Modified Aminoglycosides Bind Nucleic Acids in High-Molecular-Weight Complexes.

Aminoglycosides represent a large group of antibiotics well known for their ability to target the bacterial ribosome. In studying 6"-substituted variants of the aminoglycoside tobramycin, we serendipitously found that compounds with C12 or C14 linear alkyl substituents potently inhibit reverse transcription in vitro. Initial observations suggested specific inhibition of reverse transcriptase. However, further analysis showed that these and related compounds bind nucleic acids with high affinity, forming high-molecular weight complexes. Stable complex formation is observed with DNA or RNA in single- or double-stranded form. Given the amphiphilic nature of these aminoglycoside derivatives, they likely form micelles and/or vesicles with surface-bound nucleic acids. Hence, these compounds may be useful tools to localize nucleic acids to surfaces or deliver nucleic acids to cells or organelles.

Combining Chalcones with Donepezil to Inhibit Both Cholinesterases and Aß Fibril Assembly.

The fact that the number of people with Alzheimer's disease is increasing, combined with the limited availability of drugs for its treatment, emphasize the need for the development of novel effective therapeutics for treating this brain disorder. Herein, we focus on generating 12 chalcone-donepezil hybrids, with the goal of simultaneously targeting amyloid-ß (Aß) peptides as well as cholinesterases (i.e., acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)). We present the design, synthesis, and biochemical evaluation of these two series of novel 1,3-chalcone-donepezil (15a-15f) or 1,4-chalcone-donepezil (16a-16f) hybrids. We evaluate the relationship between their structures and their ability to inhibit AChE/BChE activity as well as their ability to bind Aß peptides. We show that several of these novel chalcone-donepezil hybrids can successfully inhibit AChE/BChE as well as the assembly of N-biotinylated Aß(1-42) oligomers. We also demonstrate that the Aß binding site of these hybrids differs from that of Pittsburgh Compound B (PIB).

Novel zafirlukast derivatives exhibit selective antibacterial activity against Porphyromonas gingivalis.

Periodontal disease is an oral chronic immune-inflammatory disease highly prevalent worldwide that is initiated by specific oral bacterial species leading to local and systemic effects. The development of new preventive/therapeutic strategies to specifically target oral periodontopathogens without perturbing oral microbiome species normally colonizing the oral cavity is needed. The fast and affordable strategy of repositioning of already FDA-approved drugs can be an answer to the development of novel treatments against periodontal pathogens such as Porphyromonas gingivalis. Herein, we report the synthesis and antibacterial activity of novel zafirlukast derivatives, their bactericidal effect, and their cytotoxicity against oral epithelial cell lines. Many of these derivatives exhibited superior antibacterial activity against P. gingivalis compared to the parent drug zafirlukast. The most promising compounds were found to be selective against P. gingivalis and they were bactericidal in their activity. Finally, we demonstrated that these potent derivatives of zafirlukast provided a better safety profile against oral epithelial cells compared to zafirlukast.

Investigating the promiscuity of the chloramphenicol nitroreductase from Haemophilus influenzae towards the reduction of 4-nitrobenzene derivatives.

Chloramphenicol nitroreductase (CNR), a drug-modifying enzyme from Haemophilus influenzae, has been shown to be responsible for the conversion of the nitro group into an amine in the antibiotic chloramphenicol (CAM). Since CAM structurally bears a 4-nitrobenzene moiety, we explored the substrate promiscuity of CNR by investigating its nitroreduction of 4-nitrobenzyl derivatives. We tested twenty compounds containing a nitrobenzene core, two nitropyridines, one compound with a vinylogous nitro group, and two aliphatic nitro compounds. In addition, we also synthesized twenty-eight 4-nitrobenzyl derivatives with ether, ester, and thioether substituents and assessed the relative activity of CNR in their presence. We found several of these compounds to be modified by CNR, with the enzyme activity ranging from 1 to 150% when compared to CAM. This data provides insights into two areas: (i) chemoenzymatic reduction of select compounds to avoid harsh chemicals and heavy metals routinely used in reductions of nitro groups and (ii) functional groups that would aid CAM in overcoming the activity of this enzyme.

