coordinates_for_supmat.zip

Supplementary Material for "Thioamide substitution to probe the hydroxyproline recognition of VHL ligand"<br><br>Coordinates of (DFT) PBF (water) MN15-L/aug-cc-pVTZ(-F) optimized geometries in xyz format.<br><br>Publication to be added upon acceptance.<br

The structure of DMSO-treated SOCS2:EloC:EloB.

<p>(A) Anomalous difference map (<i>black mesh</i>) shown at 5σ calculated from As-Peak diffraction data in proximity to SOCS2_C111 residue of Chain J (<i>CPK colored sticks</i>) with the dimethylarsenic modification. (B) Structure of the two most conformationally different complexes of DMSO-treated SOCS2-EloBC (A/B/C and M/N/O), aligned via the backbone atoms of EloB. Protein chains are shown as ribbons, with EloB (<i>cyan</i>), EloC (<i>green</i>), SOCS2 SOCS box (<i>blue</i>) and SOCS2 SH2 domain (<i>red</i>). Chains A/B/C are in darker colors and M/N/O lighter. The linker/hinging region between SOCS2 SOCS box and SH2 domain is shown in black/grey and residue Pro161 as spheres. (C) SH2 domains of DMSO-treated SOCS2-EloBC illustrating the five different conformations observed in the ASU. Chains A/B/C (<i>red</i>), D/E/F (<i>green</i>), G/H/I (<i>blue</i>), J/K/L (<i>yellow</i>) and M/N/O (<i>magenta</i>). Complexes have been aligned via the backbone atoms of EloB from each complex and superposed. Complex P/Q/R is not shown as its conformation is identical to that of D/E/F. (D) Root mean square deviation (RMSD) values (in Å) for the backbone atoms of EloB, EloC, SOCS2 SOCS-box domain (residues 162–198) and SOCS2 SH2 domain (32–134) when complexes are superposed as in <i>C</i>. RMSD values are between each complex and complex A/B/C.</p

X-ray diffraction data and model statistics for DMSO-treated SOCS2:EloC:EloB.

<p>Values in parentheses are for the highest resolution shell.</p><p>Values for the highest resolution shell are given in parentheses.</p><p>^a<i>R</i>_merge = Σ_<i>hkl</i> Σ_<i>i</i>|(<i>I</i>_<i>i</i>(<i>hkl</i>)–[<i>I</i>(<i>hkl</i>)]|/Σ_<i>hkl</i> Σ_<i>i</i><i>I</i>_<i>i</i>(<i>hkl</i>)</p><p>^b<i>R</i>_work = |<i>F</i>_obs–<i>F</i>_calc|/|<i>F</i>_obs|, where <i>F</i>_obs and <i>F</i>_calc are the observed and calculated structure amplitudes, respectively.</p><p>^c<i>R</i>_free is <i>R</i>_work for the 5% validation set.</p><p>X-ray diffraction data and model statistics for DMSO-treated SOCS2:EloC:EloB.</p

Mechanism for dimethylarsenic modification of cysteine residues proposed by Maignan <i>et al</i>. [29].

<p>DTT reduces As(V) of cacodylate in its acid form (p<i>K</i>_a = 6.3) into an As(III)-containing dimethylarsenic-dithiothreitol conjugate. This adduct then reacts with a reduced cysteine sidechain to result in dimethylarsenic cysteine and a replenished molecule of DTT.</p

Identification of arsenic as a source of experimental phasing power.

<p>(A) The 2<i>F</i>_o−<i>F</i>_c (<i>blue mesh</i>) and <i>F</i>_o−<i>F</i>_c (<i>green mesh</i>) maps around modelled SOCS2_C111 (<i>CPK colored sticks</i>) and adjacent Ni(II) (<i>green sphere</i>) from the PDB structure 2C9W. (B) As in A, with maps recalculated following removal of Ni(II). (C) As in A, with SOCS2_C111 remodelled as dimethylarsenic cysteine. 2<i>F</i>_o−<i>F</i>_c maps are contoured at 1σ and <i>F</i>_o−<i>F</i>_c at 3σ. (D) A fluorescence scan (<i>green line</i>) and measurements of <i>f</i>≠ and <i>f</i>≡ (<i>red</i> and <i>blue lines</i>, respectively) identifying the presence of arsenic and resulting anomalous signals from DMSO-treated SOCS2:EloC:EloB crystals. (E) Observed anomalous signal [Δ<i>F</i>/σ(Δ<i>F</i>)] in diffraction data plotted against resolution for the As-Peak datasets collected. The cut-off of useful signal (1.2) is indicated by a dashed line [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131218#pone.0131218.ref022" target="_blank">22</a>].</p

