Interactions of designer antibiotics and the bacterial ribosomal aminoacyl-tRNA site.

The X-ray crystal structures for the complexes of three designer antibiotics, compounds 1, 2, and 3, bound to two models for the ribosomal aminoacyl-tRNA site (A site) at 2.5-3.0 Angstroms resolution and that of neamine at 2.8 Angstroms resolution are described. Furthermore, the complex of antibiotic 1 bound to the A site in the entire 30S ribosomal subunit of Thermus thermophilus is reported at 3.8 Angstroms resolution. Molecular dynamics simulations revealed that the designer compounds provide additional stability to bases A1492 and A1493 in their extrahelical forms. Snapshots from the simulations were used for free energy calculations, which revealed that van der Waals and hydrophobic effects were the driving forces behind the binding of designer antibiotic 3 when compared to the parental neamine.

Fluorinated aminoglycosides and their mechanistic implication for aminoglycoside 3'-phosphotransferases from Gram-negative bacteria.

Aminoglycoside 3'-phosphotransferases [APH(3')s] are important bacterial resistance enzymes for aminoglycoside antibiotics. These enzymes phosphorylate the 3'-hydroxyl of these antibiotics, a reaction that inactivates the drug. A series of experiments were carried out to shed light on the details of the turnover chemistry by these enzymes. Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3') types Ia and IIa with kanamycin A, neamine, and their respective difluorinated analogues 4'-deoxy-4',4'-difluorokanamycin A and 4'-deoxy-4',4'-difluoroneamine were carried out, in conjunction with measurements of thio effect and viscosity studies. The fluorinated analogues were shown to be severely impaired as substrates for these enzymes. The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3')-Ia was studied with nonfluorinated substrates. Residue 198 is the proposed active site base that promotes the aminoglycoside hydroxyl for phosphorylation. These findings collectively argue that the Gram-negative APH(3')s show significant nucleophilic participation in the transition state for the phosphate transfer reaction.

The complex of a designer antibiotic with a model aminoacyl site of the 30S ribosomal subunit revealed by X-ray crystallography.

The ribosome is an important target for aminoglycoside antibiotics; however, the clinical effectiveness of aminoglycosides has diminished due to bacterial resistance mechanisms. Here we report the X-ray structure of a novel synthetic aminoglycoside bound to the A site of the ribosome, its target for manifestation of activity. The structure validates the in silico design paradigms for the antibiotic and reveals the molecular interactions made by this novel antibiotic in prokaryotes.

Design of novel antibiotics that bind to the ribosomal acyltransfer site.

The structure of neamine bound to the A site of the bacterial ribosomal RNA was used in the design of novel aminoglycosides. The design took into account stereo and electronic contributions to interactions between RNA and aminoglycosides, as well as a random search of 273 000 compounds from the Cambridge structural database and the National Cancer Institute 3-D database that would fit in the ribosomal aminoglycoside-binding pocket. A total of seven compounds were designed and subsequently synthesized, with the expectation that they would bind to the A-site RNA. Indeed, all synthetic compounds were found to bind to the target RNA comparably to the parent antibiotic neamine, with dissociation constants in the lower micromolar range. The synthetic compounds were evaluated for antibacterial activity against a set of important pathogenic bacteria. These designer antibiotics showed considerably enhanced antibacterial activities against these pathogens, including organisms that hyperexpressed resistance enzymes to aminoglycosides. Furthermore, analyses of four of the synthetic compounds with two important purified resistance enzymes for aminoglycosides indicated that the compounds were very poor substrates; hence the activity of these synthetic antibiotics does not appear to be compromised by the existing resistance mechanisms, as supported by both in vivo and in vitro experiments. The design principles disclosed herein hold the promise of the generation of a large series of designer antibiotics uncompromised by the existing mechanisms of resistance.

Highly Efficient and Practical Syntheses of Lavendamycin Methyl Ester and Related Novel Quinolindiones.

The novel 7-(N-formyl-, 7-(N-acetyl-, and 7-(N-isobutyrylamino)-2-methylquinoline-5,8-diones were synthesized in excellent overall yields in three steps via the nitration of the commercially available 8-hydroxy-2-methylquinoline followed by a reduction-acylation step and then oxidation. Acid hydrolysis of 7-(N-acetylamino)-2-methylquinoline-5,8-dione (14a) afforded the novel 7-aminoquinoline-5,8-dione 7 in excellent yields. Due to our efficient preparation of dione 14a, we now report a short and practical method for the total synthesis of the potent antitumor agent lavendamycin methyl ester (1b) with an excellent overall yield.