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Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside-modifying enzymes has, however, dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators that work by inhibiting aminoglycoside N-6'-acetyltransferase, one of the most common determinants of aminoglycoside resistance. Among these, prodrugs that combine the structure of an aminoglycoside with that of pantothenate into one molecule are especially promising. We report here a series of cellular studies to investigate the activity and mechanism of action of these prodrugs further. Our results reveal a new aminoglycoside resistance inhibitor, as well as the possibility that these prodrugs are transformed into more than one inhibitor in bacteria. We also report that the onset of the potentiators is rapid. Their low cell cytotoxicity, good stability, and potentiation of various aminoglycosides, against both Gram-positive and Gram-negative bacteria, make them interesting compounds for the development of new drugs., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Enzyme activity is commonly controlled by allostery, where ligand binding at one site alters the activities of distant sites. Classical explanations for multisubunit proteins involve conformational transitions that are fundamentally deterministic. For example, in the Monod-Wyman-Changeaux (MWC) paradigm, conformational transitions occur simultaneously in all subunits. In the Koshland-Nemethy-Filmer (KNF) paradigm, conformational transitions only occur in ligand-bound subunits. In contrast, recent models predict conformational changes that are governed by probabilities rather than absolute rules. To better understand allostery at the molecular level, we applied a recently developed spectroscopic and calorimetric method to the interactions of a dimeric enzyme with two different ligands. We found that conformational transitions appear MWC-like for a ligand that binds at the dimer interface and KNF-like for a distal ligand. These results provide strong experimental support for probabilistic allosteric theory predictions that an enzyme can exhibit a mixture of MWC and KNF character, with the balance partly governed by subunit interface energies.

A convenient synthesis of 4'-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4'-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6'-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA.

Allostery has been studied for many decades, yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6')-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homodimeric enzyme is modulated by opposing mechanisms. One follows a classical Koshland-Némethy-Filmer (KNF) paradigm, whereas the other follows a recently proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding and conformational changes represents a new way to govern energetic communication between binding sites.

Just five steps! The synthesis of a phosphonate-linked aminoglycoside-coenzyme A derivative (see scheme) that includes a Michael addition in water has been realized in just five steps. Aminoglycoside N-6'-acetyltransferases (AAC(6')s) are important determinants of antibiotic resistance. A good mechanistic understanding of these enzymes is essential to overcome aminoglycoside resistance. We have previously reported the synthesis of amide- and sulfonamide-linked aminoglycoside-coenzyme A conjugates, which were useful mechanistic and structural probes of AAC(6')s. We report here the synthesis of a phosphonate-linked aminoglycoside-coenzyme A variant, which is expected to be a superior mimic of the tetrahedral intermediate proposed for catalysis by AAC(6')s. This synthetic target is especially challenging for a number of reasons, including the presence of multiple functional groups, the water solubility of both starting materials, and incompatibility of P(III) chemistry with water. We have overcome these challenges by adding the expensive coenzyme A in the last step by means of an elegant Michael-type addition onto a vinylphosphonate in water. Overall, a single protection step was needed. The decreased inhibitory potency of this bisubstrate compared to that of the amide-linked analogue suggests that Enterococcus faecium AAC(6')-Ii may not stabilize the proposed tetrahedral intermediate, and may act mainly through proximity catalysis.

Aminoglycoside-coenzyme A conjugates are challenging synthetic targets because of the wealth of functional groups and high polarity of the starting materials. We previously reported a one-pot synthesis of amide-linked aminoglycoside-CoA bisubstrates. These molecules are nanomolar inhibitors of aminoglycoside N-6'-acetyltransferase Ii (AAC(6')-Ii), an important enzyme involved in bacterial resistance to aminoglycoside antibiotics. We report here the synthesis and biological activity of five new aminoglycoside-CoA bisubstrates containing sulfonamide, sulfoxide, or sulfone groups. Interestingly, the sulfonamide-linked bisubstrate, which was expected to best mimic the tetrahedral intermediate, does not show improved inhibition when compared with amide-linked bisubstrates. On the other hand, most of the sulfone- and sulfoxide-containing bisubstrates prepared are nanomolar inhibitors of AAC(6')-Ii.

Aminoglycosides are antibacterial compounds that act by binding to the A site of the small 30S bacterial ribosomal subunit and inhibiting protein translation. Clinical resistance to aminoglycosides is generally the result of the expression of enzymes that covalently modify the antibiotic, including phosphorylation, adenylylation, and acetylation. Bisubstrate analogs for the aminoglycoside N-acetyltransferases are nanomolar inhibitors of Enterococcus faecium AAC(6')-Ii. However, in the case of the Salmonella enterica aac(6')-Iy-encoded aminoglycoside N-acetyltransferase, we demonstrate that a series of bisubstrate analogs are only micromolar inhibitors. In contrast to studies with AAC(6')-Ii, the inhibition constants toward AAC(6')-Iy are essentially independent of both the identity of the aminoglycoside component of the bisubstrate and the number of carbon atoms that are used to link the CoA and aminoglycoside components. The patterns of inhibition suggest that the CoA portion of the bisubstrate analog can bind to the enzyme-aminoglycoside substrate complex and that the aminoglycoside portion can bind to the enzyme-CoA product complex. However, at the high concentrations of bisubstrate analog used in crystallization experiments, we could crystallize and solve the three-dimensional structure of the enzyme-bisubstrate complex. The structure reveals that both the CoA and aminoglycoside portions bind in essentially the same positions as those previously observed for the enzyme-CoA-ribostamycin complex, with only a modest adjustment to accommodate the "linker". These results are compared to previous studies of the interaction of similar bisubstrate analogs with other aminoglycoside N-acetyltransferases.

Aminoglycoside antibiotics act by binding to 16S rRNA. Resistance to these antibiotics occurs via drug modifications by enzymes such as aminoglycoside 6'-N-acetyltransferases (AAC(6')s). We report here the regioselective and efficient synthesis of N-6'-acylated aminoglycosides and their use as probes to study AAC(6')-Ii and aminoglycoside-RNA complexes. Our results emphasize the central role of N-6' nucleophilicity for transformation by AAC(6')-Ii and the importance of hydrogen bonding between 6'-NH(2) and 16S rRNA for antibacterial activity.

Truncated aminoglycoside-coenzyme A bisubstrate analogues were efficiently prepared using a convergent approach where the amine and the thiol are coupled in one pot with the addition of a linker, without the need for protecting groups. These derivatives were tested for their effect on the activity of the resistance-causing enzyme aminoglycoside 6'-N-acetyltransferase Ii, and key structure-activity relationships are reported. Moreover, one of the inhibitors is able to block aminoglycoside resistance in cells expressing this enzyme.