Molecular Interactions between SARS-CoV-2 SPIKE protein and host targets Restricted; Files Only

Abstract

The SARS-CoV-2 virus was identified over two years ago and has claimed thousands of lives, demanding mechanistic understanding of the viral entry and infection process to rapidly advance therapeutic agent discovery and development. In particular, rapid understanding of the host-virus interface is a critical step in uncovering therapeutics, and readily developed small molecule screening assays can expedite this process. TMPRSS2 was identified early in the pandemic as an important SPIKE interactor at the viral-host interface, critical for viral entry. Thus, identification of inhibitors of the interaction between SPIKE and TMPRSS2 may be a step in therapeutic development. Here we show the development of two ultra-high throughput screening assays for screening of inhibitors of the SPIKE and TMPRSS2 protein-protein interaction and identification of potential novel inhibitors of the interaction. These assays may be used as a model for development of future screening assays in a pandemic setting as well as for further screening themselves. The compounds found may serve as the basis for future therapeutic and chemical probe development for the treatment and study of the SARS-CoV-2 pathogenesis.

Table of Contents

Table of Contents

Chapter 1: Introduction

1.1  The impact of COVID-19

1.2  SARS-CoV-2 viral structure and method of pathogenesis

1.3  Current treatments and repurposing drug for expedited therapies

1.4  SARS-CoV-2 entry mechanism: the role of SPIKE, ACE2, and TMPRSS2

1.5  SPIKE protein in SARS-CoV-2

1.5.1    Structure

1.5.2    Role in COVID-19 pathogenesis

1.5.3    Mutations important in COVID-19 pathogenesis

1.5.4    Targeting SPIKE for COVID-19 treatment

1.6  ACE2 protein in SARS-CoV-2

1.6.1    Structure

1.6.2    Function and expression

1.6.3    Role in COVID-19 pathogenesis

1.6.4    Polymorphisms important in COVID-19 pathogenesis

1.6.5    Targeting ACE2 for COVID-19 treatment

1.7  The role of ACE2/SPIKE interaction in SARS-CoV-2 entry

1.8  TMPRSS2 protein in SARS-CoV-2

1.8.1    Structure

1.8.2    Function and Expression

1.8.3    Role in COVID-19 Pathogenesis

1.8.4    Polymorphisms important in COVID-19 pathogenesis

1.8.5    Targeting TMPRSS2 for COVID-19 treatment

1.9  The role of TMPRSS2/SPIKE interaction in SARS-CoV-2 entry

1.10      Summary and Goals for the Project

Chapter 2: Time-Resolved Fluorescence Energy Transfer Assay for SARS-CoV-2 Inhibitor Screening

           2.1 Introduction

           2.2 Results

                       2.2.1 Expression of constructs generated for SPIKE and TMPRSS2

           2.2.2 Confirmation of PPI in HEK-293T cells with affinity chromatography assays 

2.2.3 Development of TR-FRET assay for monitoring SARS-CoV-2 SPIKE and TMPRSS2 interaction

                       2.2.4 Optimization and performance evaluation of TR-FRET assay for HTS

                       2.2.5 Miniaturization of TR-FRET Assay into 1536-well ultra-HTS format

                       2.2.6 TR-FRET Pilot Screening

                       2.2.7 Confirm the positives from pilot uHTS

                       2.2.8 Validate confirmed TR-FRET positives with Thermal Shift assay

                       2.2.9 Molecular docking of TMPRSS2/SPIKE binding site

                       2.3.0 Molecular docking of top hit compounds

           2.3 Method

           2.4 Discussion

Chapter 3: Large-scale diversity screening for inhibitors of TMPRSS2/SPIKE interaction

           3.1 Introduction

           3.2 Results

           3.3 Method

                       3.3.1 Live-cell NanoBiT assay development, optimization, and evaluation

                       3.3.2 Diversity Library Screening with the TR-FRET assay

                       3.3.3 Narrowing of PPI inhibitor compounds

                       3.3.4 Thermal Shift Validation

                       3.3.5 Molecular docking of top hit compounds

3.4 Discussion

Chapter 4: Conclusions and Future Directions

Chapter 5: References

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular
Keyword	HTS COVID-19 TMPRSS2 SPIKE
Committee Chair / Thesis Advisor	Fu, Haian, Emory University
Committee Members	Zhou, Wei, Emory University Hall, Randy, Emory University Kang, Sumin, Emory University

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