Cross-utilization of ß-galactosides and cellobiose in Geobacillus stearothermophilus.

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-ß-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-ß-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

Towards Catalytic Antibiotics: Redesign of Aminoglycosides To Catalytically Disable Bacterial Ribosomes.

The emergence of multidrug-resistant pathogens that are resistant to the majority of currently available antibiotics is a significant clinical problem. The development of new antibacterial agents and novel approaches is therefore extremely important. We set out to explore the potential of catalytic antibiotics as a new paradigm in antibiotics research. Herein, we describe our pilot study on the design, synthesis, and biological testing of a series of new derivatives of the natural aminoglycoside antibiotic neomycin B for their potential action as catalytic antibiotics. The new derivatives showed significant antibacterial activity against wild-type bacteria and were especially potent against resistant and pathogenic strains including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Selected compounds displayed RNase activity even though the activity was not as high and specific as we would have expected. On the basis of the observed chemical and biochemical data, along with the comparative molecular dynamics simulations of the prokaryotic rRNA decoding site, we postulate that the rational design of catalytic antibiotics should involve not only their structure but also a comprehensive analysis of the rRNA A-site dynamics.

New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism.

Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes.

Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells.

Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity.

Covalently linked kanamycin - Ciprofloxacin hybrid antibiotics as a tool to fight bacterial resistance.

To address the growing problem of antibiotic resistance, a set of 12 hybrid compounds that covalently link fluoroquinolone (ciprofloxacin) and aminoglycoside (kanamycin A) antibiotics were synthesized, and their activity was determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The hybrids were antagonistic relative to the ciprofloxacin, but were substantially more potent than the parent kanamycin against Gram-negative bacteria, and overcame most dominant resistance mechanisms to aminoglycosides. Selected hybrids were 42-640 fold poorer inhibitors of bacterial protein synthesis than the parent kanamycin, while they displayed similar inhibitory activity to that of ciprofloxacin against DNA gyrase and topoisomerase IV enzymes. The hybrids showed significant delay of resistance development in both E. coli and B. subtilis in comparison to that of component drugs alone or their 1:1 mixture. More generally, the data suggest that an antagonistic combination of aminoglycoside-fluoroquinolone hybrids can lead to new compounds that slowdown/prevent the emergence of resistance.

Structural basis for selective targeting of leishmanial ribosomes: aminoglycoside derivatives as promising therapeutics.

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL.

Designer aminoglycosides that selectively inhibit cytoplasmic rather than mitochondrial ribosomes show decreased ototoxicity: a strategy for the treatment of genetic diseases.

There is compelling evidence that aminoglycoside (AG) antibiotics can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, prolonged AG treatment can cause detrimental side effects in patients, including most prominently, ototoxicity. Recent mechanistic discussions have considered the relative contributions of mitochondrial and cytoplasmic protein synthesis inhibition to AG-induced ototoxicity. We show that AGs inhibit mitochondrial protein synthesis in mammalian cells and perturb cell respiration, leading to a time- and dose-dependent increase in superoxide overproduction and accumulation of free ferrous iron in mitochondria caused by oxidative damage of mitochondrial aconitase, ultimately leading to cell apoptosis via the Fenton reaction. These deleterious effects increase with the increased potency of AG to inhibit the mitochondrial rather than cytoplasmic protein synthesis, which in turn correlates with their ototoxic potential in both murine cochlear explants and the guinea pig in vivo. The deleterious effects of AGs were alleviated in synthetic derivatives specially designed for the treatment of genetic diseases caused by nonsense mutations and possessing low affinity toward mitochondrial ribosomes. This work highlights the benefit of a mechanism-based drug redesign strategy that can maximize the translational value of "readthrough therapy" while mitigating drug-induced side effects. This approach holds promise for patients suffering from genetic diseases caused by nonsense mutations.

Synthetic aminoglycosides efficiently suppress cystic fibrosis transmembrane conductance regulator nonsense mutations and are enhanced by ivacaftor.

New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

Long-term nonsense suppression therapy moderates MPS I-H disease progression.

Nonsense suppression therapy is a therapeutic approach aimed at treating genetic diseases caused by in-frame premature termination codons (PTCs; also commonly known as nonsense mutations). This approach utilizes compounds that suppress translation termination at PTCs, which allows translation to continue and partial levels of deficient protein function to be restored. We hypothesize that suppression therapy can attenuate the lysosomal storage disease mucopolysaccharidosis type I-Hurler (MPS I-H), the severe form of α-L-iduronidase deficiency. α-L-iduronidase participates in glycosaminoglycan (GAG) catabolism and its insufficiency causes progressive GAG accumulation and onset of the MPS I-H phenotype, which consists of multiple somatic and neurological defects. 60-80% of MPS I-H patients carry a nonsense mutation in the IDUA gene. We previously showed that 2-week treatment with the designer aminoglycoside NB84 restored enough α-L-iduronidase function via PTC suppression to reduce tissue GAG accumulation in the Idua(tm1Kmke) MPS I-H mouse model, which carries a PTC homologous to the human IDUA-W402X nonsense mutation. Here we report that long-term NB84 administration maintains α-L-iduronidase activity and GAG reduction in Idua(tm1Kmke) mice throughout a 28-week treatment period. An examination of more complex MPS I-H phenotypes in Idua(tm1Kmke) mice following 28-week NB84 treatment revealed significant moderation of the disease in multiple tissues, including the brain, heart and bone, that are resistant to current MPS I-H therapies. This study represents the first demonstration that long-term nonsense suppression therapy can moderate progression of a genetic disease.