Crystals of the SOCS2:EloC:EloB complex grown after 7 d.

<p>The crystals shown here are 200–300 μm in the longest dimension.</p

Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach

Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of <i>in silico</i> fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers

Commentaire bibliographique [Herman Van Impe, Het Belgisch Grondwettelijk Recht in kort bestek, Anvers, Kluwer rechtswetenschappen, 1982]

The glycoproteins of selected microbial pathogens often include highly modified carbohydrates such as 2,4-diacetamidobacillosamine (diNAcBac). These glycoconjugates are involved in host–cell interactions and may be associated with the virulence of medically significant Gram-negative bacteria. In light of genetic studies demonstrating the attenuated virulence of bacterial strains in which modified carbohydrate biosynthesis enzymes have been knocked out, we are developing small molecule inhibitors of selected enzymes as tools to evaluate whether such compounds modulate virulence. We performed fragment-based and high-throughput screens against an amino-sugar acetyltransferase enzyme, PglD, involved in biosynthesis of UDP-diNAcBac in Campylobacter jejuni. Herein we report optimization of the hits into potent small molecule inhibitors (IC₅₀ < 300 nM). Biophysical characterization shows that the best inhibitors are competitive with acetyl coenzyme A and an X-ray cocrystal structure reveals that binding is biased toward occupation of the adenine subpocket of the AcCoA binding site by an aromatic ring

Group-Based Optimization of Potent and Cell-Active Inhibitors of the von Hippel–Lindau (VHL) E3 Ubiquitin Ligase: Structure–Activity Relationships Leading to the Chemical Probe (2<i>S</i>,4<i>R</i>)‑1-((<i>S</i>)‑2-(1-Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy‑<i>N</i>‑(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH298)

The von Hippel–Lindau tumor suppressor protein is the substrate binding subunit of the VHL E3 ubiquitin ligase, which targets hydroxylated α subunit of hypoxia inducible factors (HIFs) for ubiquitination and subsequent proteasomal degradation. VHL is a potential target for treating anemia and ischemic diseases, motivating the development of inhibitors of the VHL:HIF-α protein–protein interaction. Additionally, bifunctional proteolysis targeting chimeras (PROTACs) containing a VHL ligand can hijack the E3 ligase activity to induce degradation of target proteins. We report the structure-guided design and group-based optimization of a series of VHL inhibitors with low nanomolar potencies and improved cellular permeability. Structure–activity relationships led to the discovery of potent inhibitors <b>10</b> and chemical probe VH298, with dissociation constants <100 nM, which induced marked HIF-1α intracellular stabilization. Our study provides new chemical tools to probe the VHL-HIF pathways and new VHL ligands for next-generation PROTACs

Targeting the von Hippel–Lindau E3 Ubiquitin Ligase Using Small Molecules To Disrupt the VHL/HIF-1α Interaction

E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degradation, are attractive drug targets due to their exquisite substrate specificity. However, the development of small-molecule inhibitors has proven extraordinarily challenging as modulation of E3 ligase activities requires the targeting of protein–protein interactions. Using rational design, we have generated the first small molecule targeting the von Hippel–Lindau protein (VHL), the substrate recognition subunit of an E3 ligase, and an important target in cancer, chronic anemia, and ischemia. We have also obtained the crystal structure of VHL bound to our most potent inhibitor, confirming that the compound mimics the binding mode of the transcription factor HIF-1α, a substrate of VHL. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of chronic anemia and ischemia