Restoring susceptibility to aminoglycosides: identifying small molecule inhibitors of enzymatic inactivation

Abstract

Growing resistance to antimicrobial medicines is a critical health problem that must be urgently addressed. Adding to the increasing number of patients that succumb to infections, there are other consequences to the rise in resistance like the compromise of several medical procedures and dental work that are heavily dependent on infection prevention. Since their introduction in the clinics, aminoglycoside antibiotics have been a critical component of the armamentarium to treat infections. Still, the increase in resistance and their side effects led to a decline in their utilization. However, numerous current factors, like the urgent need for antimicrobials and their favorable properties, led to renewed interest in these drugs. While efforts to design new classes of aminoglycosides refractory to resistance mechanisms and with fewer toxic effects are starting to yield new promising molecules, extending the useful life of those already in use is essential. For this, numerous research projects are underway to counter resistance from different angles, like inhibition of expression or activity of resistance components. This review focuses on selected examples of one aspect of this quest, the design or identification of small molecule inhibitors of resistance caused by enzymatic modification of the aminoglycoside. These compounds could be developed as aminoglycoside adjuvants to overcome resistant infections.

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Introduction

Aminoglycoside antibiotics became a viable therapy to treat infections in 1944 when streptomycin was introduced for clinical use. In November of that year, a woman with tuberculosis became the first patient to be treated with an aminoglycoside. The treatment was successful, and the patient lived an active life.¹ From that moment until now, new aminoglycosides, discovered or generated by modification of natural ones (semisynthetic), were used alone or in combination therapies to treat tuberculosis and numerous other infections.^2–4 Aminoglycoside antibiotics are primarily used against infections caused by Gram-negative aerobic bacilli, staphylococci, and other Gram-positives.^4–6 Examples of causative agents of infections that are treated with aminoglycosides include, but are not limited to, Enterobacterales, Pseudomonas, Acinetobacter, Yersinia pestis, Francisella tularensis, and Staphylococcus.^4–8 Besides their utilization against bacterial infection, some aminoglycosides are also used to treat cryptosporidiosis, leishmaniasis, and other diseases caused by protozoa and cestodes.^9,10 The ability of aminoglycosides to suppress premature translation-termination codon caused by missense mutations led to their use as a treatment for other conditions like cystic fibrosis, Duchenne muscular dystrophy, Ménière's disease, and others.^11–18 Aminoglycosides are also being researched as gene delivery vectors for gene therapies.^19,20

The chemical structure of aminoglycosides consists of an aminocyclitol nucleus (streptamine, 2-deoxystreptamine, or streptidine) linked to amino sugars through glycosidic bonds. Exceptions to this rule are spectinomycin, an aminocyclitol not linked to amino sugars, or compounds that include the aminocyclitol fortamine.^4,6,21,22 Initially, all aminoglycosides were of natural origin, isolated from soil bacteria belonging to the genera Streptomyces (named with the suffix “-mycin”) or Micromonospora (named with the suffix “-micin”). However, the development of resistance together with the aminoglycosides' relatively high toxicity, led to the development of semisynthetic molecules that are less toxic or refractory to one or more resistance mechanisms (see the representative referenced articles and reviews for a more extensive description).^23–36 Examples of aminoglycoside structures are shown in Fig. 1.

Fig. 1 Chemical structures of representative aminoglycosides. Representative structures of aminoglycosides, which contain an aminocyclitol nucleus (colored) linked to amino sugars through glycosidic bonds.⁶ Spectinomycin contains an aminocyclitol not linked to amino sugars.²²

When aminoglycosides are in the environment that surrounds bacterial cells, they are attracted to the negatively charged bacterial surface. The interactions between the polycationic aminoglycosides and the anionic cell surface components result in increased permeability, permitting the energy-independent internalization of a limited number of molecules into the periplasm.³⁷ Two energy-dependent phases follow this phase. The energy-dependent phase I is characterized by the penetration to the cytoplasm of a small number of aminoglycoside molecules. This process requires a functional electron transport system.^38–40 The need for a functional electron transport system determines that, generally, aminoglycosides are ineffective against anaerobic bacteria.⁴¹ Once inside the cytosol, the aminoglycoside molecules bind the ribosomes and induce mistranslation. In the energy-dependent phase II, faulty membrane proteins damage the cytoplasmic membrane integrity allowing the access of a large number of aminoglycoside molecules to the cytosol.⁴⁰ As the number of aminoglycoside molecules increases within the cytosol, the number of mistranslated proteins grows to enhance the membrane's damage, resulting in a yet faster uptake that ultimately results in cell death.^40,42,43

