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Home , Sultamicillin, Tedizolid, Triclosan

Review

Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX pubs.acs.org/journal/aidcbc

Signed, Sealed, Delivered: Conjugate and Prodrug Strategies as Targeted Delivery Vectors for † † ‡ Ana V. Cheng and William M. Wuest*, , † Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States ‡ Emory Resistance Center, Emory School of Medicine, 201 Dowman Drive, Atlanta, Georgia 30322, United States

ABSTRACT: Innate and developed resistance mechanisms of bacteria to antibiotics are obstacles in the design of novel drugs. However, antibacterial prodrugs and conjugates have shown promise in circumventing resistance and tolerance mechanisms via directed delivery of antibiotics to the site of or to specific species or strains of bacteria. The selective targeting and increased permeability and accumu- lation of these prodrugs not only improves efficacy over unmodified drugs but also reduces off-target effects, toxicity, and development of resistance. Herein, we discuss some of these methods, including sideromycins, antibody-directed prodrugs, cell penetrating peptide conjugates, and codrugs. KEYWORDS: oligopeptide, sideromycin, antibody−antibiotic conjugate, cell penetrating peptide, dendrimer, transferrin

inding new and innovative methods to treat bacterial F comes with many inherent challenges in addition to those presented by the evolution of resistance mechanisms. The ideal antibiotic is nontoxic to host cells, permeates bacterial cells easily, and accumulates at the site of infection at high concentrations. Narrow spectrum drugs are also advantageous, as they can limit resistance development and leave the host commensal microbiome undisturbed.1 However, various resistance mechanisms make pathogenic infections difficult to eradicate: Many bacteria respond to antibiotic pressure by decreasing expression of active transporters and porins2 and 3,4 Downloaded via EMORY UNIV on April 18, 2019 at 12:34:17 (UTC). increasing expression of efflux pumps, making it difficult for drugs to infiltrate cells and achieve killing concentrations. Furthermore, several species of bacteria sequester themselves in human macrophages5 or in biofilms,6 leading to persistent ffi

See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. infections, which are di cult to with traditional . These problems can be complicated or even impossible to address with simple small molecules. An emerging strategy to overcome these challenges is the employment of antibiotic conjugates. In this Review, we will focus on the application of sophisticated conjugate strategies to enhance antibacterial activity. The conjugates covered enable temporary masking of activity through a cleavable prodrug linkage, directed delivery of a drug through conjugation, improved pharmacoki- netic and pharmacodynamic (PK/PD) profiles, or some combination of the three. Figure 1. Standard prodrugs.

■ STANDARD PRODRUGS revealed as precursor compounds, such as phenacetin (the By definition, a prodrug is an inactive compound that undergoes precursor to acetaminophen) and chloral hydrate (which is a chemical transformation in vivo to remove a covalently linked moiety and release the active form of the drug. Adrien Albert Received: January 15, 2019 coined the term in 1958 in reference to drugs that had been

© XXXX American Chemical Society A DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX ACS Infectious Diseases Review

