Development of Imaging Probes for Risk Assessment of Alzheimer's Disease using Phage Display

项目总结

Project Summary Parent Grant Summary: Proteases represent one of the largest and most well characterized families of enzymes in the human genome. Furthermore, there are many human health conditions such as cancer that are associated with alterations in protease activity and function. Therefore, specific molecular probes that allow individual protease activities to be imaged during disease progression in vivo would both be transformative in our understanding of the roles of proteolytic events that contribute to disease pathology while also providing a direct methods for early disease monitoring and response to therapy. The past decade has produced many diverse classes of molecular probes that can be used for imaging applications. Perhaps one of the most powerful of these reagents is the activity- based probe (ABP). However, the broad application of ABPs is typically limited by the need to painstakingly optimize probes using synthetic chemistry and often probes lack absolute specificity for a given target enzyme. This proposal will focus on establishing an innovative technology that will allow rapid design of ABPs with exceptional specificity for any given protease target of interest. This will involve application of a phage display method to screen diverse libraries of chemically constrained bi-cyclic peptides linked to a protease reactive electrophile to iteratively screen for covalent binding elements with high potency and selectivity. We propose to establish and validate the phage screening method using two stromal-cell derived protease targets, cathepsin S (cat S) and fibroblast activation protein (FAP), involved in key aspects of tumorigenesis. These new probes will then be validated for imaging applications in mouse models of cancer. The technology developed in this proposal will result in not only a new general method for protease ABP development but also will produce probes with potential future clinical applications in cancer imaging. Supplement Project Summary: Alzheimer’s Disease (AD) is a progressive neurodegenerative disease characterized by inflammation and cell death in the brain that leads to loss of cognitive function. Although a number of genetic links have been established and many cellular events associated with disease pathology have been established, very little is known about key risk factors and non-genetic triggers of this debilitating disease. Recent findings that one of the primary proteins linked to AD pathogenesis, b-amyloid, can function as an anti-microbial peptide motivated studies to identify a role for an infectious agent as a possible cause for the disease. To this end, a number of recent efforts have identified the presence of the pathogenic bacteria, Porphyromonas gingivalis, in greater than 90% of postmortem brains of patients with AD. This pathogen is typically found in the mouth where it is involved in the progression of chronic periodontitis (CP) but can also be found in other locations within the body including coronary arteries, placenta and liver. P. gingivalis produces and secretes several proteases called gingipains that act as virulence factors necessary for effective colonization by the bacteria. These enzymes have been identified in the brains of AD patients and their levels strongly correlate with disease markers such as tau and ubiquitin. Gingipains were also found to be neurotoxic in vitro and in vivo and oral infection of mice with P. gingivalis leads to brain exposure and increased accumulation of amyloid plaques. Recently, a company, Cortexyme, developed a small molecule inhibitor of lysine-specific gingipain (Kgp) and showed that it can effectively treat P. gingivalis infection in the brain in mice, leading to protection of hippocampal neurons. These results led to a phase I trial (NCT03418688) in patients with AD which showed positive protective value. A current phase II/III trial (NCT03823404) is underway with results expected in mid to late 2021. As a result of these recent findings, there is a current unmet need for non-invasive imaging methods that will enable further studies of the link between gingipain activity in the brain and AD risk as well as to identify patients who would likely benefit from treatment with gingipain inhibitors. Therefore, in this supplement, we plan use the phage display technology currently being developed in the parent grant to identify highly selective covalent binding cyclic peptides that target the gingipains. The major benefit of the phage display approach is that the resulting cyclic peptides show highly specific target binding necessary for an effective imaging agent. Selective covalent inhibitors can be converted to compounds containing a fluorescent and PET radiotracer for imaging applications. The primary aims for the supplement are as follows: Aim 1- screen cyclic peptide phage libraries against lysine-specific (Kgp) and arginine-specific gingipains (RgpA and RgpB) to identify selective covalent binding sequences. Aim 2 Convert sequences from phage screening into selective covalent binding cyclic peptide probes for each gingipain target. Aim 3 Demonstrate that fluorescent probes can label gingipains in cells and in a mouse model of P. gingivalis infection. At the completion of the one-year supplement, we will have at least one probe that can be labeled with 18F for PET imaging in mice, advancement into pre-IND studies and eventually clinical studies in humans.