Chloe Gerak is a PhD candidate in the Department of Biochemistry and Molecular Biology at the Life Sciences Institute. She is co-supervised by Dr. Lawrence McIntosh and Dr. Michel Roberge, and uses a combination of high-throughput screening, computational modeling, and NMR to find and characterize small molecule inhibitors of protein-protein interactions. We sat down with Chloe to discuss her research and the challenges she has faced throughout her time as a scientist.
What have you been studying throughout your time as a PhD student?
I study a family of transcription factors known as the ETS transcription factors. One of these, ETV6, is important for embryonic development and hematopoietic regulation. Structurally, ETV6 has two protein domains: an ETS DNA-binding domain and a PNT domain. The PNT domain of ETV6 is particularly interesting, as it’s able to self-polymerize, allowing for multiple PNT domains to come together, which then allows the ETS DNA-binding domain to bind to DNA with higher affinity.
Interestingly, the ETV6 PNT domain has been found in numerous chromosomal translocations, typically fused with either a receptor tyrosine kinase or another transcription factor. These translocations have been found in over 40 different types of cancer. The oncogenic mechanism is thought to be related to the PNT domain’s self-polymerization property, as this brings the fusion proteins produced from the translocations together. If the fusion is to a receptor tyrosine kinase, this results in their dimerization, which is the mechanism that activates their kinase activity. This in turn stimulates downstream signaling pathways, often resulting in uncontrollable cell division and cancer. This makes the PNT domain a great target for a chemotherapy drug, and the goal of my research is to find a molecule that will inhibit PNT domain polymerization.
How do you plan to go about finding inhibitors of PNT domain polymerization?
I’ve designed a high throughput split luciferase screening assay in which I inserted mutant PNT domains that are able to dimerize, but not polymerize, into the split luciferase system. When two PNT domains dimerize, the luciferase fragments comes together, resulting in an emission of light. I’ve currently screened over 17,000 compounds for a reduction in light (and therefore an inhibition in dimerization), and while I got a few hits, they also resulted in the same decrease in emission in the control cell line.
At this point I turned to the literature. Interestingly, I found a number of papers from about ten years ago that all claimed that protein-protein interactions were undruggable. Since then, some compounds have been identified that actually do target protein-protein interactions. So this is very much a new area of research, which makes it that much more interesting, but also difficult. Fortunately, I’ve got another library that I plan to screen that specifically targets protein-protein interactions.
I’ve also been taking a computational approach. I recently visited and collaborated with Dr. Richard Sessions at the University of Bristol to do some computational screening. I screened 8.2 million compounds that were each in 20 different conformations to either interface of the PNT domains, and ranked the top thousand hits. I’m now going through them and narrowing the list by identifying variations of the same compound, and looking at the potential electrostatics of the binding interaction.
Will you be able to get your hands on these molecules so that you can eventually test them in vivo?
Absolutely. All of the molecules come from the ZINC8 computational library from UCSF and most of these compounds can be ordered from different vendors. Once I’ve narrowed my potential inhibitory compounds down to 50, I plan to order and test them in my screening assay. I can also test for binding using NMR and isothermal titration calorimetry.
What advice would you give to new graduate students?
Have a side project! Especially one with low hanging fruit. Having a project you can do on the side really helps when you’re having one of those days when your project is being really difficult. It also helps to have interests or hobbies outside of the lab. I’ve been fortunate enough to be involved in TAing, the grad student association, Let’s Talk Science, and other activities around school, and having these activities has not only made me a better scientist, but has also given me something else to focus on when things aren’t going so well in the lab.