Figure 2. Codrugs. Sites of cleavage are marked in red. converted by alcohol dehydrogenase to trichloroethanol, a drug to its eventual FDA approval in 2014.18 Other strategies for sedative) (Figure 1).7 Throughout history, several drugs have increased aqueous solubility include the addition of an ionizable been discovered in their prodrug form and were only later amine,21,22 succinic acid,23 sugars,24 and polyethylene glycol25 realized to be a precursor to the active compound. For instance, and the transformation of sulfides to sulfoxides.26 These groups Gerhard Domagk won a Nobel prize for the development of yield the active compounds upon O → N acyl migration,21 Prontosil (Figure 1), one of the earliest modern antibiotics.8 enzymatic ester23,27 or glycosidic hydrolysis,24 proteolytic However, it was later identified as a precursor to the actual active cleavage,28 or reduction.26 Conversely, when the lipophilicity compound, sulfanilamide, likely metabolized by azoreductases of a drug must be increased for improved passive permeability, produced either in the liver or by gut microbiota.9 Today, − hydrophobic promoieties that can be hydrolyzed by esterases or prodrugs are used for the treatment of cancer,10 12 neuro- peptidases may be appended to polar or ionized groups.29,30 logical13,14 and cardiovascular diseases,15,16 and bacterial Commonly, alkyl and aryl esters15,31 or N-acylated groups32 are infections. In the past decade, the US Food and Drug synthesized to this effect. These moieties can also aid in Administration (FDA) has approved 30 prodrugs, 10% of 17 − improving the metabolic stability of a drug, prolonging its which are for antibacterial applications.17 19 duration of action,33 or transport across the blood−brain Prodrug strategies have traditionally been applied to rescue barrier.34 20 prospective drugs with disfavorable PK/PD profiles; the The principles of simple prodrug design described above have interplay between the steric and electronic influences of a prompted the development of much more sophisticated chemical structure and its PK/PD profiles is not always antibiotic conjugates. In fact, many of the approaches described straightforward, so designing improved drug derivatives often hereafter take advantage of similar mechanisms of linker becomes a juggling act guided by the chemical intuition of cleavage (highlighted throughout): enzyme- or environment- medicinal chemists. Additionally, the optimization process can promoted drug release proves crucial to the success of many involve years of modification after each round of in vitro or in conjugates. vivo results. Prodrugs allow chemists to install a temporary moiety in a molecule, removable by enzymes or other ■ CODRUGS environmental conditions, to address one of the PK/PD 35 variables without affecting the activity and binding of the active Sometimes called mutual prodrugs, codrugs consist of two compound, saving time and resources in the drug development covalently linked entities whose cleavage releases two different process. active drugs. Codrugs benefit from improved PK/PD profiles in Depending on the active molecule under investigation, it may the same way that simple prodrugs do. Choosing the site of be desirable to increase hydrophilicity or lipophilicity to linkage on each compound carefully can mask labile groups, improve solubility or passive permeability, respectively. The which are otherwise prone to degradation. They also enjoy promoieties used in these situations tend to be small, simple in advantages over joint administration of separate therapies. For function, and relatively easy to introduce synthetically. For instance, the type of linker allows control over the site of release example, the addition of phosphate groups has greatly improved and can confer additional metabolic stability. The intertwined the aqueous solubility of several compounds; two of the three PK/PD properties of the linked drugs ensure the equal dosing of antibacterial prodrugs approved by the FDA in the past decade the two entities for full utilization of their tandem or synergistic are phosphate esters. (Figure 1)isanN- functions. Codrugs are an intriguing innovation, especially as phosphono prodrug of the novel derivative T-91825 combination therapies gain popularity. In addition to the for the treatment of -resistant benefits listed above, they have the potential to limit resistance (MRSA).17 The addition of an N-phosphono group, cleaved in development by inhibiting multiple targets. Unfortunately, their the body by plasma phosphatases, boosted the solubility of their application is limited to compatible therapies, which benefit lead compound from 2.3 mg/mL to >100 mg/mL. Similarly, from release in the same environment. Additionally, they are addition of a phosphate to tedizolid (Figure 1), an oxazolidinone mostly limited to 1:1 combinations, as it can become difficult to antibiotic for the treatment of several Gram-positive infections, rationally link more than two drug units. Consequently, this improved water solubility and facilitated the advancement of the limits combinations to drugs with similar potencies. There are

B DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX ACS Infectious Diseases Review limited examples of this technique, making codrugs a prime area membranes and resultant internalization. Unfortunately, hydro- for development. lytic drug release yielded squalenic acid as a byproduct, leading Codrugs were first introduced in 1980 with the development to high toxicity. Abed et al. hypothesized that shorter terpenes of a β-lactam/β-lactamase inhibitor (/) would limit cytotoxicity and instead synthesized acid labile codrug, sultamicillin (Figure 2).36 Ampicillin and sulbactam geranyl- and farnesyl- G (Figure 3).43 Indeed, the are poorly absorbed when administered separately, but geranyl-penG prodrug showed decreased toxicity in murine μ sultamicillin displayed fast absorption and enabled the joint macrophages (IC50 = 72.5 g/mL) compared to the equivalent β μ and equal administration of a -lactam and an inhibitor of its squalene conjugate (IC50 =18 g/mL). However, farnesyl-penG μ fi bacterial resistance mechanism. It also decreased and (IC50 =22 g/mL) displayed a similar pro le to squalene. , common side effects of the separately dosed drugs. Today, sultamicillin is prescribed for the treatment of a myriad ■ DENDRIMERS of infections, including skin, respiratory, and urinary tract Derived from the Greek word “dendron” for tree, den- infections caused by S. aureus, , 44,45 , Klebsiella, and Enterobacter. drimers pose an extremely versatile macromolecular drug Recently, Cacciatore et al. have investigated codrugs using the delivery system for hydrophobic small molecules or molecules phenolic monoterpenoid carvacrol as one of the that do not easily cross mammalian membranes. Mostly used in therapies (Figure 2).37 Known for its antifungal and antitumor cancer treatment, they are branching structures composed of activity, carvacrol also disrupts bacterial membranes. Through repeating units with many functionalizable sites. Dendrimers are conjugation with various sulfur compounds via an enzymatically named using several structural features: generations, core, and cleavable ester bond, antibacterial activity in S. aureus, Staph- monomers, although sometimes the core is omitted. The number ylococcus epidermidis, E. coli,andPseudomonas aeruginosa, of generations refers to the number of layers radiating from the fi core, and the type of monomer used to build the branches including some bio lm inhibition, was achieved. However, “ ” minimum inhibitory concentrations (MICs) were suboptimal, determines the family in which the dendrimer belongs (Figure 4a). There are three methods commonly used to conjugate requiring further analog development. Ester-linked codrug 47 fl drugs to dendrimers: noncovalent micellar encapsulation, strategies have also been applied to a eroxacin-desacetylcefo- 48 taxime conjugate (Ro 23-9424)38 and a ciprofloxacin- ionic coordination or chelation, and covalent prodrug desacetylcefotaxime conjugate (Ro-24-6392),39 which both attachment to the dendrimer edges (Figure 4a). While improved displayed broad antibacterial activity (Figure 2). intracellular accumulation is useful in itself, dendrimers can also be polyconjugated to both drugs and targeting moieties, ■ TERPENOYL NANOMEDICINES enabling the directed delivery of antibiotics. Kumar et al. accomplished this by noncovalently encapsulating rifampicin, a Although few examples exist, terpenoylation of antibiotics via hydrophobic antitubercular drug with low macrophage pene- pH-sensitive ester bonds takes advantage of the hydrophobic tration, in mannosylated dendrimers with five generations of polypropyleneimine branches building off an ethylene diamine core (G5 EDA-PPI) (Figure 4b).45 This strategy takes advantage of surface-bound mannose-binding proteins, which then facilitate receptor-mediated endocytosis. The conjugate experienced significantly increased concentrations of rifampicin in alveolar macrophages in comparison to free rifampicin, as well as superb drug release at pH 5.0 (pH of phagolysosomes). Additionally, researchers observed decreased toxicity in a Vero Figure 3. Terpenoyl prodrugs. Cleavage site marked in red. cell line. In another example of creative dendrimer engineering, a G5 ff poly(amidoamine) (PAMAM) dendrimer was covalently e ect to induce self-assembly of drug . This conjugated to photocaged ciprofloxacin and lipopolysaccharide strategy debuted in 2006 when squalene was used with the 40 (LPS)-binding groups (either B or ethanolamine) anticancer drug, gemcitabine. Squalene was later used in (Figure 4c).46 The LPS-binding groups direct the conjugate to conjugation with penicillin G (penG) for improved targeting of 41 membranes of Gram-negative bacteria, and then, exposure to intracellular S. aureus infections (Figure 3). Although S. aureus UVA light (365 nm) cleaves the photolabile ortho-nitrobenzyl is typically an extracellular pathogen, some subpopulations are fl 42 linker between the dendrimer and cipro oxacin. Although able to sequester themselves within phagolytic cells and use antibacterial activity was lower for the conjugate than for free them as vehicles to spread infection throughout the body. By ciprofloxacin, the lack of UV exposure had a limited effect on forming small colony variants and persisters, S. aureus can bacterial viability, validating the photocontrolled release of survive the hydrolytic enzymes that kill most bacteria in these antibiotic. Dendrimers have been utilized in many antifungal, phagolysosomes. Antibiotics cannot easily penetrate phagocytes antiviral, and anticancer applications, such as a dual drug and must be administered at unattainable concentrations to delivery dendrimer for leukemia.49 Inspiration can be taken from eradicate infection. The ∼140 nm nanoparticles created by ́ these innovations for the design of future antibacterial Semiramoth et al. were stable in water and decreased dendrimer conjugates. intracellular bacterial populations by 87%, compared to the 56% decrease accomplished by penG alone.41 Authors suggested the nanoparticles entered murine macrophages via both ■ CELL PENETRATING PEPTIDES clathrin-dependent and independent endocytosis. Squalene is Cell penetrating peptides (CPPs) are short 5−30 residue a precursor of sterols and can adopt a sterol-like conformation, cationic, amphipathic, or hydrophobic peptides capable of perhaps also contributing to its interaction with mammalian infiltrating cells without lysing (unlike antimicrobial peptides),

C DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX ACS Infectious Diseases Review

Figure 4. Dendrimer antibiotic conjugates. (a) General dendrimer structure and conjugation methods. (b) G5 EDA-PPI dendrimer.45 (c) Gram- negative targeting G5 PAMAM dendrimer/ciprofloxacin prodrug.46 making them especially useful for intracellular infections. First a noncleavable variant, demonstrated the need for drug release discovered in 1988,50 CPPs have been shown to smuggle various to realize full synergistic bacterial killing. Additionally, P14KanS molecules into cells through either covalent linkage or caused significant reduction in S. aureus biofilms, intracellular electrostatic binding. These advances have opened the door to S. aureus, and planktonic MRSA and methicillin-susceptible a variety of payload-delivering CPP applications, including the Staphylococcus aureus (MSSA) strains.55 delivery of imaging agents,51 tumor therapies,52 and antibiotic Similarly, Li and co-workers were able improve the targeting − prodrugs.53 57 However, they still display low cell specificity and of intracellular Salmonellae Typhimurium with marine anti- are susceptible to proteases. Furthermore, to the best of our microbial peptide N6 by attaching a CPP (Tat11) via a 56 knowledge, little is known of resistance development to CPPs, cathepsin-cleavable linker. They also observed improved which will inevitably be a hurdle in the advancement of these stability of the conjugate with C-terminal amidation of the antimicrobial peptide. The Kelley group used two peptides to prodrugs. 57 Using a previously developed CPP, P14LRR,53 to target achieve the killing of intracellular infection. was ffi intracellular bacteria (discussed in the previous section), the covalently a xed to a cationic delivery peptide to facilitate fi bacterial cell penetration. However, an additional anionic Seleem group created a disul de-linked P14LRR-kanamycin β prodrug conjugate,54 dubbed P14KanS (Figure 5a). The peptide, attached by a -lactamase-cleavable linker, enabled endocytosis in host macrophages (Figure 5b). proline-rich CPP adopts a helical conformation, and added − cationic and hydrophobic character from guanidino and isobutyl The delivery peptide methotrexate hybrid (dpMtx) also served groups enables it to penetrate mammalian membranes without to temper the toxicity of methotrexate to murine macrophages and was able to eradicate intracelluluar Mycobacterium smegmatis lysing, reaching intracellular bacterial infections. P14KanS was while methotrexate alone was not. designed to be cleaved in the reducing environment of mammalian cells to release P14LRR and kanamycin, both of which have antimicrobial activity. Thus, P14KanS may also be ■ OLIGOPEPTIDES − classified as a codrug. Clearance of macrophage-inhabiting Membrane-bound oligopeptide permeases58 60 facilitate the Mycobacterium tuberculosis, Salmonella enteritidis, and Brucella uptake of a wide range of 2−8 length L-amino acid residues from abortus was observed in addition to reduction of Salmonella in an the environment. As a result, bacteria have cleverly taken in vivo Caenorhabditis elegans model. A comparison to P14KanC, advantage of this active transport by synthesizing and excreting

D DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX ACS Infectious Diseases Review

Figure 5. Cell penetrating peptide conjugates. (a) P14KanS. (b) Double peptide methotrexate prodrug.

Figure 6. Oligopeptide prodrugs. (a) Natural oligopeptide prodrugs. (b) Synthetic oligopeptide prodrugs. small antibacterial molecules linked to short peptides that are glucosamine 6-phosphate synthase. Bacilysin is produced by transported by the permeases of other species and then typically Bacillus subtilis and displays activity in many bacteria and fungi. (but not always) hydrolyzed by intracellular peptidases to Another B. subtilis oligopeptide, Rhizocticin A, and release the active drug. Unfortunately, resistance to these plumbeus’ Plumbemycin A are both phosphono- prodrugs is quickly developed as the permeases are not essential oligopeptides with a C-terminal (Z)-L-2-amino-5-phosphono-3- to survival. pentenoic acid (APPA) moiety.62 APPA is an irreversible A multitude of naturally occurring oligopeptide−antibiotic inhibitor of threonine synthase, an enzyme critical to bacteria, conjugates (Figure 6a) have been discovered and studied, such plants, and fungi but not found in humans. Once internalized, 61 as bacilysin, a simple dipeptide prodrug with an N-terminal L- these oligopeptides are hydrolyzed by peptidases to release alanine linked to L-anticapsin, a nonproteinogenic amino acid active (S,Z)-APPA. Other natural oligopeptide drugs include that functions as an analog of glutamine to covalently inhibit alafosfalin63 (prodrug,cellwallsynthesisinhibitor)and

E DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX ACS Infectious Diseases Review

Figure 7. Sideromycins. (a) Natural sideromycins. (b) Biscatecholate siderophore−oxazolidinone prodrug.82 (c) Enterobactin−ciprofloxacin prodrug.83

Figure 8. Siderophore ligands. (a) Three main classes of siderophore ligands. (b) Mixed β-lactam sideromycin.

64 tabtoxin (prodrug, glutamine synthetase inhibitor), among potent inhibitors of 1-deoxy-D-xylulose-5-phosphate synthase. others. However, their use was hindered by low bacterial cell uptake. By Utilization of active transport of oligopeptides has also synthesizing a peptidic enamide-alkyl acetylphosphonate inspired synthetic conjugates (Figure 6b). As early as 1989, di- prodrug, they achieved an impressive 2000-fold activity increase and tripeptide conjugates of sulfanilic acid were synthesized to in E. coli.66 These results are a testament to the power of increase its permeation into E. coli, resulting in a 207-fold antibiotic conjugates to improve in vivo activity of small enhancement in activity.65 More recently, Bartee et al. identified molecules.

F DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX ACS Infectious Diseases Review

Figure 9. Antibody−antibiotic conjugates. (a) Mechanism of AAC action in intracellular bacterial infections. (b) Cathepsin-cleavable AAC linker.101 ■ SIDEROMYCINS >125-fold improvement in MIC (0.4 μM) over oxazolidinone μ β Living organisms require iron to serve as an agent in redox alone (>50 M) when evaluated against -lactamase-expressing metabolic pathways; however, bacteria often inhabit environ- Acinetobacter baumannii. These results are especially impressive ments that are especially iron poor. To obtain iron, they because oxazolidinones cannot typically permeate the outer biosynthesize and secrete small iron-chelating compounds membranes of Gram-negative cells to reach their ribosomal (siderophores)67 to retrieve Fe3+ from their surroundings and target, limiting their activity to Gram-positive species. Addi- 68 tionally, β-lactamase dependence confers extra selectivity for return it to the cell. The iron-bound siderophores are fi recognized by membrane-bound receptors and then imported drug-resistant targets. For even more speci c intracellular − fl by active transporters. For a competitive advantage, bacteria also release, Neumann et al. designed an enterobactin cipro oxacin conjugate (Figure 7c) selectively hydrolyzed by certain express receptors for siderophores produced by other micro- 83 organisms (xenosiderophores).69 In response to this “theft,” pathogenic strains of E. coli. This prodrug is not activated by some microbes have evolved to excrete siderophore-linked the common enterobactin hydrolase, Fes, but instead by the fi antibiotics called sideromycins, which competitors unwittingly pathogenic strain-associated hydrolase IroD. Speci c linkers like import, causing cell death.70,71 Although resistance may develop those above enable selective targeting of resistant and through mutations in siderophore receptors, it comes at a pathogenic strains using broad-spectrum drugs. significant fitness cost, as bacteria with deficient siderophore Typical siderophores are split into three main classes uptake pathways suffer iron starvation.72 Inspired by this according to their iron-binding motifs: catecholates, hydrox- strategy, researchers have developed synthetic “Trojan horse” amates, or α-hydroxy carboxylates (Figure 8a). Mixed ligand drugs73 to counter resistance mechanisms that decrease siderophores can be recognized by multiple receptors, antibiotic uptake. Because mammals do not use siderophores, mitigating the risk of resistance development. The Miller these prodrug conjugates selectively target bacteria. Further, group has demonstrated that mixed ligand biscatecholate− species-specific siderophores may confer an added level of monohydroxamate antibiotic conjugates (Figure 8b) had selectivity.74 comparable or better MICs than single-type ligands.72,84,85 Albomycins (Figure 7a) are naturally occurring sideromycins Although these conjugates were not prodrugs, as many β-lactam comprised of a ferrichrome-like siderophore covalently linked drugs do not require cleavage for antibacterial activity,79 they via a hydrolyzable serine to a thioribosyl pyrimidine antibiotic. present an interesting future avenue for sideromycin explora- Upon cleavage by bacterial peptidase N, the antibiotic inhibits tion.86 Further, artificial siderophores (“sideromimics”) have transfer ribonucleic acid (tRNA) synthetase.70 Deletion of been synthesized in nonlabile conjugation with monocarbams,87 either the uptake machinery or hydrolytic enzyme results in sulfactams,88 ,89 and lactivicin.90 resistance to albomycin.75 Isolated from Streptomyces sp., they have shown activity against Enterobacteriaceae, S. aureus, and ■ TRANSFERRINS S. pneumoniae.76 Another class of natural sideromycins, the salmycins (Figure 7a), were isolated from Streptomyces violaceus Transferrins (Tfs) are glycoproteins involved in the transport in 1995.77 These conjugates kill Staphylococci and Streptococci and sequestration of iron found in vertebrates. They can exhibit through the release of an antibiotic. bacteriostatic activity due to iron chelation, which withholds 91 The investigation of synthetic siderophore−antibiotic con- essential iron from growing bacteria. However, bacterial jugates with cytoplasmic targets has demonstrated the necessity transferrin binding protein A (TbpA) recognizes Tf and actively of drug cleavage for full antibacterial activity,78,79 leading to the removes and internalizes its iron. As it turns out, Tf and TbpA employment of various linkers. Some favorable results have been have a long history of competitive evolution as detailed in a obtained using esterase-cleavable thiol-maleimide80 and “tri- review by Barber and Elde.92 Nonetheless, researchers have used methyl-lock”-based linkers.81 However, these may still be TbpA to their advantage by using Tfs as drug carriers through activated by acid or extracellular esterases before entering the either covalent linkage or encapsulation of hydrophobic and target bacteria. Recently, Liu et al. designed a siderophore− aromatic small molecules. However, it is important to note that, cephalosporin−oxazolidinone conjugate that addresses this without protein engineering, covalent linkage runs the risk of issue (Figure 7b).82 By utilizing a covalent cephalosporin linker attaching antibiotic to a position important for Tf−receptor that can be cleaved by periplasmic β-lactamases, they achieved a interaction. Additionally, Tfs are useful for the treatment of

G DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX ACS Infectious Diseases Review

Table 1. Miscellaneous Prodrugs and Conjugates

intracellular infections93 since host cells perform Tf−receptor- limiting their usefulness. Triclosan is similarly restricted. mediated endocytosis.94 However, noncovalent complexation of these antibiotics with Sulfonamides, some of the earliest antibiotics, target ovotransferrin (OTf, found in bird and reptile eggs), achieved dihydrofolate reductase. Unfortunately, they are poorly soluble through incubation of OTf in excess antibiotic over 24 h, and highly toxic with painful side effects such as kidney stones, improved activity against various bacteria, including E. coli,

H DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX ACS Infectious Diseases Review P. aeruginosa, S. epidermidis, S. aureus, Neisseria mucosa, and ■ MISCELLANEOUS PRODRUGS AND DELIVERY some intracellular infections.95 OTf alone lacked activity at STRATEGIES − equivalent concentrations to the antibiotic OTf complexes, Many other novel conjugates and prodrug methods have been indicating that the antibiotics are internalized by the bacteria fi ’ explored, including several that do not t the categories separate from OTf, possibly through a similar mechanism as Tf s described thus far or have not yet been applied to antibacterials. native substrate, iron. So as not to omit these innovations and to provide inspiration An analogous approach was used to tackle Chlamydia for future drug delivery development, we have summarized them trachomatis, an obligate intracellular pathogen responsible for in Table 1. sexually transmitted infections. This bacterium’s clever life cycle permits it to reproduce only in vacuoles within host cells, while it ■ CONCLUSIONS exists as a metabolically inert form when traveling from cell to ffi cell. Thus, the metabolically active form of C. trachomatis is Antibiotic prodrugs and conjugates aid in addressing di cult-to- treat bacterial infections, such as intracellular and persistent shielded within a vacuole inside host cells, requiring antibiotics infections. By carefully choosing a linker or a directing moiety to with exceptional cell penetration ability. Hai et al. addressed this − 93 build an antibiotic prodrug or conjugate, it is possible to induce with a covalent serum Tf conjugate. Coupling was fi fi site-speci c release of active drug and/or improved accumu- con rmed with mass spectrometry, which indicated the lation at the site of infection, opening doors to many would-lead presence of one antibiotic per Tf. The resulting conjugate was ff fi ff compounds su ering from PK/PD limitations or lack of signi cantly more e ective than amoxicillin against intracellular specificity. In doing so, we can also limit off-target effects and C. trachomatis infections. toxicity or expand the spectrum of activity of known drugs. Although some of the described strategies have been used for ■ ANTIBODY-DIRECTED PRODRUGS several decades and are popular in anticancer, antiviral, and By directing broad-spectrum drugs to antigen-possessing antifungal therapies, their employment in antibiotics is less species, antibody−antibiotic conjugates (AACs) have the extensive but promising. Prodrugs and directing strategies pose a potential to avoid unnecessary killing of commensal bacteria. supplementary strategy for addressing the increasingly urgent issue of antibiotic resistance. We can take inspiration from These conjugates have also proven useful in the treatment of fi intracellular infections (Figure 9a). Although there are few existing approaches