Linda Váradi

Synthesis and evaluation of fluorogenic 2-amino-1,8-naphthyridine derivatives for the detection of bacteria

Several novel fluorogenic N-aminoacylnaphthyridine substrates were synthesized in good yield and tested for their ability to detect pathogenic bacteria in agar-based cell culture. Simple 2-N-(β-alanyl)amino-5,7-dialkylnaphthyridine substrates were selectively hydrolysed by β-alanylaminopeptidase expressing bacteria, but were subject to diffusion in the agar medium. Diffusion was reduced in the 2-N-(β-alanyl)amino-7-alkylnaphthyridine substrates with longer alkyl chains, but inhibition of growth was increased. 2-N-(β-Alanyl)amino-7-octylnaphthyridine inhibited the growth of all species tested, except for strains resistant to colistin/polymyxin, providing a rationale for the development of substrates for the selective detection of drug resistant species in clinical samples.

New Delhi metallo-β-lactamase-1: structure, inhibitors and detection of producers.

Since its discovery in 2008, New Delhi metallo-β-lactamase-1 (NDM-1)-producing Enterobacteriaceae have disseminated globally, facilitated predominantly by gut colonization and the spread of plasmids carrying the bla NDM-1 gene. With few effective antibiotics against NDM-1 producers, and resistance developing to those which remain, there is an urgent need to develop new treatments. To date, most drug design in this area has been focused on developing an NDM-1 inhibitor and has been aided by the wealth of structural and mechanistic information available from high resolution x-ray crystallography and molecular modeling. This review aims to summarize current knowledge regarding the detection of NDM-1 producers, the mechanism of action of NDM-1 and to highlight recent attempts toward the development of clinically useful inhibitors.

A comparison of the experimental and theoretical charge density distributions in two polymorphic modifications of piroxicam

Experimental charge density distribution studies of two polymorphic forms of piroxicam, β-piroxicam (1) and piroxicam monohydrate (2), were carried out via high-resolution single crystal X-ray diffraction experiments and multipole refinement. The asymmetric unit of (2) consists of two discrete piroxicam molecules, (2a) and (2b), and two water molecules. Geometry differs between (1) and (2) due to the zwitterionic nature of (2) which results in the rotation of the pyridine ring around the C(10)-N(2) bond by approximately 180°. Consequently, the pyridine and amide are no longer co-planar and (2) forms two exclusive, strong hydrogen bonds, H(3)O(4) and H(2)O(3), with bond energies of 66.14 kJ mol-1 and 112.82 kJ mol-1 for (2a), and 58.35 kJ mol-1 and 159.51 kJ mol-1 for (2b), respectively. Proton transfer between O(3) and N(3) in (2) results in significant differences in surface electrostatic potentials. This is clarified by the calculation of atomic charges in the zwitterion that shows the formally positive charge of the pyridyl nitrogen which is redistributed over the whole of the pyridine ring instead of concentrating at N-H. Similarly, the negative charge of the oxygen is distributed across the benzothiazine carboxamide moiety. The multipole derived lattice energy for (1) is -304 kJ mol-1 and that for (2) is -571 kJ mol-1, which is in agreement with the experimentally determined observations of higher solubility and dissolution rates of (1) compared to (2).

Identification of dual PPARα/γ agonists and their effects on lipid metabolism.

The three peroxisome proliferator-activated receptor (PPAR) isoforms; PPARα, PPARγ and PPARδ, play central roles in lipid metabolism and glucose homeostasis. Dual PPARα/γ agonists, which stimulate both PPARα and PPARγ isoforms to similar extents, are gaining popularity as it is believed that they are able to ameliorate the unwanted side effects of selective PPARα and PPARγ agonists; and may also be used to treat dyslipidemia and type 2 diabetes mellitus simultaneously. In this study, virtual screening of natural product libraries, using both structure-based and ligand-based drug discovery approaches, identified ten potential dual PPARα/γ agonist lead compounds (9-13 and 16-20). In vitro assays confirmed these compounds to show no statistically significant toxicity to cells, with the exception of compound 12 which inhibited cell growth to 74.5%±3.5 and 54.1%±3.7 at 50μM and 100μM, respectively. In support of their potential as dual PPARα/γ agonists, all ten compounds upregulated the expression of cholesterol transporters ABCA1 and ABCG1 in THP-1 macrophages, with indoline derivative 16 producing the greatest elevation (2.3-fold; 3.3-fold, respectively). Furthermore, comparable to the activity of established PPARα and PPARγ agonists, compound 16 stimulated triacylglycerol accumulation during 3T3-L1 adipocyte differentiation as well as fatty acid β-oxidation in HuH7 hepatocytes.

An analysis of the experimental and theoretical charge density distributions of the piroxicam–saccharin co-crystal and its constituents

Experimental and theoretical charge density analyses of piroxicam (1), saccharin (2) and their 1 : 1 co-crystal complex (3) have been carried out. Electron density distribution (EDD) was determined through the use of high-resolution single crystal X-ray diffraction and the data were modelled using the conventional multipole model of electron density according to the Hansen–Coppens formalism. A method for optimising the core density refinement of sulfur atoms is discussed, with emphasis on the reduction of residual electron density that is typically associated with this atom. The asymmetric unit of complex (3) contains single molecules of saccharin and the zwitterionic form of piroxicam. These are held together by weak interactions (hydrogen bonds, π–π and van der Waals interactions), ranging in strength from 4 to 160 kJ mol−1, working together to stabilise the complex; analysis of the molecular electrostatic potential (MEP) of the complexes showed electron redistribution within the co-crystal, facilitating the formation of these generally weak interactions. Interestingly, in the zwitterionic form of piroxicam, the charge distribution reveals that the positive and negative charges are not associated with the formal charges normally associated with this description, but are distributed over adjacent molecular fragments. The use of anisotropic displacement parameters (ADPs) for hydrogen atoms in the multipole model was also investigated but no improvement in the quality of the topological analysis was found.

β-Alanyl aminopeptidase-activated fluorogenic probes for the rapid identification of Pseudomonas aeruginosa in clinical samples

The fluorogenic self-immolative substrates 8 are specifically hydrolyzed by β-alanyl aminopeptidase, resulting in a 1,6-elimination and the release of the highly fluorescent hydroxycoumarins 6. 7-{4-(β-Alanylamino)}benzyloxy-3-ethoxycarbonylcoumarin trifluoroacetate 8b has advantages over another fluorogenic substrate, 7-N-β-alanylamino-4-methylcoumarin 9, as it is retained by bacterial colonies in solid agar applications, and results in similar times to detection, stronger fluorescence intensities, and no decrease in signal over time in liquid media. Although 7-{4-(β-alanylamino)}benzyloxy-4-methylcoumarin trifluoroacetate 8a produces a weaker signal than substrate 8b, its use allowed better discrimination between the BAP producers P. aeruginosa (positive) and S. marcescens (negative).

A latent green fluorescent styrylcoumarin probe for the selective growth and detection of Gram negative bacteria

A novel, green fluorescent β-alanylstyrylcoumarin derivative was synthesized and evaluated for its performance as a fluorogenic enzyme substrate on a range of clinically relevant microorganisms. The substrate was selectively hydrolysed by β-alanyl aminopeptidase producing P. aeruginosa resulting in an on-to-off fluorescent signal. Growth inhibitory effect of the substrate was observed on Gram positive bacteria and yeasts. Meanwhile, Gram negative species, despite their extremely protective cell envelope, showed ready uptake and accumulation of the substrate within their healthy growing colonies displaying intense green fluorescence.