Exploring the use of conformationally locked aminoglycosides as a new strategy to overcome bacterial resistance

The emergence of bacterial resistance to the major classes of antibiotics has become a serious problem over recent years. For aminoglycosides, the major biochemical mechanism for bacterial resistance is the enzymatic modification of the drug. Interestingly, in several cases, the oligosaccharide conformation recognized by the ribosomic RNA and the enzymes responsible for the antibiotic inactivation is remarkably different. This observation suggests a possible structure-based chemical strategy to overcome bacterial resistance; in principle, it should be possible to design a conformationally locked oligosaccharide that still retains antibiotic activity but that is not susceptible to enzymatic inactivation. To explore the scope and limitations of this strategy, we have synthesized several aminoglycoside derivatives locked in the ribosome-bound "bioactive" conformation. The effect of the structural preorganization on RNA binding, together with its influence on the aminoglycoside inactivation by several enzymes involved in bacterial resistance, has been studied. Our results indicate that the conformational constraint has a modest effect on their interaction with ribosomal RNA. In contrast, it may display a large impact on their enzymatic inactivation. Thus, the work presented herein provides a key example of how the conformational differences exhibited by these ligands within the binding pockets of the ribosome and of those enzymes involved in bacterial resistance can, in favorable cases, be exploited for designing new antibiotic derivatives with improved activity in resistant strains.     

Glycopeptide antibiotics and development of inhibitors to overcome vancomycin resistance

This review outlines the history of vancomycin and other key glycopeptide antibiotics and the molecular basis of vancomycin resistance, and focuses on the development of synthetic inhibitors of vancomycin-resistant enzymes VanR, S, A, H and X to overcome resistance. The literature up to July 2001 is reviewed and 119 references are cited.     

Mathematical modelling of bacterial growth at subinhibitory levels of aminoglycosides

The subinhibitory effect of antibiotics has often been studied without any clear theoretical framework; we have chosen to use mathematical bacterial growth modelling as a useful tool to analyse these biological states in a more rigorous manner. Since their mode of action and molecular target are relatively well known, aminoglycosides were well suited for this more sophisticated study of subinhibitory action. We have shown that two models (the Monod and the logistic models) regularly used in bacteriology, were adequate to describe these effects in a glucose-limited medium.A change of model, according to antibiotic concentration, revealed the existence of two separate actions. At lower concentrations, inhibition affected mainly glucose use, the substrate remained limiting and growth mode did not change. As soon as the concentration exceeded a threshold, growth was totally disturbed, probably through a physiological ⪡catastrophe⪢. This threshold can be used to estimate bacterial susceptibility to these antibiotics.     

Development of a polymer system for the delivery of daunorubicin to tumor cells to overcome drug resistance

Method for the synthesis of polymeric nanoparticles (NP) with encapsulated daunorubicin (DNR) was developed on the basis of double emulsion solvent evaporation technique using biodegradable poly(lactide-co-glycolide) (PLGA), which is aimed at customization of pharmacokinetic properties of the preparation, enhanced accumulation of DNR in tumor cells and prolongation of its action. The obtained polymer nanoparticles (DNR-PLGA) had average size ranging around 138±36 nm, with zeta-potential of –25.3 mV and the polydispersity index (PDI) of 0.072. The release kinetics of DNR from polymer nanoparticles at pH 7.4 and 5.0 has been studied. In vitro studies showed similar specific activity of DNR- PLGA in K562 and MCF-7 cancer cell lines together with an increase in activity in K562 Adr and MCF-7 Adr cell lines, which are anthracycline resistant, by 1.6 and 3.4 times. The study demonstrated the efficacy of the developed PLGA-based DNR delivery system in the improvement of antitumor effect of DNR, overcoming multidrug resistance in cancer cells, and also in the decrease in nonspecific toxicity of the preparation.     