and apply them to bacteria-speci c receptors and active transport. Combined with the constant examples of antibody−antibiotic prodrugs, there has been study search for new antibiotics, this research will expand our of the site-dependent stability of antibody-linker linkage,96,97 − repertoire of antibacterial therapies. and novel linkers are in development.98 100 In 2015, a group at Genentech successfully applied an ■ AUTHOR INFORMATION antibody−antibiotic conjugate (AAC) prodrug to the treatment of intracellular S. aureus in mice,101 which has now completed Corresponding Author phase I clinical trials. Lehar et al. administered , *E-mail: [email protected]. , , and rifampicin and, although these drugs ORCID handily defeated extracellular MRSA, they were unable to treat William M. Wuest: 0000-0002-5198-7744 murine macrophage intracellular bacteria even at the maximum Author Contributions serum concentration.101 However, application of an optimized A.V.C. conceptualized the manuscript. A.V.C. and W.M.W. AAC prodrug composed of THIOMAB (an anti-S. aureus wrote the manuscript. antibody) covalently tethered via a cathepsin-cleavable linker (Figure 9b) to rifampicin decreased amounts of MRSA in Notes fi murine macrophages to below the detectable limit. While The authors declare no competing nancial interest. rifampicin cannot penetrate the membranes of macrophages due to insufficient lipophilicity, the AAC can easily accumulate at ■ ACKNOWLEDGMENTS killing concentrations. It has been suggested that this approach We gratefully acknowledge funding from the National Institute could also be applied to M. tuberculosis infections.102 of General Medical Sciences (GM119426), National Science In another antibody-based approach, the use of photo- Foundation (CHE1755698), and the Georgia Research Alliance dynamic therapy to target P. aeruginosa was preliminarily based in Atlanta, Georgia. We also would like to thank Dr. investigated.103 The conjugate is composed of an antibody William Shafer, Dr. Justin Shapiro, Dr. Taylor Hari, and Dr. linked via an ethylenediamine spacer to several photosensitizers Colleen Keohane for their feedback on the manuscript. which, upon irradiation with light, release singlet oxygen and trigger death in nearby cells. Despite being highly reactive, ■ ABBREVIATIONS singlet oxygen has a short lifetime that limits its activity to an AAC, antibody−antibiotic conjugate; AMP, antimicrobial area within 1000 Å of its generation, and the use of an antibody peptide; CPP, cell penetrating peptide; EDA, ethylene diamine; directs the formation of this unstable species to the site of FDA, US Food and Drug Administration; LPS, lipopolysac- infection. The conjugates were stable both in vitro and in an in charide; MIC, minimum inhibitory concentration; MRSA, vivo rat thigh abscess model. However, bacterial killing required methicillin-resistant Staphylococcus aureus; MSSA, methicillin- saturated concentrations of conjugate due to the difficulty of susceptible Staphylococcus aureus;OTf,ovotransferrin; lysing cells with thick cell walls. Another in vivo study of a PAMAM, poly(amidoamine); PD, pharmacodynamic; penG, different photosensitizer immunoconjugate demonstrated 75% penicillin G; PK, pharmacokinetic; PPI, polypropyleneimine; killing of P. aeruginosa in mice,104 and two additional antibody− TbpA, transferrin binding protein A; Tf, transferrin; tRNA, photosensitizer conjugates have since been tested.105,106 transfer ribonucleic acid; UV,

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M DOI: 10.1021/acsinfecdis.9b00019 ACS Infect. Dis. XXXX, XXX, XXX−XXX