this research can provide. I believe being able to see the direct application of research is incredible important.

A Talk with the PI

Dr. Mike Tadross has always liked tinkering with things and looking to solve difficult problems. For his undergraduate years studied to become an electrical and computer engeerir at Rutgers. He liked electrical engeering because of viewing circuits as puzzles that he could solve.

After undergraduate he started his work on M.D. P.H.D in Biomedical engeering at Johns Hopkins. His P.H.D ended up taking three years longer than he expected because of a project that he could not get to work. This project has still yet to be solved. Following his P.H.D he started working on DART (Drugs Acutely Restricted by Tethering) before he had an established lab of his own. After the publication of paper with DART he was offered a faculty position at duke and to establish the Tadross lab. The Tadross lab focuses on making tools to study the brain whether that be chemical or electrical wiring. He believed that since the brain and computers are composed of circuits then one would be able to design computers to interact with each other. He came to the conclusion that one needs to be able to put all of pieces together to understand the brain. Dr. Tadross when putting together his lab embraced this. He brought in people that focused on the chemical, electrical and the biology of the brain.

His path has not been as linear as one would expect. At Johns Hopkins, he joined a lab that focused on a topic that was not particularly interesting to him. During his time in the lab, he would spend three years on a project that would never actually end up working out. Because of this he worked on a project that he was not particularly interested in, but this gave him the experiencing failure firsthand in the lab. After graduate school, he worked in a pre-faculty position where he was able to work on his own projects but did not have his own lab. This eventually led to him figuring out his greatest invention using halo tag technology to restrict binding for drugs nears the channels.

Talking with Dr. Tadross, I had learned that if you are flexible and are willing to work hard that you will eventually stumble upon success. That while diving deep into topics is important having a broader understanding of what is occurring at all levels is just as crucial. Overall, my talk with Dr. Tadross made me more comfortable and adjusted to his lab.

My day in the Tadross Lab

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.

HaloTag Development

My research project is working with a HaloTags ligand and a variety of HaloTags receptors. What are HaloTags? The HaloTags ligand is customizable and but there are a few consistent features a base, a long flexible linker, and a halogen head at the end of the linker.  The base can be composed of a wide variety of compounds. It can be composed of aromatic rings that allow for detection of ligand binding, or it can be a drug that can spatially linker near its respective channel allowing for a faster uptake of the drug. The linker itself can be composed of different atoms, traditionally a long carbon chain. The linker is used as a tether to get the halogen head into the enzymatic pocket of the enzyme. The linker is a connector between the base and the halogen. The part that binds to the HaloTags receptor is a halogen at the end of the linker. The ligand’s halogen is attacked by a nucleophilic residue in the enzyme allowing the reaction to occur.

There are a few things that make this a great system. The first is that most eukaryotic systems do not have a mechanism to digest HaloTags ligands. This makes the HaloTag ligand binding specific as only HaloTags Ligands are only able to bind to the receptor. Another benefit of this system is that it can be differentiated into different cell types. The ability to differentiate between cell types allows for a better analysis of the effects pharmaceuticals have on a system of cells. The specificity of HaloTags also enables it to react in low concentrations allowing for ease of binding to a receptor for the pharmaceutical base.

What exactly am doing with HaloTags?  I am looking at a single ligand and then finding the receptor that binds onto that ligand the best. There is a wide variety of different ligands that have few different amino acid sequences. The strongest binding receptor over time is extracted and then sequenced.

Finding Familiarity in Uncertainty

What did I expect from my summer research experience? The only thing expected was uncertainty. Uncertainty in what working in a lab would consist of, in the expectations in what life would entail, and if I would even like working in a lab.

I was taking the first step into the unknown for me. I haven’t worked in a college research lab before and have only worked on research in high school. My previous research was in environmental science, which is unrelated to biomolecular and biochemistry lab. While doing my previous research, I was the person in charge, and I choose how much I worked, and everything was my project design. One of my hopes for this summer was that working in a lab would help clarify that. For most of my time in college, I was looking to find a lab that I could join, and I am excited to start researching in a lab.

I found familiarity in uncertainty was through the process of getting the concentration of DNA in solution. Part of my day on Thursday was getting the concentration of DNA in using the intensity at the peak absorbances of the DNA. While getting the concentration of DNA was repetitive, I found the connection to one of my physical chemistry labs. The objective of the experiment was to calculate the change in concentration of a substrate as is catalyzed by an enzyme. I found it interesting how I found a connection to a physical chemistry lab in a biomolecular process. It made me realize how interdisciplinary science research can be and that while there are defined research areas ideas from other fields can be used.

With my first week done in the Tadross lab, I’ve learned to embrace the uncertainty. That uncertainty and unknown is just part of the process. I have two hopes for the rest of the summer that eventually, there is less uncertainty and That I will become more confident in the lab. With my first days being over in the Tadross lab I am sure that there are great things on the horizon, and I’m looking forward to the uncertainty.