Structural Basis of APH(3′)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

Fong, D.H.; Berghuis, Albert M.

doi:10.1128/aac.00062-09

Cited by 25 publications

(22 citation statements)

References 54 publications

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“…APH(3′)-IIa pdb id: 1ND4 (Nurizzo et al, 2003). APH(3′)-IIIa pdb id: 1J7I, 1J7L (in complex with ADP), 1J7U (in comlex with APPNP) (Burk et al, 2001), 1L8T (in complex with ADP and kanamycin A) (Fong and Berghuis, 2002) 3H8P (in complex with AMPPNP and butirosin A) (Fong and Berghuis, 2009), 2BKK (in complex with the inhibitor AR_3A) (Kohl et al, 2005). APH(2″)-IIa pdb id: 3HAV (in complex with ATP and streptomycin), 3HAM (in complex with gentamicin) (Young et al, 2009).…”

Section: Figures and Tablesmentioning

confidence: 99%

Aminoglycoside modifying enzymes

Ramírez

Tolmasky

2010

Drug Resistance Updates

1,088

1,060

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Aminoglycosides have been an essential component of the armamentarium in the treatment of lifethreatening infections. Unfortunately, their efficacy has been reduced by the surge and dissemination of resistance. In some cases the levels of resistance reached the point that rendered them virtually useless. Among many known mechanisms of resistance to aminoglycosides, enzymatic modification is the most prevalent in the clinical setting. Aminoglycoside modifying enzymes catalyze the modification at different −OH or −NH 2 groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be nucleotidyltranferases, phosphotransferases, or acetyltransferases. The number of aminoglycoside modifying enzymes identified to date as well as the genetic environments where the coding genes are located is impressive and there is virtually no bacteria that is unable to support enzymatic resistance to aminoglycosides. Aside from the development of new aminoglycosides refractory to as many as possible modifying enzymes there are currently two main strategies being pursued to overcome the action of aminoglycoside modifying enzymes. Their successful development would extend the useful life of existing antibiotics that have proven effective in the treatment of infections. These strategies consist of the development of inhibitors of the enzymatic action or of the expression of the modifying enzymes. Keywordsantibiotic resistance; aminoglycoside; aminoglycoside modifying enzyme; acetyltransferase; nucleotidyltransferase; phosphotransferase; kinase; antisense; RNase P; RNase H; bacterial infection 1. A brief overview of aminoglycoside antibiotics General aspectsAminoglycoside antibiotics are a complex family of compounds characterized for having an aminocyclitol nucleus (streptamine, 2-deoxystreptamine, or streptidine) linked to amino sugars through glycosidic bonds. In addition, other compounds such as spectinomycin, which is an aminocyclitol not linked to amino sugars, or compounds that include the aminocyclitol fortamine are also included in this family (Bryskier, 2005;Veyssier and Bryskier, 2005). Aminoglycosides are primarily used in the treatment of infections caused by gram-negative aerobic bacilli, staphylococci, and other gram-positives (Yao and

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Section: Figures and Tablesmentioning

confidence: 99%

Aminoglycoside modifying enzymes

Ramírez

Tolmasky

2010

Drug Resistance Updates

1,088

1,060

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“…To date, the structures of three aminoglycoside APH enzymes have been reported: APH(3Ј)-IIa (15), APH(3Ј)-IIIa (12,14,37), and APH(2Љ)-IIa (16). The two APH(3Ј) isozymes share 33% sequence identity, identical phosphorylation targets, nearly identical substrate specificity profiles, and possess an essentially identical fold.…”

Section: Aph(9)-ia Is a Novel Member Of The Aph Family Of Enzymes-mentioning

confidence: 99%

“…This loop is highly flexible and can adopt different shapes to accommodate the structurally different substrates bound to different zones of the active site (13,37). It has also been indicated that aminoglycoside binding in APH(3Ј)-IIIa does not make use of distinguishing functional groups of its various substrates for binding.…”

Section: Aph(9)-ia Is a Novel Member Of The Aph Family Of Enzymes-mentioning

confidence: 99%

Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

Fong

Lemke

Hwang

et al. 2010

Journal of Biological Chemistry

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Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3) isozymes and an APH(2؆) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3) and APH(2؆) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.Aminoglycosides are a class of commonly used broadspectrum antibiotics that target the bacterial ribosome. They are characterized by their signature chemical structure, an aminocyclitol nucleus. These antibiotics can be further categorized into two groups: the first group includes those that contain a 2-deoxystreptamine core, such as kanamycin, and the second, smaller group includes those that contain a non-2-deoxystreptamine core (Fig. 1A). Spectinomycin has a streptamine core and it is used in the treatment of acute gonococcal infections (1). Unfortunately, the efficacy of aminoglycosides has been compromised due to the continuous rise of drug resistance in pathogens. Resistance to aminoglycosides can be attributed to several mechanisms, of which enzymatic inactivation of the aminoglycoside is the most prevalent in the clinical setting. Aminoglycosides can be inactivated by the addition of an acetyl, a nucleotidyl, or a phosphate group by acetyltransferases, nucleotidyltransferases, or phosphotransferases (kinases; APHs), 5 respectively (2). Nomenclature of aminoglycoside-modifying enzymes follows the convention proposed by Shaw et al. (2): the type of modifying enzyme is identified by acetyltransferase, nucleotidyltransferase, or APH; this is followed by, in parentheses, the enzyme regiospecificity; then the substrate profile is designated by a roman numeral, and the unique amino acid sequence is denoted by a small letter.APHs generally yield high levels of resista...

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“…Each ATP molecule adopted a bidentate chelating conformation with respect to two ions, which were modeled as Ca 2+ due to the prevalence of these ions in the crystallization buffer, the magnitude of the electron density peaks and the enzyme-ion atomic bond distances. The position of the Ca 2+ ions in the APH(3’)-Ia•ATP complex corresponded to that of the two Mg 2+ ions in the APH(3’)-IIIa-AMPPNP-Mg 2+ complex [17,21]. The adenine moiety of ATP occupied a hydrophobic pocket formed by Phe53, Ile205 and Ile215 and establishes a number of hydrogen bonds with backbone atoms of the hinge, specifically between N6 and the carbonyl oxygen of Thr99, between N1 and the amide nitrogen of Ile101, and a weaker bond between the C2-H and the carbonyl oxygen of Ile101 (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance

Stogios

Spanogiannopoulos

Evdokimova

et al. 2013

Biochemical Journal

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SYNOPSIS Activity of the aminoglycoside phosphotransferase APH(3’)-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. Previously we demonstrated that eukaryotic protein kinase (ePK) inhibitors could inhibit APH enzymes, due to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. As well, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. Here, we structurally and functionally characterize inhibition of APH(3’)-Ia by three diverse chemical scaffolds – anthrapyrazolone, 4-anilinoquinazoline and pyrazolopyrimidine (PP) – and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3’)-Ia versus ePKs. Using this observation, we identify PP-derivatives that select against ePKs, attenuate APH(3’)-Ia activity and rescue aminoglycoside antibiotic activity against a resistant E. coli strain. The structures presented here and these inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.

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Structural Basis of APH(3′)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

Cited by 25 publications

References 54 publications

Aminoglycoside modifying enzymes

Aminoglycoside modifying enzymes

Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance

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