Multifunctional Donepezil Analogues as Cholinesterase and BACE1 Inhibitors.

A series of 22 donepezil analogues were synthesized through alkylation/benzylation and compared to donepezil and its 6-O-desmethyl adduct. All the compounds were found to be potent inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), two enzymes responsible for the hydrolysis of the neurotransmitter acetylcholine in Alzheimer's disease patient brains. Many of them displayed lower inhibitory concentrations of EeAChE (IC50 = 0.016 ± 0.001 µM to 0.23 ± 0.03 µM) and EfBChE (IC50 = 0.11 ± 0.01 µM to 1.3 ± 0.2 µM) than donepezil. One of the better compounds was tested against HsAChE and was found to be even more active than donepezil and inhibited HsAChE better than EeAChE. The analogues with the aromatic substituents were generally more potent than the ones with aliphatic substituents. Five of the analogues also inhibited the action of ß-secretase (BACE1) enzyme.

Differential Effects of Linkers on the Activity of Amphiphilic Tobramycin Antifungals.

As the threat associated with fungal infections continues to rise and the availability of antifungal drugs remains a concern, it becomes obvious that the need to bolster the antifungal armamentarium is urgent. Building from our previous findings of tobramycin (TOB) derivatives with antifungal activity, we further investigate the effects of various linkers on the biological activity of these aminoglycosides. Herein, we analyze how thioether, sulfone, triazole, amide, and ether functionalities affect the antifungal activity of alkylated TOB derivatives against 22 Candida, Cryptococcus, and Aspergillus species. We also evaluate their impact on the hemolysis of murine erythrocytes and the cytotoxicity against mammalian cell lines. While the triazole linker appears to confer optimal activity overall, all of the linkers incorporated into the TOB derivatives resulted in compounds that are very effective against the Cryptococcus neoformans species, with MIC values ranging from 0.48 to 3.9 µg/mL.

Zinc transporters YbtX and ZnuABC are required for the virulence of Yersinia pestis in bubonic and pneumonic plague in mice.

A number of bacterial pathogens require the ZnuABC Zinc (Zn2+) transporter and/or a second Zn2+ transport system to overcome Zn2+ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn2+ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn2+ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic plague while a ybtX mutant retains high virulence in both plague models. While sequestration of host Zn is a key nutritional immunity factor, excess Zn appears to have a significant antimicrobial role in controlling intracellular bacterial survival. Here, we demonstrate that ZntA, a Zn2+ exporter, plays a role in resistance to Zn toxicity in vitro, but that a zntA zur double mutant retains high virulence in both pneumonic and bubonic plague models and survival in macrophages. We also confirm that Ybt does not directly bind Zn2+in vitro under the conditions tested. However, we detect a significant increase in Zn2+-binding ability of filtered supernatants from a Ybt+ strain compared to those from a strain unable to produce the siderophore, supporting our previously published data that Ybt biosynthetic genes are involved in the production of a secreted Zn-binding molecule (zincophore). Our data suggest that Ybt or a modified Ybt participate in or promote Zn-binding activity in culture supernatants and is involved in Zn acquisition in Y. pestis.

Differential Effects of Structural Modifications on the Competition of Chalcones for the PIB Amyloid Imaging Ligand-Binding Site in Alzheimer's Disease Brain and Synthetic Aß Fibrils.

