Directory Entry For: Thomas Sabo
Biography
The unifying theme of my research concerns elucidating the relationship between biomolecular motion, structure, and functionality. My fascination with these concepts was initially cultivated during my graduate studies under the mentorship of Muriel Maurer at the University of Louisville. This invaluable graduate experience was a significant stepping-stone to my post-doctoral studies with Christian Griesinger at the Max-Planck Institute for Biophysical Chemistry (MPI-BPC) in Göttingen, Germany. It was here where I honed my skills in applying Nuclear Magnetic Resonance (NMR) spectroscopy for characterizing the amplitudes, the timescales, and the nature of motion within biomolecules. In February of 2015, I established my independent career at the Brown Cancer Center, University of Louisville (UofL) with a primary focus on applying the techniques and methodology that I have developed toward anti-neoplastic targets and on providing researchers with the tools necessary to characterize structurally silent, yet functionally important, conformational states. The idea is to eventually exploit these “alternative” structures and functional dynamics as unique targets for future drug development efforts.
Research Interests
Project 1. Human Guanylate Kinase (hGMPK): functional investigation of a new biomolecular target for lung cancer. Being the only identified enzyme responsible for cellular GDP production, hGMPK is essential for cellular viability and proliferation of cancer cells, yet, only few studies on hGMPK exist. Recently, we solved the first structure of a human, unliganded GMPK with NMR spectroscopy, illustrating the dynamic nature of the two nucleotide-binding regions in the absence of substrates. Also, we characterized ten cancer-associated hGMPK non-synonymous single-nucleotide variants (nsSNVs) and, quite surprisingly, observe increased catalytic activity for six nucleotide binding-site distant nsSNVs located in the CORE domain of the kinase. Importantly, we have recent data, in collaboration with Levi Beverly, that inhibition of hGMPK in lung adenocarcinoma cell lines causes cell vulnerability without adversely affecting normal lung cells. Our findings strongly suggest that hGMPK has the potential to be a promising biomolecular target and, with this in mind, we now have the structural data for initiating the development of novel cancer therapeutics targeting hGMPK.
Project 2. Direct Detection of Functional Sub-States in Proteins. Physiological processes, such as biomolecular recognition and enzymatic catalysis, harness protein structural dynamics for functionality. However, the routine characterization of the biologically important and structurally silent functional states within a conformational ensemble remains elusive and is currently applicable to only the most well-behaved systems. We are attempting to define the minimal amount of NMR data that is necessary for generating high quality structural protein ensembles. We anticipate this methodology offering unprecedented possibilities for application to a wide range of biological systems in order to identify distinct and/or unique functional states. Currently, we are applying the framework to ubiquitin, the third immunoglobulin domain of protein G (GB3), and hen-egg white lysozyme.
Project 3. ZEB1 (Zinc finger E-box binding homeobox 1) is a member of the homeobox transcription factor family and plays a key role in regulating the epithelial mesenchymal transition (EMT) in carcinoma cells. Direct inhibition of ZEB1 with small molecule therapeutics has not yet been achieved. Another avenue for regulating ZEB1 is through the stabilization of DNA G-quadruplexes (G4s) found within the ZEB1 promoter with small molecules. G4-small molecule binding can influence the transcription of adjacent genes, particularly cancer-related protein promoters like ZEB1, by repressing proteins that are difficult to target or considered “undruggable”. In collaboration with Robert Monsen, we have identified and structurally characterized with solution nuclear magnetic resonance spectroscopy (NMR) a unique and first of its kind, 5-tetrad, interlocked, intramolecular higher-order G4 fold using a thymidine scanning of the ZEB1 promoter. The two-stack and the three-stack G4 systems are essentially adopting a 5'-5' stacking interface, which is predicted as the most stable form of G4 dimerization. The high thermodynamic stability is confirmed using CD melting analysis. The interlocking of nucleotide G21 into the three stacked G4 domain is shown to be essential for its formation. The unique folding of the ZEB1 G4 offers a unique targetable landscape for the indirect inhibition of ZEB1 in carcinoma, specifically at the junction of the two G4 units that forms a pocket with the interlocking G21.
Project 4. Isotropic labeling of larger proteins (> 30 kDa) with deuterium is a well-established technique for decreasing relaxation rates in a range suitable for Nuclear Magnetic Resonance (NMR) spectroscopy. Although uniform perdeuteration can be achieved to near completion, specific labeling of individual proton sites can be a challenge. Many different chemical approaches have been developed to make specifically protonated/deuterated labelled amino acids and use them in the bacterial cell cultures to produce specifically labeled proteins. Such an endeavor can be very expensive, time consuming and are typically only partially successful in labeling all the intended positions. For NMR relaxation measurements on larger proteins involving the HαCα bond, maintaining the proton at the α-position while the proximal protons are deuterated will greatly benefit the signal-to-noise of the experiment since it will reduce relaxation due to large dipolar interactions with proximal protons. We are developing a new approach to achieve this goal, which is an extension of an enzymatic approach published recently wherein a Pyridoxal 5’-Phospahte-Dependent (PLP) Mannich Cyclase (LolT) was used to stereospecifically deuterate Hα positions of individual L-amino acids. Through this enzymatic reaction, almost all the amino acids are specifically deuterated at the α-position except for Ile. Instead of deuterating the α-position, we used LoIT to protonate the α-position in a mixture of deuterated amino acids (Silantes’ 2H, 13C and 15N media powder for E.coli).
Degrees and Certifications
University of Louisville