Small Molecule Preparation for Differential Scanning Calorimetry from Concentrated Stock Solutions

Author: Chloe Hutchison | Major: Biology

My name is Chloe Hutchison, and I am a Senior Biology Major with future plans in pursuing a career in medicine. I attended the Arkansas Summer Research Institute in 2020 and watched a guest lecture by Dr. Adams which piqued my interest as his work that largely focused on small molecule binding interactions with proteins that are important in modulating cell growth and differentiation and human disease. We met the following semester and he introduced me to his collaborative research project on Ruthenium-Polypyridyl Complexes (RPCs) and I began preparing and planning my Thesis.

My project is focused on characterizing the binding of small Ruthenium-based Polypyridyl complexes to the dynamic polymers of Tubulin. Tubulin is a heterodimeric protein that exhibits spontaneous GTP-dependent polymerization at 37^oC. Microtubules (MT) play a crucial role in cell cycle division by separating duplicated chromosomes through their dynamic instability, cycling through rounds of polymerization and depolymerization. Small molecules can bind the MT’s and stabilize or destabilize their polymerized state. In previous research, a small molecule, RPC2, has shown to stabilize this dynamic feature of MTs therefore disrupting their function which can lead to apoptosis. Taxol is a current chemotherapeutic agent which exploits this mechanism by acting as a Microtubule Stabilizing Agent clearly showing the importance of further work with this RPC2 small molecule and others with similar structure.

The RPC2 molecule has large aromatic ring structures and it’s solubility was limited to hydrophobic solvents which can make applications in hydrophillic cellular systems complex and challenging. Within the proposal and time frame of starting experiments, organic chemists working in conjugation with a previous collaborator of Dr. Adams synthesized novel Ruthenium-Polypridal Complexes with similar structures that were hypothesized to be more soluble in aqueous solvents and sent to us for further work. The novel small molecules used are DB ([Ru(dip)₂bpy]Cl₂) and DP ([Ru(dip)₂phen]Cl₂). I established these molecules were water soluble.  This property will allow for characterization devoid of organic conditions previously required for the RPC small molecules and experiments were continued with the DP and DB RPC’s. Triplicate Fluorescence Titrations were performed for each small molecule with the MT’s. The K_d for the DP molecule was 15.61×10^-6 M and K_d for the DB molecule was 19.1×10^-6 M. The hyperbolic curve and K_d in the micromolar range show that these molecules are binding to the MTs and binding in ranges similar to other RPCs. These results support the notion that other Ruthenium-based compounds can bind Microtubules and highlight the need for further characterization in the hopes that these molecules can be used in anti-mitotic chemotherapeutic treatments.

This semester I completed Differential Scanning Calorimetry (DSC). This technique characterizes the enthalpic changes as the drug and protein of interest interact over a range of temperatures to establish thermal stability and how the small molecule alters this intrinsic property of the protein. The DSC was run under conditions with final concentrations of the protein as 100uM. First, a DSC was performed of the Tubulin protein. The results showed a melting temperature of 68^oC with a large range in enthalpic change of 700kcal/mol K. DSC was performed with the tubulin + small molecule in triplicates for each small molecule with final concentrations of 100uM of the protein and 20uM of the RPC. The results show variation in the thermograms and gave a multitude of melting temperatures. The graph with and without the small molecule show significant differences with the endothermic and exothermic peaks shifting and overall magnitude of enthalpic change. These results reveal the complex and dynamic nature of the protein and the temperature dependence of polymerization. Other research also found a large variation in the enthalpic change that varied with scan rate, concentration and other factors. My research is congruent with other work on Tubulin protein using DSC and reveal that the small molecules do alter the Tubulin stability in a complex way that will be investigated in other methods that should provide more clear data.

Future work will include Isothermal Titration Calorimetry (ITC) and Polymerization Assays. ITC is a binding experiment that measures the heat exchange over time at a specific temperature and gives important thermodynamic parameters such as the stoichiometry of binding (n), enthalpy (ΔH), entropy (ΔS), Gibb’s Free Energy of the reaction (ΔG) and the binding affinities (K_d). The experiment will be done at 4^oC for free Tubulin or at 37^oC for preformed MTs. This will inform us of important binding characteristics void of the complexities of the temperature dependence of polymerization. Additionally, polymerization assays will be performed to determine if the novel RPC’s (DP and DB) have any Microtubule stabilizing activity. The protein kit I purchased contains a MSA (paclitaxel) which will act as the positive control while the protein alone will act as another control. This experiment is essential in determining the specific influence the novel RPC’s have on MT activity and thus their potential in anti-mitotic chemotherapy use as an MSA.

During the Grant period, I grew an exceptional amount in my scientific approach and perspective, and I was challenged by Dr. Adams and the graduate students I worked with to make this research my own. This experience with Dr. Adams and his graduate students was not only foundational in my future, but something I consider to be one of the most important. I have always known that medicine and leading through a compassionate presence was something I was going to do; however, I have taken a whole new interest in the field of research after my two years as an undergraduate researcher with Dr. Adams. It has pushed me to apply for the NIH Postbaccalaureate Intramural Research Program and even consider training in a fellowship after medical school. I know that I would not have come close to all that I have accomplished with my research without the strong continued support of Dr. Adams, his graduate students and the Honors College funding and for that, I am exceptionally grateful.