Probing the dynamics of dihydrofolate reductase with small molecule inhibitors Public Deposited

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  • March 21, 2019
Creator
  • Carroll, Mary J.
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
Abstract
  • High-resolution protein crystal structures have served as the template from which to devise small molecule modulators of protein function through structure-based drug design for many decades. However, protein function, such as ligand binding, dissociation and catalysis, necessitates structural flexibility of the macromolecule. Protein dynamics are often ignored in structure-based design, largely due to limited information on protein flexibility in the presence of bound ligands (e.g., peptides or small molecules). To gain insight into how protein dynamics are modulated by the binding of small molecule inhibitors, nuclear magnetic resonance spectroscopy has been used herein to characterize both fast and slow timescale motions in the pharmaceutical target protein Escherichia coli dihydrofolate reductase. A series of inhibitors of the enzyme (antifolates) that cover a broad range of binding affinity have been studied while bound to the holoenzyme complex. In the presence of two high-affinity inhibitors, dynamics appear to be essentially identical on both the fast and slow timescales. Specifically, these two inhibitors decouple functional loop switching motions on the slow timescale. One medium-affinity antifolate from the series is shown to be conformationally flexible while bound to the enzyme, eliciting a functional switching motion in a loop distal to its binding. In considering the entire panel of eight antifolates (two of high affinity, one of medium affinity, and a homologous series five of reduced-affinity antifolates), the rates of conformational switching of the enzyme on the slow timescale correlate well with ligand binding affinity and the kinetic rate of ligand dissociation. Within the sites exhibiting slow motions, a consensus set of residues is identified to be sampling a novel and structurally identical excited state that differs from physiological complexes bound to folate-derived ligands. This group of antifolate consensus residues is posited to serve as a mechanical initiator of ligand dissociation, an observation that may be quite prevalent in receptor-ligand interactions. Fast timescale motions across the series of antifolate complexes studied also suggest a correlation of flexibility with ligand binding affinity and a possible linkage between motions on the fast and slow timescales.
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  • In Copyright
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  • " ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Eshelman School of Pharmacy (Pharmaceutical Sciences)."
Advisor
  • Lee, Andrew
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Chapel Hill, NC
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  • Open access
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