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.

Kinase-mediated resistance to antibiotics is a significant clinical challenge. These enzymes share a common protein fold characteristic of Ser/Thr/Tyr protein kinases. We screened 14 antibiotic resistance kinases against 80 chemically diverse protein kinase inhibitors to map resistance kinase chemical space. The screens identified molecules with both broad and narrow inhibition profiles, proving that protein kinase inhibitors offer privileged chemical matter with the potential to block antibiotic resistance. One example is the flavonol quercetin, which inhibited a number of resistance kinases in vitro and in vivo. This activity was rationalized by determination of the crystal structure of the aminoglycoside kinase APH(2″)-IVa in complex with quercetin and its antibiotic substrate kanamycin. Our data demonstrate that protein kinase inhibitors offer chemical scaffolds that can block antibiotic resistance, providing leads for co-drug design.

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.

Aminoglycoside-2″-O-nucleotidyltransferase ANT(2″)-Ia is an aminoglycoside resistance enzyme prevalent among Gram-negative bacteria, and is one of the most common determinants of enzyme-dependant aminoglycoside-resistance. The following report outlines the use of our recently described oxidopyrylium cycloaddition/ring-opening strategy in the synthesis and profiling of a library of synthetic α-hydroxytropolones against ANT(2″)-Ia. In addition, we show that two of these synthetic constructs are capable of rescuing gentamicin activity against ANT-(2″)-Ia-expressing bacteria.

Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3)-IIIa, APH(2؆)-Ib, and MPH(2)-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3)-IIIa exclusively uses ATP, MPH(2)-I exclusively uses GTP, and APH(2؆)-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.Antibiotic modification is a major mechanism of resistance that impacts the efficacy of numerous antimicrobial drug classes. The enzymes that catalyze these group transfer mechanisms have evolved from precursor genes that encode proteins used to accomplish numerous metabolic tasks no doubt unrelated to drug resistance. Borrowing nomenclature from the oncogene field, we term such elements protoresistance genes. Biochemical and structural evidence has shown that protoresistance genes can be found in a number of key metabolic pathways, including cell wall biosynthesis and signal transduction (23). With respect to group transfer antibiotic resistance enzymes, these protoresistance genes encode protein and other small-molecule kinases, acetyltransferases, adenylyltransferases, ADP-ribosyltransferases, and glycosyltransferases. The addition of phosphoryl, acyl, adenyl, ribosyl, and glycosyl groups to the antibiotic scaffold alters the interaction with the cellular target to such a degree that the resistance phenotype results (6).The unifying biochemical logic of group transfer in antibiotic resistance is the co-opting of the normal cellular function of the protoresistance element by natural selection to include the modification of antibiotic molecules. Because group transfer enzymes require a second substrate (e.g., ATP, acetyl coenzyme A, or thymidine diphosphate glucose), these should retain the original specificity of the protoresistance enzyme since natural selection would not normally be expected to act on this site during resistance gene evolution.Antibiotic kinases represent a large superfamily of enzymes that covalently modify antibiotic...