Synthesis and study of new siderophore analog-ciprofloxacin conjugates with antibiotic activities against Pseudomonas aeruginosa and Burkholderia spp.

Antibacterial resistance is a healthcare burden. Among Gram-negative bacteria, Pseudomonas aeruginosa belongs to the first list of antibiotic-resistant "priority pathogens" described by the World Health Organization. Formerly Pseudomonas pseudomallei, Burkholderia pseudomallei, responsible for melioidosis, is considered as a potential bioterrorist weapon by the Centers of Diseases Control and Prevention. We are interested in the development of new ways to combat these bacteria, targeted due to their high level of resistance to antibiotics via a lack of membrane permeability or efflux. Using iron transport systems is a promising strategy to bypass the bacteria cell membrane and restore the activity of conventional antibiotics such as ciprofloxacin. Specific outer membrane receptors are necessary to most microbes as they allow iron uptake, essential for their survival through siderophore-dependent mechanisms. These systems may allow the introduction of antibacterial agents, chemically coupled to a natural or synthetic siderophore molecule to form siderophore-antibiotic conjugates. In this work, we describe the synthesis of six new siderophore analog-ciprofloxacin conjugates including cleavable linker or not. The siderophore analogs correspond to a mono-catechol or a hydroxypyridinone moiety recognized by both Pseudomonas and Burkholderia species. Physico-chemical studies showed that (i) conjugates were unable to interact or cross the membrane by passive diffusion and (ii) conjugates with cleavable linker are stable in physiologic environment. Biological evaluations have highlighted a promising compound 2d, bearing an hydroxypyridinone moiety with a cleavable linker, active on a large panel of strains of Pseudomonas aeruginosa, Burkholderia pseudomallei and Burkholderia thailandensis without toxicity observed in vitro.

Development of H1e histone linker-specific antibodies by means of synthetic peptides.

A large body of data suggests that the linker histones family (H1) affects gene expression. Investigation of the linker histones role is then of a major interest in cell cycle studies with implications in gene therapy. Indeed, it has been shown that in most tissues a switch of histone subtypes occurs when the cells cease to divide. To investigate linker histone role in gene or transgene expression, an antibody against subtypes of H1 would be useful for immunoprecipitation experiments and further assays measuring H1subtypes-DNA interactions in living cells. In order to produce an antibody against the H1e subtype of linker histones, two synthetic peptides derived from two regions of the H1e mouse histone protein were examined for their potential, [as keyhole limpet hemocyanin (KLH) conjugates] to elicit polyclonal anti-H1e antibodies in New Zealand white rabbits. Selection of the peptide sequences was based on amino acid differences within the different classes of histones and between mice and rabbit histones as well. The evaluation of their potential immunogenic properties was based on examination of peptide hydropathy using predicting algorithms. Immunoglobulins (IgG) obtained from immunized and nonimmunized rabbits were tested using enzyme-linked immunosorbent assay (ELISA) procedures, Western immunoblot, and immunofluorescence experiments. Results showed that the selected synthetic peptides gave rise to a high-titer polyclonal antibody able to recognize the H1e histone under various conditions. This polyclonal antibody did not cross-react with other histones. To our knowledge, this is the first antibody produced against the mouse H1e linker histone.

Reduction of opioid dependence by the CB(1) antagonist SR141716A in mice: evaluation of the interest in pharmacotherapy of opioid addiction.

Several compounds, mainly opioid agonists such as methadone, are currently used for long term medication of heroin addicts. Nevertheless, these maintenance treatments have the disadvantage to induce a dependence to another opiate. As interactions between opioid and cannabinoid systems have been demonstrated, the ability of the CB(1) antagonist, SR141716A to reduce morphine-induced addiction was investigated. The effects of SR141716A on the rewarding responses of morphine were evaluated in the place conditioning paradigm. No significant conditioned preference or aversion were observed after repeated treatment with the CB(1) antagonist alone. However, SR141716A was able to antagonize the acquisition of morphine-induced conditioned place preference. SR141716A was co-administered with morphine for 5 days, and the withdrawal syndrome was precipitated by naloxone administration. A reduction in the incidence of two main signs of abstinence: wet dog shakes and jumping was observed while the other were not significantly modified. In contrast, an acute injection of the CB(1) antagonist just before naloxone administration was unable to modify the incidence of the behavioural manifestations of the withdrawal, suggesting that only chronic blockade of CB(1) receptors is able to reduce morphine-induced physical dependence. Several biochemical mechanisms could explain the reduction of opioid dependence by CB(1) antagonists. Whatever the hypotheses, this study supports the reported interaction between the endogenous cannabinoid and opioid systems, and suggests that SR 141716A warrants further investigations for a possible use in opioid addiction.

Binding properties of a highly potent and selective iodinated aminopeptidase N inhibitor appropriate for radioautography.

Aminopeptidase N (APN) is a zinc metallopeptidase involved in the inactivation of biologically active peptides. The knowledge of its precise distribution is crucial to investigate its physiological role. This requires the use of appropriate probes such as the recently developed highly potent and selective radiolabeled APN inhibitor 2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ (12 5)I]iodotyrosine ([(125)I]RB 129). Its binding properties were investigated using rat brain homogenates (K(d)=3.4 nM) or APN expressed in COS-7 cells (K(d)=0.9 nM). The specific binding was 95% at [K(d)], and preliminary autoradiography in intestine is promising. The decreased affinity of [(125)I]RB 129 (=10(-6) M) for the E(350)D APN mutant, supports the critical role of E(350) in the amino-exopeptidase action of APN.

The cholecystokininB receptor is coupled to two effector pathways through pertussis toxin-sensitive and -insensitive G proteins.

Previous binding studies have suggested the existence of two affinity states for type B cholecystokinin receptors (CCK(B)R), which could correspond to different coupling states of the receptor to G proteins. To test this hypothesis, we have further investigated signal transduction pathways coupled to rat CCK(B)R stably transfected in Chinese hamster ovary cells. We show that CCK(B)R are coupled to two distinct transduction pathways involving two different G proteins, a pertussis toxin-insensitive/phospholipase C pathway leading to the production of inositol phosphate and arachidonic acid, and a pertussis toxin-sensitive/phospholipase A2 pathway leading to the release of arachidonic acid. We further demonstrate that the relative degree of activation of each effector pathway by different specific CCK(B)R agonists is the same, and that a specific CCK(B)R antagonist, RB213, can differentially antagonize the two signal transduction pathways elicited by these agonists. Taken all together, these data could be explained by the recently proposed theory assuming that the receptor can exist in a three-state model in which two active conformations corresponding to the complex formed by the receptor with two different G proteins coexist. According to this model, agonists or antagonists could recognize preferentially either conformation of the activated receptor, leading to variable behavior in a system containing a single receptor type.

Novel constrained CCK-B dipeptoid antagonists derived from pipecolic acid.

A new series of 4-substituted pipecolic acid derivatives was prepared and incorporated into dipeptoids. The resulting products behave as moderately potent CCK-B antagonists but their constrained structure and its comparison with structurally related compounds yield valuable information about the conformational requirements for optimal recognition of the CCK-B receptor by antagonists.