Keith D. Green

The Future of Aminoglycosides: The End or Renaissance?

Although aminoglycosides have been used as antibacterials for decades, their use has been hindered by their inherent toxicity and the resistance that has emerged to these compounds. It seems that such issues have relegated a formerly front‐line class of antimicrobials to the proverbial back shelf. However, recent advances have demonstrated that novel aminoglycosides have a potential to overcome resistance as well as to be used to treat HIV‐1 and even human genetic disorders, with abrogated toxicity. It is not the end for aminoglycosides, but rather, the challenges faced by researchers have led to ingenuity and a change in how we view this class of compounds, a renaissance.

Exploring the Substrate Promiscuity of Drug-Modifying Enzymes for the Chemoenzymatic Generation of N-Acylated Aminoglycosides

Aminoglycosides are broad‐spectrum antibiotics commonly used for the treatment of serious bacterial infections. Decades of clinical use have led to the widespread emergence of bacterial resistance to this family of drugs limiting their efficacy in the clinic. Here, we report the development of a methodology that utilizes aminoglycoside acetyltransferases (AACs) and unnatural acyl coenzyme A analogues for the chemoenzymatic generation of N‐acylated aminoglycoside analogues. Generation of N‐acylated aminoglycosides is followed by a simple qualitative test to assess their potency as potential antibacterials. The studied AACs (AAC(6′)‐APH(2′′) and AAC(3)‐IV) show diverse substrate promiscuity towards a variety of aminoglycosides as well as acyl coenzyme A derivatives. The enzymes were also used for the sequential generation of homo‐ and hetero‐di‐N‐acylated aminoglycosides. Following the clinical success of the N‐acylated amikacin and arbekacin, our chemoenzymatic approach offers access to regioselectively N‐acylated aminoglycosides in quantities that allow testing of the antibacterial potential of the synthetic analogues making it possible to decide which molecules will be worth synthesizing on a larger scale.

6′′-Thioether Tobramycin Analogues: Towards Selective Targeting of Bacterial Membranes†

Decades of widespread clinical use of the bacterial ribosome A-site targeting aminoglycosides (AGs) enhanced the evolution of resistance to these antibiotics and reduced their clinical efficacy.[1] Three modes of action lead to bacterial resistance to AGs: reduction in the intracellular concentration of the antibiotics by efflux pump proteins or through reduced membrane permeability; structural modifications of the 16S ribosomal RNA leading to reduced target affinity; and deactivation by AG-modifying enzymes (AMEs).[1c, 2] AMEs are divided into three families: AG nucleotidyltransferases (ANTs), AG phosphotransferases (APHs), and AG acetyltransferases (AACs).[1b, 3]

Identification and characterization of inhibitors of the aminoglycoside resistance acetyltransferase Eis from Mycobacterium tuberculosis.

With an anticipated 9.8 million new cases this year,[1] the tuberculosis (TB) epidemic is one of the most serious health problems worldwide. The continuous emergence and global spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB, underscore the pressing clinical need for novel treatments of this deadly infectious disease and for new solutions to alleviate the resistance problem.[2, 3]

Aminoglycoside multiacetylating activity of the enhanced intracellular survival protein from Mycobacterium smegmatis and its inhibition.

The enhanced intracellular survival (Eis) protein improves the survival of Mycobacterium smegmatis (Msm) in macrophages and functions as the acetyltransferase responsible for kanamycin A resistance, a hallmark of extensively drug-resistant (XDR) tuberculosis, in a large number of Mycobacterium tuberculosis (Mtb) clinical isolates. We recently demonstrated that Eis from Mtb (Eis_Mtb) efficiently multiacetylates a variety of aminoglycoside (AG) antibiotics. Here, to gain insight into the origin of substrate selectivity of AG multiacetylation by Eis, we analyzed AG acetylation by Eis_Msm, investigated its inhibition, and compared these functions to those of Eis_Mtb. Even though for several AGs the multiacetylation properties of Eis_Msm and Eis_Mtb are similar, there are three major differences. (i) Eis_Msm diacetylates apramycin, a conformationally constrained AG, which Eis_Mtb cannot modify. (ii) Eis_Msm triacetylates paromomycin, which can be only diacetylated by Eis_Mtb. (iii) Eis_Msm only monoacetylates hygromycin, a structurally unique AG that is diacetylated by Eis_Mtb. Several nonconserved amino acid residues lining the AG-binding pocket of Eis are likely responsible for these differences between the two Eis homologues. Specifically, we propose that because the AG-binding pocket of Eis_Msm is more open than that of Eis_Mtb, it accommodates apramycin for acetylation in Eis_Msm, but not in Eis_Mtb. We also demonstrate that inhibitors of Eis_Mtb that we recently discovered can inhibit Eis_Msm activity. These observations help define the structural origins of substrate preference among Eis homologues and suggest that Eis_Mtb inhibitors may be applied against all pathogenic mycobacteria to overcome AG resistance caused by Eis upregulation.

