title: Development of tag-free photoprobes for studies aimed at identifying the target of novel Group A Streptococcus antivirulence agents.
abstract: We previously reported the identification and development of novel inhibitors of streptokinase (SK) expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic screen. Although phenotypic screening is well-suited to identifying compounds that exert desired biological effects in potentially novel ways, it requires follow-up experiments to determine the macromolecular target(s) of active compounds. We therefore designed and synthesized several classes of chemical probes for target identification studies, guided by previously established structure-activity relationships. The probes were designed to first irreversibly photolabel target proteins in the intact bacteria, followed by cell lysis and click ligation with fluorescent tags to allow for visualization on SDS-PAGE gels. This stepwise, 'tag-free' approach allows for a significant reduction in molecular weight and polar surface area compared to full-length fluorescent or biotinylated probes, potentially enhancing membrane permeability and the maintenance of activity. Of the seven probes produced, the three most biologically active were employed in preliminary target identification trials. Despite the potent activity of these probes, specific labeling events were not conclusively observed due to a considerable degree of nonspecific protein binding. Nevertheless, the successful synthesis of potent biologically active probe molecules will serve as a starting point for initiating more sensitive methods of probe-based target identification.
authors.affiliation: Vahlteich Medicinal Chemistry Core, Department of Medicinal Chemistry, University of Michigan, 428 Church St., Ann Arbor, MI 48109, United States.
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Yestrepsky
authors.affiliation: Department of Human Genetics, Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109, United States.
authors.forename: Colin A
authors.initials: CA
authors.lastname: Kretz
authors.affiliation: Department of Internal Medicine, School of Medicine, University of Missouri-Columbia, 1 Hospital Dr., DC043.00, Columbia, MO 65212, United States.
authors.forename: Yuanxi
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Human Genetics, Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109, United States.
authors.forename: Autumn
authors.initials: A
authors.lastname: Holmes
authors.affiliation: Department of Internal Medicine, School of Medicine, University of Missouri-Columbia, 1 Hospital Dr., DC043.00, Columbia, MO 65212, United States.
authors.forename: Hongmin
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Human Genetics, Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109, United States.
authors.forename: David
authors.initials: D
authors.lastname: Ginsburg
authors.affiliation: Vahlteich Medicinal Chemistry Core, Department of Medicinal Chemistry, University of Michigan, 428 Church St., Ann Arbor, MI 48109, United States. Electronic address: sdlarsen@umich.edu.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Larsen
mesh_terms: D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D001386:Azides / Q000737:chemistry; D001577:Benzophenones / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D057930:Click Chemistry; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D013297:Streptococcus pyogenes / Q000187:drug effects*; D013329:Structure-Activity Relationship; D037521:Virulence Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001386:Azides; D001577:Benzophenones; D015335:Molecular Probes; D037521:Virulence Factors; C047723:benzophenone
keywords: Antibiotic; Click chemistry; Group A Streptococcus; Phenotypic screening; Photo-crosslinking; Streptokinase; Tag-free photoprobes; Target identification; Virulence inhibitor