Chapter 1. A brief history of the use of ionizing radiation for cancer therapy is presented, as the motivation for modern clinical investigations on alpha therapy and the use and development of bifunctional chelators in targeted medicine. Recent studies on targeted therapy with alpha-emitters, such as 223Ra and 227Th, are described. The chemistry of Th(IV) is presented with a survey of important coordination compounds, with special emphasis on those developed by the Raymond group.

Chapter 2. The rational design of a ligand as a suitable bifunctional chelator for 227Th therapy is described in the context of ligands developed by the Raymond group for actinide sequestration. A novel Φ ligand topology incorporating macrocyclic and pendant terephthalamide binding groups aims to address the kinetic and thermodynamic requirements of targeted alpha therapy. The syntheses of the model ligands Φ(2,2)moeTAM and Φ(3,3)moeTAM are detailed, from which a derivative of Φ(2,2)moeTAM is developed. Functionalization of a pendant terephthalamide of the model ligand gives the asymmetric bifunctional chelate Φ(2,2)NBuTAM via the strategic use of protecting groups.

Chapter 3. The evaluation of the solution thermodynamic behavior of Φ(2,2)moeTAM is extensively detailed. The protonation constants of the ligand and its affinity for Th(IV) is measured by spectrophotometric titration. The thorium complex was found to have a remarkably high thermodynamic stability, with a formation constant of 1054, surpassing the stabilities of previously measured complexes. Efforts to elucidate the effects of the Φ topology on the coordination of Th(IV) using thermodynamic studies of the linear analog 3,4,3-LiMeTAM as well as the shape analyses of the crystal structure of Th[Φ(2,2)moeTAM] are described. The surprising geometry adopted by Φ(2,2)moeTAM in the thorium complex relative to its structure as the free ligand is further investigated with DFT studies.

Chapter 4. While the measurement of the thermodynamic parameters of the thorium complex provides an assessment of its inertness, its formation kinetics are also of paramount importance due to the inherent time sensitivity of radiotherapeutic agents. Difficulties encountered in preliminary kinetic experiments with existing octadentate ligands and Φ(2,2)moeTAM lead to the use of indirect kinetics. Dye-displacement kinetic studies with the Φ ligands, 3,4,3-LiMeTAM, and the aminocarboxylic acid ligands provide a useful qualitative comparison of the rates of complexation of these ligands with Th(IV). Φ(2,2)moeTAM is observed to be a much more rapid chelator than the prevailing DTPA and DOTA, and its association (a second-order reaction with a rate constant of k2 = 1.8(1) x 104 M-1s-1) can be directly measured using stopped-flow kinetics.

Chapter 5. The application of Φ(2,2)moeTAM toward the coordination of the lanthanide analogs cerium and praseodymium is presented. The crystal structure of the Ce(IV) complex with Φ(2,2)moeTAM is isomorphous to that of the Th(IV) complex, prompting solution thermodynamic and electrochemical studies to investigate the stability of the Ce(IV) complex. Spectrophotometric titration indicates an unprecedented formation constant on the order of 1061, and a Nernstian shift of the Ce(III)-(IV) couple of 2.0 V upon complexation is observed by cyclic voltammetry. The strong preference of Φ(2,2)moeTAM for the +4 oxidation state relative to the +3 is further highlighted by the solution thermodynamics of Pr(III).

Abstract

Siderophore-mediated Competition for Iron Between Humans and Pathogenic Bacteria

by

Allyson Kawile Sia

Doctor of Philosophy in Chemistry

University of California, Berkeley

Professor Kenneth N. Raymond, Chair

Iron is the most abundant and essential transition metal used by many living organisms. To understand the means by which organisms acquire, transport and use iron expands the understanding we humans have of life. Organisms evolved multiple strategies to solubilize iron hydroxides from the environment and to increase iron bioavailability. Most bacteria, fungi and plants solubilize iron from the environment by synthesis and secretion of siderophores, or small molecules that have a high affinity for ferric iron. Alternative iron acquisition strategies are often employed simultaneously which include piracy of iron stores from other organisms and exploitative uptake of nonnative siderophores and hemes.

Generally this work is meant to provide detailed information for mechanisms and strategies involved in iron competition between pathogenic bacteria and human hosts. Detailed structural and kinetic investigations of iron acquisition pathways in the Bacillus cereus group of bacteria are discussed in Chapters 1-4. The B. cereus group of bacteria includes threatening pathogens such as the anthrax pathogen (B. anthracis) and enterotoxic bacteria (B. cereus). Iron complexes of molecules as small as citrate and as large as a polymeric bacterial capsule are probed for structural and kinetic data related to iron acquisition and transport. Studies of the iron binding ability and subsequent transport of synthetic analogs of the B. anthracis stealth siderophore petrobactin are also included. Taken together, these studies shed light on multiple mechanisms and structure-function relationships necessary for the pathogenic bacteria to obtain an adequate iron supply and regulate virulence.

