Quantitative characterization of the mechanism of action and impact of a 'proteolysis-permitting' anti-PCSK9 antibody.

A recent report described a novel mechanism of action for an anti-proprotein convertase subtilisin-kexin type 9 (PCSK9) monoclonal antibody (LY3015014, or LY), wherein the antibody has improved potency and duration of action due to the PCSK9 epitope for LY binding. Unlike other antibodies, proteolysis of PCSK9 can occur when LY is bound to PCSK9. We hypothesized that this allowance of PCSK9 cleavage potentially improves LY efficiency through two pathways, namely lack of accumulation of intact PCSK9 and reduced clearance of LY. A quantitative modeling approach is necessary to further understand this novel mechanism of action. We developed a mechanism-based model to characterize the relationship between antibody pharmacokinetics, PCSK9 and LDL cholesterol levels in animals, and used the model to better understand the underlying drivers for the improved efficiency of LY. Simulations suggested that the allowance of cleavage of PCSK9 resulting in a lack of accumulation of intact PCSK9 is the major driver of the improved potency and durability of LY. The modeling reveals that this novel 'proteolysis-permitting' mechanism of LY is a means by which an efficient antibody can be developed with a total antibody dosing rate that is lower than the target production rate. We expect this engineering approach may be applicable to other targets and that the mathematical models presented herein will be useful in evaluating similar approaches.

Characterization and quantification of oxyntomodulin in human and rat plasma using high-resolution accurate mass LC-MS.

BACKGROUND: A thorough understanding of the biological role of oxyntomodulin (OXM) has been limited by the availability of sensitive and specific analytical tools for reliable in vivo characterization. Here, we utilized immunoaffinity capture coupled with high-resolution accurate mass LC-MS detection to quantify OXM and its primary catabolites. RESULTS: Quantification of intact OXM 1-37 in human and rat plasma occurred in pre- and post-prandial samples. Profiles for the major catabolites were observed allowing kinetic differences to be assessed between species. CONCLUSION: A validated assay in human and rat plasma was obtained for OXM 1-37 and its catabolites, 3-37 and 4-37. The value of full scan high-resolution accurate mass detection without selected reaction monitoring for low-abundance peptide quantification was also demonstrated.

The interactome of the atypical phosphatase Rtr1 in Saccharomyces cerevisiae.

The phosphatase Rtr1 has been implicated in dephosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) during transcription elongation and in regulation of nuclear import of RNAPII. Although it has been shown that Rtr1 interacts with RNAPII in yeast and humans, the specific mechanisms that underlie Rtr1 recruitment to RNAPII have not been elucidated. To address this, we have performed an in-depth proteomic analysis of Rtr1 interacting proteins in yeast. Our studies revealed that hyperphosphorylated RNAPII is the primary interacting partner for Rtr1. To extend these findings, we performed quantitative proteomic analyses of Rtr1 interactions in yeast strains deleted for CTK1, the gene encoding the catalytic subunit of the CTD kinase I (CTDK-I) complex. Interestingly, we found that the interaction between Rtr1 and RNAPII is decreased in ctk1Δ strains. We hypothesize that serine-2 CTD phosphorylation is required for Rtr1 recruitment to RNAPII during transcription elongation.

Detection of left ventricular hypertrophy in rats administered a peroxisome proliferator-activated receptor alpha/gamma dual agonist using natriuretic peptides and imaging.

Chronic treatment with suprapharmacologic doses of peroxisome proliferator-activated receptor (PPAR) agonists has a known potential for causing left ventricular hypertrophy (LVH). The mechanism by which LVH develops is not well understood nor are biomarkers of it well characterized. Natriuretic peptides are important regulators of cardiac growth, blood volume, and arterial pressure and may be useful biomarkers of LVH and hemodynamic changes that precede it. We measured amino-terminal pro-atrial natriuretic peptide (NTproANP), amino-terminal pro-brain natriuretic peptide (NTproBNP), and cardiac troponin I (cTnI) concentrations in serum and plasma, as well as transcripts in left ventricular heart tissue for atrial natriuretic peptide precursor (Nppa), brain natriuretic peptide precursor (Nppb), and myosin heavy chain-beta (Myh7) as potential biomarkers of LVH induced by a PPARalpha/gamma dual agonist in Sprague-Dawley rats. We used magnetic resonance imaging, echocardiography, and hemodynamics to identify structural and functional cardiovascular changes related to the biomarkers. Heart-to-brain weight ratios (HW:BrW) were correlated with NTproANP, NTproBNP, and cTnI concentrations in serum as well as fold change in expression of Nppa and Nppb. LVH was characterized by increased left ventricular wall thickness and inner diameter, increased cardiac output, decreased arterial blood pressure, and increased heart rate. In these studies, each end point contributed to the early detection of LVH, the ability to monitor its progression, and demonstrated the ability of NTproANP concentration in serum to predict LVH and hemodynamic changes.

