Graduation Year

2021

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Yu Chen, Ph.D.

Committee Member

Robert J. Deschenes, Ph.D.

Committee Member

James W. Leahy, Ph.D.

Committee Member

Gloria C. Ferreira, Ph.D.

Committee Member

Sophie E. Darch, Ph.D.

Committee Member

Thomas G. Bernhardt, Ph.D.

Keywords

penicillin-binding protein, diazabicyclooctane, X-ray crystallography, molecular modelling, pathogens, bacteria, viruses, antibiotic resistance

Abstract

Since the first living organisms appeared on earth, their very existence was dependent on their ability to contend for resources. To this day, organisms are perpetually competing for resources, and in doing so, have evolved highly effective defense mechanisms and systems for propagation. Although invisible to the eye, we live in a world teeming with microbes and viruses. While many are harmless, others cause widespread disease. One of the key niches for these pathogens, particularly bacteria, are health-care facilities. These health-care facilities have a high concentration of immunocompromised patients and rely heavily on antibiotics, which strongly contributes to antibiotic resistance. Almost immediately after the introduction of an antibiotic to the clinic, resistant strains are spawned, and their associated resistance elements are disseminated throughout the bacterial kingdom. Likewise, viruses, which are non-living organisms, have also developed mechanisms that enable rapid infection. Respiratory viruses are notorious for widespread transmission, with the most recent being the COVID-19 pandemic. The understanding that a virus can kill millions while disrupting everyday life at the drop of a hat is a troubling prospect. However, unlike our primordial ancestors, we have a strong basis of scientific knowledge at our disposal. Modern scientific knowledge and techniques allow us to prevent and treat infection almost instantaneously, a feat that would otherwise take hundreds of generations. It is important to constantly monitor and characterize emerging pathogens, while addressing existing ones. In the work presented herein, I describe the use of modern scientific techniques to dissect promising drug targets in clinically relevant pathogens and design novel small-molecule inhibitors that disrupt their function. My dissertation is divided into five chapters: 1. A brief introduction, 2. Studies on Pseudomonas aeruginosa PBP3, 3. Characterization of the Clostridioides difficile PBPs, 4. Targeting LPS biosynthesis in Pseudomonas aeruginosa and 5. Structure and inhibition of SARS-CoV-2 Mpro

In the first chapter I provide background for certain subjects discussed in this proposal, including hospital-acquired infections, penicillin-binding proteins (PBPs), the bacterial divisome/elongasome, β-lactam antibiotics, mechanism of resistance to β-lactams and SARS-CoV-2.In the second chapter I describe the structural features of the essential penicillin-binding protein PBP3 in P. aeruginosa. Encoded by the ftsI gene, PBP3 is the central transpeptidase of the divisome in P. aeruginosa, and its enzymatic activity is required for cell division. I characterize important interactions required for inhibition and design novel inhibitors that are resistant to β-lactamase inactivation, a resistance element that challenges the efficacy of existing PBP3 inhibitors.

In the second chapter I, for the first time, characterize the PBPs of the most common hospital-acquired pathogen, C. difficile. Independently, C. difficile infection and penicillin-binding proteins (PBPs) have been the subject of exhaustive research, yet the PBPs of C. difficile remain almost entirely uncharacterized. I present the crystal structures and biochemical inhibition profiles of all four transpeptidase PBPs in this organism. In doing so, I identify a novel family of Zn-binding PBPs belonging to spore-forming firmicutes. One of which, PBP2, is a monofunctional transpeptidase that is the primary target for β-lactams, but is insensitive to inhibition by most cephalosporins, revealing a key cause of the well documented but poorly understood C. difficile cephalosporin resistance. Crystal structures of PBP2 in its unbound form and with different β-lactams reveal the structural basis for inhibition. Notably, of the hundreds of PBP structures in the Protein Data Bank (PDB), PBP2 is by far the largest to date and reveals several novel domains. We also observe unique global conformational changes in response to ligand binding that involves regions that interact with other cell wall enzymes. Additionally, I show that sporulation can be impaired by inhibiting the sporulation specific PBPs. As an anaerobic bacterium, spore-formation is absolutely critical for C. difficile transmission, it also increases the likelihood of recurrent infection. I further explore these promising drug targets by describing a series of novel diazabicylooctane (DBO) inhibitors that impair sporulation in C. difficile through their inhibition of these PBPs. The described compounds, which are derivatives of the β-lactamase inhibitor avibactam, are mostly non-bactericidal, have no cytotoxicity, and are intrinsically stable to β-lactamases secreted by enteric bacteria.

In the third chapter I characterize two enzymes required for lipid A biosynthesis in P. aeruginosa, LpxA and LpxD. Lipid A is the minimal component required for the essential lipopolysaccharide of the Gram-negative bacterial outer membrane. Lipid A is also a potent immunogen and is the antigenic component that induces septic shock in humans. Hence, inhibitors of enzymes that synthesize lipid A would likely be good antibiotics. In these studies, I find that shared features of the LpxA and LpxD active sites can be leveraged for the design of dual-binding inhibitors. Using molecular docking, surface plasmon resonance, and dynamic scanning fluorimetry, we use a rational drug design approach to develop these initial hits into low µM binders of LpxA.

In the fourth chapter, building upon our previous work, I determine the crystal structure of SARS-CoV-2 Mpro with a series on inhibitors that were designed to probe the chemical recognition features of the active-site subpockets and different covalenty warheads. We find that ligand binding is plastic and observe a highly unique, inverted binding pose for calpain inhibitor XII. We also find these calpain inhibitors, despite being weaker than other Mpro-specific inhibitors, also inhibit the lysosomal protease cathepsin L, thus improving their antiviral properties. These findings were published in the early stages of the COVID-19 pandemic and provided a platform for extensive molecular modelling and medicinal chemistry campaigns against this bona fide drug target.

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