Through binding the 30S ribosome subunit, aminoglycosides induce a modification of the conformation of the A site, making it closer to that adopted after the interaction between an mRNA and its cognate tRNA. This process reduces the ribosome proofreading properties, increasing mistranslation.^44–48 Although all aminoglycosides cause mistranslation, not all bind to identical sites of the 16S rRNA. Furthermore, aminoglycosides produce other effects like inhibition of assembly of the 30S ribosomal subunit (detected for the aminoglycosides neomycin and paromomycin), various disruptions of ribozyme activities, modification of transcription rates, and interference with the formation of the Z ring, these latter two effects at sublethal concentrations of the antibiotic.^49–53

Aminoglycosides have numerous advantages as anti-infectives. Besides being highly efficacious, their properties include broad-spectrum, rapid action, low allergies, and low interactions with other medicines. However, a combination of factors like their side effects, development of resistance, and inconvenient routes of administration diminished the interest in this kind of antimicrobials.²⁵ Aminoglycosides' main side effects are ototoxicity, nephrotoxicity, and, less commonly, neuromuscular blockade.^{5,25,28,54–56} Ototoxicity may result in permanent hearing loss caused by damage to the inner ear sensory hair cells.⁵⁶ Nephrotoxicity is usually reversible; the most common manifestation is nonoliguric acute kidney injury (due to decreased glomerular filtration).⁵⁵ Fortunately, several studies are being conducted to find drugs to protect against these undesirable effects.^57–59

Aminoglycoside resistance mechanisms, which in many cases coexist in the same bacterial cells,^48,60,61 include enzymatic inactivation by modifying enzymes;^5,62–64 methylation of 16S rRNA;^65,66 mutation of the 16S rRNA or ribosomal proteins to prevent binding;⁶⁵ reduced uptake that can occur by reduced inner membrane transport or modification of outer membrane's permeability;⁶⁷ export outside the cell by efflux pumps,⁶⁸ and other more rare mechanisms like active swarming or sequestration of the drug by tightly binding to an acetyltransferase of deficient activity.⁶⁹ Of all these mechanisms, the antibiotic's enzymatic inactivation mediated by aminoglycoside modifying enzymes, which catalyze the transfer of acetyl, nucleotidyl, or phosphate groups to –OH or –NH₂ positions of the aminocyclitol or the sugar moieties of the molecules, is the most prevalent in the clinical setting.^{5,28,62–64,70}

The current antibiotic resistance crisis has reawakened interest in aminoglycoside antibiotics.^6,25,60 There are renewed efforts to repurpose aminoglycosides not yet used in humans like apramycin⁷⁰ or design new molecules refractory to resistant mechanisms that also have lower toxicity. A product of these efforts is the recent approval for human use of plazomicin.⁷¹ Another strategy to preserve aminoglycosides as part of the armamentarium against infections is the development of inhibitors of the resistance that, in combination with an antimicrobial, would be active against resistant pathogens.⁶¹ Although aminoglycoside resistance inhibitors have not progressed beyond the experimental laboratory phase, the success obtained with similar strategies in the case of inhibitors of β-lactamases, which are already part of combination therapies with β-lactams,⁷² strongly suggests that it is a viable option in the fight against aminoglycoside resistance. The main strategies to overcome aminoglycoside modifying enzymes-mediated resistance consist of designing compounds that prevent the expression of resistance genes, such as antisense compounds, or inhibit the enzymatic inactivation of aminoglycosides (Fig. 2). However, other possibilities like identifying compounds such as the proline-rich antimicrobial peptide OM19r, which augment the antibacterial activity of aminoglycosides turn them effective against resistant strains are also being researched.⁷³

Fig. 2 Strategies to reduce aminoglycoside modifying enzymes-mediated resistance to aminoglycoside antibiotics. The yellow circle points to the focus of this review article.

Numerous antisense-mediated mechanisms of inhibition of gene expression have been explored to interfere with translation by steric hindrance or inducing degradation of the mRNA.^74–82 While most attempts to use antisense in bacterial therapeutics target essential genes, i.e., the antisense would be the antibiotic, another less utilized path is to target resistance genes and combine the antisense with the cognate antibiotic. Antisense oligonucleotide analogs were used to silence genes coding for resistance to numerous antibiotics like β-lactams,^83–85 carbapenems,^86,87 chloramphenicol,^88,89 fluoroquinolones,^86,90 aminoglycosides,^5,75 and others. Experiments on antisense inhibition of resistance to aminoglycosides mediated by the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] produced promising results.^75,91,92

Reversal of resistance mediated by aminoglycoside modifying acetyltransferases was observed in the presence of some cations (Fig. 2), which interfere with the acetylating reaction.^93,94 In the cases of Cu²⁺ and Zn²⁺, the concentrations required to observe a phenotypic conversion to susceptibility were significantly reduced when coordination complexes of the cations and ionophores were added to the culture medium.^94–99 The mechanism of inhibition of resistance remains to be understood. However, an attractive hypothesis is that the antibiotic in complex with the metal ion becomes a poor substrate.⁶¹

A third strategy to counter resistance mediated by aminoglycoside modifying enzymes is the design or identification of enzymatic inhibitors. Several research groups made valuable contributions, and numerous inhibitors with diverse effectiveness have been studied. This review describes representative recent advances in the quest for identifying or designing enzymatic inhibitors of aminoglycoside modifying enzymes. For more extensive descriptions of inhibitors of resistance to aminoglycosides, the reader is referred to other recent review articles.^{28,29,60,61,63,64,100–106}

Bisubstrate prodrugs

The aminoglycoside 6′-N-acetyltransferase type Ii [AAC(6′)-Ii] is an enzyme coded for by a gene located in the chromosome of Enterococcus faecium, which makes this Gram-positive pathogen resistant to several aminoglycosides.¹⁰⁷E. faecium, together with E. faecalis, causes the vast majority of enterococcal infections.¹⁰⁸ Early strategies to characterize AAC(6′)-Ii led to the synthesis of aminoglycoside-CoA bisubstrates,¹⁰⁹ valuable compounds in prior studies on other GCN5-related acetyltransferases that were also inhibitors of their cognate enzymes.^110–113 Several aminoglycoside-CoA bisubstrates were not only instrumental in characterizing AAC(6′)-Ii, but they were also potent inhibitors of its activity.^29,114 For example, selected neamine-containing bisubstrates (Fig. 3) can inhibit the enzymatic acetylation of aminoglycosides at nanomolar levels.¹¹⁵ However, these compounds cannot reach the bacterial cytosol since the bacterial cell surface is negatively charged. An ingenious way around this problem was to generate prodrugs whose chemical structures are truncated. These compounds do not show inhibitory activity but are internalized and subsequently processed by the host cell's CoA biosynthetic pathway enzymes to produce the active inhibitor.^115,116 The proposed mechanism for this conversion consists of a phosphorylation step catalyzed by the pantothenate kinase followed by adenylylation and phosphorylation at the 3′ position of the ribose mediated by phosphopantetheine adenylyltransferase and dephosphocoenzyme A kinase (Fig. 3).¹¹⁵ Bisubstrate inhibitors were also researched for the AAC(6′)-Iy from Salmonella enterica.¹¹⁷ Prodrug bisubstrates are a promising alternative to design adjuvants that inhibit resistance to aminoglycosides mediated by AAC(6′)-Ii and maybe other acetyltransferases.

Fig. 3 Intracellular prodrug to active bisubstrate inhibitor conversion. Proposed pathway of intracellular conversion of a neamine-containing bisubstrate prodrug (inactive) to an active bisubstrate inhibitor by enzymes of the CoA biosynthetic pathway.¹¹⁵ The donor substrate in all three reactions is ATP. The region of the molecules modified in each enzymatic reaction is circled. The neamine is shown in blue.

Peptide inhibitors of aminoglycoside phosphotransferases and acetyltransferases

The presence of anionic locations that interact with positively charged aminoglycosides was taken advantage of to screen cationic peptides as enzymatic inhibitors.¹¹⁸ Clinically relevant enzymes that confer resistance to aminoglycosides in Gram-positives, APH(3′)-IIIa, AAC(6′)-Ii, and the bifunctional AAC(6′)-Ie-APH(2′′)-Ia, were used in a study to test the inhibitory capabilities of indolicidin, an antimicrobial peptide of bovine origin, and synthetic analogs. Although not all those peptides that showed an inhibitory effect were active against all enzymes tested, all three were susceptible to inhibition by one or more peptides. Determination of the mechanisms showed competitive and noncompetitive inhibition with respect to the aminoglycoside and acetyl CoA, respectively, in the case of AAC(6′)-Ii. Conversely, inhibition of the phosphotransferases occurred through noncompetitive inhibition patterns for both the antibiotic and the donor ATP. Unfortunately, none of the peptides tested in this work reversed resistance in bacterial cells in culture, probably because of their inability to penetrate the cell wall.¹¹⁸

