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Neamine Derivatives

Neamine is a poor antibiotic and is not clinically useful. However, it serves as an invaluable template for the design of new antibiotics. It has been shown that neamine is the minimal structural motif required for binding to the A-site of 16S subunit of ribosomal RNA (Fourmy et al. 1996, 1998). Hence, it is sensible, in designing new aminoglycosides, to preserve the minimum structural motif required for RNA binding and antibiotic activity, but to deviate from typical aminoglycoside structures, in order to elude the various modifying enzymes.

Previous studies showed that the antibiotic activity can be retained when ring IV of neomycin B is substituted with a diaminoalkane group, even though the analogue binds to RNA with diminished specificity (Alper et al. 1998). Subsequently, Greenberg et al. synthesized derivatives of neamine by appending various poly amino, amino alcohol, or aromatic substitutions at the O5-position (Greenberg et al. 1999). The results showed that the compounds substituted with a diaminoalkyl group enhanced the binding to RNA while exhibiting antibiotic activity equivalent to neamine.

More recently, several neamine derivatives were synthesized based on the interactions observed in the nuclear magnetic resonance (NMR) solution structure of paromomycin bound to an A-site rRNA template, as well as extensive searches in the Cambridge Structural Database and the National Cancer Institute 3-D Database (Haddad et al. 2002). These compounds are composed of a neamine core, with an AHB group or its analogue at the N1-position of the 2-deoxystreptamine (as in butirosin and amikacin), plus an diaminoalkane group of various lengths at the O6-position Fig. 7a). An AHB group was selected as a substituent, since aminoglycosides such as butirosin and amikacin, which possess this structure at N1, have reduced affinity for aminoglycoside-modifying enzymes (see Sect. 4.1.2). The terminal amine-containing aliphatic component was added in order to improve the interaction between the O6 and the phosphate backbone of the target rRNA. Many of the designed compounds were shown to be capable of binding to a fragment modelling the A-site of the E. coli rRNA, and demonstrated broad spectrum antibiotic activity that is much higher than their parent compound or equal to that of other commonly used aminoglycosides. Some of these designed antibiotics were shown to be poor substrates of APH(30)-Ia and AAC(6')-Ie-APH(2")-Ia, the bifunctional enzyme. The crystal structure of the A-site rRNA template in complex with a designer neamine derivative of high antibiotic activity has recently been reported (Russell et al. 2003). The structure showed that the binding mode of the designer compound is essentially identical to that of paromomycin (Carter et al. 2000). Comparison

Bifunctional Dimeric Neamine

Fig. 7a-d Four synthetic aminoglycosides based on the structure of neamine, the minimal structure required for binding to the bacterial ribosome. a A neamine derivative with AHB and diaminoalkane substitutions at the N1 and O6 positions, respectively. b Amine-linked neamine dimers. c 1,2-hydroxyamine-linked neamine dimers. d A bromoacetylat-ed neamine

Fig. 7a-d Four synthetic aminoglycosides based on the structure of neamine, the minimal structure required for binding to the bacterial ribosome. a A neamine derivative with AHB and diaminoalkane substitutions at the N1 and O6 positions, respectively. b Amine-linked neamine dimers. c 1,2-hydroxyamine-linked neamine dimers. d A bromoacetylat-ed neamine of neamine derivatives to the kanamycin A bound to APH(30)-IIIa (Fong and Berghuis 2002) also explains the basis of the designer molecules' ability to elude inactivation by aminoglycoside-modifying enzymes. The AHB moiety at the N1-position forms steric clashes with the antibiotic binding loop of

APH(3')-IIIa, impeding the formation of an active ternary complex (Russell et al. 2003).

It has been shown that neamine binds to the A-site model of prokaryotic rRNA in a 2:1 ratio (Sucheck et al. 2000). A series of neamine dimers were constructed in order to identify bivalent aminoglycosides that would interact with the model target site of aminoglycosides in bacteria, and at the same time resist modification by aminoglycoside-modifying enzymes due to its dramatically altered and unusual structure. Two neamine molecules are joined by either amides or 1,2-hydroxyamine with methylene bridges of variable lengths (Fig. 7b,c). These compounds were found to possess antibiotic activity that is comparable or even superior to that of neamine. They are also potent competitive inhibitors of APH(2") activity of the bifunctional enzyme AAC(6')-Ie-APH(2")-Ia and are poor substrates for APH(3')-IIIa and the AAC(6')-Ie activity of the bifunctional enzyme.

Four derivatives of neamine have also been synthesized by regiospecifi-cally appending a bromoacetyl group to the various amines of the antibiotic (Roestamadji and Mobashery 1998) (Fig. 7d). The affinity of the bro-moacetylated compounds for APH(3')-IIa is significantly reduced. In the presence of ATP, the phosphorylation reaction would proceed but at an attenuated rate, whereas in the absence of ATP, the bromoacetylated neamines would inactivate APH(3')-IIa in a time-dependent and saturable manner. Moreover, the activity of the enzyme could not be recovered despite extensive dialysis, in an attempt to remove the modified neamine molecules. This observation suggests that the electrophilic bromoacetyl group could form covalent bonds with different nucleophilic residues in the active site. As a result, the modified neamine becomes irreversibly bound to the enzyme and prevents it from binding and inactivating aminoglycosides.

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