Biosynthetic Origin of the Octose Core and Its Mechanism of Assembly during Apramycin Biosynthesis.

Apramycin is an aminoglycoside antibiotic isolated from Streptoalloteichus tenebrarius and S. hindustanus that has found clinical use in veterinary medicine. The apramycin structure is notable for its atypical eight-carbon bicyclic dialdose (octose) moiety. While the apramycin biosynthetic gene cluster (apr) has been identified and several of the encoded genes functionally characterized, how the octose core itself is assembled has remained elusive. Nevertheless, recent gene deletion studies have hinted at an N-acetyl aminosugar being a key precursor to the octose, and this hypothesis is consistent with the additional feeding experiments described in the present report. Moreover, bioinformatic analysis indicates that AprG may be structurally similar to GlcNAc-2-epimerase and hence recognize GlcNAc or a structurally similar substrate suggesting a potential role in octose formation. AprG with an extended N-terminal sequence was therefore expressed, purified, and assayed in vitro demonstrating that it does indeed catalyze a transaldolation reaction between GlcNAc or GalNAc and 6'-oxo-lividamine to afford 7'-N-acetyldemethylaprosamine with the same 6'-R and 7'-S stereochemistry as those observed in the apramycin product. Biosynthesis of the octose core in apramycin thus proceeds in the [6 + 2] manner with GlcNAc or GalNAc as the two-carbon donor, which has not been previously reported for biological octose formation, as well as novel inverting stereochemistry of the transferred fragment. Consequently, AprG appears to be a new transaldolase that lacks any apparent sequence similarity to the currently known aldolases and catalyzes a transaldolation for which there is no established biological precedent.

Two Cryptic Self-Resistance Mechanisms in Streptomyces tenebrarius Reveal Insights into the Biosynthesis of Apramycin.

Apramycin is a clinically promising aminoglycoside antibiotic (AGA). To date, mechanisms underlying the biosynthesis and self-resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps, in which a novel aminoglycoside phosphotransferase (AprU), a putative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5-OH of a pseudotrisaccharide intermediate, whose N-7' acetyl group is subsequently hydrolyzed by AprP. AprZ is located extracellularly where it removes the phosphate group from a pseudotetrasaccharide intermediate, leading to the maturation of apramycin. Intriguingly, 7'-N-acetylated and 5-O-phosphorylated apramycin that were accumulated in ΔaprU and ΔaprZ respectively exhibited significantly reduced antibacterial activities, implying Streptomyces tenebrarius employs C-5 phosphorylation and N-7' acetylation as two strategies to avoid auto-toxicity. Significantly, this study provides insight into the design of new generation AGAs to circumvent the emergence of drug-resistant pathogens.

Apralogs: Apramycin 5-O-Glycosides and Ethers with Improved Antibacterial Activity and Ribosomal Selectivity and Reduced Susceptibility to the Aminoacyltranserferase (3)-IV Resistance Determinant.

Apramycin is a structurally unique member of the 2-deoxystreptamine class of aminoglycoside antibiotics characterized by a monosubstituted 2-deoxystreptamine ring that carries an unusual bicyclic eight-carbon dialdose moiety. Because of its unusual structure, apramycin is not susceptible to the most prevalent mechanisms of aminoglycoside resistance including the aminoglycoside-modifying enzymes and the ribosomal methyltransferases whose widespread presence severely compromises all aminoglycosides in current clinical practice. These attributes coupled with minimal ototoxocity in animal models combine to make apramycin an excellent starting point for the development of next-generation aminoglycoside antibiotics for the treatment of multidrug-resistant bacterial infections, particularly the ESKAPE pathogens. With this in mind, we describe the design, synthesis, and evaluation of three series of apramycin derivatives, all functionalized at the 5-position, with the goals of increasing the antibacterial potency without sacrificing selectivity between bacterial and eukaryotic ribosomes and of overcoming the rare aminoglycoside acetyltransferase (3)-IV class of aminoglycoside-modifying enzymes that constitutes the only documented mechanism of antimicrobial resistance to apramycin. We show that several apramycin-5-O-ß-d-ribofuranosides, 5-O-ß-d-eryrthofuranosides, and even simple 5-O-aminoalkyl ethers are effective in this respect through the use of cell-free translation assays with wild-type bacterial and humanized bacterial ribosomes and of extensive antibacterial assays with wild-type and resistant Gram negative bacteria carrying either single or multiple resistance determinants. Ex vivo studies with mouse cochlear explants confirm the low levels of ototoxicity predicted on the basis of selectivity at the target level, while the mouse thigh infection model was used to demonstrate the superiority of an apramycin-5-O-glycoside in reducing the bacterial burden in vivo.