Rational modification of a selection strategy leads to deoxyribozymes that create native 3'-5' RNA linkages

We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5'-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2'-position of an RNA substrate, leading to 2',5'-branched RNA. To obtain deoxyribozymes that instead create linear 3'-5'-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. This approach should favor formation of linear rather than branched RNA because the two RNA termini are spatially constrained by Watson-Crick base pairing during the ligation reaction. Furthermore, because native 3'-5' linkages are more stable in a duplex than isomeric non-native 2'-5' linkages, this strategy is predicted to favor the formation of 3'-5' linkages. All of the new deoxyribozymes indeed create only linear 3'-5' RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA with k(obs) values up to 0.5 h(-1) at 37 degrees C and 40 mM Mg2+, pH 9.0, with up to 41% yield at 3 h incubation. They require several specific RNA nucleotides on either side of the ligation junction, which may limit their practical generality. These RNA ligase deoxyribozymes are the first that create native 3'-5' RNA linkages, which to date have been highly elusive via other selection approaches.     

Rational design of platinum(IV) compounds to overcome glutathione-S-transferase mediated drug resistance

A rationally designed Pt(IV) anticancer compound is described, employing the novel concept of tethering an inhibitor of glutathione-S-transferase, an enzyme associated with Pt-based drug-resistance, to cisplatin. Its enzyme inhibition activity, investigated using spectrophotometric and mass spectrometry-based techniques, and cytotoxic profile in resistant cancer cells are described.     

Peptide derivatization as a strategy to form fixed-charge peptide radicals

As a means of generating fixed-charge peptide radicals in the gas phase we have examined the collision-induced dissociation (CID) chemistry of ternary [Cu(II)(terpy)(TMPP-M)]2+ complexes, where terpy = 2,2':6'2'-terpyridine and TMPP-M represents a peptide (M) modified by conversion of the N-terminal amine to a [tris(2,4,6-trimethoxyphenyl)phosphonium]acetamide (TMPP-) fixed-charge derivative. The following modified peptides were examined: oligoglycines, (Gly)n (n = 1-5), alanylglycine, glycylalanine, dialanine, trialanine and leucine-enkephaline (YGGFL). The [Cu(II)(terpy)(TMPP-M)]2+ complexes are readily formed upon electrospray ionization (ESI) of a mixture of derivatized peptide and [Cu(II)(terpy)(NO3)2] and generally fragment to form transient peptide radical cations, TMPP-M+*, which undergo rapid decarboxylation for the simple aliphatic peptides. This is contrasted with the complexes containing the unmodified peptides, which predominantly undergo fragmentation of the coordinated peptide. These differences demonstrate the importance of proton mobility in directing fragmentation of ternary copper(II) peptide complexes. In the case of leucine-enkephaline, a sufficient yield of the radical cation was obtained to allow further CID. The TMPP-YGGFL+* ion showed a rich fragmentation chemistry, including CO2 loss, side-chain losses of an isopropyl radical, 2-methylpropene and p-quinomethide, and *a1 and *a4 sequence ion formation. In contrast, the even-electron TMPP-YGGFL+ ion fragments to form *a(n) and *b(n) sequence ions as well as the [*b4 + H2O]+ rearrangement ion.     

From spirolactam mixtures to regioisomerically pure 5- and 6-rhodamines: a chemodosimeter-inspired strategy

Inspired by the ring-open reaction of rhodamine spriolactams as typical chemodosimeters, a general strategy is proposed to conveniently and efficiently synthesize isomerically pure 5- and 6-R-tetramethylrhodamine on a larger scale.     

Ramoplanin inhibits bacterial transglycosylases by binding as a dimer to lipid II

Ramoplanin is a lipglycodepsipeptide antibiotic that inhibits peptidoglycan biosynthesis. Its mechanism of action has been the subject of debate. It was originally proposed to inhibit the MurG step of peptidoglycan synthesis by binding Lipid I. In this paper, we report that ramoplanin inhibits bacterial transglycosylases by binding to Lipid II, the substrate for these enzymes. The inhibition curves reveal that the inhibitory species has a stoichiometry of 2:1 ramoplanin:Lipid II. A Job titration confirms that ramoplanin binds as a dimer to Lipid II. The apparent dissociation constant is in the nanomolar range, which is unusually low given the nature of the interacting species. We show that Lipid II binding is coupled to the formation of a higher order species, which may explain the tight binding. We also present a testable model for the binding-competent dimeric conformation of ramoplanin.     