Exploring Kinase Cosubstrate Promiscuity: Monitoring Kinase Activity through Dansylation

Kinases catalyze protein phosphorylation, which is a key event in cell signalling. Importantly, kinases are targeted by a number of drugs in clinical trials for cancer, rheumatoid arthritis, and immunosuppression.[1] Because kinases and protein phosphorylation play fundamental roles in disease, methods to monitor kinase activity and substrates are needed. We recently described a phosphoprotein-labelling reaction that couples kinases with an analogue of the adenosine 5′-tri-phosphate (ATP, 1a) cosubstrate. Specifically, ATP conjugated to biotin through the γ-phosphate (ATP-biotin) served as a kinase cosubstrate, which allowed phosphoprotein biotinylation in vitro and in cell lysates.[2] The studies with ATP-biotin raise the possibility that cellular kinases generally tolerate γ-phosphate-modified ATP analogues (Scheme 1A) as cosubstrates. Cosubstrate promiscuity is documented with multiple protein-modifying enzymes, including farnesyltransferase,[3] transglutaminase,[4] galactosyltransferase,[5] N-acetyltransferase,[6] DNA methyltransferase,[7] and phosphopantetheinyl transferase (PPtase).[8] To explore kinase cosubstrate promiscuity, we hypothesized that additional γ-phosphate-modified ATP analogues would be compatible with kinase-catalyzed labelling. Scheme 1 Kinase-catalyzed dansylation. A) General structures of γ-phosphate-modified ATP analogues. B) ATP-dansyl (1c) acts as a kinase cosubstrate to enable phosphorylation-dependent dansylation of peptides and proteins. Incubation with acid (2n HCl or ... We describe here a kinase-catalyzed dansylation reaction for fluorophore labelling of phosphopeptides and phosphoproteins; this reaction substantiates the cosubstrate promiscuity of kinases. Specifically, a commercially available γ-phosphate-modified ATP analogue, ATP-dansyl (1c; dansyl=5(-dimethyl-amino)naphthalene-1-sulfonyl), was coupled with three kinase substrate peptides containing serine (3), threonine (4), or tyrosine (5), and three kinases, cAMP-dependent protein kinase (PKA), casein kinase II (CK2) and Abelson tyrosine kinase (Abl). In all cases, ATP-dansyl served as a kinase cosubstrate, converting each peptide to its corresponding dansylated phosphopep-tide (Scheme 1B), as assessed by MALDI-TOF MS (Table 1). To determine the efficiency of the dansylation reaction, we employed quantitative MS analysis (Scheme S1 in the Supporting Information). After incubation with the corresponding kinase and either ATP (1a) or ATP-dansyl (1c), the phosphopeptide products were isotopically labelled through esterification as previously described,[2,9] and the dansyl group was removed with acid (Scheme 1B). By comparing the two isotopically differentiated phosphopeptides using MALDI-TOF MS, the ATP-dansyl reactions demonstrated 81–91% conversion compared to the ATP reactions (Table 1 and Figures S1–S3 in the Supporting Information). The data indicate that kinase-catalyzed dansylation is compatible with the three natural hydroxyl-containing residues and three kinase enzymes, similar to results previously observed with ATP-biotin.[2] Table 1 MALDI-TOF MS data of peptides 3–5 after incubation with ATP-dansyl and PKA, CK2, or Abl kinase. We next tested compatibility of the kinase-catalyzed dansylation reaction with a full-length protein substrate. Full-length β-casein was incubated with ATP-dansyl (1c) and CK2, trypsin-digested, and analyzed by quantitative MALDI-TOF MS analysis. Dansylation of the β-casein peptide (FQpSEEQQQpTEDELQDK) occurred with 54% conversion compared to the ATP reaction (Figure S4 in the Supporting Information). The data indicate that kinase-catalyzed dansylation is compatible with a full-length protein substrate. To characterize the efficiency of the dansylation reaction, kinetics measurements were obtained with the kemptide peptide (LRRASLG) and PKA using an enzyme-coupled assay (Figure S5 in the Supporting Information).[10,11] ATP demonstrated KM and kcat values consistent with previous reports.[12,13] ATP-dansyl (1c) displayed a similar KM compared to ATP (Table 2). Table 2 Kinetic constants of PKA and kemptide with ATP, ATP-γS or ATP-dansyl as the cosubstrate.[a] The data indicate that the γ-phosphate modification does not interfere significantly with kinase binding. In contrast, ATP-dansyl displayed a 9.1-fold reduced kcat value compared to ATP; this reduction suggests that phosphoramidate transfer with ATP-dansyl is not as rapid as phosphate transfer with ATP, or the dansyl group at the γ-phosphate interferes with substrate binding or the catalytic mechanism. While ATP-dansyl is a less efficient cosubstrate than ATP, dansylation with ATP-dansyl demonstrated catalytic efficiency (kcat/KM) similar to thio-phosphorylation with ATP-γS (1b- Table 2).