Structure-Based Design and Synthesis of Apramycin-Paromomycin Analogues: Importance of the Configuration at the 6'-Position and Differences between the 6'-Amino and Hydroxy Series.

The preparation of a series of four analogues of the aminoglycoside antibiotics neomycin and paromomycin is described in which ring I, involved in critical binding interactions with the ribosomal target, is replaced by an apramycin-like dioxabicyclo[4.4.0]octane system. The effect of this modification is to lock the hydroxymethyl side chain of the neomycin or paromomycin ring I, as part of the dioxabicyclooctane ring, into either the gauche-gauche or the gauche-trans conformation (respectively, axial or equatorial to the bicyclic system). The antiribosomal activity of these compounds is investigated with cell-free translation assays using both bacterial ribosomes and recombinant hybrid ribosomes carrying eukaryotic decoding A site cassettes. Compounds substituted with an equatorial hydroxyl or amino group in the newly formed ring are considerably more active than their axial diastereomers, lending strong support to crystallographically derived models of aminoglycoside-ribosome interactions. One such bicyclic compound carrying an equatorial hydroxyl group has activity equal to that of the parent yet displays better ribosomal selectivity, predictive of an enhanced therapeutic index. A paromomycin analog lacking the hydroxymethyl ring I side chain is considerably less active than the parent. Antibacterial activity against model Gram negative and Gram positive bacteria is reported for selected compounds, as is activity against ESKAPE pathogens and recombinant bacteria carrying specific resistance determinants. Analogues with a bicyclic ring I carrying equatorial amino or hydroxyl groups mimicking the bound side chains of neomycin and paromomycin, respectively, show excellent activity and, by virtue of their novel structure, retain this activity in strains that are insensitive to the parent compounds.

Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis.

Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products.

Oncogenic MicroRNAs Biogenesis as a Drug Target: Structure-Activity Relationship Studies on New Aminoglycoside Conjugates.

MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers and that the inhibition of these oncogenic miRNAs could find application in the therapy of different types of cancer. Herein, we describe the synthesis and biological evaluation of new small-molecule drugs that target oncogenic miRNAs production. In particular, we chose to target two miRNAs (i.e., miRNA-372 and -373) implicated in various types of cancer, such as gastric cancer. Their precursors (pre-miRNAs) are overexpressed in cancer cells and lead to mature miRNAs after cleavage of their stem-loop structure by the enzyme Dicer in the cytoplasm. Some of the newly synthesized conjugates can inhibit Dicer processing of the targeted pre-miRNAs in vitro with increased efficacy relative to our previous results (D.D. Vo et al., ACS Chem. Biol. 2014, 9, 711-721) and, more importantly, to inhibit proliferations of adenocarcinoma gastric cancer (AGS) cells overexpressing these miRNAs, thus representing promising leads for future drug development.

Identification of the molecular attributes required for aminoglycoside activity against Leishmania.

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.

Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin.

