High-Throughput Assessment of Bile Salt Export Pump Inhibition Using RapidFire-MS and DESI-MS.

The bile salt export pump (BSEP) assay is widely used to evaluate the potential for drug-induced liver injury (DILI) early in the drug discovery process. While traditional liquid chromatography-mass spectrometry (LC-MS)-based approaches have been utilized for BSEP activity testing, they have intrinsic limitations in either throughput or the requirement for sample preparation and are difficult to scale up in order to screen drug candidates. Here we demonstrate the use of two different high-throughput MS methods based on solid-phase extraction (SPE) and desorption electrospray ionization (DESI) for high-throughput BSEP activity assessment in a label-free manner, with minimal needs for sample workup, at sampling rates of ∼11 and ∼5.

From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9.

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound.

Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.

HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform.

Use of high-throughput mass spectrometry to reduce false positives in protease uHTS screens.

As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)-MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥ 60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5-7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates.

The host-range, genomics and proteomics of Escherichia coli O157:H7 bacteriophage rV5.

Background: Bacteriophages (phages) have been used extensively as analytical tools to type bacterial cultures and recently for control of zoonotic foodborne pathogens in foods and in animal reservoirs.

Methods: We examined the host range, morphology, genome and proteome of the lytic E. coli O157 phage rV5, derived from phage V5, which is a member of an Escherichia coli O157:H7 phage typing set.

1,3,8-Triazaspiro[4.5]decane-2,4-diones as efficacious pan-inhibitors of hypoxia-inducible factor prolyl hydroxylase 1-3 (HIF PHD1-3) for the treatment of anemia.

The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors).

Application of affinity selection/mass spectrometry to determine the structural isomer of parnafungins responsible for binding polyadenosine polymerase.

To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans.

Affinity-based ranking of ligands for DPP-4 from mixtures.

Affinity-based selection strategies have recently emerged as a complement to traditional high throughput screening for the rapid discovery of lead compounds for the large number of protein targets emerging from--omics technologies. Herein, we describe a method for the ranking of mixtures of ligands by affinity selection and apply it to rank order a set of inhibitors for the enzyme dipeptidyl peptidase IV.

Affinity mass spectrometry from a tailored porous silicon surface.

The development of chemically stable porous silicon (pSi) materials for DIOS (Desorption/Ionization on Silicon) mass spectrometry, covalent linkers cleaved in the DIOS laser pulse, and efficient methods for bond formation to immobilized species, allows for on-chip affinity purification and mass detection.

New catalysts for the asymmetric hydrosilylation of ketones discovered by mass spectrometry screening.

A method for determining enantiomeric excess by mass spectrometry was employed to screen a family of chiral phosphite P,N-ligands for activity in the rhodium-catalyzed asymmetric hydrosilylation of ketones. The identification of an effective set of ligands was followed by preliminary studies of the reaction scope and mechanism. Asymmetric induction of 84-88% ee for larger-scale reactions was observed, which is close to the level of the best alternative catalysts previously discovered.