Balancing Nonsense Mutation Readthrough and Toxicity of Designer Aminoglycosides for Treatment of Genetic Diseases.

New derivatives of aminoglycosides with a side chain 1,2-aminoalcohol at the 5" position of ring III were designed, synthesized, and biologically evaluated. The novel lead structure (compound 6), exhibiting substantially enhanced selectivity toward eukaryotic versus prokaryotic ribosome, high readthrough activity, and considerably lower toxicity than the previous lead compounds, was discovered. Balanced readthrough activity and toxicity of 6 were demonstrated in three different nonsense DNA-constructs underlying the genetic diseases, cystic fibrosis and Usher syndrome, and in two different cell lines, baby hamster kidney and human embryonic kidney cells. Molecular dynamics simulations within the A site of the 80S yeast ribosome demonstrated a remarkable kinetic stability of 6, which potentially determines its high readthrough activity.

The designer aminoglycoside NB84 significantly reduces glycosaminoglycan accumulation associated with MPS I-H in the Idua-W392X mouse.

Suppression therapy utilizes compounds that suppress translation termination at in-frame premature termination codons (PTCs) to restore full-length, functional protein. This approach may provide a treatment for diseases caused by nonsense mutations such as mucopolysaccharidosis type I-Hurler (MPS I-H). MPS I-H is a lysosomal storage disease caused by severe α-L-iduronidase deficiency and subsequent lysosomal glycosaminoglycan (GAG) accumulation. MPS I-H represents a good target for suppression therapy because the majority of MPS I-H patients carry nonsense mutations, and restoration of even a small amount of functional α-L-iduronidase may attenuate the MPS I-H phenotype. In this study, we investigated the efficiency of suppression therapy agents to suppress the Idua-W392X nonsense mutation in an MPS I-H mouse model. The drugs tested included the conventional aminoglycosides gentamicin, G418, amikacin, and paromomycin. In addition, the designer aminoglycosides NB54 and NB84, two compounds previously designed to mediate efficient PTC suppression with reduced toxicity, were also examined. Overall, NB84 suppressed the Idua-W392X nonsense mutation much more efficiently than any of the other compounds tested. NB84 treatment restored enough functional α-L-iduronidase activity to partially reverse abnormal GAG accumulation and lysosomal abundance in mouse embryonic fibroblasts derived from the Idua-W392X mouse. Finally, in vivo administration of NB84 to Idua-W392X mice significantly reduced urine GAG excretion and tissue GAG storage. Together, these results suggest that NB84-mediated suppression therapy has the potential to attenuate the MPS I-H disease phenotype.

Dynamic Intramolecular Cap for Preserving Metallodrug Integrity─A Case of Catalytic Fluoroquinolones.

A library of eight new fluoroquinolone-nuclease conjugates containing a guanidinoethyl or aminoethyl auxiliary pendant on the cyclen moiety was designed and synthesized to investigate their potential for overcoming the general issue of "metallodrug vulnerability" under physiological conditions. The Cu(II) and Co(III) complexes of the new designer compounds were synthesized, and their potential to operate a dynamic, intramolecular cap with DNase activity was explored. The lead Co(III)-cyclen-ciprofloxacin conjugate showed excellent in vitro hydrolytic DNase activity, which was retained in the presence of strong endogenous chelators and exhibited enhanced antibacterial activity relative to the metal-free ligand (in the absence of any adjuvants), thereby demonstrating a "proof of concept" in vitro and ex vivo, respectively, for the dynamic cap hypothesis. The lead conjugate nicked supercoiled plasmid DNA within the fluoroquinolone-gyrase-DNA ternary complex and thereby disabled the function of gyrase, a new mode of action not previously reported for any fluoroquinolone.

Toward Catalytic Antibiotics: Redesign of Fluoroquinolones to Catalytically Fragment Chromosomal DNA.

A library of ciprofloxacin-nuclease conjugates was designed and synthesized to investigate their potential as catalytic antibiotics. The Cu(II) complexes of the new designer compounds (i) showed excellent in vitro hydrolytic and oxidative DNase activity, (ii) showed good antibacterial activity against both Gram-negative and Gram-positive bacteria, and (iii) proved to be highly potent bacterial DNA gyrase inhibitors via a mechanism that involves stabilization of the fluoroquinolone-topoisomerase-DNA ternary complex. Furthermore, the Cu(II) complexes of two of the new designer compounds were shown to fragment supercoiled plasmid DNA into linear DNA in the presence of DNA gyrase, demonstrating a "proof of concept" in vitro. These ciprofloxacin-nuclease conjugates can therefore serve as models with which to develop next-generation, in vivo functioning catalytic antimicrobials.

