Carolacton and Honokiol: using natural product inspiration to target the oral microbiome Open Access

Solinski, Amy (Spring 2020)

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Streptococcus mutans, one of the cariogenic species within the oral microbiome, has become a model organism for the formation of bacterial biofilm. In contrast to free floating, planktonic cells, biofilm cells use various methods to attach to environmental surfaces. Investigating biofilm has become more prevalent as it has been connected to antibiotic resistance. Scientists are searching for new compounds that can inhibit, or eradicate the formation of these disease causing biofilms. Carolacton and honokiol are natural products that have been found to target S. mutans biofilm. Carolacton demonstrates lethal effects towards cells in S. mutans biofilm but a full understanding of the biological target and mechanism of action have remained elusive for the past decade. Herein we present two generations of novel carolacton analogs that were developed into tool compounds and used to probe the target and mechanism of action. From these molecules we have gained a better understanding of the antibiofilm mechanism of carolacton. In contrast, we have proven that honokiol lacks potent biofilm activity in physiologically relevant conditions. We were able to develop a library of approximately 80 compounds that led to the identification of a new, potent antimicrobial bisphenolic scaffold, C2. Furthermore, from our structure-activity relationship study, we have identified a secondary modification that appears to change the mechanism of the inhibition against S. mutans. The investigation of these two natural products has led to multiple discoveries that are impactful for the field of antibiotic development. The data provided in this dissertation will influence the future of antibiofilm research.

Table of Contents

Chapter 1     Introduction

1.1     Oral Microbiome

1.1.1   Introduction

1.1.2   Commensal vs pathogenic bacteria: dysbiosis in the oral microbiome

1.1.3   Dental cavities

1.2     Biofilm Formation

1.2.1   Global impact

1.2.2   Mechanism of biofilm formation

1.2.3   A model organism – Streptococcus mutans

1.3     Anti-biofilm small molecules against S. mutans

1.3.1   Introduction – Prevention vs Treatment

1.3.2   Sucrose-dependent mechanisms

1.3.3   Sucrose-independent mechanisms

1.3.4   Cellular signaling interference

1.3.5   Next generation of molecules

1.4     References

Chapter 2     Natural Product Carolacton

2.1     Background

2.1.1   Carolacton’s isolation and biofilm specific activity

2.2     Total syntheses

2.2.1   Kirschning Group

2.2.2   Wuest/Phillips Group

2.2.3   Goswami Group

2.2.4   Carolacton derivatives

2.3     Progress towards target identification

2.3.1   Carolacton: a decade of biological investigation

2.3.2   Target or targets?

2.3.3   Rationale and design for simplified analogs

2.4     Generation 1: Carylacton Analogs

2.4.1   Synthesis of carylacton analogs

2.4.2   Discovery of new phenotypes

2.4.3   Computational modeling

2.4.4   Generation 1 conclusions

2.5     Generation 2: Simplified Sidechain Analogs

2.5.1   Analog development

2.5.2   Synthesis of simplified analogs

2.5.3   Biofilm-specific inhibition

2.5.4   LIVE/DEAD® Staining with Confocal Imaging

2.5.5   Colony Forming Unit Assay

2.5.6   Acid tolerance based activity

2.5.7   Genetic Screen with Analog 2

2.5.8   Conclusions

2.6     References

Chapter 3     Analog 2 Target Elucidation

3.1     Affinity-based proteomic probe

3.1.1   Affinity-based Protein Profiling

3.1.2   Synthesis of analog 2 probe

3.1.3   Developing the biofilm workflow

3.1.4   Identified protein targets

3.1.5   Competition assay

3.1.6   Proteomic pulldown results

3.2     Proteomic result validation

3.2.1   Resistance assay development

3.2.2   ΔftsA, ΔftsX, ΔbrpA, and ΔfolD screen

3.2.3   ΔfolD

3.2.4   GbpB: the main biofilm target?

3.3     Future directions

3.4     Conclusions

3.5     References

Chapter 4     Honokiol

4.1     Background

4.1.1   Isolation

4.1.2   Anti-bacterial activity

4.2     Generation 1

4.2.1   Synthesis of Generation 1

4.2.2   Micro-aerophilic vs aerophilic growth conditions

4.2.3   Structure-activity relationships of Generation 1

4.2.4   Lead compound C2 and Generation 2 design

4.3     Generation 2

4.3.1   Structure-activity relationships of generation 2

4.3.2   Hemolysis results

4.3.3   Mechanism of action – C2

4.4     Mechanism of action – C2-propyl

4.5     Future Directions

4.5.1   Sortase A Hypothesis

4.6     Conclusions

4.7     References

Chapter 5     Experimental Details

5.1     Supplemental Figures

5.2     Biofilm Replicates Generation 1 (Aryl)

5.2.1   Concentrations 500 μM or 250 μM

5.2.2   Concentrations 125 μM to 0.5 μM

5.3     Biofilm Replicates Generation 2 (Simplified)

5.4     Carolacton Generation 2 IC50 Curves

5.5     Analog 2 Mutant Screen

5.6     Honokiol Master Compound List

5.7     Membrane Assay Data

5.8     Biological Procedures

5.9     Chemistry General Notes

5.10    Procedures and Characterization

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