Inhibitors of the Mycobacterium tuberculosis Eis acetyltransferase

Tuberculosis, one of the oldest known diseases, continues to be a scourge to humans, and despite multiple efforts to control it, about 1.6 million people succumb to the disease every year.^119–121 Aminoglycosides such as kanamycin or amikacin are second-line drugs for treating tuberculosis. Unfortunately, numerous M. tuberculosis strains developed resistance to these antibiotics, mainly through the acetyltransferase known as enhanced intracellular survival (Eis).¹²² This protein is encoded by the chromosomal gene eis, which is part of the WhiB7 regulon.¹²³ Aminoglycoside resistance M. tuberculosis strains overexpress Eis, a phenotype caused by mutations in the eis promoter region or the 5′-untranslated segment of whiB7.^124–126 Unlike most aminoglycoside modifying enzymes, the Eis enzyme catalyzes the acetylation of multiple positions on the antibiotic molecule.^122,127 Over ten years of intensive research applying a high throughput screening methodology followed by optimization based on structure–activity relationship studies yielded numerous Eis inhibitors with diverse chemical structures that decrease the resistance to aminoglycosides in resistant M. tuberculosis strains at different levels.^128–135 An initial high throughput study screened 123 000 compounds, of which 617 molecules containing an isothiazole S,S-dioxide heterocyclic core (Fig. 4) were selected for further studies that ended up individualizing 17 that were potent inhibitors of resistance when tested in combination with kanamycin.¹²⁸ This pioneering work showed that it is possible to identify small molecules that inhibit aminoglycoside resistance caused by acetyltransferases. A library of compounds with a sulfonamide scaffold (Fig. 4) contained a compound including a substituted aniline ring that showed strong inhibition of the Eis activity. Analogs to this compound with modifications to the aniline ring were subsequently synthesized and tested, that one consisting of the sulfonamide scaffold with a 2-naphthyl substitution was a potent inhibitor of resistance to kanamycin mediated by Eis.¹²⁹ Crystal structure analysis showed that the inhibitor binds the Eis protein at the aminoglycoside-binding pocket. Further high throughput screening followed by optimization, by synthesizing compounds with different substituents, in some cases guided by structural information, led to identification of other Eis inhibitors containing the scaffolds methyl 4H-furo[3,2-b]pyrrole-5-carboxylate,¹³⁰ 3-(1,3-dioxolano)-2-indolinone,¹³⁰ pyrrolo[1,5-a]pyrazine containing a phenyl ring (where one of the substituents is attached) adjacent to the pyrrolo[1,5-a]pyrazine moiety and an acetophenone group where another substituent binds,¹³¹ 1,2,4-triazino[5,6b]indole-3-thioether,¹³² thieno[2,3d]pyrimidines,¹³³ and benzyloxy-benzylamine¹³⁴ (Fig. 4).

Fig. 4 Chemical structures of scaffolds that originated Eis inhibitors.^128–135 Analogs were generated changing substituents at the positions highlighted in color.

The approaches described in the previous paragraph were complemented with drug repurposing studies, i.e., the search for inhibitors among compounds already approved for human use or hybrid strategies like designing and testing analogs of existing drugs that showed Eis inhibitory properties.^120,135 These studies permitted the identification of inhibitors and their rational modification to generate even more potent compounds that could be used as adjuvants to aminoglycosides to treat resistant M. tuberculosis infections.¹³⁵ Haloperidol (Fig. 5), an antipsychotic drug, exhibited inhibition of the reaction catalyzed by Eis with an IC₅₀ of 0.39 ± 0.08 μM. Numerous analogs were designed on the basis of structural studies of the complex formed by haloperidol and Eis. The modifications to the haloperidol molecule were carried out at the positions indicated in colors in Fig. 5. Analysis of these analogs determined that eight also have potent inhibition properties. However, none of them was more powerful than haloperidol.¹³⁵ Three of these compounds showed additive effect with spectinomycin against resistant M. tuberculosis. These studies proved the potential of repurposing existing drugs.

