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  • The fluorescence in a fluorescent antibiotic

    2024-04-16

    The fluorescence in a fluorescent antibiotic can be derived either from functional groups with intrinsic fluorescence in an existing antibiotic, or via synthetic conjugation of a fluorophore to an antibiotic core to enable visualisation. Attachment of fluorophore to an antibiotic should ideally not alter binding to the cellular target or the pharmacokinetics of the drug. The key design parameters affecting these properties are usually the size and lipophilicity of the fluorophore, and the site of functionalization of the antibiotic, although this varies from drug to drug. The most commonly used fluorophores are boron-dipyrromethene (BODIPY), fluorescein, dansyl, 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), and pyrene (Figure 2), which all have relatively small molecular weights.
    Intrinsically Fluorescent Antibiotics Historically, the most widely studied fluorescent EIPA were mithramycin, chromamycin A3, and olivomycin, which all have a fluorescent anthraquinone core. These drugs bind specifically to DNA and, as such, have been used to stain DNA in cell cultures 2, 3, 4 and for flow cytometry 5, 6, 7, 8. Other studies using intrinsically fluorescent antibiotics include the use of aurovertin to study the mechanism of oxidative phosphorylation [9]; tracking the localisation and microbial incorporation of heliomycin [10]; studying the mode of action (MOA) of 7-aminoactinomycin [11]; and examining the movement of the clinically relevant antibiotic tetracycline across bacterial membranes 12, 13. More recently, the clinical quinolone antibiotic levofloxacin was tethered to a porous silica membrane. Upon heating, the solid support released the antibiotic [14], an approach designed for use in wound care, where an increase in temperature is linked to the response of the body to infection. The quinolone fluorescence was used to monitor the release of levofloxacin. When used therapeutically, the intrinsic fluorescence of some antibiotics, such as fluoroquinolones, has the potential to interfere with other diagnostic techniques (not necessarily related to infection) that rely on fluorescence, such as detection of abnormal tissue [15]. In general, antibiotics with intrinsic fluorescence have been used for specialised assays, and have limited universal utility. Given the limited number of intrinsically fluorescent antibiotics (all mentioned EIPA above), most studies using fluorescent antibiotics have focussed on the development on conjugates of fluorophores with different classes of antibiotic.
    Fluorescent Conjugates of Antibiotics that Target the Bacterial Cell Wall There are several antibiotic classes whose targets are proximal to the cell wall, namely the β-lactams, glycopeptides, lipopeptides, and polypeptides. Examples of each of these classes have been conjugated to fluorophores. Bacteria that are resistant to β-lactams or glycopeptides dominate the lists of the most-urgent threats published by organisations such as the Centers for Disease Control and Prevention (CDC) [16]. Accordingly, there is a comparatively large body of fluorescent antibiotic research utilising these antibiotics to investigate antibiotic resistance. An advantage of basing probes on antibiotics targeting cell surface features, such as the peptidoglycan layer of Gram-positive bacteria, or outer cell membrane (e.g., lipid A) of Gram-negative bacteria, is that penetration into the bacterial cell is not required. Given that molecular size is known to adversely influence intracellular entry and accumulation, attachment of fluorophores to antibiotics with intracellular targets is more likely to ablate activity.
    Fluorescent Antibiotic Conjugates with Intracellular Targets There are several different classes of antibiotic that act by targeting various aspects of DNA and/or protein synthesis within the bacterial cell, including macrolides, aminoglycosides, quinolones, sulfonamides, and oxazolidinones. Fluorescent derivative representatives of most of these antibiotic classes have been prepared, mainly to study the MOA of the parent drug. However, the ability of these conjugates to penetrate the cell and avoid active efflux can be impaired, particularly for Gram-negative bacteria, because the outer membrane limits passage of large hydrophobic molecules [56]. Many studies have instead focussed on in vitro studies of the isolated targets (see below).