Aminoglycosides are potent antibacterials, but therapy is compromised by substantial toxicity causing, in particular, irreversible hearing loss. Aminoglycoside ototoxicity occurs both in a sporadic dose-dependent and in a genetically predisposed fashion. We recently have developed a mechanistic concept that postulates a key role for the mitochondrial ribosome (mitoribosome) in aminoglycoside ototoxicity. We now report on the surprising finding that apramycin, a structurally unique aminoglycoside licensed for veterinary use, shows little activity toward eukaryotic ribosomes, including hybrid ribosomes which were genetically engineered to carry the mitoribosomal aminoglycoside-susceptibility A1555G allele. In ex vivo cultures of cochlear explants and in the in vivo guinea pig model of chronic ototoxicity, apramycin causes only little hair cell damage and hearing loss but it is a potent antibacterial with good activity against a range of clinical pathogens, including multidrug-resistant Mycobacterium tuberculosis. These data provide proof of concept that antibacterial activity can be dissected from aminoglycoside ototoxicity. Together with 3D structures of apramycin-ribosome complexes at 3.5-Å resolution, our results provide a conceptual framework for further development of less toxic aminoglycosides by hypothesis-driven chemical synthesis.

Redesign of substrate specificity and identification of the aminoglycoside binding residues of Eis from Mycobacterium tuberculosis.

The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis . Eis from M. tuberculosis (Eis_Mtb) and M. smegmatis (Eis_Msm) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis_Mtb is divided into two narrow channels, whereas Eis_Msm contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis_Mtb, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis_Msm, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis_Mtb and for the functional differences from Eis_Msm, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis_Mtb by mutating them into their Eis_Msm counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis_Mtb. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis_Mtb nomenclature) dictate the differences between the acetylation efficiencies of Eis_Mtb and Eis_Msm for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis_Mtb into Ala plays a role in APR acetylation.

Importance of Co-operative Hydrogen Bonding in the Apramycin-Ribosomal Decoding A-Site Interaction.

An intramolecular hydrogen bond between the protonated equatorial 7'-methylamino group of apramycin and the vicinal axial 6'-hydroxy group acidifies the 6'-hydroxy group leading to a strong hydrogen bond to A1408 in the ribosomal drug binding pocket in the decoding A site of the small ribosomal subunit. In 6'-epiapramycin, the trans-nature of the 6'-hydroxy group and the 7'-methylamino group results in a much weaker intramolecular hydrogen bond, and a consequently weaker cooperative hydrogen bonding network with A1408, resulting overall in reduced inhibition of protein synthesis and antibacterial activity.

ApmA Is a Unique Aminoglycoside Antibiotic Acetyltransferase That Inactivates Apramycin.

Apramycin is an aminoglycoside antibiotic with the potential to be developed to combat multidrug-resistant pathogens. Its unique structure evades the clinically widespread mechanisms of aminoglycoside resistance that currently compromise the efficacy of other members in this drug class. Of the aminoglycoside-modifying enzymes that chemically alter these antibiotics, only AAC(3)-IVa has been demonstrated to confer resistance to apramycin through N-acetylation. Knowledge of other modification mechanisms is important to successfully develop apramycin for clinical use. Here, we show that ApmA is structurally unique among the previously described aminoglycoside-modifying enzymes and capable of conferring a high level of resistance to apramycin. In vitro experiments indicated ApmA to be an N-acetyltransferase, but in contrast to AAC(3)-IVa, ApmA has a unique regiospecificity of the acetyl transfer to the N2' position of apramycin. Crystallographic analysis of ApmA conclusively showed that this enzyme is an acetyltransferase from the left-handed ß-helix protein superfamily (LßH) with a conserved active site architecture. The success of apramycin will be dependent on consideration of the impact of this potential form of clinical resistance.IMPORTANCE Apramycin is an aminoglycoside antibiotic that has been traditionally used in veterinary medicine. Recently, it has become an attractive candidate to repurpose in the fight against multidrug-resistant pathogens prioritized by the World Health Organization. Its atypical structure circumvents most of the clinically relevant mechanisms of resistance that impact this class of antibiotics. Prior to repurposing apramycin, it is important to understand the resistance mechanisms that could be a liability. Our study characterizes the most recently identified apramycin resistance element, apmA We show ApmA does not belong to the protein families typically associated with aminoglycoside resistance and is responsible for modifying a different site on the molecule. The data presented will be critical in the development of apramycin derivatives that will evade apmA in the event it becomes prevalent in the clinic.

