Label Free Fragment Screening Using Surface Plasmon Resonance as a Tool for Fragment Finding – Analyzing Parkin, a Difficult CNS Target

<div><p>Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.</p></div

Affinities K_D (μM) of protein ligands to different Parkin proteins.

1<p><b>Ubiquitin-like domain of Parkin; ²Ubiquitin conjugating enzyme E2; ³Ubiquitin; ⁴ Full-length.</b></p

Functionally active FL-Parkin binds three different protein ligands.

<p>FL-FLAG Parkin was captured on a CM5 sensor chip with immobilized anti-FLAG antibody at a stoichiometry of (3∶1) (Parkin:Ab). Each of the protein ligands was injected at concentrations above 10-fold K_D if possible: (a) Ubiquitin at 500, 250, 125, 62.5, 31.25 and 15.62 μM (b) His-UblD and (c) UbcH7 were injected at 140, 46.7, 15.6, 5.2, 1.7 and 0.6 μM over FL-FLAG Parkin. All data was fitted to 1∶1 binding model. Each binding test was repeated at different test occasions (n≥3).1∶1 binding isotherms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066879#pone.0066879.s004" target="_blank">Figure S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066879#pone.0066879.s006" target="_blank">6</a>.</p

Thermal treated FL-FLAG-Parkin is more active than non-thermal treated FL-FLAG-Parkin independent of reducing agent.

<p>TR-FRET S5a assay using 150 nM FL-FLAG Parkin and 200nM biotinylated S5a substrate in the presence or absence of 5 mM reducing agent. FL-FLAG Parkin was incubated at 56°C for 30 min and then cooled to RT (thermal treated). Thermal treated FL-FLAG Parkin exhibit significantly different levels of Parkin activity in presence of each of the three reducing agents p<0.0001 (hatched bar) (n = 4). Non thermal treated FL-FLAG Parkin showed similar levels of activity in presence of either DTT or TCEP with p = 0.5368 and both activities are significantly higher than in presence of BME with p<0.0001 (white bar) (n = 4).</p

Ligand efficiencies of confirmed SPR hits.

<p>Plot of ligand efficiencies (LE) of SPR confirmed hits vs. number of heavy atoms <i>N_h.</i> Binding affinities (K_D) were obtained from injection of fragments in dose response manner from 50 uM in two-fold dilutions. One very interesting hit with LE of 0.5 and <i>HAC</i> of 13 is A (red dot) with a mid micro-molar affinity (K_D of 18.6 uM).</p

Physico-chemical properties of SPR hits vs. fragment library.

<p>The distribution of compounds in the fragment library is shown as circles; the SPR hits as triangles. The distribution of SPR hits is consistent with the fragment library (though noisier because of the small number of compounds), with the exception of hydrogen bond donors, which are over-represented in the SPR hits compared to fragment library.</p

Dithiothreitol (DTT) binds to non-thermal treated FL-FLAG Parkin.

<p>(A) SPR data: DTT was injected at 62.5, 31.25, 15.62, 7.8, 3.9, 1.95 and 0.98, 1.9 μM in 50 mM HEPES pH 8.8, 0.005% Tween-20, 0.01% PF-127. The kinetic fits are shown in red. The affinity was determined to 1.4 μM at 47% Rmax (Rmax: 34 RU). DTT dissociates very slowly and the sensorgrams show ill-behavior above 16.5 μM. SPR data for thermal treated FL-FLAG Parkin was similar (data not shown) (B) NMR-STD: (a) Proton spectrum of 0.25 mM compound Z (black); (b) STD spectrum of 0.25 mM compound Z (green); (c) STD spectrum of 0.25 mM compound Z in the presence of 4 µM FL Parkin protein (blue); (d) STD spectrum of 0.25 mM compound Z in the presence of 4 µM FL Parkin protein and 0.5 mM DTT (red).</p

Parkin fragment screen data.

<p>(A) Graphical representation of a typical run of the Parkin fragment screen representing 10% of the total number of fragments screened (560 fragments). The binding level of each fragment of the run is shown in Response Units (RU) on the x-axis and the number of fragments on the y-axis. Experiments were performed on a Biacore 4000 instrument w buffer containing 2% DMSO. FL-FLAG Parkin was captured on a CM5 sensor chip with immobilized anti-FLAG antibody at a stoichiometry of (3∶1) (Parkin:Ab). Fragments were injected at 25 μM in buffer containing 2% DMSO All data were reference subtracted, solvent corrected and adjusted for changes in surface activity during a run.(B) Fragment binding levels as %Rmax of single concentration SPR hits at 25 uM of a screen of 5260 fragments. Only hits with a binding level greater than 3-fold standard deviation (SD) and acceptable sensorgrams are shown.</p

Discovery of (<i>R</i>)‑4-Cyclopropyl-7,8-difluoro-5-(4-(trifluoromethyl)phenylsulfonyl)-4,5-dihydro‑1<i>H</i>‑pyrazolo[4,3‑<i>c</i>]quinoline (ELND006) and (<i>R</i>)‑4-Cyclopropyl-8-fluoro-5-(6-(trifluoromethyl)pyridin-3-ylsulfonyl)-4,5-dihydro‑2<i>H</i>‑pyrazolo[4,3‑<i>c</i>]quinoline (ELND007): Metabolically Stable γ‑Secretase Inhibitors that Selectively Inhibit the Production of Amyloid‑β over Notch

Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aβ generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds <b>30</b> (ELND006) and <b>34</b> (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aβ generation in vivo between <b>30</b>, <b>34</b>, Semagacestat <b>41</b>, Begacestat <b>42</b>, and Avagacestat <b>43</b> in mice is made. <b>30</b> lowered Aβ in the CSF of healthy human volunteers