Protein and Ligand Dynamics in Drug Development and Resistance

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2020

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Abstract

Biomolecules such as proteins are highly dynamic, and undergo a wide variety of motions at different timescales. Movements as small as a bond vibration or as large as a domain rearrangement can be critical for the function of a protein, making consideration and investigation of protein dynamics necessary for understanding biological systems and developing therapeutics. In this work, we describe the development and implementation of novel techniques to study dynamics in proteins and protein-bound ligands, and discuss our investigation of the crucial role of dynamics in two disease-relevant systems.

First, we have expanded the utility of Chemical Exchange Saturation Transfer (CEST) NMR techniques to aid in the characterization of dynamics for nitrogen- and carbon-attached protons, as well as fluorine nuclei. Protons and fluorine nuclei can be exceptionally sensitive to their chemical environment, allowing detection and measurement of protein motions which may not be readily identified by conventional heteronuclear experiments. Additionally, we discovered the motion of a protein-bound ligand and utilized such information to improve the potency of an antibiotic molecule.

Next, we undertook the investigation and optimization of an inhibitor targeting translesion synthesis, a process that cancer cells can employ to resist the killing action of chemotherapeutics. Early work on this project demonstrated that inhibition of Rev1, an important scaffold in the translesion synthesis process, by the compound JH-RE-06 sensitizes cancer cells to cisplatin chemotherapy and prevents drug resistance. Surprisingly, we found that this inhibition occurs through inhibitor-induced dimerization of Rev1, which masks the protein-protein interface required for assembly of the translesion machinery. We further investigated a transient conformational change in the C-terminal tail of Rev1 and validated dimerization in solution using NMR. Our structure- activity relationship investigation of JH-RE-06 yielded a number of insights into how to develop more potent inhibitors. Most significantly, we found that small changes in the chemical structure of the inhibitor resulted in improved inhibitory activity and also led to a novel dimer arrangement. Our combination of Rev1 crystal structures and dynamics studies has led to a deeper understanding of the inhibitory mechanism of JH-RE-06 and will guide the optimization of this potential chemotherapy adjuvant.

Finally, we have investigated a mechanism of resistance to beta-lactam antibiotics in Neisseria gonorrhoeae, which relies on modulation of conformational dynamics. Neisseria gonorrhoeae is a major growing health concern due to the rapid spread of multi-drug resistance. We have discovered conformational exchange in PBP2, the target of beta-lactam antibiotics in Neisseria gonorrhoeae, between a low-affinity state and a high- affinity state. A histidine residue was found to be the key mediator of interconversion between these states via a network of molecular interactions, and we found that drug resistance-conferring mutations shift the equilibrium toward the low-affinity state by modulating these interactions. This work describes a novel mechanism of drug resistance in bacteria in which conformational dynamics are restricted.

This document illustrates a small sample of the important roles molecular motions have in biology, and the power of dynamics studies in understanding protein function, developing drugs, and elucidating resistance mechanisms.

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Fenton, Benjamin (2020). Protein and Ligand Dynamics in Drug Development and Resistance. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/21499.

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