Applying TERS to Questions in Drug Discovery

When scientists are trying to develop a new drug, they desperately want it to succeed. But if a new drug candidate is going to fail, it’s best if it does so as early in the process as possible-before a lot of time and money have been spent developing it. Figuring out whether a drug will fail, and why it might fail, is a complex problem, however, and one that many scientists are trying to address in different ways. Zachary Schultz of The Ohio State University is investigating how tip-enhanced Raman spectroscopy (TERS) can help with this process, particularly in terms of studying binding between membrane receptors and ligands. He recently spoke to us about this work.

You have been using TERS to study the binding between membrane receptors and ligands, for use in drug discovery. First, why is this an important area to study, and what are some of the key questions that still need to be answered?

What caught our attention was the late-stage failure of drugs in clinical trials due to adverse side-effects. We were interested to see if we could provide chemical information to identify which cellular receptors were interacting with the drugs, and other molecules, and potentially identify low-occurrence interactions at an early stage of drug development that might cause significant complications later on. That is still the long-term goal.

My laboratory discovered a TERS method that provides molecular information about how proteins interact with short peptides and small molecules attached to nanoparticles.  Specifically, the amino acids in the protein receptor near the nanoparticle probe contribute to the observed enhanced Raman signal. This suggests that binding sites on the protein can be characterized from the observed signal, perhaps enabling localized structure analysis of protein binding sites using surface-enhanced Raman spectroscopy (SERS). Different proteins show distinct spectra arising from subtle structural changes.

There is still plenty of work to be done in this area. We are looking for computational scientists to help model the observed spectra to see how well our data correlate with other methods for structure determination. However, the origin of the signal in proteins is not fully understood. Part of this ambiguity is associated with the technical challenges currently associated with TERS and part may be our understanding of what is enhanced. Improvements in TERS instrumentation and complementary models may dramatically improve what we can learn from TERS experiments.

Why is TERS a good technique for this type of analysis? What other spectroscopic techniques have been used to study ligand–receptor binding?

The cell membrane is an incredibly complex environment. The more we study it, the more we realize that the interaction between membrane components appears to be as important as the identity of each individual biomolecule. The methods currently used to study proteins are X-ray crystallography, nuclear magnetic resonance, and more recently cryo-electron microscopy; however, these methods are complicated to use on proteins in intact cells. The tools used to study biomolecules in cells are super-resolution and other advanced optical microscopy techniques, but chemical identity and low-occurrence events are difficult to capture. TERS is not without challenges, but it provides unique information that can complement these other methods. The ability to determine the binding site of a protein receptor and then monitor the cellular response could dramatically change how we study drug interactions. Our TERS work may provide this possibility. Overall, I think it is important that techniques complement and support each other to provide the best picture of what is occurring.

In a recent study (1), you combined TERS with single-particle tracking (SPT). How are you harnessing the combination of the two techniques?

What we were able to show in that publication was that increases in the diffusion coefficient determined from SPT of the peptide-functionalized nanoparticles correlated with variance in the Raman signal from the TERS experiments. This discovery was exciting because it suggests that a peptide that is not specifically recognized by the protein receptor will interact with various locations on the cell, and spend more time unbound where it will move faster (larger diffusion coefficient). The different spectra observed in the TERS data may provide insight into other receptors, or allosteric interactions, that might be relevant to unwanted side-effects, but this approach still needs further testing to validate.

You used TERS with SPT to study the binding interactions between an integrin receptor-a membrane receptor that regulate cellular migration, invasion, and proliferation in tumors-and three cyclic peptides in a human metastatic colon cancer cell line. What were you able to see that had not been seen before with previous approaches?

To our knowledge, no one has used enhanced Raman spectroscopy in this way before. This is exciting, but it also raises significant challenges to verify our claims. The ability to reproducibly enhance nanoparticles bound to cells using TERS detection has enabled this research in ways that would otherwise have been very difficult. One of the reasons we chose the integrin–RGD system was that it had been studied pretty thoroughly, which lets us see if our methodology is consistent with previous reports. So far, our findings largely support what had been previously reported. We see this agreement as a good thing, because it validates our approach. With this new method in hand, we are looking for observations that may further elucidate receptor–ligand recognition that might be useful for the treatment of cancer. One of the exciting opportunities of my recent move to The Ohio State University is the opportunity to work more closely with experts at the James Cancer Hospital and Solove Research Institute. We are looking forward to contributing to this community to further cancer research.

What are your next steps in this work?

As I mentioned earlier, TERS is a technically demanding technique. We would like to demonstrate this approach in living cells, where it may be possible to follow changes in phenotype or biochemical pathways that result from interaction with the functionalized nanoparticles. We are exploring ways to facilitate this methodology and increase the throughput. We have a manuscript under review that is a first step in this direction. Increasing the throughput will allow us to increase the number of data points and also to look for low-occurring events that may be important in drug development.

We are also interested in exploring other membrane receptor signaling pathways. Now that we understand our method, we hope to apply it to other membrane receptor systems that are less well understood.

Reference

(1)  L. Xiao, K.A. Bailey, H. Wang, and Z.D. Schultz, Anal. Chem.89, 9091−9099 (2017). DOI: 0.1021/acs.analchem.7b01796

Zachary D. Schultz, PhD, is an associate professor of chemistry and biochemistry at The Ohio State University, where his research focuses on developing innovative approaches utilizing the unique properties of nanostructured materials for near field imaging, ultrasensitive label-free spectroscopic detection, and controlling chemical reactions. Schultz earned his PhD from the University of Illinois at Urbana-Champaign in 2005. Upon completing his PhD, he did a postdoctoral fellowship at the National Institute of Standards and Technology, where his research was performed largely in collaboration with Ira Levin at the National Institutes of Health (NIH). Schultz then continued as a research fellow with Levin at NIH using vibrational spectroscopy and microscopy to study biomembrane systems. While at the NIH, Schultz was awarded an NIH Pathway to Independence Award. Schultz began his independent career as an assistant professor at the University of Notre Dame in 2009 and was promoted with tenure to associate professor in 2015. In January of 2018, Schultz moved his research program to The Ohio State University. Schultz was named a Cottrell Scholar in 2013 in recognition of his outstanding teaching and research.