Alzheimer's disease (AD) is a complex brain disorder that still remains ill defined. In order to understand the significance of binding of different clinical in vivo imaging ligands to the polymorphic pathological features of AD brain, the molecular characteristics of the ligand interacting with its specific binding site need to be defined. Herein, we observed that tritiated Pittsburgh Compound B ((3)H-PIB) can be displaced from synthetic Aß(1-40) and Aß(1-42) fibrils and from the PIB binding complex purified from human AD brain (ADPBC) by molecules containing a chalcone structural scaffold. We evaluated how substitution on the chalcone scaffold alters its ability to displace (3)H-PIB from the synthetic fibrils and ADPBC. By comparing unsubstituted core chalcone scaffolds along with the effects of bromine and methyl substitution at various positions, we found that attaching a hydroxyl group on the ring adjacent to the carbonyl group (ring I) of the parent member of the chalcone family generally improved the binding affinity of chalcones toward ADPBC and synthetic fibrils F40 and F42. Furthermore, any substitution on ring I at the ortho-position of the carbonyl group greatly decreases the binding affinity of the chalcones, potentially as a result of steric hindrance. Together with the finding that neither our chalcones nor PIB interact with the Congo Red/X-34 binding site, these molecules provide new tools to selectively probe the PIB binding site that is found in human AD brain, but not in brains of AD pathology animal models. Our chalcone derivatives also provide important information on the effects of fibril polymorphism on ligand binding.

A combination approach to treating fungal infections.

Azoles are antifungal drugs used to treat fungal infections such as candidiasis in humans. Their extensive use has led to the emergence of drug resistance, complicating antifungal therapy for yeast infections in critically ill patients. Combination therapy has become popular in clinical practice as a potential strategy to fight resistant fungal isolates. Recently, amphiphilic tobramycin analogues, C12 and C14, were shown to display antifungal activities. Herein, the antifungal synergy of C12 and C14 with four azoles, fluconazole (FLC), itraconazole (ITC), posaconazole (POS), and voriconazole (VOR), was examined against seven Candida albicans strains. All tested strains were synergistically inhibited by C12 when combined with azoles, with the exception of C. albicans 64124 and MYA-2876 by FLC and VOR. Likewise, when combined with POS and ITC, C14 exhibited synergistic growth inhibition of all C. albicans strains, except C. albicans MYA-2876 by ITC. The combinations of FLC-C14 and VOR-C14 showed synergistic antifungal effect against three C. albicans and four C. albicans strains, respectively. Finally, synergism between C12/C14 and POS were confirmed by time-kill and disk diffusion assays. These results suggest the possibility of combining C12 or C14 with azoles to treat invasive fungal infections at lower administration doses or with a higher efficiency.

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 structural modifications on the metal binding, anti-amyloid activity, and cholinesterase inhibitory activity of chalcones.

As the number of individuals affected with Alzheimer's disease (AD) increases and the availability of drugs for AD treatment remains limited, the need to develop effective therapeutics for AD becomes more and more pressing. Strategies currently pursued include inhibiting acetylcholinesterase (AChE) and targeting amyloid-ß (Aß) peptides and metal-Aß complexes. This work presents the design, synthesis, and biochemical evaluation of a series of chalcones, and assesses the relationship between their structures and their ability to bind metal ions and/or Aß species, and inhibit AChE/BChE activity. Several chalcones were found to exhibit potent disaggregation of pre-formed N-biotinyl Aß1-42 (bioAß42) aggregates in vitro in the absence and presence of Cu(2+)/Zn(2+), while others were effective at inhibiting the action of AChE.

Tobramycin Variants with Enhanced Ribosome-Targeting Activity.

With the increased evolution of aminoglycoside (AG)-resistant bacterial strains, the need to develop AGs with 1) enhanced antimicrobial activity, 2) the ability to evade resistance mechanisms, and 3) the capability of targeting the ribosome with higher efficiency is more and more pressing. The chemical derivatization of the naturally occurring tobramycin (TOB) by attachment of 37 different thioether groups at the 6''-position led to the identification of generally poorer substrates of TOB-targeting AG-modifying enzymes (AMEs). Thirteen of these displayed better antibacterial activity than the parent TOB while retaining ribosome-targeting specificity. Analysis of these compounds in vitro shed light on the mechanism by which they act and revealed three with clearly enhanced ribosome-targeting activity.