Access to L- and D-iminosugar C-glycosides from a D-gluco-derived 6-azidolactol exploiting a ring isomerization/alkylation strategy

A flexible synthetic access to six-membered L- and D-iminosugar C-glycosides is reported starting from the easily available 6-azido-6-deoxy-2,3,4-tri-O-benzyl-D-glucopyranose precursor. This methodology involves a highly diastereoselective tandem ring enlargement/alkylation and a stereocontrolled ring contraction. It allows an efficient synthesis of iminosugar C-glycosides displaying structural diversity at both C-1 and C-6.     

Nature-inspired dimerization as a strategy to modulate neuropeptide pharmacology exemplified with vasopressin and oxytocin

Vasopressin (VP) and oxytocin (OT) are cyclic neuropeptides that regulate fundamental physiological functions via four G protein-coupled receptors, V_1aR, V_1bR, V₂R, and OTR. Ligand development remains challenging for these receptors due to complex structure–activity relationships. Here, we investigated dimerization as a strategy for developing ligands with novel pharmacology. We regioselectively synthesised and systematically studied parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimer constructs of VP, OT and dVDAVP (1-deamino-4-valine-8-d-arginine-VP). All disulfide-linked dimers, except for the head-to-tail cyclized constructs, retained nanomolar potency despite the structural implications of dimerization. Our results support a single chain interaction for receptor activation. Dimer orientation had little impact on activity, except for the dVDAVP homodimers, where an antagonist to agonist switch was observed at the V_1aR. This study provides novel insights into the structural requirements of VP/OT receptor activation and spotlights dimerization as a strategy to modulate pharmacology, a concept also frequently observed in nature.

Structural and pharmacological study of parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimers of vasopressin and oxytocin. This study spotlights dimerization as a strategy to modulate the pharmacology of neuropeptides.     

Random copolymerization of macromonomers as a versatile strategy to synthesize mixed-graft block copolymers

The random copolymerization of norbornene-functionalized macromonomers was explored as a method of synthesizing mixed-graft block copolymers (mGBCPs). The copolymerization kinetics of a model system of polystyrene (PS) and poly(lactic acid) (PLA) macromonomers was first analyzed, revealing a gradient composition of side chains along the mGBCP backbone. The phase separation behavior of mGBCPs with PS and PLA side chains of various backbone lengths and side chain molar ratios was investigated, and increasing the backbone length was found to stabilize the phase-separated nanostructures. The graft architecture was also demonstrated to improve the processability of the mGBCP, compared to a linear counterpart. Investigations of mGBCPs comprised of polydimethylsiloxane and poly(ethylene oxide) side chains exemplified the diverse self-assembled morphologies, including a Frank-Kasper A15 phase, that can be obtained with mGBCPs synthesized by random copolymerization of macromonomers. Lastly, a ternary mGBCP was synthesized by the copolymerization of three macromonomers.     

Origin of the bathochromically shifted optical spectra of meso-tetrathien-2'- and 3'-ylporphyrins as compared to meso-tetraphenylporphyrin

The UV-Vis and fluorescence spectra of free base and diprotonated meso-tetrathien-2'-ylporphyrins are, when compared to the spectra of meso-tetra-phenyl- or even -thien-3'-ylporphyrins, characterized by surprisingly large red-shifts. A comparison of the optical spectra and the computed rotational barriers for these meso-aryl-substituted porphyrins and a detailed conformational analysis of the single crystal X-ray structure of a diprotonated meso-tetrathien-2'-ylporphyrin suggest that the origin of the altered electronic properties of meso-tetrathien-2'-ylporphyrins are mainly due to the contribution of conformations in which the thienyl groups adopt idealized co-planar arrangements with the porphyrin ring. These conformations allow an efficient extension of the porphyrinic pi-system through conjugation. We synthesized a meso-tetrathien-2'-ylporphyrin with methyl groups in the o-position, thus preventing the formation of conformers with co-planar thienyl groups and a corresponding thien-2'-ylporphyrin with methyl substituents in a distal position that possesses the same steric requirements for thienyl group rotation as the parent compound, to conclusively deduce the influence of the conformers on the electronic structure. A MNDO-PSDCI computation of their optical spectra further supports our key hypothesis. DFT computations of the total energies of the hypothetical diprotonated thien-2'-ylporphyrin conformer with perpendicular thienyl groups and the conformer containing near-co-planar thienyl groups quantify the resonance stabilization energy. Our results support and complement recent photophysical and theoretical studies by Gupta and Ravikanth and Friedlein et al. on thien-2'-yl-substituted core-modified porphyrins and [meso-tetra(5'-bromothien-2'-yl)porphyrinato]Zn(ii), respectively.     

