2017 — 2018 |
Garcia, Brandon Lee Skare, Jonathan T |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Classical Complement Pathway Evasion Mechanisms of Lyme Disease Spirochetes @ Kansas State University
PROJECT SUMMARY The human complement system is a collection of ~30 membrane-bound or serum proteins which form a tightly regulated proteolytic cascade with antimicrobial and homeostatic effector functions. Complement dysregulation contributes significantly to a wide array of human diseases including arthritis, age-related macular degeneration, and transplant rejection. Although only two complement-directed drugs are currently FDA approved, therapeutic intervention of complement has gained traction with the advent of several new drugs in the preclinical and clinical pipeline. Nature has provided an unexpected source of novel complement inhibitory mechanisms in the form of microbial immune evasion proteins, and strides have recently been made in translating these evolutionarily-optimized inhibitory templates into novel therapeutics. However, relatively few examples of classical pathway specific immune evasion strategies have been described. Given the causal link of the classical pathway in neurodegenerative conditions such as Alzheimer?s disease and schizophrenia, this represents a critical deficit in our knowledge which stands to be addressed by the scientific community. The etiological agent of Lyme disease, Borrelia burgdorferi, is transmitted to humans by the bite of infected Ixodes ticks where it disseminates and colonizes remote tissues. Untreated B. burgdorferi infection causes disease in immune competent hosts, in part, by evading innate immune systems such as complement. Recently we reported that the lipoprotein B. burgdorferi BBK32 specifically prevents activation of the complement classical pathway by acting as a nanomolar inhibitor of the first component of complement, C1. We hypothesize that in addition to BBK32, B. burgdorferi expresses additional factors which also recognize and interfere with C1, and that one of these immunomodulators is the lipoprotein CspA. The overarching goal of this project is to shed light on the interaction of B. burgdorferi with the classical pathway of complement as outlined by three Specific Aims. In the first Aim, we will investigate the structural and molecular basis for C1 inhibition by BBK32 and follow-up on interesting preliminary data which suggests that other Lyme disease associated Borrelia BBK32?s have differential activity. In the second aim, we will use a multi-platform approach to quantitatively measure the novel interaction between CspA and the pattern recognition protein of the classical pathway, C1q. In Aim 3, we will use in vivo imaging in mouse models of Lyme borreliosis to explore the hypothesis that the presence of multiple overlapping classical pathway evasion proteins in B. burgdorferi contributes significantly to its ability to cause infection. By completing this research plan, we will greatly improve our understanding of how an important human pathogen causes disease while simultaneously laying the groundwork for the development of novel classical pathway complement-directed therapeutics for the treatment of devastating human autoimmune, inflammatory, and neurodegenerative diseases.
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0.978 |
2017 — 2018 |
Garcia, Brandon Lee |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of Small Molecule Inhibitors of the Classical Complement Pathway @ Kansas State University
The human complement system is a tightly regulated set of ~30 serum or membrane-bound proteins which is best known for its role as a ?first-line-of-defense? against microbial intruders. A modern view places complement at the center of a number of important physiological processes including adaptive immunity crosstalk, developmental roles, and as a critical player in maintaining homeostasis. A large number of human autoimmune, inflammatory, and neurodegenerative diseases are now linked to the loss of the fine-tuned control of the complement cascade. Recently, the dysregulation of the classical complement pathway has been shown to play a causal role in murine models of Alzheimer?s disease. With 5 million Americans currently suffering from Alzheimer?s disease, and a predicted 14 million by 2050, development of new treatments is desperately needed. Unfortunately, the clinical pipeline of complement-directed drugs is currently inadequately positioned to produce therapies for classical pathway-driven neurodegenerative conditions. To meet this need, the fundamental goal of this project is to develop high quality, high specificity small molecule inhibitors of the classical complement pathway. The first component of complement, C1, is the multi-subunit zymogen of the classical pathway and consists a single molecule of C1q in complex with the serine protease heterotetramer C1r2C1s2. C1r is the initiator protease of the pathway and has the unique feature of requiring the molecular context of C1 to carry out its only known physiological function (i.e. activation of the classical pathway). In this project we will attempt to exploit this molecular provision by identifying C1r-binding small molecules which disrupt the stability of C1. To achieve this we will use fragment based drug design and natural product-inspired chemical libraries in combination with an surface plasmon resonance-based screening methodology. We will then implement a novel strategy to isolate compounds with high C1r-specificty and high complement inhibitory potential. Finally, x-ray crystallography will be used to reveal the binding mode of prioritized hit compounds. This project will provide the framework for structure-based drug design efforts for the development of novel complement-directed therapeutics for treatment of classical pathway-related human diseases such as Alzheimer?s disease.
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0.978 |
2019 — 2021 |
Garcia, Brandon Lee Skare, Jon T (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Virulence Mechanisms of Multifunctional Borrelial Proteins @ East Carolina University
PROJECT SUMMARY Spirochetes of the Borrelia genus are the cause of several prevalent vector-borne diseases. The most well- known pathogen from this group is Borrelia burgdorferi sensu stricto, which causes over 300,000 cases of Lyme disease in the United States each year. B. garinii and B. afzelii, which belong to the B. burgdorferi sensu lato complex, are the primary agent of Lyme disease in Europe and Asia. Borrelia spirochetes are also the etiological agent of the ancient human disease relapsing fever, as well as a newly recognized infectious condition called Borrelia miyamotoi disease. Lyme-associated, relapsing fever-associated, and B. miyamotoi spirochetes have differing lifecycles and their infections are accompanied by distinct clinical presentations. However, each of these pathogens are known to encode multifunctional surface-expressed lipoproteins that interact with vertebrate host molecules. Among these proteins are a small arsenal of immunomodulators that specifically target and inactivate a primary arm of innate immunity known as the complement system. We have recently reported two independent lines of evidence that support the hypothesis that one of these pathways, known as the classical pathway, is important in controlling B. burgdorferi infections. First, we have shown that mice deficient in the pattern recognition molecule of the classical pathway, C1q, are significantly more susceptible to B. burgdorferi infection. Secondly, we have shown that the lipoprotein B. burgdorferi BBK32 is a high-affinity inhibitor of the initiating protease of the classical pathway, C1r. In Aim 1 of this project we seek to understand the C1r inhibitory activity of BBK32 sensu lato proteins at the molecular level. In Aim 2 we will determine the immunomodulatory roles and virulence contribution of three BBK32 orthologues known as FbpA, FbpB, and FbpC which are found uniquely in relapsing fever and B. miyamotoi spirochetes. In Aim 3 we will delineate the role of C1r inhibition in borrelial pathogenesis using in vivo models of disease. To achieve this, we propose a multi-disciplinary strategy that employs x-ray crystallography, biophysical approaches, and complement functional assays to pinpoint key ?hot-spot? residues on BBK32 that give rise to its potent anti-C1r activity. These data will inform the design of bbk32 mutants which will be used in mouse infectivity studies to connect structural features of BBK32, at the amino-acid level, to an in vivo phenotype. Parallel studies will use genetic deletion mutants of fbp genes from the relapsing fever- associated spirochetes B. turicatae and B. hermsii. These studies will be paired with experimental models of Lyme and relapsing fever borrelioses using C1r-/- mice to better understand the role of the classical pathway initiating protease in the control of borrelial infections. By addressing fundamental questions of how medically important Borrelia spirochetes recognize and evade host immunity, the studies proposed here stand to have a broad and significant impact on the field of bacterial pathogenesis.
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0.939 |