Detection of Noncovalent Protein-Ligand Complexes by IR-MALDESI-MS.

Native mass spectrometry (MS) is a powerful analytical technique to directly probe noncovalent protein-protein and protein-ligand interactions. However, not every MS platform can preserve proteins in their native conformation due to high energy deposition from the utilized ionization source. Most small molecules approved as drugs and in development interact with their targets through noncovalent interactions. Therefore, rapid methods to analyze noncovalent protein-ligand interactions are necessary for the early stages of the drug discovery pipeline. Herein, we describe a method for analyzing noncovalent protein-ligand complexes by IR-MALDESI-MS with analysis times of ∼13 s per sample. Carbonic anhydrase and the kinase domain of Bruton's tyrosine kinase are paired with known noncovalent binders to evaluate the effectiveness of native MS by IR-MALDESI.

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.

Recent Developments in Mass Spectrometry to Support Next-Generation Synthesis and Screening.

The complexity of new therapeutics continues to increase and the timeline for the discovery of these therapeutics continues to shrink. This creates demand for new analytical techniques to facilitate quicker discovery and development of novel drugs. Mass spectrometry is one of the most prolific analytical techniques that has been applied across the entire drug discovery pipeline. New mass spectrometers and the associated methods for sampling have been introduced at a rate that keeps pace with new chemistries, therapeutic types, and screening practices used by modern drug hunters. This microperspective covers application and implementation of new mass spectrometry workflows that enable current and future efforts in screening and synthesis for drug discovery.

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.

Evaluation of Quantitative Platforms for Single Target Mass Spectrometry Imaging.

(1) Imaging of pharmaceutical compounds in tissue is an increasingly important subsection of Mass Spectrometry Imaging (MSI). Identifying proper target engagement requires MS platforms with high sensitivity and spatial resolution. Three prominent categories of drugs are small molecule drugs, antibody-drug conjugate payloads, and protein degraders. (2) We tested six common MSI platforms for their limit of detection (LoD) on a representative compound for each category: a Matrix-Assisted Laser Desorption/Ionization (MALDI) Fourier Transform Ion Cyclotron, a MALDI-2 Time-of-Flight (ToF), a MALDI-2 Trapped Ion Mobility Spectrometry ToF, a Desorption Electrospray Ionization Orbitrap, and 2 Atmospheric Pressure-MALDI Triple Quadrupoles. Samples were homogenized tissue mimetic models of rat liver spiked with known concentrations of analytes. (3) We found that the AP-MALDI-QQQ platform outperformed all 4 competing platforms by a minimum of 2- to 52-fold increase in LoD for representative compounds from each category of pharmaceutical. (4) AP-MALDI-QQQ platforms are effective, cost-efficient mass spectrometers for the identification of targeted analytes of interest.

Optimization of Zebrafish Larvae Sectioning for Mass Spectrometry Imaging.

The utility of zebrafish is becoming more frequent due to lower costs and high similarities to humans. Zebrafish larvae are attractive subjects for drug screening and drug metabolism research. However, obtaining good quality zebrafish larvae sections for batch samples at designated planes, angles, and locations for comparison purposes is a challenging task. We report here the optimization of fresh frozen zebrafish larvae sectioning for mass spectrometry imaging. We utilized the gelatin solutions that were created at two different temperatures (50 and 85 °C) as embedding media. Gelatin-50 (gelatin created under 50 °C, solid gel under room temperature) was used to make a larvae-shaped mold and gelatin-85 (gelatin created under 85 °C, liquid under room temperature) was used to embed the larvae. H&E staining of sections shows well-preserved morphology and minimal histological interference. More importantly, the position of the larvae was well controlled resulting in more consistent sectioning of the larvae.

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.

A Pilot Study of the Impact of Microwave Ablation on the Dielectric Properties of Breast Tissue.

Percutaneous microwave ablation (MWA) is a promising technology for patients with breast cancer, as it may help treat individuals who have less aggressive cancers or do not respond to targeted therapies in the neoadjuvant or pre-surgical setting. In this study, we investigate changes to the microwave dielectric properties of breast tissue that are induced by MWA. While similar changes have been characterized for relatively homogeneous tissues, such as liver, those prior results are not directly translatable to breast tissue because of the extreme tissue heterogeneity present in the breast. This study was motivated, in part by the expectation that the changes in the dielectric properties of the microwave antenna's operation environment will be impacted by tissue composition of the ablation target, which includes not only the tumor, but also its margins. Accordingly, this target comprises a heterogeneous mix of malignant, healthy glandular, and adipose tissue. Therefore, knowledge of MWA impact on breast dielectric properties is essential for the successful development of MWA systems for breast cancer. We performed ablations in 14 human ex-vivo prophylactic mastectomy specimens from surgeries that were conducted at the UW Hospital and monitored the temperature in the vicinity of the MWA antenna during ablation. After ablation we measured the dielectric properties of the tissue and analyzed the tissue samples to determine both the tissue composition and the extent of damage due to the ablation. We observed that MWA induced cell damage across all tissue compositions, and found that the microwave frequency-dependent relative permittivity and conductivity of damaged tissue are lower than those of healthy tissue, especially for tissue with high fibroglandular content. The results provide information for future developments on breast MWA systems.

The Performance of Higher Frequency Microwave Ablation in the Presence of Perfusion.

