Biology subjects

Park, K. W.

Publications and source records attributed to Park, K. W..

4 recordsLinked to original sources

bioRxiv2024

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

microbiology↗

bioRxiv2024

Remote ischemic conditioning attenuates transneuronal degeneration and promotes stroke recovery via CD36-mediated efferocytosis.

BACKGROUNDRemote ischemic limb conditioning (RIC) has been implicated in cross-organ protection in cerebrovascular disease, including stroke. However, the lack of a consensus protocol and controversy over the clinical therapeutic outcomes of RIC suggest inadequate mechanistic understanding of RIC. The current study identifies RIC-induced molecular and cellular events in the blood that enhance long-term functional recovery in experimental cerebral ischemia METHODSNaive mice or mice subjected to transient ischemic stroke were randomly selected to receive sham conditioning or RIC in the hind limb at 2 h post-stroke. At 3d post-stroke, monocyte composition in the blood was analyzed, and brain tissue was examined for monocyte-derived macrophages (M{varphi}), levels of efferocytosis, and CD36 expression. Mouse with conditional deletion of CD36 in M{varphi} (cKOMM{varphi}) was used to establish the role of CD36 in RIC-mediated modulation of efferocytosis, transneuronal degeneration, and recovery following stroke. RESULTSRIC applied 2h after stroke increased entry of monocytes into the injured brain. In the post-ischemic brain, M{varphi} had increased levels of CD36 expression and efferocytosis. These changes in brain M{varphi} were derived from RIC-induced changes in circulating monocytes. In the blood, RIC increased CD36 expression in circulating monocytes and shifted monocytes to a proinflammatory LY6CHigh state. Conditional deletion of CD36 in M{varphi} abrogated the RIC-induced monocyte shift in the blood and efferocytosis in the brain. During the recovery phase of stroke, RIC rescued the loss of the volume and of tyrosine hydroxylase+ neurons in substantia nigra (SN) as well as behavioral deficits in WT mice, but not in cKOMM{varphi} mice. CONCLUSIONSRIC induces a shift in monocytes to a proinflammatory state with elevated CD36 levels, and this is associated with CD36-dependent efferocytosis in M{varphi}s that rescues delayed transneuronal degeneration in the post-ischemic brain and promotes stroke recovery. Together, these findings provide novel insight into our mechanistic understanding of how RIC improves in post-stroke recovery. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIInfiltrated monocyte-derived macrophages (M{varphi}) into the post-ischemic brain cause neural inflammation, but they also engage in efferocytosis that promotes tissue repair in the injured CNS. C_LIO_LIRemote ischemic limb conditioning (RIC) changes monocyte composition and enhances functional recovery in experimental brain ischemia. C_LIO_LIThe application of RIC is safe, feasible, and tolerable in stroke patients, but clinical outcomes remain inconsistent. C_LI What New Information Does This Article Contribute?O_LIWe provide experimental evidence that RIC modifies peripheral monocyte composition and molecular expression, and leads to favorable changes in debris clearance, structure integrity, transneuronal degeneration, and behavior following stroke. C_LIO_LIProtective effects of RIC disappear in the absence of CD36 in M{varphi}, suggesting an essential mechanistic role for CD36 in RIC-induced endogenous protective outcomes. C_LIO_LIThe current study demonstrates that immune-mediated RIC mechanisms facilitate inflammatory and recovery processes in the injured CNS. Given the challenges in directly manipulating the brain after stroke, the study suggests that RIC is a promising alternative strategy by inducing changes in peripheral monocytes that can influence injury progression and recovery. Moreover, RIC-induced peripheral changes uncovered by this study may serve as biomarkers to establish an optimal RIC protocol. C_LI

neuroscience↗

bioRxiv2024

Polymer-tethered quenched fluorescent probes for enhanced imaging of tumor associated proteases

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

bioengineering↗

bioRxiv2023

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of S. aureus Infections

Staphylococcus aureus is a major human pathogen responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologs in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling and mouse models of infection we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

microbiology↗