Ligand-Orientation Based Fragment Selection in STD NMR Screening

While saturation transfer difference (STD) is a widely used NMR method for ligand screening, the selection of specific binders requires the validation of the hits through competition experiments or orthogonal biophysical techniques. We show here that the quantitative STD analysis is a reliable and robust approach to discriminate between specific and nonspecific ligands, allowing selection of fragments that bind proteins with a privileged binding mode, in the absence of any structural data for the protein

Comparison of NMR and X-ray protein-fragment structures.

<p>(A) Superposition of the X-ray structures of the complexes for fragments 1 (red), 2 (blue), 3 (green) and 4 (orange). (B) The NMR-derived binding modes (cyan) are compared to the X-Ray structures (yellow) for fragments 2 (B), 3 (C) and 4 (D). Fragment positions were extracted from the solved X-Ray structure and are displayed in the 3MNG protein structure (used for docking).</p

STD investigation of fragment binding to PRDX5.

<p>1D ¹H NMR spectra (in red) are superimposed to STD NMR spectra (in blue). (A) NMR experiments in the presence of PRDX5, (B) NMR experiments in the presence of HSA. The relative STD effects (R ratio, see Material and Methods) measured for the aromatic protons are indicated. The proton in position HA, labelled with an asterisk (*), exhibits a weak STD effect for fragments 2, 3 and 4, upon binding to PRDX5, indicating that the proton HA is solvent exposed. This effect is not observed in the presence of HSA. STD spectra were scaled by setting the largest ratio to 100%. Positions A and B are displayed on the catechol (top right corner).</p

Calculated CSP sign differences between binding modes of analogous fragments.

<p>Comparative CSP sign analysis in case of binding mode conservation (left) or binding mode change (right) of the catechol group for the fragment pairs 1/4 (A) and 1/5 (B) demonstrating a conserved binding mode between fragments 1 and 4 and a binding mode modification between fragments 1 and 5. (A) CSP signs differences are expected for residues located in the loop 113–125 in case of superimposition of the catechol moieties of fragments 1 (green) and 4 (blue) (in agreement with experimental CSP data), while CSP signs differences are expected in numerous protein regions in case of a change of the catechol orientation. (B) No CSP signs differences are expected in case of superimposition of the catechol moieties of fragment 1 (green) and 5 (blue), while CSP signs differences are expected in the active site region in case of a change of the catechol orientation (in agreement with experimental CSP data). Spheres are displayed only if one of the two compared fragment protons displayed calculated CSPs larger than 0.02 ppm.</p

Comparative experimental CSP analysis.

<p>CSP observed on PRDX5 spectra when bound to fragments 2, 3, 4 and 5 are compared to CSP observed in the presence of fragment 1. Residues displaying CSP magnitude (A) or CSP sign (B) differences are displayed with small or large red spheres, for protons and nitrogens, respectively. (A) Comparison of the CSP magnitudes. Spheres are displayed in the case of the absolute CSP differences are larger than 0.02 ppm for protons and 0.1 ppm for nitrogens. (B) Comparison of the CSP signs. Spheres indicate experimental CSP signs differences. The loop 113–125 is coloured in blue.</p

Affinities of fragments 1 to 5 to the PRDX5 protein.

<p>*Average values ± standard error of the mean.</p><p>**LE = ΔG/(number of heavy atoms).</p

Chemical shift perturbations and affinity measurement for the PRDX5-fragment 3 complex.

<p>(A) Section of the ¹⁵N-HSQC spectrum, with the superimposition of the free protein spectrum (black) and spectra with increasing fragment concentration (110 µM blue, 220 µM violet, 330 µM red, 550 µM light red, 880 µM orange, and 2 mM yellow). (B) Titration curves obtained from ¹⁵N-HSQC spectra. Combined CSP_(H+N) were measured for each fragment concentration. Curves obtained for residues S48, G148, C47, and T50 are shown.</p

Binding modes of the fragments determined by CSP calculation and STD.

<p>(A) 200 ligand orientations generated by docking (B) CSP filter: binding modes of the fragments obtained by filtering the positions according to their agreement between experimental and calculated CSP. (C) Combined CSP and STD filter: binding modes of the fragments in agreement with both CSP calculation and STD data. Hydrogen bonds, identified using Ligplot + <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102300#pone.0102300-Laskowski1" target="_blank">[41]</a>, are displayed in blue lines.</p

The Colloidal State of Tannins Impacts the Nature of Their Interaction with Proteins: The Case of Salivary Proline-Rich Protein/Procyanidins Binding

While the definition of tannins has been historically associated with its propensity to bind proteins in a nonspecific way, it is now admitted that specific interaction also occurs. The case of the astringency perception is a good example to illustrate this phenomenon: astringency is commonly described as a tactile sensation induced by the precipitation of a complex composed of proline-rich proteins present in the human saliva and tannins present in beverages such as tea or red wines. In the present work, the interactions between a human saliva protein segment and three different procyanidins (B1, B3, and C2) were investigated at the atomic level by NMR and molecular dynamics. The data provided evidence for (i) an increase in affinity compared to shortest human saliva peptides, which is accounted for by protein “wraping around” the tannin, (ii) a specificity in the interaction below tannin critical micelle concentration (CMC) of ca. 10 mM, with an affinity scale such that C2 > B1 > B3, and (iii) a nonspecific binding above tannin CMC that conducts irremediably to the precipitation of the tannins/protein complex. Such physicochemical findings describe in accurate terms saliva protein–tannin interactions and provide support for a more subtle description by oenologists of wine astringency perception in the mouth

Virtual and Biophysical Screening Targeting the γ-Tubulin Complex – A New Target for the Inhibition of Microtubule Nucleation

<div><p>Microtubules are the main constituents of mitotic spindles. They are nucleated in large amounts during spindle assembly, from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). With the aim of developing anti-cancer drugs targeting these nucleating complexes, we analyzed the interface between GCP4 and γ-tubulin proteins usually located in a multiprotein complex named γ-TuRC (γ-Tubulin Ring Complex). 10 ns molecular dynamics simulations were performed on the heterodimers to obtain a stable complex <i>in silico</i> and to analyze the residues involved in persistent protein-protein contacts, responsible for the stability of the complex. We demonstrated <i>in silico</i> the existence of a binding pocket at the interface between the two proteins upon complex formation. By combining virtual screening using a fragment-based approach and biophysical screening, we found several small molecules that bind specifically to this pocket. Sub-millimolar fragments have been experimentally characterized on recombinant proteins using differential scanning fluorimetry (DSF) for validation of these compounds as inhibitors. These results open a new avenue for drug development against microtubule-nucleating γ-tubulin complexes.</p></div