[12] Because ATP-γS is documented for phosphoprotein labelling,[14–16] the kinase-catalyzed dansylation occurs with catalytic efficiencies appropriate for future applications. Having established the kinase-catalyzed dansylation reaction, we developed a kinase activity assay in which dansylation of a fluorophore-labelled peptide substrate promotes fluorescence resonance energy transfer (FRET; Figure 1A). Specifically, the Abl substrate peptide 5 (Table 1) was synthesized with a 5(6)-carboxy-X-rhodamine (ROX) fluorophore attached at the amino terminus (ROX 5, Figure 1A). The expectation was that kinase-catalyzed dansylation would position the two fluorophores appropriately for FRET (peptide 6, Figure 1A). To test the assay, the ROX 5 peptide was incubated with ATP-dansyl (1c) in the presence and absence of Abl kinase and FRET was monitored. In the presence of recombinant Abl, a 30 ± 5% increase in emission fluorescence was observed compared to the reaction without kinase activity (Figure 1B, columns 1 and 2; Table S1 and Figure S7 in the Supporting Information), which indicates that the FRET assay is sensitive to kinase activity. The 30% increase is consistent with previous FRET-based kinase assays in which 10–60% signal changes are reported.[17–19] To further confirm the quantitative nature of the FRET assay, kinetics measurements were performed. The catalytic efficiency (kcat/KM) was similar with ATP-dansyl whether using the enzyme-coupled or FRET assay (Table 3). Consistent with results observed with PKA, ATP-dansyl displayed a 6.2-fold reduced kcat value compared to ATP (Table 3, Figure S6 in the Supporting Information). The combined data indicate that the FRET assay based on kinase-catalyzed dansylation reproducibly and quantitatively monitors kinase activity. Figure 1 FRET-based kinase activity assay. A) The ROX-peptide 5 (ROX-5, Table 1) was dansylated by Abl and ATP-dansyl to create peptide 6; B) and C) Normalized fluorescence intensity at 595 nm after excitation at 360 nm with ROX 5, ATP-dansyl, and the indicated ... Table 3 Kinetic constants of Abl with ATP or ATP-dansyl as the cosubstrate.[a] Kinases are the targets of multiple small-molecule inhibitor drugs. As a result, identification of inhibitors of kinase activity is an important area of pharmaceutical research. To determine if the FRET assay is appropriate for drug screening, known kinase inhibitors were preincubated with Abl before addition of the ROX 5 peptide and ATP-dansyl. While a 30% increase in emission fluorescence was observed in the absence of inhibitor (Figure 1B, column 2), no increase in emission fluorescence was observed in the presence of staurosporine, a general kinase inhibitor[20] (Figure 1B, column 3; Table S1 in the Supporting Information). Similar results were seen with imatinib (STI-571; Figure 1B, column 4), which has preference for Abl[20] and is used clinically to treat chronic myeloid leukemia.[21] In contrast, the emission fluorescence was similar in the absence or presence of 5,6-dichloro-1-δ-d-ribofuranosyl benzimidazole (DRB), an inhibitor with preference for CK2 kinase[22] (Figure 1B, compare columns 2 and 5). These results indicate that the FRET assay is sensitive and selective for Abl kinase inhibitors, and thus, it is appropriate for drug screening. Fluorescence-based kinase assays have been used to detect kinase activity in mammalian cells and lysates for drug design and disease characterization.[23–29] To further assess the utility of the FRET assay, we monitored Abl kinase activity in HeLa cell lysates. In this case, a 32 ± 2% increase in emission fluorescence was observed compared to the reaction without lysates (Figure 1C, columns 6 and 7; Table S2 in the Supporting Information). Similar to experiments with recombinant Abl, staurosporine inhibited kinase activity and resulted in a loss of fluorescence emission (Figure 1C, column 8; Table S2). These studies indicate that the FRET assay is capable of monitoring cellular kinase activity and is compatible with drug screening using lysates. Significantly, these studies establish that cellular kinases accept ATP-dansyl to label substrates, which corroborates the cosubstrate promiscuity of kinases. In summary, we have established a kinase-catalyzed dansylation reaction with ATP-dansyl as a cosubstrate. The reaction is compatible with peptide or full-length protein substrates and kinases from cell lysates. In addition, the kinase-catalyzed dansylation reaction is appropriate for use in FRET-based kinase activity and inhibition assays. Because kinases are the targets of a variety of clinical drugs, the FRET assay can be applied towards monitoring kinase activity in diseased states and facilitating drug screening efforts. Combined with the kinase-catalyzed biotinylation reaction,[2] the results establish that cellular kinases tolerate γ-phosphate-modified ATP analogues as co-substrates. With the critical role of kinases in signalling and disease, these studies pioneer development of chemical tools monitoring kinase activity and protein phosphorylation.

