Crystallization to obtain protein–ligand complexes for structure-aided drug design

Antimicrob Agents Chemother

Rybtke

Jakobsen

et al. 2009

213

173

Attenuation of Pseudomonas aeruginosa virulence by the use of small-molecule quorum-sensing inhibitors (referred to as the antipathogenic drug principle) is likely to play a role in future treatment strategies for chronic infections. In this study, structure-based virtual screening was used in a search for putative quorumsensing inhibitors from a database comprising approved drugs and natural compounds. The database was built from compounds which showed structural similarities to previously reported quorum-sensing inhibitors, the ligand of the P. aeruginosa quorum-sensing receptor LasR, and a quorum-sensing receptor agonist. Six top-ranking compounds, all recognized drugs, were identified and tested for quorum-sensing-inhibitory activity. Three compounds, salicylic acid, nifuroxazide, and chlorzoxazone, showed significant inhibition of quorumsensing-regulated gene expression and related phenotypes in a dose-dependent manner. These results suggest that the identified compounds have the potential to be used as antipathogenic drugs. Furthermore, the results indicate that structure-based virtual screening is an efficient tool in the search for novel compounds to combat bacterial infections.

Section: Discussionmentioning

confidence: 99%

Computer-Aided Identification of Recognized Drugs as Pseudomonas aeruginosa Quorum-Sensing Inhibitors

Antimicrob Agents Chemother

Rybtke

Jakobsen

et al. 2009

213

173

“…[15] It has also been reported that structures of ligand-binding proteins can be employed in computational protein engineering to generate mutants with artificial functions. [16] Therefore, the inhibitors described here will contribute to our efforts to generate a ligand library that could be used to enhance PhaC crystallization for its first structure.…”

Section: Introductionmentioning

confidence: 99%

Inhibitors of Polyhydroxyalkanoate (PHA) Synthases: Synthesis, Molecular Docking, and Implications

et al. 2014

Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered as an ideal alternative to nonbiodegradable synthetic plastics. However, study of PhaC has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty along with lack of a structure has become the main hurdle to understand and engineer PhaCs for economical PHA production. Here we reported the synthesis of two carbadethia CoA analogs, sT-CH2-CoA 26a and sTet-CH2-CoA 26b as well as sT-aldehyde 29 as new PhaC inhibitors. Study of these analogs with PhaECAv revealed that 26a/b and 29 are competitive and mixed inhibitors, respectively. It was observed that CoA moiety and PHA chain extension can increase binding affinity, which is consistent with the docking study. Estimation from Kic of 26a/b predicts that a CoA analog attached with an octameric-HB chain may facilitate the formation of a kinetically well-behaved synthase.

“…For 90% occupancy, the amount of added ligands must be greater than the amount of protein so that free ligands at equilibrium is not depleted to less than about 10 × K d . 30 In practice, ratios of ligands to a given protein up to 10:1 or more are commonly used, but large excesses should be avoided owing to the possibility of ligand binding to nonspecific sites. In the case of a weak binding affinity, the concentrations of ligands (or ligand solubility) may have to be 10 mM or higher in order to observe crystallographic occupancy.…”

Section: Crystallization and Diffraction Of Lg-vitamin D 3 Complex Prmentioning

confidence: 99%

“…For 90% occupancy, the amount of added ligands must be greater than the amount of protein so that the free ligands at equilibrium are not depleted. 30 In this work, we reported a rationally designed approach for preparing the complex of LG and vitamin D 3 at various pH and vitamin D 3 /LG ratios to optimize the occupancy of vitamin D 3 and improve the electron density of the secondary binding site. Finally, we identified an exosite for vitamin D binding to be located near the R-helix and -strand I of LG using a crystal prepared at pH 8 with a vitamin D 3 /LG ratio of 3:1.…”

Section: Introductionmentioning

confidence: 99%

Rational Design for Crystallization of β-Lactoglobulin and Vitamin D₃ Complex: Revealing a Secondary Binding Site

Guan

Crystal Growth & Design

et al. 2008

-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D 3 binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D 3 to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was "disrupted" by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D 3 in the complex by altering the pH and initial ratios of vitamin D 3 /LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D 3 /LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D 3 in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.