Identification of Anti-Inflammatory and Anti-Hypertensive Drugs as Inhibitors of Bacterial Diguanylate Cyclases

<div><p>Biofilms are widely present in many human chronic infections, often more resistant to treatment with antibiotics. Bacterial diguanylate cyclases (DGCs) synthesize cyclic dimeric guanosine monophosphate (c-di-GMP) from two guanosine-5'-triphosphate (GTP) molecules. c-di-GMP is a central second messenger controlling biofilm formation, turning this class of enzymes an attractive target to prevent and disrupt biofilms of pathogenic bacteria. Here, we apply an in silico ligand- and target-based hybrid method to screen potential DGC inhibitors from an FDA-approved drug databank. Mass spectrometry assays confirmed that seven screened compounds selectively bound to the GTP active site of P. aeruginosa WspR GGDEF domain. Four out of those, including the anti-inflammatory sulfasalazine and the anti-hypertensive eprosartan, inhibited distinct DGCs (P. aeruginosa WspR and E. coli YdeH) in the micromolar range. Sulfasalazine and eprosartan reduced aggregation in solution of E. coli overexpressing WspR or YdeH. Similar anti-aggregation effects were also observed for sulfasalazine-related anti-inflammatory drugs sulfadiazine and sulfathiazole, the latter a previously described anti-biofilm agent. The optimized pharmacokinetic properties and toxicological profiles of the DGC inhibitors could be promising candidates for new anti-microbial agents based on the drug reposition strategy.</p></div

Luminescent Ruthenium Complexes for Theranostic Applications

The water-soluble and visible luminescent complexes <i>cis-</i>[Ru­(L-L)₂(L)₂]²⁺ where L-L = 2,2-bipyridine and 1,10-phenanthroline and L= imidazole, 1-methylimidazole, and histamine have been synthesized and characterized by spectroscopic techniques. Spectroscopic (circular dichroism, saturation transfer difference NMR, and diffusion ordered spectroscopy NMR) and isothermal titration calorimetry studies indicate binding of <i>cis-</i>[Ru­(phen)₂(ImH)₂]²⁺ and human serum albumin occurs via noncovalent interactions with <i>K</i>_b = 9.8 × 10⁴ mol^–1 L, Δ<i>H</i> = −11.5 ± 0.1 kcal mol^–1, and <i>T</i>Δ<i>S</i> = −4.46 ± 0.3 kcal mol^–1. High uptake of the complex into HCT116 cells was detected by luminescent confocal microscopy. Cytotoxicity of <i>cis-</i>[Ru­(phen)₂(ImH)₂]²⁺ against proliferation of HCT116p53^+/+ and HCT116p53^–/– shows IC₅₀ values of 0.1 and 0.7 μmol L^–1. Flow cytometry and western blot indicate RuphenImH mediates cell cycle arrest in the G1 phase in both cells and is more prominent in p53^+/+. The complex activates proapoptotic PARP in p53^–/–, but not in p53^+/+. A cytostatic mechanism based on quantification of the number of cells during the time period of incubation is suggested

Luminescent Ruthenium Complexes for Theranostic Applications

The water-soluble and visible luminescent complexes <i>cis-</i>[Ru­(L-L)₂(L)₂]²⁺ where L-L = 2,2-bipyridine and 1,10-phenanthroline and L= imidazole, 1-methylimidazole, and histamine have been synthesized and characterized by spectroscopic techniques. Spectroscopic (circular dichroism, saturation transfer difference NMR, and diffusion ordered spectroscopy NMR) and isothermal titration calorimetry studies indicate binding of <i>cis-</i>[Ru­(phen)₂(ImH)₂]²⁺ and human serum albumin occurs via noncovalent interactions with <i>K</i>_b = 9.8 × 10⁴ mol^–1 L, Δ<i>H</i> = −11.5 ± 0.1 kcal mol^–1, and <i>T</i>Δ<i>S</i> = −4.46 ± 0.3 kcal mol^–1. High uptake of the complex into HCT116 cells was detected by luminescent confocal microscopy. Cytotoxicity of <i>cis-</i>[Ru­(phen)₂(ImH)₂]²⁺ against proliferation of HCT116p53^+/+ and HCT116p53^–/– shows IC₅₀ values of 0.1 and 0.7 μmol L^–1. Flow cytometry and western blot indicate RuphenImH mediates cell cycle arrest in the G1 phase in both cells and is more prominent in p53^+/+. The complex activates proapoptotic PARP in p53^–/–, but not in p53^+/+. A cytostatic mechanism based on quantification of the number of cells during the time period of incubation is suggested

Data collection and refinement statistics.

a<p>Values in parenthesis represents the highest resolution shells.</p>b<p>Calculated for both molecules in the asymmetric units.</p

Preliminary trypanocidal activity of compounds Neq42 and Neq37 evaluated against the Tulahuen lacZ strain.

<p>Preliminary trypanocidal activity of compounds Neq42 and Neq37 evaluated against the Tulahuen lacZ strain.</p

(A) Dose-response and (B) Linewaver-Burk curves for Neq30 from Table S1.

<p>Non-linear fit method was employed in the analysis.</p

Trypanocidal activity and cytotoxicity of cruzain inhibitors evaluated against Tulahuen lacZ strain.

<p>Bz: benznidazole. N.d., not determined. See text for explanation.</p

2D structural representation of (A) K11777 and (B) WRR-483 inhibitors.

<p>2D structural representation of (A) K11777 and (B) WRR-483 inhibitors.</p

A scheme of the multi-step virtual screening protocol used for the identification of cruzain inhibitors.

<p>A scheme of the multi-step virtual screening protocol used for the identification of cruzain inhibitors.</p

(A) Structure of the co-crystallized cruzain inhibitor K11777 and (B–D) examples of complex structures predicted by our molecular docking (Glide XP).

<p>Figure prepared using CCP4mg software <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002370#pntd.0002370-McNicholas1" target="_blank">[43]</a>.</p