Amphiphilic tobramycin analogues as antibacterial and antifungal agents

ABSTRACT In this study, we investigated the in vitro antifungal activities, cytotoxicities, and membrane-disruptive actions of amphiphilic tobramycin (TOB) analogues. The antifungal activities were established by determination of MIC values and in time-kill studies. Cytotoxicity was evaluated in mammalian cell lines. The fungal membrane-disruptive action of these analogues was studied by using the membrane-impermeable dye propidium iodide. TOB analogues bearing a linear alkyl chain at their 6″-position in a thioether linkage exhibited chain length-dependent antifungal activities. Analogues with C12 and C14 chains showed promising antifungal activities against tested fungal strains, with MIC values ranging from 1.95 to 62.5 mg/liter and 1.95 to 7.8 mg/liter, respectively. However, C4, C6, and C8 TOB analogues and TOB itself exhibited little to no antifungal activity. Fifty percent inhibitory concentrations (IC50s) for the most potent TOB analogues (C12 and C14) against A549 and Beas 2B cells were 4- to 64-fold and 32- to 64-fold higher, respectively, than their antifungal MIC values against various fungi. Unlike conventional aminoglycoside antibiotics, TOB analogues with alkyl chain lengths of C12 and C14 appear to inhibit fungi by inducing apoptosis and disrupting the fungal membrane as a novel mechanism of action. Amphiphilic TOB analogues showed broad-spectrum antifungal activities with minimal mammalian cell cytotoxicity. This study provides novel lead compounds for the development of antifungal drugs.

Unexpected N-acetylation of capreomycin by mycobacterial Eis enzymes

OBJECTIVES The enhanced intracellular survival (Eis) protein from Mycobacterium tuberculosis (Eis_Mtb), a regio-versatile N-acetyltransferase active towards many aminoglycosides (AGs), confers resistance to kanamycin A in some cases of extensively drug-resistant tuberculosis (XDR-TB). We assessed the activity of Eis_Mtb and of its homologue from Mycobacterium smegmatis (Eis_Msm) against a panel of anti-tuberculosis (TB) drugs and lysine-containing compounds. METHODS AND RESULTS Both enzymes acetylated capreomycin and some lysine-containing compounds, but not other non-AG non-lysine-containing drugs tested. Modelling studies predicted the site of modification on capreomycin to be one of the two primary amines in its β-lysine side chain. Using Eis_Mtb, we established via nuclear magnetic resonance (NMR) spectroscopy that acetylation of capreomycin occurs on the ε-amine of the β-lysine side chain. Using Msm, we also demonstrated for the first time to our knowledge that acetylation of capreomycin results in deactivation of the drug. CONCLUSIONS Eis is a unique acetyltransferase capable of inactivating the anti-TB drug capreomycin, AGs and other lysine-containing compounds.

Synthesis and Bioactivities of Kanamycin B-Derived Cationic Amphiphiles.

Cationic amphiphiles derived from aminoglycosides (AGs) have been shown to exhibit enhanced antimicrobial activity. Through the attachment of hydrophobic residues such as linear alkyl chains on the AG backbone, interesting antibacterial and antifungal agents with a novel mechanism of action have been developed. Herein, we report the design and synthesis of seven kanamycin B (KANB) derivatives. Their antibacterial and antifungal activities, along with resistance/enzymatic, hemolytic, and cytotoxicity assays were also studied. Two of these compounds, with a C12 and C14 aliphatic chain attached at the 6″-position of KANB through a thioether linkage, exhibited good antibacterial and antifungal activity, were poorer substrates than KANB for several AG-modifying enzymes, and could delay the development of resistance in bacteria and fungi. Also, they were both relatively less hemolytic than the known membrane targeting antibiotic gramicidin and the known antifungal agent amphotericin B and were not toxic at their antifungal MIC values. Their oxidation to sulfones was also demonstrated to have no effect on their activities. Moreover, they both acted synergistically with posaconazole, an azole currently used in the treatment of human fungal infections.

Effects of Altering Aminoglycoside Structures on Bacterial Resistance Enzyme Activities

ABSTRACT Aminoglycoside-modifying enzymes (AMEs) constitute the most prevalent mechanism of resistance to aminoglycosides by bacteria. We show that aminoglycosides can be doubly modified by the sequential actions of AMEs, with the activity of the second AME in most cases unaffected, decreased, or completely abolished. We demonstrate that the bifunctional enzyme AAC(3)-Ib/AAC(6′)-Ib′ can diacetylate gentamicin. Since single acetylation does not always inactivate the parent drugs completely, two modifications likely provide more-robust inactivation in vivo.