OBJECTIVE: In this paper, we investigate the impact of perfusion on the performance of microwave ablation across a large frequency range. METHODS: We designed multiple microwave ablation antennas to operate in liver tissue at discrete frequencies in the range 1.9-18 GHz. We performed electromagnetic simulations to calculate microwave power absorption patterns. Five-minute, 25 W ablation experiments were performed at each frequency in perfused and nonperfused ex vivo porcine livers, and thermal lesion dimensions were measured. RESULTS: The volume of greatest microwave power absorption shrinks by two orders of magnitude as the frequency is increased from 1.9 to 18 GHz. Mean thermal lesion volumes are consistent across the frequency range for a given perfusion state and are about three times smaller under active perfusion. Typical thermal lesion diameters (perpendicular to the antenna axis) were 24 mm and 16 mm for nonperfused and perfused ablations, respectively. No significant differences in axial ratio were observed among different frequency groups in active-perfusion experiments. CONCLUSION: Higher-frequency microwave ablation produces thermal lesions with volumes comparable to those achieved at lower frequencies, even in strongly perfused environments. SIGNIFICANCE: Higher-frequency microwave ablation is appealing because it allows for more flexibility in antenna design. A critical issue concerning the feasibility of higher frequency microwave ablation, considering its strong dependence on heat diffusion to grow thermal lesions, is its performance in strongly perfused environments. This paper shows that higher frequency microwave ablation achieves thermal lesions comparable to those from microwave ablation performed at conventional frequencies in both non- and strongly perfused environments.

The impact of frequency on the performance of microwave ablation.

PURPOSE: The use of higher frequencies in percutaneous microwave ablation (MWA) may offer compelling interstitial antenna design advantages over the 915 MHz and 2.45 GHz frequencies typically employed in current systems. To evaluate the impact of higher frequencies on ablation performance, we conducted a comprehensive computational and experimental study of microwave absorption and tissue heating as a function of frequency. METHODS: We performed electromagnetic and thermal simulations of MWA in ex vivo and in vivo porcine muscle at discrete frequencies in the 1.9-26 GHz range. Ex vivo ablation experiments were performed in the 1.9-18 GHz range. We tracked the size of the ablation zone across frequency for constant input power and ablation duration. Further, we conducted simulations to investigate antenna feed line heating as a function of frequency, input power, and cable diameter. RESULTS: As the frequency was increased from 1.9 to 26 GHz the resulting ablation zone dimensions decreased in the longitudinal direction while remaining relatively constant in the radial direction; thus at higher frequencies the overall ablation zone was more spherical. However, cable heating at higher frequencies became more problematic for smaller diameter cables at constant input power. CONCLUSION: Comparably sized ablation zones are achievable well above 1.9 GHz, despite increasingly localised power absorption. Specific absorption rate alone does not accurately predict ablation performance, particularly at higher frequencies where thermal diffusion plays an important role. Cable heating due to ohmic losses at higher frequencies may be controlled through judicious choices of input power and cable diameter.

Metabolism and Disposition of a Novel B-Cell Lymphoma-2 Inhibitor Venetoclax in Humans and Characterization of Its Unusual Metabolites.

Venetoclax (ABT-199), a B-cell lymphoma-2 (Bcl-2) protein inhibitor, is currently in clinical development for the treatment of hematologic malignancies. We characterized the absorption, metabolism, and excretion of venetoclax in humans. After a single oral dose of [14C]venetoclax to healthy volunteers, the recovery of total radioactive dose was 100%, with feces being the major route of elimination of the administered dose, whereas urinary excretion was minimal (<0.1%). The extent of absorption was estimated to be at least 65%. Venetoclax was primarily cleared by hepatic metabolism (∼66% of the administered dose). ∼33% of the administered dose was recovered as the parent drug and its nitro reduction metabolite M30 [2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-amino-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)benzamide] (13%) in feces. Biotransformation of venetoclax in humans primarily involves enzymatic oxidation on the dimethyl cyclohexenyl moiety, followed by sulfation and/or nitro reduction. Nitro reduction metabolites were likely formed by gut bacteria. Unchanged venetoclax was the major drug-related material in circulation, representing 72.8% of total plasma radioactivity. M27 (oxidation at the 6 position of cyclohexenyl ring followed by cyclization at the α-carbon of piperazine ring; 4-[(10aR,11aS)-7-(4-chlorophenyl)-9,9-dimethyl-1,3,4,6,8,10,10a,11a-octahydropyrazino[2,1-b][1,3]benzoxazin-2-yl]-N-[3-nitro-4-(tetrahydropyran-4-ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) was identified as a major metabolite, representing 12% of total drug-related material. M27 was primarily formed by cytochrome P450 isoform 3A4 (CYP3A4). Steady-state plasma concentrations of M27 in human and preclinical species used for safety testing suggested that M27 is a disproportionate human metabolite. M27 is not expected to have clinically relevant on- or off-target pharmacologic activities.

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.

Ni-catalyzed alkenylation of triazolopyridines: synthesis of 2,6-disubstituted pyridines.

A synthetic strategy to access 2,6-disubstituted pyridines from triazolopyridines through a regioselective nickel-catalyzed alkenylation reaction of the C7-H bond is described. The N2 fragment embedded in the resulting C-H functionalized triazolopyridine can be readily excised using acidic or oxidative conditions to unmask the pyridine.