Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2��-IVa

Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2″-IVa (APH(2″)-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2″)-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (k_cat/K_m) of 1.5 × 10³ to 1.2 × 10⁶ (M⁻¹ s⁻¹). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2″)-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes.     

Discovery of two aminoglycoside antibiotics as inhibitors targeting the menin–mixed lineage leukaemia interface

Menin functions as an oncogenic cofactor of mixed lineage leukaemia (MLL) fusion proteins in leukaemogenesis. The menin–MLL interface is a potential therapeutic target in acute leukaemia cases. In this study, approximately 900 clinical compounds were evaluated and ranked using pharmacophore-based virtual screening, the top 29 hits were further evaluated by biochemical analysis to discover the inhibitors that target the menin–MLL interface. Two aminoglycoside antibiotics, neomycin and tobramycin, were identified as menin–MLL inhibitors with binding affinities of 18.8 and 59.9 μM, respectively. The results of thermal shift assay validated the direct interactions between the two antibiotics and menin. The results of isothermal titration calorimetry showed that the equilibrium dissociation constant between menin and neomycin was approximately 15.6 μM. We also predicted the binding modes of inhibitors at the menin–MLL interface through molecular docking analysis. The results indicated that neomycin and tobramycin competitively occupy the binding site of MLL. This study has shed light on the development of powerful probes and new therapies for MLL-mediated leukaemogenesis.     

The Pseudomonas aeruginosa membranes: a target for a new amphiphilic aminoglycoside derivative?

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.     

Defining RNA motif–aminoglycoside interactions via two-dimensional combinatorial screening and structure–activity relationships through sequencing

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3 × 3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure–activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif–aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site.     

Does aminoglycoside-acetyltransferase in rapidly growing mycobacteria have a metabolic function in addition to aminoglycoside inactivation?

All the rapidly growing mycobacteria tested, Mycobacterium fortuitum complex, M. smegmatis, M. phlei, and M. vaccae, contained one of two characteristics, but were different from previously recognized aminoglycoside-acetyltransferases. The acetylation reaction of both the enzymes from M. fortuitum and Pseudomonas aeruginosa (3-N-acetyltransferase-III) with radiolabeled acetyl coenzyme A was inhibited severely by oxalacetate. It was suggested that the inhibitory effect of oxalacetate is due to the condensation reaction between oxalacetate and acetyl coenzyme A resulting in the generation of citrate.     

Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside–RNA hairpin loop interactions

Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5′UNNNC3′ loops for the kanamycin A derivative (where N is any nucleotide); 5′UNNC3′ loops for the tobramycin derivative; 5′UNC3′ loops for the neamine derivative; and 5′UNNG3′ loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand–hairpin loop interactions were determined. The selected interactions have K_d values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule–RNA motif interactions, which could be useful to design ligands targeting RNA.     

Is combination therapy with betalactam plus aminoglycoside (AGL) improving the outcome of nosocomial meningitis in children?

We have been unable to document a benefit of a combination of aminoglycosides with betalactam or carbapenem antibiotics in nosocomial meningitis. This was similar to the cases of sepsis, where survival of patients did not improve with combination therapy. Combination therapy did not increase the chance of appropriateness of the therapy. 30% of those on combination therapy were considered as inappropriately treated in comparison to 2.8% of those on monotherapy (p<0.01).     

Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic,structural and kinetic study

BackgroundAminoglycoside O-phosphotransferases make up a large class of bacterial enzymes that is widely distributed among pathogens and confer a high resistance to several clinically used aminoglycoside antibiotics. Aminoglycoside 2″-phosphotransferase IVa, APH(2″)-IVa, is an important member of this class, but there is little information on the thermodynamics of aminoglycoside binding and on the nature of its rate-limiting step.MethodsWe used isothermal titration calorimetry, electrostatic potential calculations, molecular dynamics simulations and X-ray crystallography to study the interactions between the enzyme and different aminoglycosides. We determined the rate-limiting step of the reaction by the means of transient kinetic measurements.ResultsFor the first time, K_d values were determined directly for APH(2″)-IVa and different aminoglycosides. The affinity of the enzyme seems to anti-correlate with the molecular weight of the ligand, suggesting a limited degree of freedom in the binding site. The main interactions are electrostatic bonds between the positively charged amino groups of aminoglycosides and Glu or Asp residues of APH. In spite of the significantly different ratio K_d/K_m, there is no large difference in the transient kinetics obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction.ConclusionsAPH(2″)-IVa has a higher affinity for aminoglycosides carrying an amino group in 2′ and 6′, but tighter bindings do not correlate with higher catalytic efficiencies. As with APH(3′)-IIIa, an intermediate containing product is preponderant during the steady state.General significanceThis intermediate may constitute a good target for future drug design.     

