Microbial esterases and ester prodrugs: An unlikely marriage for combating antibiotic resistance

Abstract

The rise of antibiotic resistance necessitates the search for new platforms for drug development. Prodrugs are common tools for overcoming drawbacks typically associated with drug formulation and delivery, with ester prodrugs providing a classic strategy for masking polar alcohol and carboxylic acid functionalities and improving cell permeability. Ester prodrugs are normally designed to have simple ester groups, as they are expected to be cleaved and reactivated by a wide spectrum of cellular esterases. However, a number of pathogenic and commensal microbial esterases have been found to possess significant substrate specificity and can play an unexpected role in drug metabolism. Ester protection can also introduce antimicrobial properties into previously nontoxic drugs through alterations in cell permeability or solubility. Finally, mutation to microbial esterases is a novel mechanism for the development of antibiotic resistance. In this review, we highlight the important pathogenic and xenobiotic functions of microbial esterases and discuss the development and application of ester prodrugs for targeting microbial infections and combating antibiotic resistance. Esterases are often overlooked as therapeutic targets. Yet, with the growing need to develop new antibiotics, a thorough understanding of the specificity and function of microbial esterases and their combined action with ester prodrug antibiotics will support the design of future therapeutics.

Keywords: Mycobacterium tuberculosis; antibiotic resistance; drug delivery; drug design; esterases; prodrugs.

Figure 1:

Bacterial esterase structure and function. A) Classic α/β-hydrolase structural fold present in many bacterial esterases.[Carr and Ollis 2009; Holmquist 2000] This α/β-hydrolase structural fold is composed of a left-handed, twisted β-sheet surrounded by α-helices and encircling a centrally located catalytic triad.[Carr and Ollis 2009] The bacterial esterase, LipW from M. tuberculosis (PDB ID: 3QH4) is shown as an example with its β-sheets in tan, its α-helices in purple, and its catalytic triad in sticks and labeled. The catalytic serine of LipW was observed in dual rotameric conformations.[McKary et al. 2016] These conformations have been observed in other α/β-hydrolase structures and have been related to the catalytic mechanism of esterases, including facilitating product release and inhibiting the reverse reaction.[McKary et al. 2016] B) Close up of the catalytic triad from LipW with the hydrogen bonding network that facilitates the nucleophilicity of the catalytic serine shown with black dotted lines.[McKary et al. 2016] Colored identically to A. C and D) Substrate differentiating cap/lid structure of α/β-hydrolases. Surface structures of LipW (C)[McKary et al. 2016] and Rv0045c (D; PDB ID: 3P2M) [Zheng et al. 2011]; two Mtb esterases with varying substrate specificity and binding pocket structures.[Lukowski et al. 2014; McKary et al. 2016] Cap/lid regions are shown in blue with the remaining residues in gray. The catalytic triad is shown in sticks and is colored by atom type. E) Full-length autotransporter EstA from P. aeruginosa (PDB ID: 3KVN).[van den Berg 2010] The N-terminal domain (green) encodes the esterase functionality with the membrane spanning β-barrel (red) domain connected to the esterase domain through an extended kinked helix (blue). F) The GSDL protein fold. The GSDL protein fold exemplified by the esterase domain of EstA contains a three-layered α/β/α structure with five β-strands and at least four α-helices.[van den Berg 2010; Wilhelm et al. 2011] EstA is colored identically to E. The catalytic triad is shown in sticks and is colored by atom type.

Figure 2:

