A High-Throughput Screening Assay for Pyruvate Carboxylase

Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potentsmall molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC

Design and Evaluation of Heterobivalent PAR1–PAR2 Ligands as Antagonists of Calcium Mobilization

A novel class of bivalent ligands targeting putative protease-activated receptor (PAR) heteromers has been prepared based upon reported antagonists for the subtypes PAR1 and PAR2. Modified versions of the PAR1 antagonist RWJ-58259 containing alkyne adapters were connected via cycloaddition reactions to azide-capped polyethylene glycol (PEG) spacers attached to imidazopyridazine-based PAR2 antagonists. Initial studies of the PAR1–PAR2 antagonists indicated that they inhibited G alpha q-mediated calcium mobilization in endothelial and cancer cells driven by both PAR1 and PAR2 agonists. Compounds of this novel class hold promise for the prevention of restenosis, cancer cell metastasis, and other proliferative disorders

Inhibition of a viral enzyme by a small-molecule dimer disruptor.

We identified small-molecule dimer disruptors that inhibit an essential dimeric protease of human Kaposi's sarcoma-associated herpesvirus (KSHV) by screening an alpha-helical mimetic library. Next, we synthesized a second generation of low-micromolar inhibitors with improved potency and solubility. Complementary methods including size exclusion chromatography and 1H-13C HSQC titration using selectively labeled 13C-Met samples revealed that monomeric protease is enriched in the presence of inhibitor. 1H-15N HSQC titration studies mapped the inhibitor binding site to the dimer interface, and mutagenesis studies targeting this region were consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of a dynamic intermediate. These results validate the interface of herpesvirus proteases and other similar oligomeric interactions as suitable targets for the development of small-molecule inhibitors

Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4 μM), suramin sodium salt (IC50 = 3.6 μM), NF 023 hydrate (IC50 = 6.2 μM) and tyrphostin AG 538 (IC50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.National Institutes of Health [RO1 AI088001]; Research Growth Initiative Award [101X219] from the University of Wisconsin-Milwaukee Research Foundation; National Institutes of Health Molecular Libraries Initiative [U54 HG005031]. Funding for open access charge: University of Wisconsin-Milwaukee Research Foundation

The Parmodulin NRD-21 is an Allosteric Inhibitor of PAR1 Gq Signaling with Improved Anti-Inflammatory Activity and Stability

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications