A high density assay format for the detection of novel cytotoxic agents in large chemical libraries

In response to the need for inexpensive high throughput assays for anti-cancer drug screening, a 1536-well microtiter plate based assay utilizing the Alamar Blue fluorescent dye as a measure of cellular growth was validated in 10 μL assay volume. Its robustness was assessed in a screen against a library of 2000 known bioactives; with an overall Z′ value of 0.89 for assay robustness, several known cytotoxic agents were identified including and not limited to anthracyclines, cardiac glycosides, gamboges, and quinones. To further test the sensitivity of the assay, IC₅₀ determinations were performed in both 384-well and 1536-well formats and the obtained results show a very good correlation between the two density formats. These findings demonstrate that this newly developed assay is simple to set up, robust, highly sensitive and inexpensive. It could potentially provide a rapid way to screen established and primary tumor cell lines against large chemical libraries.     

Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure.     

Affinity-based screening of MDM2/MDMX–p53 interaction inhibitors by chemical array: Identification of novel peptidic inhibitors

MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein–protein interactions (PPIs) of MDM2–p53 and MDMX–p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2–p53 and MDMX–p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2–p53 and MDMX–p53 interactions.     

Identification of potent Yes1 kinase inhibitors using a library screening approach

Yes1 kinase has been implicated as a potential therapeutic target in a number of cancers including melanomas, breast cancers, and rhabdomyosarcomas. Described here is the development of a robust and miniaturized biochemical assay for Yes1 kinase that was applied in a high throughput screen (HTS) of kinase-focused small molecule libraries. The HTS provided 144 (17% hit rate) small molecule compounds with IC₅₀ values in the sub-micromolar range. Three of the most potent Yes1 inhibitors were then examined in a cell-based assay for inhibition of cell survival in rhabdomyosarcoma cell lines. Homology models of Yes1 were generated in active and inactive conformations, and docking of inhibitors supports binding to the active conformation (DFG-in) of Yes1. This is the first report of a large high throughput enzymatic activity screen for identification of Yes1 kinase inhibitors, thereby elucidating the polypharmacology of a variety of small molecules and clinical candidates.     

Facile synthesis and biological evaluation of 3,3-diphenylpropanoyl piperazines as T-type calcium channel blockers

We have developed a facile synthesis of 3,3-diphenylpropanamides using Meldrum’s acid derivatives as amide coupling components. The in vitro biological evaluation of the title compounds led to the identification of compound 1h, which has good inhibitory activity against T-type calcium channel (IC₅₀ = 0.83 μM).     

Substrate analysis of homoserine acyltransferase from Bacillus cereus

Substrate specificity within the family of enzymes designated as homoserine transsuccinylases is variable, with some organisms utilizing succinyl-CoA and other organisms utilizing acetyl-CoA. In this study it is shown that the enzyme from Bacillus cereus uses acetyl-CoA as its acyl donor, but its catalytic rate is significantly lower than other HTS family members. BcHTS is inactivated by both iodoacetamide and diethyl pyrocarbonate and the enzyme can be partially protected from inactivation by the presence of succinyl-CoA. This leads to the conclusion that BcHTS can bind both acetyl-CoA and succinyl-CoA and suggests that it may represent an intermediate between the succinate-transferring HTS family members and the acetate-transferring HTS family members. The B. cereus enzyme was unable to rescue growth of an Escherichia coli strain lacking a functional transsuccinylase, however.     

Identification of a Compound That Disrupts Binding of Amyloid-β to the Prion Protein Using a Novel Fluorescence-based Assay

The prion protein (PrP) has been implicated both in prion diseases such as Creutzfeldt-Jakob disease, where its monomeric cellular isoform (PrP^C) is recruited into pathogenic self-propagating polymers of misfolded protein, and in Alzheimer disease, where PrP^C may act as a receptor for synaptotoxic oligomeric forms of amyloid-β (Aβ). There has been considerable interest in identification of compounds that bind to PrP^C, stabilizing its native fold and thereby acting as pharmacological chaperones to block prion propagation and pathogenesis. However, compounds binding PrP^C could also inhibit the binding of toxic Aβ species and may have a role in treating Alzheimer disease, a highly prevalent dementia for which there are currently no disease-modifying treatments. However, the absence of a unitary, readily measurable, physiological function of PrP makes screening for ligands challenging, and the highly heterogeneous nature of Aβ oligomer preparations makes conventional competition binding assays difficult to interpret. We have therefore developed a high-throughput screen that utilizes site-specifically fluorescently labeled protein to identify compounds that bind to PrP and inhibit both Aβ binding and prion propagation. Following a screen of 1,200 approved drugs, we identified Chicago Sky Blue 6B as the first small molecule PrP ligand capable of inhibiting Aβ binding, demonstrating the feasibility of development of drugs to block this interaction. The interaction of Chicago Sky Blue 6B was characterized by isothermal titration calorimetry, and its ability to inhibit Aβ binding and reduce prion levels was established in cell-based assays.     

Inhibition of Anopheles gambiae Odorant Receptor Function by Mosquito Repellents

The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca²⁺-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET.     

High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase

The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening.     

High-throughput Assays for Superoxide and Hydrogen Peroxide: DESIGN OF A SCREENING WORKFLOW TO IDENTIFY INHIBITORS OF NADPH OXIDASES*

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.