Repairing faulty genes by aminoglycosides: development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations.

New pseudo-di- and pseudo-trisaccharide derivatives of the aminoglycoside drug G418 were designed, synthesized and their ability to readthrough nonsense mutations was examined in both in vitro and ex vivo systems, along with the toxicity tests. Two novel lead structures, NB74 and NB84, exhibiting significantly reduced cell toxicity and superior readthrough efficiency than those of gentamicin, were discovered. The superiority of new leads was demonstrated in six different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Duchenne muscular dystrophy, Usher syndrome and Hurler syndrome.

Crystal structures of Escherichia coli KDO8P synthase complexes reveal the source of catalytic irreversibility.

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS) catalyses the condensation of arabinose 5-phosphate (A5P) and phosphoenol pyruvate (PEP) to obtain 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P). We have elucidated initial modes of ligand binding in KDO8PS binary complexes by X-ray crystallography. Structures of the apo-enzyme and of binary complexes with the substrate PEP, the product KDO8P and the catalytically inactive 1-deoxy analog of arabinose 5-phosphate (1dA5P) were obtained. The KDO8PS active site resembles an irregular funnel with positive electrostatic potential situated at the bottom of the PEP-binding sub-site, which is the primary attractive force towards negatively charged phosphate moieties of all ligands. The structures of the ligand-free apo-KDO8PS and the binary complex with the product KDO8P visualize for the first time the role of His202 as an active-site gate. Examination of the crystal structures of KDO8PS with the KDO8P or 1dA5P shows these ligands bound to the enzyme in the PEP-binding sub-site, and not as expected to the A5P sub-site. Taken together, the structures presented here strengthen earlier evidence that this enzyme functions predominantly through positional catalysis, map out the roles of active-site residues and provide evidence that explains the total lack of catalytic reversibility.

Design of Novel Aminoglycoside Derivatives with Enhanced Suppression of Diseases-Causing Nonsense Mutations.

New pseudotrisaccharide derivatives of aminoglycosides that exploit additional interaction on the shallow groove face of the decoding-site rRNA of eukaryotic ribosome were designed, synthesized and biologically evaluated. Novel lead structures (6 and 7 with an additional 7'-OH), exhibiting enhanced specificity to eukaryotic cytoplasmic ribosome, and superior nonsense mutation suppression activity than those of gentamicin, were discovered. The comparative benefit of new leads was demonstrated in four different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Usher syndrome, and Hurler syndrome.

The identification of the acid-base catalyst of alpha-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase.

The alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid-base catalytic residue. AbfA T-6 exhibits residual activity towards aryl beta-D-xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid-base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid-base catalyst of AbfA T-6.

A new class of branched aminoglycosides: pseudo-pentasaccharide derivatives of neomycin B.

[reaction: see text] The clinically important antibiotic neomycin B was modified at position C5' ' by adding one extra sugar ring in the beta-configuration, and the observed pseudo-pentasaccharides were tested against various bacterial strains, including pathogenic and resistant strains. The designed antibiotics show antibacterial activity superior to that of neomycin B against pathogenic and resistant bacterial strains.

Synthesis and evaluation of a mechanism-based inhibitor of KDO8P synthase.

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. In attempts to investigate the lack of antibacterial activity of the most potent inhibitor of KDO8P synthase, the amino phosphonophosphate 3, we have synthesized its hydrolytically stable isosteric phosphonate analogue 4 and tested it as an inhibitor of the enzyme. The synthesis of 4 was accomplished in a one step procedure by employing the direct reductive amination in aqueous media between unprotected sugar phosphonate and glyphosate. The analogue 4 proved to be a competitive inhibitor of KDO8P synthase with respect to both substrates A5P and PEP binding. In vitro antibacterial tests against a series of different Gram-negative organisms establish that both inhibitors (3 and 4) lack antibacterial activity probably due to their reduced ability to penetrate the bacterial cell membrane.

Aminoglycoside-Induced Premature Stop Codon Read-Through of Mucopolysaccharidosis Type I Patient Q70X and W402X Mutations in Cultured Cells.

The premature stop codon mutations, Q70X and W402X, are the most common α-L-iduronidase gene (IDUA) mutations in mucopolysaccharidosis type I (MPS I) patients. Read-through drugs have been used to suppress premature stop codons, and this can potentially be used to treat patients who have this type of mutation. We examined the effects of aminoglycoside treatment on the IDUA mutations Q70X and W402X in cultured cells and show that 4,5-disubstituted aminoglycosides induced more read-through for the W402X mutation, while 4,6-disubstituted aminoglycosides promoted more read-through for the Q70X mutation: lividomycin (4,5-disubstituted) induced a 7.8-fold increase in α-L-iduronidase enzyme activity for the W402X mutation; NB54 (4,5-disubstituted) induced a 3.7 fold increase in the amount of α-L-iduronidase enzyme activity for the W402X mutation, but had less effect on the Q70X mutation, whereas gentamicin (4,6-disubstituted) had the reverse effect on read-through for both mutations. The predicted mRNA secondary structural changes for both mutations were markedly different, which may explain these different effects on read-through for these two premature stop codons.