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

Identification of a peptide inhibitor for the histone methyltransferase WHSC1

<div><p>WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.</p></div

Representative sensorgram for PTD2 binding to Avi-tagged WHSC1 941–1240 from single-cycle kinetic SPR measurements.

<p>WHSC1 was immobilized on a streptavidin-coated chip and peptide inhibitor was co-injected with SAM utilizing a 3-fold, 5-point dilution series ending at a 20 μM top concentration. Data reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#pone.0197082.t002" target="_blank">Table 2</a> is presented as the standard deviation of three independent experiments.</p

Norleucine-containing peptides can inhibit WHSC1 and WHSC1L1 activity in vitro.

<p>Representative peptide inhibitor biochemical dose-response curves for (A) WHSC1 941–1240 and (B) WHSC1L1 1054–1285. Error bars represent the standard deviation of three independent replicates. Resulting IC₅₀ values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#pone.0197082.t002" target="_blank">Table 2</a>.</p

Biochemical and biophysical peptide inhibitor potency values for WHSC1 941–1240 and WHSC1L1 1054–1285^a.

<p>Biochemical and biophysical peptide inhibitor potency values for WHSC1 941–1240 and WHSC1L1 1054–1285<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#t002fn001" target="_blank">^a</a>.</p

WHSC1L1 1054–1285 has a similar overall structure in relation to other NSD family proteins and can form a ternary complex with SAM and PTD2.

<p>(A) Superposition of NSD family proteins (green/yellow = WHSC1L1-PTD2 (PDB code = 6CEN); cyan = WHSC1L1 (PDB code = 5UPD); magenta = WHSC1 (PDB code = 5LSU); purple = NSD1 (PDB code = 3OOI). All protein chains are shown as ribbons; SAM and PTD2 are depicted in stick representation. (B) Structure of WHSC1L1-PTD2-SAM ternary complex. Hydrogen bonds are indicated with dashed lines. (C) Superposition of WHSC1L1-PTD2 and SETD2-H3.3 K36M (grey; PDB code = 5JJY).</p

Isothermal titration calorimetry of PTD2 binding to WHSC1 941–1240.

<p>Upper panel, calorimetric trace for ligand titration; lower panel, binding isotherm from calorimetric trace. WHSC1 concentration in the cell was 10 μM supplemented with 100 μM SAM. PTD2 concentration in the syringe was 100 μM.</p

Crystallographic data collection and refinement statistics for WHSC1L1 1054–1285 with PTD2 and SAM.

<p>Crystallographic data collection and refinement statistics for WHSC1L1 1054–1285 with PTD2 and SAM.</p

Representative SPR sensorgrams for PTD2 binding to Avi-tagged WHSC1 941–1240 in the absence or presence of SAM analogs.

<p>WHSC1 was immobilized on a streptavidin-coated chip and peptide inhibitor was either injected in the absence of cofactor (left panel), co-injected with SAH (middle panel), or co-injected with SFG (right panel) utilizing a 2-fold, 10-point dilution series ending at a 100 μM top concentration.</p