Amphiphilic Tobramycin Analogues as Antibacterial and Antifungal Agents.

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.

Structure-activity relationships for antibacterial to antifungal conversion of kanamycin to amphiphilic analogues.

Novel fungicides are urgently needed. It was recently reported that the attachment of an octyl group at the O-4″ position of kanamycin B converts this antibacterial aminoglycoside into a novel antifungal agent. To elucidate the structure-activity relationship (SAR) for this phenomenon, a lead compound FG03 with a hydroxyl group replacing the 3″-NH2 group of kanamycin B was synthesized. FG03's antifungal activity and synthetic scheme inspired the synthesis of a library of kanamycin B analogues alkylated at various hydroxyl groups. SAR studies of the library revealed that for antifungal activity the O-4″ position is the optimal site for attaching a linear alkyl chain and that the 3″-NH2 and 6″-OH groups of the kanamycin B parent molecule are not essential for antifungal activity. The discovery of lead compound, FG03, is an example of reviving clinically obsolete drugs like kanamycin by simple chemical modification and an alternative strategy for discovering novel antimicrobials.

New trends in aminoglycosides use.

Despite their inherent toxicity and the acquired bacterial resistance that continuously threaten their long-term clinical use, aminoglycosides (AGs) still remain valuable components of the antibiotic armamentarium. Recent literature shows that the AGs' role has been further expanded as multi-tasking players in different areas of study. This review aims at presenting some of the new trends observed in the use of AGs in the past decade, along with the current understanding of their mechanisms of action in various bacterial and eukaryotic cellular processes.

Synthesis and anticancer structure activity relationship investigation of cationic anthraquinone analogs.

We have synthesized a series of novel 4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium salts, which can be viewed as analogs of cationic anthraquinones. Unlike the similar analogs that we have reported previously, these compounds show relatively weak antibacterial activities but exert strong anticancer activities (low µM to nM GI50), in particular, against melanoma, colon cancer, non-small cell lung cancer and central nervous system (CNS) cancer. These compounds are structurally different from their predecessors by having the aromatic group, instead of alkyl chains, directly attached to the cationic anthraquinone scaffold. Further investigation in the structure-activity relationship (SAR) reveals the significant role of electron donating substituents on the aromatic ring in enhancing the anticancer activities via resonance effect. Steric hindrance of these groups is disadvantageous but is less influential than the resonance effect. The difference in the attached groups at N-1 position of the cationic anthraquinone analog is the main structural factor for the switching of biological activity from antibacterial to anticancer. The discovery of these compounds may lead to the development of novel cancer chemotherapeutics.

Membrane lipid-modulated mechanism of action and non-cytotoxicity of novel fungicide aminoglycoside FG08.

A novel aminoglycoside, FG08, that differs from kanamycin B only by a C8 alkyl chain at the 4″-O position, was previously reported. Unlike kanamycin B, FG08 shows broad-spectrum fungicidal but not anti-bacterial activities. To understand its specificity for fungi, the mechanism of action of FG08 was studied using intact cells of the yeast Saccharomyces cerevisiae and small unilamellar membrane vesicles. With exposure to FG08 (30 µg mL(-1)), 8-fold more cells were stained with fluorescein isothiocyanate, cells had 4 to 6-fold higher K(+) efflux rates, and 18-fold more cells were stained with SYTOX Green in comparison to exposure to kanamycin B (30 µg mL(-1)). Yeast mutants with aberrant membrane sphingolipids (no sphingoid base C4 hydroxyl group, truncated very long fatty acid chain, or lacking the terminal phosphorylinositol group of mannosyl-diinositolphosphorylphytoceramide were 4 to 8-fold less susceptible to growth inhibition with FG08 and showed 2 to 10-fold lower SYTOX Green dye uptake rates than did the isogenic wild-type strain. FG08 caused leakage of pre-loaded calcein from 50% of small unilamellar vesicles with glycerophospholipid and sterol compositions that mimic the compositions of fungal plasma membranes. Less than 5 and 10% of vesicles with glycerophospholipid and sterol compositions that mimic bacterial and mammalian cell plasma membranes, respectively, showed calcein leakage. In tetrazolium dye cytotoxicity tests, mammalian cell lines NIH3T3 and C8161.9 showed FG08 toxicity at concentrations that were 10 to 20-fold higher than fungicidal minimal inhibitory concentrations. It is concluded that FG08's growth inhibitory specificity for fungi lie in plasma membrane permeability changes involving mechanisms that are modulated by membrane lipid composition.

