Investigating the Mode(s) of Action of Human B7-1 Small-Molecule Inhibitors

• Author / Creator

  Chen, Rui

• Human B7-1 (hB7-1) is the first and most characterized costimulatory protein from the B7 family, which plays a key role in regulating T-cell functions. It is, therefore, regarded as an important target for the treatment of autoimmune diseases and, potentially, cancers. Given the promising clinical benefits of targeting hB7-1, several hB7-1 specific small-molecule modulators were developed and characterized. However, the binding sites and the mode(s) of action of these molecules are not fully understood and relatively unexplored. Identifying the binding locations and understanding their mode(s) of action may help develop more selective and potent modulators rationally. This thesis, therefore, aimed at closing this knowledge gap, by studying the interactions of hB7-1 and two known small-molecule B7-1 inhibitors, namely inhibitors 1 and 2. Towards this goal, I established a workflow utilizing various advanced modelling tools. In this workflow, molecular dynamics (MD) simulations were initially performed to relax the hB7-1 protein structures and to reach a low energy state, suitable for further studies. Then I used root mean square deviation (RMSD)-based clustering methods to capture the dominant B7-1 conformations visited by the protein during the MD simulations for subsequent molecular docking studies. Following molecular docking, several docked poses were selected to closely investigate their interactions with B7-1. In this context, MD simulations of the protein/compound complexes were used to refine the structures of the generated complexes and to analyze the interactions between the protein and each tested compound. By studying the stability of the formed complexes and the compounds’ binding affinities, I identified two binding sites that were suitable for compounds’ binding within B7-1. Inhibitor 1 binds to one site with two orientations, while inhibitor 2 binds to a second site. The hydrogen bond (H-bond) analyses and the binding free energy decompositions allowed me to understand the interactions of the final selected complexes and to further confirm the findings.To experimentally validate the modelling results, I expressed and purified the extracellular domain of hB7-1 using an E. coli expression system in the form of inclusion bodies as an initial step. Then I recovered the biological function of the aggregated B7-1 through denaturing the proteins followed by refolding them slowly. Two protein purification tools were used to improve the purity of the recombinant proteins, including the immobilized metal affinity chromatography (IMAC) and the size exclusion chromatography (SEC). The preliminary binding assays proved that the recombinant hB7-1 has a limited cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) binding activity, compared to a commercial human B7-1. This could be in part due to the recombinant proteins not correctly folding because of the lack of mammalian chaperons in the bacterial expression system. Expressing the extracellular domain of hB7-1 through the mammalian expression system will be a future direction. Furthermore, using mutagenesis assays to confirm the key residues important for compounds binding predicted through modelling studies will be another future direction. To my knowledge, this thesis revealed the possible binding site(s) and potential mode(s) of action of hB7-1 small-molecule compounds for the first time. These findings can be used as a good starting point to guide the development of novel hB7-1 small-molecule modulators in the future.

• Subjects / Keywords

  • Computational Modeling
  • Molecular Dynamics Simulation
  • Protein Preparation
  • human B7-1
  • Molecular Docking

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-ccas-fh94

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.