Ultra-high-throughput mass spectrometry in drug discovery: fundamentals and recent advances.

INTRODUCTION: Ultra-high-throughput mass spectrometry, uHT-MS, is a technology that utilizes ionization and sample delivery technologies optimized to enable sampling from well plates at > 1 sample per second. These technologies do not need a chromatographic separation step and can be utilized in a wide variety of assays to detect a broad range of analytes including small molecules, lipids, and proteins. AREAS COVERED: This manuscript provides a brief historical review of high-throughput mass spectrometry and the recently developed technologies that have enabled uHT-MS. The report also provides examples and references on how uHT-MS has been used in biochemical and chemical assays, nuisance compound profiling, protein analysis and high throughput experimentation for chemical synthesis. EXPERT OPINION: The fast analysis time provided by uHT-MS is transforming how biochemical and chemical assays are performed in drug discovery. The potential to associate phenotypic responses produced by 1000's of compound treatments with changes in endogenous metabolite and lipid signals is becoming feasible. With the augmentation of simple, fast, high-throughput sample preparation, the scope of uHT-MS usage will increase. However, it likely will not supplant LC-MS for analyses that require low detection limits from complex matrices or characterization of complex biotherapeutics such as antibody-drug conjugates.

Disconnect between COX-2 selective inhibition and cardiovascular risk in preclinical models.

INTRODUCTION: Secondary pharmacology profiling is routinely applied in pharmaceutical drug discovery to investigate the pharmaceutical effects of a drug at molecular targets distinct from (off-target) the intended therapeutic molecular target (on-target). Data from a randomized, placebo-controlled clinical trial, the APPROVe (Adenomatous Polyp Prevention on VIOXX, rofecoxib) trial, raised significant concerns about COX-2 inhibition as a primary or secondary target, shaping the screening and decision-making processes of some pharmaceutical companies. COX-2 is often included in off-target screens due to cardiovascular (CV) safety concerns about secondary interactions with this target. Several potential mechanisms of COX-2-mediated myocardial infarctions have been considered including, effects on platelet stickiness/aggregation, vasal tone and blood pressure, and endothelial cell activation. In the present study, we focused on each of these mechanisms as potential effects of COX-2 inhibitors, to find evidence of mechanism using various in vitro and in vivo preclinical models. METHODS: Compounds tested in the study, with a range of COX-2 selectivity, included rofecoxib, celecoxib, etodolac, and meloxicam. Compounds were screened for inhibition of COX-2 vs COX-1 enzymatic activity, ex vivo platelet aggregation (using whole blood from multiple species), ex vivo canine femoral vascular ring model, in vitro human endothelial cell activation (with and without COX-2 induction), and in vivo cardiovascular assessment (anesthetized dog). RESULTS: The COX-2 binding assessment generally confirmed the COX-2 selectivity previously reported. COX-2 inhibitors did not have effects on platelet function (spontaneous aggregation or inhibition of aggregation), cardiovascular parameters (mean arterial pressure, heart rate, and left ventricular contractility), or endothelial cell activation. However, rofecoxib uniquely produced an endothelial mediated constriction response in canine femoral arteries. CONCLUSION: Our data suggest that rofecoxib-related cardiovascular events in humans are not predicted by COX-2 potency or selectivity. In addition, the vascular ring model suggested possible adverse cardiovascular effects by COX-2 inhibitors, although these effects were not seen in vivo studies. These results may also suggest that COX-2 inhibition alone is not responsible for rofecoxib-mediated adverse cardiovascular outcomes.

High-Throughput Intact Protein Analysis for Drug Discovery Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Mass spectrometry (MS) is the primary analytical tool used to characterize proteins within the biopharmaceutical industry. Electrospray ionization (ESI) coupled to liquid chromatography (LC) is the current gold standard for intact protein analysis. However, inherent speed limitations of LC/MS prevent analysis of large sample numbers (>1000) in a day. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI-MS), an ambient ionization MS technology, has recently been established as a platform for high-throughput small molecule analysis. Here, we report the applications of such a system for the analysis of intact proteins commonly performed within the drug discovery process. A wide molecular weight range of proteins 10-150 kDa was detected on the system with improved tolerance to salts and buffers compared to ESI. With high concentrations and model proteins, a sample rate of up to 22 Hz was obtained. For proteins at low concentrations and in buffers used in commonly employed assays, robust data at a sample rate of 1.5 Hz were achieved, which is ∼22× faster than current technologies used for high-throughput ESI-MS-based protein assays. In addition, two multiplexed plate-based high-throughput sample cleanup methods were coupled to IR-MALDESI-MS to enable analysis of samples containing excessive amounts of salts and buffers without fully compromising productivity. Example experiments, which leverage the speed of the IR-MALDESI-MS system to monitor NISTmAb reduction, protein autophosphorylation, and compound binding kinetics in near real time, are demonstrated.

Ultra-High-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method that is being developed for high-throughput, label-free, biochemical screening of large-scale compound libraries. Here, we report the development of an ultra-high-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well, and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 µM 1-hydroxymidazolam and 12 µM dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with a ∼30 s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115200 samples from 300 microtiter plate reads consecutively over 5.54 h with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high-throughput compound screening.

High-Throughput Label-Free Biochemical Assays Using Infrared Matrix-Assisted Desorption Electrospray Ionization Mass Spectrometry.

Mass spectrometry (MS) can provide high sensitivity and specificity for biochemical assays without the requirement of labels, eliminating the risk of assay interference. However, its use had been limited to lower-throughput assays due to the need for chromatography to overcome ion suppression from the sample matrix. Direct analysis without chromatography has the potential for high throughput if sensitivity is sufficient despite the presence of a matrix. Here, we report and demonstrate a novel direct analysis high-throughput MS system based on infrared matrix-assisted desorption electrospray ionization (IR-MALDESI) that has a potential acquisition rate of 33 spectra/s. We show the development of biochemical assays in standard buffers for wild-type isocitrate dehydrogenase 1 (IDH1), diacylglycerol kinase zeta (DGKζ), and p300 histone acetyltransferase (P300) to demonstrate the suitability of this system for a broad range of high-throughput lead discovery assays. A proof-of-concept pilot screen of ∼3k compounds is also shown for IDH1 and compared to a previously reported fluorescence-based assay. We were able to obtain reliable data at a speed amenable for high-throughput screening of large-scale compound libraries.

An improved procedure for the preparation of Ru(bpz)3(PF6)2 via a high-yielding synthesis of 2,2'-bipyrazine.

There has been a recent surge of interest in the use of transition metal polypyridyl complexes as visible light-absorbing photocatalysts for synthetic applications. Among the most attractive features of this approach is the availability of many known complexes with well-characterized photophysical and electrochemical properties. In particular, Ru(bpz)3 (2+) is a powerful photooxidant that has proven to be uniquely suited for oxidatively induced photoredox transformations. We present here a straightforward and high-yielding route to Ru(bpz)3(PF6)2 that features an improved Pd-catalyzed synthesis of the 2,2'-bipyrazine ligand that is amenable to gram-scale preparations.