(340bu) Design of Antimicrobial Prodrugs Against Multidrug-Resistant Bacteria

The rapid development of bacterial resistance to antibiotics presents a global health crisis and a growing need to develop new antibacterial strategies. Antimicrobial peptides (AMPs) are being researched as potential antibacterial agents due to their bacteriolytic properties, broad spectrum of activity, and high potency¹. AMPs are part of the innate host defense system and take part in the regulation of the host immune response. In general, they are facially amphiphilic and relatively small (containing fewer than 50 amino acid residues)². AMPs disrupt the structure of the bacterial cell membrane by binding to it through charge-mediated interactions, followed by membrane permeabilization via hydrophobic interactions, and subsequent cell death³. Since the bacterial cell membrane contains an abundance of anionic phospholipids, most AMPs are cationic. In comparison, the mammalian cell membrane is neutral, thus attenuating bilayer disruption⁴.

Despite this difference in membrane charge, AMPs display systemic toxicity because the abundance of mammalian cells in circulation hinders selectivity to bacteria. Coupled with proteolytic susceptibility, the clinical translation of AMPs has been limited^5,6. In response, AMP mimetics have been investigated as alternative therapeutic options. These synthetic mimetics have been designed to combine the favorable properties of AMPs, namely cationic charge and amphipathicity, while incorporating abiotic backbones to resist proteolytic degradation. One such class of AMP mimetics called oligothioetheramides (oligoTEAs) has been designed that exhibits potent antimicrobial activity against a wide array of bacterial species^7,8. OligoTEAs are synthetic, sequence-defined cationic oligomers synthesized through rapid orthogonal chemical reactions². Monomers are added in solution on a liquid phase fluorous support which provides fast reaction kinetics⁹. The antimicrobial activity of oligoTEAs can be regulated by altering overall charge via the pendant group and hydrophobicity through the backbone⁷. OligoTEAs are distinct from AMPs due to their flexible thioether backbone but are similar in that they can permeabilize bacterial cell membranes. The advantage of oligoTEAs over AMPs is that they are resistant to proteases and thus remain potent in the presence of serum proteins. However, oligoTEAs’ translation to clinical usage is limited by their systemic toxicity in mammalian cells which narrows their therapeutic window.

Thus, there is a need to increase the therapeutic window of potent oligoTEAs for their use as viable antimicrobial agents against multidrug-resistant (MDR) bacteria. The oligoTEA known as BDT-4G, which has a 1,4-butanedithiol (BDT) backbone and four guanidinium (4G) pendant groups, has displayed potent antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as polymyxin-resistant clinical isolates. However, BDT-4G alone is toxic to mammalian cells at concentrations required to be bactericidal against Pseudomonas aeruginosa, i.e., between 16 and 32 µM. Incorporating BDT-4G into a prodrug has the potential to decrease the cytotoxicity of the oligoTEA while retaining antimicrobial activity. Prodrugs are biologically inactive molecules that are converted to an active pharmacological agent upon chemical conversion by metabolic processes in vivo¹⁰. Prodrugs are used to improve the selectivity, pharmacokinetics, or physicochemical properties of the parent drug¹¹. Activity blocking can be accomplished using an anionic moiety, polyethylene glycol (PEG) polymer, biomacromolecule (protein or antibody), or charge-protecting group¹⁰. Parent drugs can be released via cleavage of a linker attached to the inactivating moiety facilitated by either enzymatic or hydrolytic cleavage or nonenzymatic chemical reactions^12,13.

In this talk, I will discuss a prodrug strategy that reactivates BDT-4G in the presence of bacterium-specific enzymes and has the potential to reduce cytotoxicity and specifically target resistant bacteria¹⁴. Coupling BDT-4G to a bulky, hydrophilic, inert polymer such as PEG is hypothesized to form a hydration layer around the oligoTEA, resulting in steric shielding and an increased hydrodynamic volume¹⁵. This conjugation, termed PEGylation, has been reported to improve the pharmacokinetics of the parent drug by increasing drug stability and the retention time of the conjugates in blood¹⁶. To compensate for the loss in antimicrobial activity that accompanies PEGylation, BDT-4G was conjugated to PEG via a bacteria-susceptible cleavable substrate, which allowed for the controlled release of the oligoTEA in the presence of a targeted bacterial enzyme. To target P. aeruginosa, triglycine (Gly₃) was chosen as the peptide linker since it is cleaved exclusively by the secreted P. aeruginosa virulence factor, LasA^17–19.

Through cell growth kinetics assays the prodrug, PEG-Gly₃-BDT-4G, demonstrated antimicrobial activity against P. aeruginosa at 32 µM but was inactive against methicillin-resistant Staphylococcus aureus, illustrating that the linker could be cleaved specifically by P. aeruginosa proteases¹⁴. Cell viability assays showed that PEGylation improved the cytotoxicity (CC₅₀) of BDT-4G from 15 µM to 280 µM against human lung epithelial (A549) cells. Next, cleavage of a fluorogenic derivative of the triglycine peptide was detected via fluorescence resonance energy transfer (FRET). Rapid cleavage occurred within 15 minutes when the substrate was incubated in P. aeruginosa supernatant, however, the peptide was stable in serum. Liquid chromatography mass spectrometry confirmed that cleavage of the substrate occurred between the second and third glycine residues when the prodrug was incubated in purified LasA. Through FRET, 6 out of 10 clinical isolates of P. aeruginosa were found to contain detectable levels of LasA, and the prodrug displayed antimicrobial activity against the high-LasA-producing isolates. Overall, these studies broaden the therapeutic implications for oligoTEAs against MDR bacteria using a prodrug methodology which reduces cytotoxicity by greater than one order of magnitude in vitro. The bacteria-susceptible linker provides species specificity and facilitates the activation of BDT-4G against P. aeruginosa. The design of a prodrug that is cleaved by bacterial proteases offers the advantage of strain specificity and localized reactivation of the potent antimicrobial agent at the infection site, and this design can be translated to target other strains of MDR bacteria by altering the linker.

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