Bacillus bacteria can infect humans and compete with the host iron supply. The consequences of iron piracy by Bacillus anthracis are especially likely to be fatal. Crucial to the human defense against bacterial infections is the immune protein siderocalin. Siderocalin is antibacterial and limits bacterial growth by intercepting bacterial siderophores. Chapter 5 highlights siderocalin and its recently established alternative role as an iron transport protein. For siderocalin to transport iron, an endogenous siderophore equivalent is required. A putative endogenous, or `mammalian' siderophore, is evaluated by solution thermodynamic analyses, inorganic spectroscopy and biochemical assays. The multipurpose role of siderocalin could have important clinical impact, and it is important that the foundation of these studies are clear and consistent at the chemical level. The elucidated roles of siderocalin in humans convey key iron-restricting strategies by humans to compete with invading pathogens for iron. The collection of chapters in this work portrays the competition for iron as dynamic, aggressive and evolving by both humans and invading bacterial pathogens and is meant to provide a snapshot of the competition in current times.

Bacterial pathogens acquire the iron they need for survival and growth in a host by using siderophores. The structures of siderophores are specialized for binding ferric ion with high affinity. Siderophore structures are also specialized to specifically interact with the proteins that mediate siderophore function. These proteins include the bacterial proteins involved in siderophore uptake and utilization, as well as host proteins that inhibit bacterial iron acquisition by intercepting siderophores.

The interactions between siderophores and iron underlie biological function. The fundamental coordination chemistry of catecholate and hydroxamate siderophores affects protein interactions during siderophore uptake and host defense. Chapter 1 reviews previous studies on siderophore coordination chemistry and the effects it has on protein interactions and biology with emphasis on research from the Raymond laboratory.

The human protein siderocalin defends against siderophore-mediated iron acquisition. Human siderocalin recognizes the metal center of catecholate siderophores, including enterobactin, with high affinity. Several pathogens modify the catecholate metal binding units to make stealth siderophores that are not recognized by human siderocalin. As presented in Chapter 2, the pathogens Vibrio fluvialis and Vibrio cholerae use the siderophores fluvibactin and vibriobactin, respectively, which have catechol-oxazoline metal-binding units. The catechol-oxazoline had been proposed to be a stealth mechanism, but the results herein presented clearly demonstrate that it is not a stealth mechanism. Catechol-oxazoline coordinates iron in either a catecholate mode or a phenolate-oxazoline mode. The phenolate-oxazoline mode is not recognized by siderocalin while the catecholate mode is. Siderocalin stabilizes the catecholate mode sufficiently to cause a shift from the phenolate-oxazoline mode at physiological pH. The high affinity recognition of the ferric triscatecholate metal center allows siderocalin to defend against iron acquisition by a large number of bacterial siderophores.

Siderocalin defense has been identified in hosts other than humans. Ex-FABP is a protein found in chickens that has high structural homology to human siderocalin. Chapter 3 reports that Ex-FABP binds many of the same siderophores that are bound by human siderocalin. Unlike human siderocalin, the binding pocket of Ex-FABP is expanded to allow it to bind glucosylated enterobactin. Many of the pathogens specific to chickens use glucosylated enterobactin siderophores known as salmochelins. Salmochelins are stealth siderophores in humans. The siderophore recognition of Ex-FABP demonstrates that siderophore binding proteins may be a general host defense mechanism, and that siderocalins have adapted to the pathogens most frequently encountered by the host. Siderocalin defense and stealth siderophores are at the edge of the arms race for iron.

Siderophores carry iron into a bacterial cell through specific transport systems. Once inside the cell, the iron must be removed from the siderophore. Bacteria that use ferric enterobactin remove the iron by hydrolyzing the backbone with an esterase followed by reduction of the ferric ion. Hydrolysis is necessary because the high stability of intact ferric enterobactin prevents biological reduction and iron release. V. cholerae had been reported to use ferric enterobactin, but it does not have an esterase to hydrolyze the backbone. Chapter 4 reports that V. cholerae does not use intact ferric enterobactin, but that it most likely uses ferric complexes of enterobactin hydrolysis products.

Uptake of ferric siderophores relies on specific cell membrane receptors. Many siderophore receptors recognize the apo-siderophores as well as the ferric complexes. Binding apo-siderophores does not directly deliver iron to the bacteria, but it plays a role in the uptake mechanism. Chapter 5 describes YxeB, the ferrichrome/ferrioxamine receptor of Bacillus cereus. YxeB transports the siderophores using a Gram-positive siderophore-shuttle in which metal exchange between a ferric siderophore and the bound apo-siderophore is facilitated by the receptor. Metal exchange is not required for uptake, but the siderophore-shuttle is faster than transport without metal exchange.

Metal exchange, iron release, and the sterics and electronics of the metal center are coordination chemistry principles that influence the interactions between siderophores and proteins. Proteins usher siderophores through the biological functions of removing iron from the host, passing through the bacterial membrane, and releasing iron to the cell. Therefore, siderophores act as the intermediaries between ferric ion and the biology of bacterial iron uptake.