Fig. 5 Chemical structures of haloperidol (antipsychotic), azelastine (antihistamine), venlafaxine (antidepressant), chloroquine (antimalarial), mefloquine (antimalarial), and proguanil (antimalarial). Analogs were generated changing substituents at the positions highlighted in color. The yellow highlight indicates that the number of methylene groups can be modified.¹³⁵

A recent study identified five FDA-approved medicines, azelastine (antihistamine), venlafaxine (antidepressant), and the antimalarial chloroquine, mefloquine, and proguanil (Fig. 5).¹²⁰ Analysis of the crystal structure of Eis in complex with these compounds found that they bind the aminoglycoside binding pocket. Based on the knowledge acquired, three new compounds were designed, and one of them, a proguanil analog, showed 3-fold more potency than the original drug.¹²⁰ Unfortunately, these compounds did not show significant activity to overcome kanamycin resistance in M. tuberculosis.¹²⁰

An inhibitor of the AAC(6′)-Ib enzyme identified using mixture-based combinatorial libraries

Aminoglycoside N-acetyltransferases (AAC) are a large family of aminoglycoside modifying enzymes of high clinical relevance found in Gram-negatives and Gram-positives.^{5,63,64,100,136} The more than sixty enzymes in this group are unequally represented in clinical strains; for example, the AAC(6′)-Ib enzyme is present in a disproportionally high number of AAC(6′)-I-producing Gram-negatives of clinical origin.¹³⁷ Its high impact on complications to treat Gram-negative infections resulted in numerous efforts to find inhibitors that can restore susceptibility to aminoglycosides such as amikacin or tobramycin.^{91–93,95–97,138–141} Most of these numerous strategies to inhibit the expression of the aac(6′)-Ib gene or the acetylation reaction have been recently reviewed.^28,61,75 Early attempts to identify small molecule inhibitors of AAC(6′)-Ib by computer docking successfully selected compounds with in vitro activity. However, they did not reverse resistance in bacterial cells in culture.^139,140 An inhibitor has recently been identified using mixture-based combinatorial libraries.¹⁴² Thirty-seven combinatorial libraries, each including tens or hundreds of thousands of compounds with different scaffolds, were ranked to determine if they contained a potential inhibitor.¹⁴³ The library containing pyrrolidine pentamine as a scaffold and four positions for functionalities was the most active. Upon deconvolution, a compound with S-phenyl at two positions, S-hydromethyl, and 3-phenylbutyl functionalities, was the most active inhibitor of in vitro acetylation (IC₅₀ = 2.1 μM) (Fig. 6). This compound, in combination with amikacin, restored susceptibility of Escherichia coli, A. baumannii, and Klebsiella pneumoniae harboring aac(6′)-Ib. Furthermore, it improved survival when in combination with amikacin, it was used to treat infected Galleria mellonella, and it was not toxic when tested on HEK 293 cells.¹⁴³ Structure–activity relationship analysis expanded the number of compounds studied and helped to illuminate some interactions between the inhibitor and enzyme and design analogs. Other modifications consisted of truncations of the molecule, which in all cases resulted in the loss of the inhibitory properties. Furthermore, none of those assayed to date was more active than the compound initially identified (Fig. 6).¹⁴⁴

Fig. 6 Chemical structure of the pyrrolidine pentamine scaffold compound with the most potent AAC(6′)-Ib-mediated acetylation inhibitory activity. The colored locations are those where different substituents were placed in the combinatorial library and analogs. The green region is modified in stereochemistry.

Small molecule inhibitors of aminoglycoside O-phosphotransferases

Aminoglycoside O-phosphotransferases of different subtypes catalyze the inactivation of numerous aminoglycosides of clinical relevance by transferring a phosphate from ATP or GTP to a hydroxyl group in the aminoglycoside molecule.^{5,101,145–147} The crystal structures of many enzymes from this family have been resolved, and interestingly, they share characteristics with eukaryotic protein kinases.¹⁴⁸ This property was exploited in early work when already known inhibitors of eukaryotic protein kinases were tested on two aminoglycoside O-phosphotransferases, APH(3′)-IIIa and the bifunctional AAC(6′)-Ie-APH(2′′)-Ia. This study identified isoquinolinesulfonamides (Fig. 7), compounds known to inhibit protein kinases by a competitive mechanism with respect to ATP that could inhibit the action of one or both enzymes. However, as happens in many cases, the chemicals failed to overcome resistance in bacterial cells in culture.¹⁴⁹