Investigation of antibacterial mode of action for traditional and amphiphilic aminoglycosides.

Aminoglycoside represents a class of versatile and broad spectrum antibacterial agents. In an effort to revive the antibacterial activity against aminoglycoside resistant bacteria, our laboratory has developed two new classes of aminoglycoside, pyranmycin and amphiphilic neomycin (NEOF004). The former resembles the traditional aminoglycoside, neomycin. The latter, albeit derived from neomycin, appears to exert antibacterial action via a different mode of action. In order to discern that these aminoglycoside derivatives have distinct antibacterial mode of action, RNA-binding affinity and fluorogenic dye were employed. These studies, together with our previous investigation, confirm that pyranmycin exhibit the traditional antibacterial mode of action of aminoglycosides by binding toward the bacterial rRNA. On the other hand, the amphiphilic neomycin, NEOF004 disrupts the bacterial cell wall. In a broader perspective, it verifies that structurally modified neomycin can exert different antibacterial mode of action leading to the revival of activity against aminoglycoside resistant bacteria.

Library synthesis and antibacterial investigation of cationic anthraquinone analogs.

We report the parallel synthesis of a series of novel 4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium chloride salts, which are analogs to cationic anthraquinones. Three synthetic protocols were examined leading to a convenient and facile library synthesis of the cationic anthraquinone analogs that contain double alkyl chains of various lengths (C(2)-C(12)) at N-1 and N-3 positions. The antibacterial activities of these compounds were evaluated against Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli. The antibacterial activities of these compounds were expected to be associated with the structural features of naphthoquinone, cation and lypophilic alkyl chain and, interestingly, they showed much higher levels of antibacterial activities against G+ than G- bacteria. In addition, when the total number of carbon atoms of the alkyl groups at both N-1 and N-3 positions lies between 9 and 18, the bactericidal activity against S. aureus increased with increasing alkyl chain length at both N-atoms with MIC ≤ 1 µg/mL.

Antibacterial to antifungal conversion of neamine aminoglycosides through alkyl modification. Strategy for reviving old drugs into agrofungicides.

Many Actinomycetes aminoglycosides are widely used antibiotics. Although mainly antibacterials, a few known aminoglycosides also inhibit yeasts, protozoans and important crop pathogenic fungal oomycetes. Here we show that attachment of a C8 alkyl chain to ring III of a neamine-based aminoglycoside specifically at the 4″-o position yields a broad-spectrum fungicide (FG08) without the antibacterial properties typical for aminoglycosides. Leaf infection assays and greenhouse studies show that FG08 is capable of suppressing wheat fungal infections by Fusarium graminearum-the causative agent of Fusarium head blight-at concentrations that are minimally phytotoxic. Unlike typical aminoglycoside action of ribosomal protein translation miscoding, FG08's antifungal action involves perturbation of the plasma membrane. This antibacterial to antifungal transformation could pave the way for the development of a new class of aminoglycoside-based fungicides suitable for use in crop disease applications. In addition, this strategy is an example of reviving a clinically obsolete drug by simple chemical modification to yield a new application.