Leveraging Chemical Tools to Target Bacterial Membranes and Combat Antimicrobial Resistance Public

Abstract

The need for antibacterials with novel mechanisms of action is dire, particularly in the face of the COVID-19 pandemic. The drastic increases in mis-prescriptions, hospital visits, and use of antibacterials have created a perfect storm for antimicrobial resistance. To combat this looming public health crisis, a diverse range of strategies must be employed, as tapping into new biological and druggable spaces enhances the likelihood of evading resistance mechanisms. The work described herein uses a multi-faceted approach that includes natural product total synthesis, diverted total synthesis, and simplification, as well as small molecule derivatization. The first project I detail focuses on the diverted total synthesis of carolacton, a natural product that exhibits a unique, acid-mediated anti-biofilm effect against Streptococcus mutans. Through the development of a second generation of simplified carolacton analogs, we discovered a more potent compound that enabled the identification of a novel Streptococcus protein target, glucan-binding protein B (GbpB). This molecule is the first compound to target an essential bacterial cell wall division hydrolase and provides a fruitful starting point for further tool and antibiotic development to treat Streptococcal infections. Additionally, we demonstrated enzymatic activity of the full length, recombinant protein for the first time, which will enable drug discovery campaigns. The second project outlines progress toward the total synthesis of α-santal-11-en-10-one, an inhibitor of S. mutans and Porphyromonas gingivalis, that likely works via a cell membrane mechanism. This molecule displays more potent activity against the Gram-negative strain, which challenges the typical paradigm and suggests a unique target. The final project aimed to develop improved quaternary phosphonium compounds (QPCs) inspired by the cannon of work on their nitrogen counterparts, the quaternary ammonium compounds (QACs). We identified a scaffold that outcompetes the best-in-class quaternary ammonium compounds (QACs) from our lab and the best-in-class antibacterials currently used.  Most importantly, this compound displays a unique resistance profile signifying a new mechanism of action. Collectively this thesis highlights the utility of chemical synthesis and chemical biology in the pursuit of antibiotic development and the fight against antimicrobial resistance.

 

Table of Contents

Table of Contents

1         Introduction    1.1

1.1      The discovery of anti-infectives         1.1

1.2      Antibiotics – Mechanisms of action and resistance  1.2

1.2.1   Nucleic acid synthesis inhibitors       1.3

1.2.2   Protein synthesis inhibitors   1.4

1.2.3   Cell membrane disruptors     1.5

1.2.4   Cell wall inhibitors      1.6

1.2.5   Folate synthesis inhibitors     1.8

1.3      Biofilms in disease     1.8

1.4      Introduction to oral disease   1.9

1.5      Oral biofilms   1.10

1.6      Streptococcus mutans           1.12

1.6.1   S. mutans pathogenicity        1.13

1.6.2   Acidogenicity 1.14

1.6.3   Acid tolerance response (ATR)         1.14

1.6.4   Two-component systems (TCS)        1.15

1.6.5   Quorum sensing        1.16

1.6.6   Prevention methods  1.17

1.6.7   Selective inhibition of S. mutans biofilm formation   1.18

1.7      Looking ahead           1.22

1.8      References    1.23

2         Carolacton – inspiration for cancer inhibitors & SMU biofilm inhibitors        2.34

2.1      Carolacton Introduction         2.34

2.1.1   Isolation and bioactivity of carolacton 2.34

2.1.2   Investigations into the mechanism and target of carolacton 2.35

2.1.3   Total and biological investigation by the Wuest lab  2.38

2.1.4   Diverted total synthesis of carolacton 2.40

2.2      Carolacton-inspired cancer inhibitors 2.42

2.2.1   Background   2.42

2.2.2   MTHFD1 & MTHFD2 as cancer targets        2.42

2.2.3   Synthesis of carylacton         2.43

2.2.4   Evaluation of carylacton as MTHFD1 inhibitor          2.44

2.3      Synthetic simplification enables chemical genetic studies   2.45

2.3.1   Second-generation design    2.45

2.3.2   Simplified analog synthesis   2.45

2.4      Preliminary biological investigation   2.52

2.5      Mechanism of action studies 2.55

2.6      Conclusion and future work   2.59

2.7      References    2.60

3         Natural Product simplification yields first inhibitor of an essential streptococcal cell wall hydrolase           3.63

3.1      Introduction:   3.63

3.2      Ruling out folate dehydrogenase      3.64

3.3      Label-free affinity-based protein profiling      3.65

3.3.1   Probe development   3.67

3.3.2   AfBPP assay optimization     3.69

3.3.3   Protein target identification via AfBPP with label-free quantification 3.71

3.4      Narrowing down targets        3.74

3.5      Exploring GbpB as the target 3.76

3.5.1   Glucan binding protein B       3.77

3.5.2   Resistance selection 3.78

3.5.3   Synergy studies         3.80

3.5.4   GbpB overexpression in S. mutans  3.85

3.5.5   Attempts to express and purify GbpB 3.86

3.5.6   Expanded bacterial screen    3.87

3.6      PcsB expression and purification      3.91

3.7      Microscale thermophoresis   3.92

3.8      In vitro crosslinking and molecular docking  3.96

3.9      Hydrolase assay development          3.102

3.10    Conclusions and future work 3.108

3.11    References    3.109

4         Progress toward the total synthesis of α-santal-11-en-10-one         4.111

4.1      Introduction    4.111

4.1.1   Porphyromonas gingivalis     4.111

4.1.2   P. gingivalis pathogenicity     4.111

4.2      Santalene-sesquiterpenoids – isolation and bioactivity        4.112

4.3      Synthesis       4.113

4.3.1   Retrosynthetic plan    4.113

4.3.2   Accessing the key intermediate        4.114

4.3.3   One-step alkylation attempts 4.116

4.3.4   Two-step alkylation routes    4.117

4.4      Conclusion and outlook         4.119

4.5      References    4.119

5         Quaternary Phosphonium Compounds         5.121

5.1      Quaternary ammonium compounds (QACs) 5.121

5.2      QACs in oral healthcare        5.122

5.3      QAC diversification    5.123

5.4      Quaternary Phosphonium Compounds         5.124

5.5      Preparation of the QPCs by the Minibole Lab          5.125

5.6      Biological investigation of QPCs       5.126

5.6.1   Evaluation of bioactivity and toxicity of the QPC library       5.126

5.6.2   Elucidation of structure-activity relationships 5.129

5.6.3   Determination of QPC cytotoxicity    5.131

5.6.4   Elucidation of QPC resistance mechanisms 5.132

5.6.5   Implication of SMR family transporters in QPC resistance   5.132

5.6.6   Investigations into efflux-mediated resistance mechanisms 5.133

5.7      Conclusions   5.135

5.8      References    5.137

6         Experimental  6.142

6.1      Supplementary figures          6.142

6.2      Biological procedures 6.164

6.2.1   General notes 6.164

6.2.2   Procedures    6.165

6.3      Chemistry experimental         6.180

6.3.1   General notes 6.180

6.3.2   Procedures and characterization      6.180

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Microbiology Chemistry, Organic Chemistry, Biochemistry
Mot-clé	natural product antibiotic total synthesis
Committee Chair / Thesis Advisor	William Wuest, Emory University
Committee Members	Katherine Davis, Emory University Jennifer Heemstra, Emory University

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