An Advanced Apralog with Increased in†vitro and in†vivo Activity toward Gram-negative Pathogens and Reduced ex vivo Cochleotoxicity.

We describe the convergent synthesis of a 5-O-ß-D-ribofuranosyl-based apramycin derivative (apralog) that displays significantly improved antibacterial activity over the parent apramycin against wild-type ESKAPE pathogens. In addition, the new apralog retains excellent antibacterial activity in the presence of the only aminoglycoside modifying enzyme (AAC(3)-IV) acting on the parent, without incurring susceptibility to the APH(3') mechanism that disables other 5-O-ß-D-ribofuranosyl 2-deoxystreptamine type aminoglycosides by phosphorylation at the ribose 5-position. Consistent with this antibacterial activity, the new apralog has excellent 30 nM activity (IC50 ) for the inhibition of protein synthesis by the bacterial ribosome in a cell-free translation assay, while retaining the excellent across-the-board selectivity of the parent for inhibition of bacterial over eukaryotic ribosomes. Overall, these characteristics translate into excellent in vivo efficacy against E. coli in a mouse thigh infection model and reduced ototoxicity vis à vis the parent in mouse cochlear explants.

Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

Two conformational states in the crystal structure of the Homo sapiens cytoplasmic ribosomal decoding A site.

The decoding A site of the small ribosomal subunit is an RNA molecular switch, which monitors codon-anticodon interactions to guarantee translation fidelity. We have solved the crystal structure of an RNA fragment containing two Homo sapiens cytoplasmic A sites. Each of the two A sites presents a different conformational state. In one state, adenines A1492 and A1493 are fully bulged-out with C1409 forming a wobble-like pair to A1491. In the second state, adenines A1492 and A1493 form non-Watson-Crick pairs with C1409 and G1408, respectively while A1491 bulges out. The first state of the eukaryotic A site is, thus, basically the same as in the bacterial A site with bulging A1492 and A1493. It is the state used for recognition of the codon/anticodon complex. On the contrary, the second state of the H.sapiens cytoplasmic A site is drastically different from any of those observed for the bacterial A site without bulging A1492 and A1493.

Determination of apramycin in animal feeds by solid-phase extraction and liquid chromatography with precolumn derivatization and fluorescence detection.

A high-performance liquid chromatographic method for determining apramycin in animal feeds was developed. Apramycin in feeds was extracted with 0.1 M HCl solution and cleaned up with an MCX solid-phase extraction column. The purified extract was derivatized with o-phthaldehyde, and components were separated on a C18 column and detected with a fluorescence detector. Mass spectrometric data confirmed that apramycin was derivatized at all the 4 primary amines on the apramycin molecule. Average recoveries at 8 included levels (5, 10, 20, 40, 80, 200, 400, and 2000 mg/kg) ranged from 92.2 to 100.5%, and the coefficients of variation were < 6.5%. Standard curves were linear over the range 0.05 to 10 microg/mL. The detection and quantitation limits were determined to be 0.2 and 1.0 mg/kg, respectively.

Glycine origin of the methyl substituent on C7'-N of octodiose for the biosynthesis of apramycin.

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicating that glycine and/or serine might be involved in the biosynthesis of apramycin. The 13C-NMR analysis of [2-13C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH3 substituent of apramycin on the C7' of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes a previously reported for the case of rapamycin. The 15N NMR spectra of [2-13C,15N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the -NH2 substituents of apramycin.

