A novel method using nuclear magnetic resonance for plasma protein binding assessment in drug discovery programs.

A new methodology based on Nuclear Magnetic Resonance (NMR) was developed to determine plasma protein binding (PPB) of drug candidates in drug discovery programs. A strong correlation was found between the attenuation of NMR signals of diverse drugs in the presence of different plasma concentrations and their fraction bound (f b ) reported in the literature. Based on these results, a protocol for a rapid calculation of f b of small molecules was established. The advantage of using plasma instead of purified recombinant proteins and the possibility of pool analysis to increase throughput were also evaluated. This novel methodology proved to be very versatile, cost-effective, fast and suitable for automation. As a plus, it contemporarily provides a quality check and solubility of the compound

Azotobacter vinelandii rhodanese Selenium loading and ion interaction studies

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]CyS, and changes in 1H and 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo

A novel method using nuclear magnetic resonance for plasma protein binding assessment in drug discovery programs

A new methodology based on Nuclear Magnetic Resonance (NMR) was developed to determine plasma protein binding (PPB) of drug candidates in drug discovery programs. A strong correlation was found between the attenuation of NMR signals of diverse drugs in the presence of different plasma concentrations and their fraction bound (f(b)) reported in the literature. Based on these results, a protocol for a rapid calculation of fb of small molecules was established. The advantage of using plasma instead of purified recombinant proteins and the possibility of pool analysis to increase throughput were also evaluated. This novel methodology proved to be very versatile, cost-effective, fast and suitable for automation. As a plus, it contemporarily provides a quality check and solubility of the compound. (C) 2019 The Authors. Published by Elsevier B.V

Structural rearrangements of the two domains of Azotobacter vinelandii rhodanese upon sulfane sulfur release: Essential molecular dynamics, 15N NMR relaxation and deuterium exchange on the uniformly labeled protein

The Azotobacter vinelandii rhodanese is a 31kDa sulfurtransferase protein that catalyzes the transfer of sulfur atom from thiosulfate to cyanide in the detoxification process from cyanide and is able to insert sulfur atom in the iron-sulfur cluster. A study of the uniformly 15N isotopic labeling by high resolution NMR, before obtaining the backbone sequential assignment, has been carried out. The sulfur loaded and the sulfur discharged forms of the enzyme show very similar HSQC spectra with a good spectral dispersion. Few resonances show changes in chemical shift between the two forms. Relaxation parameters T1, T2 and 1H-15N NOE of all amide nitrogen atoms, as well as isotope exchange kinetics, show that the two forms exhibit the same global correlation time and hydrodynamic properties. In parallel, essential dynamics studies show that formation and discharging of catalytic cysteine persulfide group has no significant impact on the overall conformation of the protein. These results, taken together, give a clearcut answer to the question if the catalytic mechanism of the enzyme involves a change in the conformation and/or in the mutual orientation of the two domains. On the contrary these results clearly indicate that upon the catalytic mechanism the two domains of the protein behave as a unique fold. © 2003 Elsevier B.V. All rights reserved

Surface changes and role of buried water molecules during the sulfane sulfur transfer in rhodanese from Azotobacter vinelandii : a fluorescence quenching and nuclear magnetic relaxation dispersion spectroscopic study

The Azotobacter vinelandii rhodanese is a sulfurtransferase Enz. that catalyzes the Transf. of the outer sulfur atom from thiosulfate to cyanide. Recently, investigations by NMR relaxation on the 15N-enriched protein reported that interdomain contacts are rigidly maintained upon the sulfane sulfur Transf. from the Enz. to the substrate. The modality of the enzymatic mechanism is then confined to a Surf. interaction, including Dynam. of water molecules buried in the Tert. structure. Thus, investigations have been carried out by fluorescence, circular dichroism,/Nucl. magnetic relaxation dispersion measurements. The comparison of circular dichroism spectra of the persulfurated Enz. /the sulfur-free form indicated that small changes occur. Fluorescence quenching Studs. have been performed to evaluate the conformational changes during Catalys. using the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid, and acrylamide, iodide, and cesium ions as quenchers. Changes in exchange dynamics of water molecules buried in the structure with bulk water, observed by nuclear magnetic relaxation dispersion, are due to local conformational transitions, likely involving residues around the active site, and are consistent with the global correlation time found by 15N relaxation. These results, taken together, provide important information for elucidating the conformational features of the mechanism of action of the enzyme either in the role of a selective donor of a sulfur atom to small-sized substrates (i.e., to cyanide, transforming it into thiocyanate) or in the role of sulfur insertase for the formation of the Fe2S2 iron-sulfur cluster in sulfur-deprived ferredoxins