Which aminoglycoside ring is most important for binding? A hydropathic analysis of gentamicin, paromomycin, and analogues

The NMR structures of gentamicin and paromomycin in complex with the A-site of Escherichia coli 16S ribosomal RNA were modified with molecular modeling to 12 analogues. The intermolecular interactions between these molecules and RNA were examined using the HINT (Hydropathic INTeractions) computational model to obtain interaction scores that have been shown previously to be related to free energy. The calculations correlated well with experimental binding data, and the interaction scores were used to analyze the specific structural features of each aminoglycoside that contribute to the overall binding with the 16S rRNA. Our calculations indicate that, while ring I binds to the main binding pocket of the rRNA A-site, ring IV of paromomycin-based aminoglycosides contributes significantly to the overall binding.     

Inhibitors of the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] identified by in silico molecular docking

AAC(6′)-Ib is an important aminoglycoside resistance enzyme to target with enzymatic inhibitors. An in silico screening approach was used to identify potential inhibitors from the ChemBridge library. Several compounds were identified, of which two of them, 4-[(2-{[1-(3-methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid and 2-{5-[(4,6-dioxo-1,3-diphenyl-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid, showed micromolar activity in inhibiting acetylation of kanamycin A. These compounds are predicted to bind the aminoglycoside binding site of AAC(6′)-Ib and exhibited competitive inhibition against kanamycin A.     

Structure of the cytosine–cytosine mismatch in the thymidylate synthase mRNA binding site and analysis of its interaction with the aminoglycoside paromomycin

The structure of a cytosine–cytosine (CC) mismatch-containing RNA molecule derived from a hairpin structure in the thymidylate synthase mRNA that binds the aminoglycoside paromomycin with high affinity was determined using nuclear magnetic resonance (NMR) spectroscopy. The cytosines in the mismatch form a noncanonical base pair where both cytosines are uncharged and stack within the stem of the RNA structure. Binding to paromomycin was analyzed using isothermal titration calorimetry (ITC) to demonstrate the necessity of the CC mismatch and to determine the affinity dissociation constant of this RNA to paromomycin to be 0.5 ± 0.3 μM. The CC mismatch, and the neighboring GC base pairs experienced the highest degree of chemical shift changes in their H6 and H5 resonances indicating that paromomycin binds in the major groove at the CC mismatch site. In comparing the structure of CC mismatch RNA with a fully Watson–Crick GC base paired stem, the CC mismatch is shown to confer a widening of the major groove. This widening, combined with the dynamic nature of the CC mismatch, enables binding of paromomycin to this RNA molecule.     

Dependence of aminoglycoside 3′-phosphotransferase VIII activity on serine/threonine protein kinases in <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

In Streptomyces rimosus, selection for resistance to the aminoglycoside antibiotic kanamycin triggers the normally silent aminoglycoside 3-phosphotransferase VIII gene (aphVIII). The expression of APHVIII is accompanied by amplification of the chromosomal DNA fragment containing the aphVIII gene. Earlier, S. rimosus aphVIII gene was isolated and sequenced. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibodies, we have demonstrated that endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 actively phosphorylate two serine residues in the APHVIII molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca²⁺ is 1.84-fold greater than that without Ca²⁺. Analysis of ingel autophosphorylation and phosphorylation of the substrate incorporated into the gel matrix has shown that modification of APHVIII is catalyzed by two serine/threonine PKs (74 kDa and 55 kDa). The activity of 55-kDa PK is dependent on Ca²⁺ and calmodulin. The specific kanamycin phosphotransferase activity of exhaustively phosphorylated APHVIII is 3.72 times higher than that of the unmodified enzyme. It is proposed that the above PKs may be involved in the regulation of kanamycin resistance in S. rimosus.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 255–263.Original Russian Text Copyright © 2005 by Elizarov, Sergienko, Sizova, Danilenko.