Fig. 7 Chemical structures of aminoglycoside O-phosphotransferase inhibitors. Left, the isoquinolinesulfamide scaffold (blue) was replaced by various chemical groups at the R position (highlighted in yellow). The compounds shown, H-7 and CKI-7 are competitive inhibitors (versus ATP) of APH(3′)-IIIa and the bifunctional AAC(6′)-Ie-APH(2′′)-Ia.¹⁴⁹ Center, two pyrazolopyrimidine scaffolds (red) showing the R positions (highlighted in colors) used to insert various chemical groups. The compounds shown, 1-NA-PP1 and the parent compound PP1, are inhibitors of APH(3′)-Ia activity in vitro and reduce resistance in hyperpermeable E. coli.¹⁵¹ Right, compounds NL6 and NL8. Both compounds are inhibitors of APH(3′)-IIIa, and NL6 is also an APH(2′)-IVa inhibitor.¹⁵²

An extensive screening of several aminoglycoside O-phosphotransferases against chemically diverse protein kinase inhibitors confirmed that protein kinase inhibitors could also inhibit this class of resistance enzymes. Among others, the authors identified the flavonoid quercetin as a potential inhibitor of resistance to aminoglycosides mediated by aminoglycoside O-phosphotransferases.¹⁵⁰ Further work utilized a selected group of these inhibitors in a study of the 3D structure of the APH(3′)-Ia in complex with the inhibitors and the aminoglycoside substrate. The results of these studies concluded that inhibitors with chemical scaffold pyrazolopyrimidine (Fig. 7) not only reduced APH(3′)-Ia activity in vitro and reversed aminoglycoside resistance in APH(3′)-Ia-harboring hyperpermeable E. coli cells in culture but also demonstrated advantages over eukaryotic protein kinase inhibitors tested before.¹⁵¹ This latter property makes them attractive chemicals that could have low toxicity to the host.

There were other attempts to identify inhibitors by computer docking. One of them led to the finding of two inhibitors, NL6 and NL8 (Fig. 7) of the APH(3′)-IIIa, and one of them, NL6, with activity towards APH(2′)-IVa.¹⁵² The compound NL6, a noncompetitive inhibitor towards the substrates ATP and kanamycin A, was studied more thoroughly by assessing the inhibitory properties of two analogs. Only one of them showed inhibition but with lower efficiency.¹⁵² In another study, molecular docking identified an inhibitor of a Bacillus phosphotransferase.^153,154 While these studies are promising, they were limited to in vitro analysis, and further work, including molecular optimization and in vivo experiments, will be required.

Concluding remarks

After a period in which the use of aminoglycosides seemed to be in decline, these antibiotics are making a comeback due to a combination of factors like the urgent need for antimicrobials and their favorable properties that outweigh the reasons they were being left aside (administration route, toxicity, resistance). Furthermore, newer aminoglycoside molecules, improved dosing schemes, and alternative administration continue minimizing the negative aspects of aminoglycosides, reinforcing the renewed interest in these drugs.^4,25,60 A major cause of failure in treating infections with aminoglycosides is the development and dissemination of resistance, which can occur through numerous mechanisms, several of which can coexist in the same bacterial cell.⁵ Besides generating molecules refractory to the existing resistance mechanisms, designing combination therapies that include the antibiotic and an inhibitor of resistance can prolong the useful life of existing aminoglycosides. Numerous efforts are underway to counter resistance from different angles, and this review described a few examples of just one aspect of this quest. Small molecules have enormous potential to become adjuvants that overcome resistant infections in combination with aminoglycosides. Although the multitude of efforts still did not result in an adjuvant approved for its use in humans, the direction and depth of the efforts by multiple research groups present an optimistic outlook. Of course, there will be challenges to overcome, like possible toxicity issues, spectrum of action, or ability of bacteria to develop enzymes that resist the action of the inhibitors. The efficacy of combinations aminoglycoside/enzyme inhibitor will most probably depend on the relationships between the pharmacokinetics and pharmacodynamics of the inhibitor and the antibiotic. Optimizing therapeutic regimens might become a challenging proposition depending on the chemical nature of the inhibitor. It may be worth mentioning that in the case of β-lactam/β-lactamase inhibitor combinations, while still complex, the dose adjustments are expected to be simpler to determine because the pharmacokinetics and pharmacodynamics of the inhibitor and antibiotic are usually similar.¹⁵⁵ Despite all challenges, it is possible that in the not-so-far future, a combination aminoglycoside/adjuvant that overcomes resistance caused by common aminoglycoside modifying enzymes will be used for human treatment.

Author contributions

MET, conceptualization and writing of the first draft. All authors contributed to the literature search, writing, and editing.

Conflicts of interest

None to declare.

Acknowledgements

Cited work made by the authors was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, grant number R15AI047115, and National Institute of General Medical Sciences, National Institutes of Health, grant number SC3GM125556.

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