A sensitive non-derivatization method for apramycin and impurities analysis using hydrophilic interaction liquid chromatography and charged aerosol detection.

A sensitive non-derivatization method was developed for the analysis of apramycin and impurites using hydrophilic interaction liquid chromatography (HILIC) and charged aerosol detection (CAD). Sample was pretreated with an effective SPE method (recovery >90%) to remove interference with apramycin impurities from sulfate, then analyzed with direct injection. Different chromatography modes of separation and choices of HILIC column were investigated in search of a direct analysis method. The HILIC-CAD method was optimized using a cysteine-bonded zwitterionic HILIC column and compared to the strong cation exchange-ultraviolet (SCX-UV) method with post-column derivatization recommended by the Chinese Pharmacopoeia (veterinary) 2010. The improved chromatographic resolution and peak shape with the HILIC-charged aerosol detection method allows for increase of sample load to 48.9 µg from only 2.8 µg with the SCX-UV approach. More than 16 impurities were detected with this method with improved resolution, and four were identified with MS, while only 7 impurities were detected with the SCX-UV method. Moreover, the current method has a good precision and reproducibility. The intra-day and inter-day of peak area variability was less than or equal to 4.760% RSD and 9.950%, respectively. The average limit of detection and quantization was 80 ng and 200 ng injected on the column, respectively. The overall results demonstrated that the presented method can be used as an alternative to SCX-UV method in the analysis of apramycin and impurities.

Pretreatment of apramycin wastewater by catalytic wet air oxidation.

The pretreatment technology of wet air oxidation (WAO) and coagulation and acidic hydrolysis for apramycin wastewater was investigated in this paper. The COD, apramycin, NH4+ concentration, and the ratio of BOD5/COD were analyzed, and the color and odor of the effluent were observed. WAO of apramycin wastewater, without catalyst and with RuO2/Al2O3 and RuO2-CeO2/Al2O3 catalysts, was carried out at degradation temperature of 200 degrees C and the total pressure of 4 MPa in a 1 L batch reactor. The result showed that the apramycin removals were respectively 50.2% and 55.0%, COD removals were 40.0% and 46.0%, and the ratio of BOD5/COD was increased to 0.49 and 0.54 with RuO2/Al2O3 and RuO2-CeO2/Al2O3 catalysts in catylytic wet air oxidation (CWAO) after the reaction of 150 min. With the pretreatment of coagulation and acidic hydrolysis, COD and apramycin removals were slight decreased, and the ratio of BOD5/COD was increased to 0.45, and the effluents was not suitable to biological treatment. The color and odor of the wastewater were effectively controlled and the reaction time was obviously shortened with WAO. HO2 may promote organic compounds oxidized in WAO of the apramycin wastewater. The addition of CeO2 could promote the activity and stability of RuO2/Al2O3 in WAO of apramycin wastewater.

The antibacterial and NMDA receptor activating properties of aminoglycosides are dissociable.

The use of aminoglycoside antibiotics is limited by side effects, the most critical of which are vestibular and cochlear toxicity. Recent evidence indicates that these effects result from an excitotoxic process mediated, at least in part, through a polyamine-like activation of NMDA receptors. This study investigated whether these positive modulatory effects of aminoglycosides at NMDA receptors are dissociable from their antibacterial properties. A group of structurally related apramycin derivatives was evaluated for the ability to enhance [3H]dizocilpine binding to rat brain membranes, and for the ability to augment agonist responses on recombinant (NR1A/2B) NMDA receptors expressed in Xenopus oocytes. Based on the antibacterial potencies of these derivatives against Staphylococcus aureus and Escherichia coli, it is concluded that there is no correlation between the ability of an aminoglycoside to produce a positive modulation of NMDA receptors and minimum inhibitory antibacterial concentrations. These findings indicate that it may be possible to develop an aminoglycoside antibiotic with reduced potential for ototoxicity.