From Serendipity to the Rational Design of Protein–Protein Interface Modulators Targeting a tRNA-Modifying Enzyme

Serendipity has played a historically significant role in science and especially in the discovery of new drugs. Chance discoveries have led to important drug developments that characterize the pharmaceutical industry and our current health care system. Over the last decades, the concept of rational...

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Bibliographic Details
Main Author: Nguyen, Dzung
Contributors: Klebe, Gerhard (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2020
Pharmazeutische Chemie
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Summary:Serendipity has played a historically significant role in science and especially in the discovery of new drugs. Chance discoveries have led to important drug developments that characterize the pharmaceutical industry and our current health care system. Over the last decades, the concept of rational drug design has developed into one of the key approaches to the research of new and specific drugs. Nevertheless, the development of inhibitors for protein–protein interactions, especially oligomeric protein contact surfaces, remains challenging. The bacterial enzyme, tRNA-guanine transglycosylase (TGT), is an example of a homodimer protein and serves as model target protein in this work. This thesis summarizes three projects that were created from serendipitous discoveries of previous studies to develop new approaches for the modulation of the TGT homodimer interface and thus lay out the basis for alternative inhibition pathways in oligomeric enzymes. A previous crystallographic study of TGT–inhibitor complexes revealed an unexpected, twisted arrangement of the TGT homodimer. However, data from the obtained X-ray crystal structures can neither exclude crystallographic artifacts resulting from crystal packing forces nor study the transition between the two dimeric end states. Therefore, a method based on electron paramagnetic resonance (EPR) was established to study this ligand-induced rearrangement mechanism of the TGT homodimer in a solution equilibrium (Chapter 2). For this purpose, paramagnetic spin markers were introduced via site-directed cysteine mutations on the protein surface. The following pulsed EPR techniques enabled the observation of inter-spin distance distributions within the TGT homodimer. During the study, a pyranose-substituted lin-benzoguanine inhibitor was identified to be superior in transforming the functional TGT dimer to its twisted state. Thus, the developed method can be used to distinguish between the functional and twisted TGT dimer species upon ligand addition in a solution equilibrium. A second project comprises the search of small molecule fragments that target a newly discovered transient binding pocket at the homodimer interface of TGT (Chapter 3). In an earlier mutational study, a new crystal form of the TGT dimer was discovered, in which the two protomers are covalently linked by an introduced disulfide bridge. This arrangement breaks up the original dimer interface and exposes a small hydrophobic binding pocket through an extended movement of the nearby β1α1-loop motif. Surprisingly, the newly formed pocket was occupied by a dimethyl sulfoxide molecule. In this work, a further stabilized variant of the disulfide-linked dimer was designed for subsequent fragment soaking studies to target the interface binding pocket. In the course of the soaking experiments, the solvent channels of the covalently linked TGT dimer were analyzed in silico to estimate the putative cutoff radii of small molecules that are capable of freely traversing through the protein crystal. The structural characterization of initial fragment hits led to the rational design of compounds with optimized functionalities which are able to modulate the β1α1-loop. Furthermore, fragment binding studies were attempted by nuclear magnetic resonance (NMR) techniques. Although no binding could be detected using a 19F-based method, the binding constants of two fragments were successfully estimated in the micromolar range using diffusion-ordered spectroscopy (DOSY). While DOSY provides binding data on two of the investigated fragments, structure determination by X-ray crystallography combined with soaking experiments proves to be superior in the identification of fragment binding events. This study highlights the difficulties in characterizing weak binders to a transient interface pocket within the TGT homodimer. The last project initiated the design of peptide-based modulators derived from the interface of the TGT homodimer (Chapter 4). The idea for this approach was inspired by a crystal structure from the second project in which N-terminal residues of TGT, which were usually ill-defined in the electron density, were structurally resolved and stabilized by the exposed dimer contact surface of a crystallographic symmetry mate. In the functional dimer, the position of this particular N-terminal tail is occupied by helix αE, which contains two aromatic residues of the dimer-stabilizing hot spot. This served as a starting point for the development of helical peptides, that are capable to compete with the native dimer partner at the contact surface. A peptide microarray was used to determine the binding epitope to TGT. Subsequently, the most promising peptides were synthesized and characterized with respect to their binding properties by fluorescence polarization. An optimization approach by peptide stapling was attempted. In order to determine the modulation of TGT dimerization, two biophysical assays each based on MicroScale thermophoresis and isothermal titration calorimetry were developed to track changes in the dimerization constants of TGT. Furthermore, a Shigella host cell invasion assay was established to investigate the invasion pathogenicity of a Shigella flexneri Δtgt mutant strain. Henceforth, the established in vitro assays can be further used to characterize potential peptides and drug candidates for TGT inhibition. In conclusion, serendipitous discoveries served as a foundation for the projects discussed in this thesis. It was demonstrated that accidental findings in basic research can be used and acted upon to uncover novel mechanisms of action or for the design of new small molecule fragments and peptides as starting points with the common goal of modulating challenging protein–protein interactions.
Physical Description:168 Pages