Profiling bacterial esterases. A – D) Activity based protein profiling (ABPP) ligands used to characterize proteome-wide esterase activity in Mycobacteria and to identify esterase activity present under disease relevant growth conditions.[Lehmann et al. 2018; Ortega et al. 2016; Ravindran et al. 2014; Tallman et al. 2016b] Each ligand contains an electrophilic moiety (labeled in red) for covalent labeling of esterases and an alkyne for isolation and identification of labeled esterases by click chemistry. A) Tetrahydrolipstatin (THL)-alkyne.[Ravindran et al. 2014] B) Modified version of THL-alkyne (EZ120) designed to mimic mycolic acids on the Mtb mycomembrane.[Lehmann et al. 2018] C) Fluorophosphonate-PEG-alkyne.[Ortega et al. 2016] D) Desthiobiotin-fluorophosphonate.[Tallman et al. 2016b] E and F) Complementary fluorogenic ester substrates used to measure dynamic esterase activity under disease-related growth conditions.[Bassett et al. 2018; Tallman and Beatty 2015; Tallman et al. 2016a; Tallman et al. 2016b] Cleavage of ester moieties (labeled red) by a bacterial esterase transitions the fluorophore from a stable non-fluorescent form into a highly fluorescent state. Changing the ester moieties and screening the resulting fluorogenic libraries have identified ester moieties with specificity to pathogenic Mycobacteria species (E)[Tallman et al. 2016a; Tallman et al. 2016b] and to dormant growth conditions (F).[Bassett et al. 2018] E) DDAO (7-hydroxy-9H-1,3-dichloro-9,9-dimethylacridin-2-one)-acyloxymethyl ether probe.[Tallman et al. 2016a; Tallman et al. 2016b] The eight-carbon acyloxymethyl ether probe was the most selective for pathogenic Mycobacteria esterases. F) Fluorescein acyloxymethyl ether esterase probe.[Bassett et al. 2018] A variety of hydrophobic and long-chain esters, including a fluorescein bis((4-methyl)valeryloxymethyl ether) derivative, were selectively activated under nutrient starvation growth conditions. G) Two anti-malarial prodrugs selectively activated by PfPARE.[Istvan et al. 2017] The ester prodrug moiety that increases cell permeability and is selectively removed by PfPare within P. falciparum is shown in red.

Figure 3:

Classic, clinical, and pre-clinical ester prodrugs. A) Colistin, colistin methanesulfonate (CMS),[Bergen et al. 2006] and the mono-aaPEG polymer (col-aaPEG).[Zhu et al. 2017] The PEG esters are hydrolyzed by plasma esterases at 37°C to yield colistin. B) Ethambutol and ethambutol esters. Ester prodrugs are inactive against mycobacteria until hydrolyzed by an esterase. R groups include alkyl chains, cycloalkyl groups and branched alkyl functionalities.[Larsen et al. 2017] C) The pyrazinamide (PZA) and pyrazinoic acid (POA) ester activation pathways. POA esters demonstrate activity against resistant strains of mycobacteria that lack pyranizamidase (PZAse), instead being activated by internal esterases. The “duplicated” prodrug 2 shows increased efficacy over 1 due to increased intracellular concentrations of POA.[Segretti et al. 2016] D) Core structures of the penicillins and cephalosporins, along with ampicillin and its ester prodrugs. The different ester moieties are designed to improve lipophilicity and oral bioavailability. [Bodin et al. 1975; Clayton et al. 1976; Sakamoto et al. 1984; Sjövall et al. 1978] E) Ciprofloxacin and various ester prodrugs and conjugates. PEGlyated prodrugs are designed to disrupt crystallinity and enhance solubility.[Assali et al. 2016] The ciprofloxacin-anhydride coating is engineered to be cleaved by extracellular esterases,[Komnatnyy et al. 2014] and the nanoantibiotic is designed to be cleaved by internal esterases following disruption of the bacterial cell wall by the carbon nanotube.[Assali et al. 2017] F) Fosimydomycin, FR900098, and two FR900098 phosphonate esters. The acetyloxyethyl ester demonstrates enhanced activity against malaria due to increased oral absorption,[Phillips et al. 2015] while the pivaloyloxymethyl (POM) ester activates FR900098 against Mtb.[Uh et al. 2011]

Figure 4:

Novel ester prodrugs and mechanisms. A) Decomposition of the ciprofloxacin-enterobactin conjugate 3. IroD-catalyzed hydrolysis breaks apart the siderophore, leaving behind the monocatecholate product 4.[Neumann et al. 2018] B) Ester prodrugs of the metalloproteinase proinhibitor PY-2: methyl ester 7, benzyl ether linked 8, and catechol linked 9. C) Mechanism of ester-responsive trigger for 8. Hydrolysis of the protecting methyl ester leads to decomposition of the benzyl ether, yielding the parent inhibitor.[Perez et al. 2013] D) ELQ-300 and its carbonate and alkoxycarbonate esters. The ester functionality disrupts the planar compound and decreases crystallinity, which enhances oral absorption.[Frueh et al. 2017] E) Release mechanism for metal-free CO prodrugs. Esterase activation opens the 7-membered lactone ring, freeing the alkyne functionality to undergo Diels-Alder cycloaddition with the cyclopentadienone core. The intermediate rearranges to release CO and the final cyclization product.[Ji et al. 2017]