115 research outputs found
AlphaScreen-Based Assays: Ultra-High-Throughput Screening for Small-Molecule Inhibitors of Challenging Enzymes and Protein-Protein Interactions
No full textHigh-throughput 1,536-well fluorescence polarization assays for alpha(1)-acid glycoprotein and human serum albumin binding
No full textTwo major plasma proteins in humans are primarily responsible for drug binding, the α1-acid-glycoprotein (AGP) and human serum albumin (HSA). The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP) of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC1280 collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets
Applications of Differential Scanning Fluorometry and Related Technologies in Characterization of Protein–Ligand Interactions
No full textA strategy to discover inhibitors of Bacillus subtilis surfactin-type phosphopantetheinyl transferase
No full text
High-throughput 1,536-well fluorescence polarization assays for α(1)-acid glycoprotein and human serum albumin binding.
No full textTwo major plasma proteins in humans are primarily responsible for drug binding, the α(1)-acid-glycoprotein (AGP) and human serum albumin (HSA). The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP) of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC(1280) collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets
Kinetic and structural investigations of novel inhibitors of human epithelial 15-lipoxygenase-2
No full textHuman epithelial 15-lipoxygenase-2 (h15-LOX-2, ALOX15B) is expressed in many tissues and has been implicated in atherosclerosis, cystic fibrosis and ferroptosis. However, there are few reported potent/selective inhibitors that are active ex vivo. In the current work, we report newly discovered molecules that are more potent and structurally distinct from our previous inhibitors, MLS000545091 and MLS000536924 (Jameson et al, PLoS One, 2014, 9, e104094), in that they contain a central imidazole ring, which is substituted at the 1-position with a phenyl moiety and with a benzylthio moiety at the 2-position. The initial three molecules were mixed-type, non-reductive inhibitors, with IC values of 0.34 ± 0.05 μM for MLS000327069, 0.53 ± 0.04 μM for MLS000327186 and 0.87 ± 0.06 μM for MLS000327206 and greater than 50-fold selectivity versus h5-LOX, h12-LOX, h15-LOX-1, COX-1 and COX-2. A small set of focused analogs was synthesized to demonstrate the validity of the hits. In addition, a binding model was developed for the three imidazole inhibitors based on computational docking and a co-structure of h15-LOX-2 with MLS000536924. Hydrogen/deuterium exchange (HDX) results indicate a similar binding mode between MLS000536924 and MLS000327069, however, the latter restricts protein motion of helix-α2 more, consistent with its greater potency. Given these results, we designed, docked, and synthesized novel inhibitors of the imidazole scaffold and confirmed our binding mode hypothesis. Importantly, four of the five inhibitors mentioned above are active in an h15-LOX-2/HEK293 cell assay and thus they could be important tool compounds in gaining a better understanding of h15-LOX-2\u27s role in human biology. As such, a suite of similar pharmacophores that target h15-LOX-2 both in vitro and ex vivo are presented in the hope of developing them as therapeutic agents
AGP Assay Miniaturization to 1536-well Format.
No full text<p>A) whole-plate scatter plot obtained from 4 µM AGP/1 µM Dipyridamole mix (•) and 1 µM Dipyridamole (○); B) concentration-response profile of Propranolol (•).</p
Structures of the AGP and HSA Validation Set.
No full text<p>Structures of the AGP and HSA Validation Set.</p
Validation Set Testing.
No full text<p>A) IC<sub>50</sub> and K<sub>d</sub> values for the Validation Set in 1,536-well (▪), 96-well (▪), and protein coated bead (□) AGP assays; B) the corresponding results for the HSA assays.</p
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