Development of an Inhibitor Screening Platform via Mass Spectrometry

Abstract

Commonly used methods for isolated enzyme inhibitor screening typically rely on fluorescent or chemiluminescent detection techniques that are often indirect and/or coupled assays. Mass spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays and has more recently been demonstrated as an alternative readout system for inhibitor screening. In this report, a high-throughput mass spectrometry (HTMS) readout platform, based on the direct measurement of substrate conversion to product, is presented. The rapid ionization and desorption features of a new generation matrix-assisted laser desorption ionization-triple quadrupole (MALDI-QqQ) mass spectrometer are shown to improve the speed of analysis to greater than 1 sample per second while maintaining excellent Z′ values. Furthermore, the readout was validated by demonstrating the ability to measure IC 50 values for several known kinase inhibitors against cyclic AMP-dependent protein kinase (PKA). Finally, when the assay performance was compared with a common ADPaccumulation readout system, this HTMS approach produced better signal-to-background ratios, higher Z′ values, and a reagent cost of about $0.03 per well compared with about $0.60 per well for the fluorescence assay. Collectively, these data demonstrate that a MALDI-QqQ-MS-based readout platform offers significant advantages over the commonly used assays in terms of speed, sensitivity, reproducibility, and reagent cost. INTRODUCTION HIGH-THROUGHPUT SCREENING (HTS) plays a central role in the drug discovery process starting at the early "hit" discovery stage then continuing through to lead optimization. As such, the screening community has continuously developed new technologies to meet the broadening assay needs and to expedite the work flow of drug discovery. Within the pharmaceutical and biotechnology enzyme screening community, fluorescent-and chemiluminescent-based detection methods continue to be the routine assay platforms of choice for isolated enzyme assays. These assays are desirable because they have been developed to be simple homogeneous assays that often offer universal methods to evaluate a variety of targets using the same reagents. However, 1 of the primary challenges for these approaches continues to be how to maintain the high speed of analysis while minimizing false-positive or false-negative readouts. Inherent to this challenge is the nature of fluorescence and chemiluminescence readout, which can lend itself to false readouts because of the properties of various compound classes that enhance or quench the signals. Thus a comparable approach in speed, robust readout, and cost that further minimizes the potential for false readouts would be a benefit to the field.One approach that offers promise in this arena is mass spectrometry. A variety of mass spectrometry (MS) techniques have been used to measure enzyme activity, enzyme kinetics, inhibition, and more recently for HTS applications (reviewed in the studies of Greis 1 and De Boer et...

Biomedical application of MALDI mass spectrometry for small‐molecule analysis

“…For quantitative analysis of, for instance, HIV protease inhibitors, analysis times for a single spot on the target plate have been reported of $3 min for MALDI-FTICR MS (20 Hz laser), $30 sec for MALDI-TOF MS (50 Hz laser), and $5 sec for MALDI-triple quadrupole MS (1,000 Hz laser; van Kampen et al, 2006Kampen et al, , 2008aKampen et al, ,b, 2009b. Recently, an analysis time of 1.75 min for an entire 384-well target plate was reported for an assay to screen for small-molecule inhibitors of enzymes with MALDI-triple quadrupole MS (Rathore et al, 2008). When the time required for vacuum pump down was included, the total analysis time for a single target plate was still just under 3.5 min.…”

“…In MALDI-MS, however, the sample analysis time is primarily determined by the number of laser shots needed to generate an average mass spectrum of high quality and the repetition rate of the laser used. Dwell time of the ions does not contribute significantly to the analysis time, although the detection time of ions can take second(s) per scan in Fourier-transform ion cyclotron resonance (FTICR) MS and Orbitrap MS. With a high repetition rate laser that fires at 1,000-2,000 Hz, an averaged mass spectrum of high quality can be obtained in a few seconds with MALDI-time-of-flight (TOF) MS or MALDI-triple quadrupole MS (Hatsis et al, 2003;McLean, Russell, & Russell, 2003;Moskovets et al, 2006;Rathore et al, 2008). For quantitative analysis of, for instance, HIV protease inhibitors, analysis times for a single spot on the target plate have been reported of $3 min for MALDI-FTICR MS (20 Hz laser), $30 sec for MALDI-TOF MS (50 Hz laser), and $5 sec for MALDI-triple quadrupole MS (1,000 Hz laser; van Kampen et al, 2006Kampen et al, , 2008aKampen et al, ,b, 2009b.…”

Determination of the antiretroviral drug tenofovir in plasma from HIV‐infected adults by ultrafast isotope dilution MALDI‐triple quadrupole tandem mass spectrometry

“…We demonstrated previously that MALDI-QqQ-MS technology has a great usability and is a versatile tool in the determination of concentrations (extra-and intracellular) of small molecules such as antiretroviral drugs (protease inhibitors), [14,15] anticancer drugs [13] but also other types of drugs [16 -18] and as screening tool in enzyme kinetic studies. [19] In a study by Volmer et al [20] the MALDI-QqQ-MS technology demonstrated equal performances compared with LC-QqQ-MS/MS instrumentation applying electrospray ionization (ESI) as ionization source when applied in pharmacokinetic studies. Previously, Notari et al [21,22] demonstrated that MALDI-TOF could be applied for the determination of low concentrations of antiretroviral drugs in plasma samples although concentrations of the antiretroviral drugs were not determined by isotope dilution mass spectrometry but by means of internal standard quantification.…”

Assay Development and Screening of Human DGAT1 Inhibitors with an LC/MS-Based Assay: Application of Mass Spectrometry for Large-Scale Primary Screening

“…[1][2][3][4] In the past several years, MS has been explored in primary screening for hit identification, including several methodologies in development for use in function-based biochemical reactions 1,5 and affinity-based binder screening (such as Dios-MS, 6 SpeedScreen, 7 and FAC-MS 8,9 ). Recently, approaches to further increase the analysis speed have been tested with matrix-assisted laser desorption/ionization (MALDI)-based MS technologies (e.g., MALDI-TOF-MS, 10 MALDI-QqQ-MS, 11 and MALDI-FTMS 12 ). However, many of the aforementioned approaches are still in proof-of-principle stages [10][11][12] or applied on relatively small-scale compound screening.…”

“…Recently, approaches to further increase the analysis speed have been tested with matrix-assisted laser desorption/ionization (MALDI)-based MS technologies (e.g., MALDI-TOF-MS, 10 MALDI-QqQ-MS, 11 and MALDI-FTMS 12 ). However, many of the aforementioned approaches are still in proof-of-principle stages [10][11][12] or applied on relatively small-scale compound screening. 5,13,14 MS is still in its early stage for primary screening application.…”