An in vitro and in vivo study of a novel zinc complex, zinc N-(2-hydroxyacetophenone)glycinate to overcome multidrug resistance in cancer

Multiple drug resistance (MDR) remains a major clinical challenge for cancer treatment. P-glycoprotein is the major contributor and they exceed their role in the chemotherapy resistance of most of the malignancies. Attempts in several preclinical and clinical studies to reverse the MDR phenomenon by using MDR modulators have not yet generated promising results. In the present study, a co-ordination complex of zinc viz., Zn N-(2-hydroxyacetophenone)glycinate (ZnNG) has been synthesized, characterized and its antitumour activity was tested in vitro against drug sensitive and resistant human T-lymphoblastic leukemic cell lines (CCRF/CEM and CEM/ADR5000 respectively) and in vivo against Ehrlich ascites carcinoma (EAC) implanted in female Swiss albino mice. To evaluate the cytotoxic potential of ZnNG, we used sensitive CCRF/CEM and drug resistant CEM/ADR 5000 cell lines in vitro. Moreover, ZnNG also has the potential ability to reverse the multidrug resistance phenotype in drug resistant CEM/ADR 5000 cell line and induces apoptosis in combination with vinblastine. ZnNG remarkably increases the life span of Swiss albino mice bearing sensitive and doxorubicin resistant subline of EAC in presence and in absence of doxorubicin. In addition, intraperitoneal application of ZnNG in mice does not show any systemic toxicity in preliminary trials in normal mice. To conclude, a novel metal chelate of zinc viz., ZnNG, may be a promising therapeutic agent against sensitive as well as drug resistant cancers.     

Modification of polyolefins as a modern strategy to designing polyolefin materials with a new complex of properties

Experimental data on multistage catalytic olefin polymerization processes are generalized. Such processes as the sequential homo-and copolymerization of ethylene and α-olefins; the copolymerization of ethylene and a cyclic monomer followed by postpolymerization polymer-analogous transformations via the ozonolysis of side vinylidene bonds; and the preparation of multilayer polyolefin compositions by polymerization filling make it possible to control the composition, molecular mass characteristics, supramolecular structure, and properties of polyolefins. The kinetic features of the sequential polymerization of olefins, namely, the monomer effect and the absence of the influence of the preliminary polymerization stage on the composition and molecular mass characteristics of polymer products isolated at subsequent stages, are examined. The mutual influence of components of multiphase polymer systems on the morphology and mechanical characteristics of final products is established.     

Two‐dimensional solid‐phase extraction strategy for the selective enrichment of aminoglycosides in milk

An orthogonal two‐dimensional solid‐phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed‐phase liquid chromatography material (C₁₈) and a weak cation‐exchange material (TGA) were integrated in a single solid‐phase extraction cartridge. The feasibility of two‐dimensional clean‐up procedure that experienced two‐step adsorption, two‐step rinsing, and two‐step elution was systematically investigated. Based on the orthogonality of reversed‐phase and weak cation‐exchange procedures, the two‐dimensional solid‐phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed‐phase solid‐phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation‐exchange solid‐phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two‐dimensional clean‐up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk.     

Cytokine loaded biopolymers as a novel strategy to study stem cells during wound-healing processes

The biopolymer matrigel loaded with cytokine can be used for the recruitment in vivo of specific cell populations and as a vector for the preparation of cell cultures. Data demonstrate that the injection of the matrigel biopolymer supplemented with interleukin-8 (IL-8) in the leech Hirudo medicinalis can be used to purify cell populations showing the same morphofunctional and molecular mechanisms of specific populations of vertebrate hematopoietic precursor cells involved in tissue repair. These cells spontaneously differentiated into myofibroblasts. This approach highlights how the innovative use of a cytokine-loaded biopolymer for an in vivo cell sorting method, applied to a simple invertebrate model, can be a tool for studying myofibroblast cell biology and its regulation, step by step.