Current applications of liquid chromatography/mass spectrometry in pharmaceutical discovery after a decade of innovation.

Current drug discovery involves a highly iterative process pertaining to three core disciplines: biology, chemistry, and drug disposition. For most pharmaceutical companies the path to a drug candidate comprises similar stages: target identification, biological screening, lead generation, lead optimization, and candidate selection. Over the past decade, the overall efficiency of drug discovery has been greatly improved by a single instrumental technique, liquid chromatography/mass spectrometry (LC/MS). Transformed by the commercial introduction of the atmospheric pressure ionization interface in the mid-1990s, LC/MS has expanded into almost every area of drug discovery. In many cases, drug discovery workflow has been changed owing to vastly improved efficiency. This review examines recent trends for these three core disciplines and presents seminal examples where LC/MS has altered the current approach to drug discovery.

Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers.

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.

Strategic use of immunoprecipitation and LC/MS/MS for trace-level protein quantification: myosin light chain 1, a biomarker of cardiac necrosis.

Myosin light chain 1 (Myl3) is a 23-kDa isoform of one of the subunits of myosin, a protein involved in muscle contraction. Myl3 is presently being studied as a biomarker of cardiac necrosis to predict drug-induced cardiotoxicity, and in the work presented here, an LC/MS/MS assay was developed and validated to measure Myl3 in rat serum. The key steps in this approach involved immunoaffinity purification of Myl3 from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified using a synthetic peptide standard and a corresponding stable isotope-labeled internal standard, and the results were stoichiometrically converted to Myl3 serum concentrations. Myl3 concentrations were corrected for peptide recovery following immunoprecipitation and digestion (85%) and showed excellent agreement with synthetic peptide standards. Both the synthetic peptide and His-Myl3 protein were used to evaluate assay accuracy (% RE) and precision (% CV), which were measured on each of 3 days. The synthetic peptide was evaluated over the range of 0.073-7.16 nM, while Myl3 protein QC samples prepared in rat serum were evaluated over the range of 0.13-6.62 nM. To prepare control matrix, endogenous Myl3 was immunodepleted from pooled rat serum. Peptide interday accuracy and precision did not exceed 7.6 and 11.1%, and Myl3 interday accuracy and precision did not exceed 12.9 and 13.2%, respectively. Data are presented from the application of this assay to establish a time course in which rats demonstrated a marked increase in Myl3 serum concentrations following administration of isoproterenol, a beta-adrenergic receptor agonist known to induce cardiac injury. This assay is an example of a larger effort in our laboratory to use LC/MS/MS in conjunction with immunoaffinity techniques to evaluate candidate biomarkers of target organ toxicity and to expedite the development of biomarker assays for drug development.

Quantification of serine enantiomers in rat brain microdialysate using Marfey's reagent and LC/MS/MS.

The ability to selectively measure serine enantiomer concentrations in rat brain microdialysate is essential during drug discovery to study the interaction of d-serine with the N-methyl-d-aspartate (NMDA) subtype of the glutamate receptor. NMDA receptor-stimulating agents, such as d-serine, have been shown to reduce the negative symptoms and cognitive dysfunction in individuals with schizophrenia when added to conventional or atypical antipsychotic drug regimens. In the work presented here, an LC/MS/MS assay was developed and validated to simultaneously measure d-serine and l-serine concentrations in rat brain microdialysate. Reverse phase chromatographic resolution of the enantiomers was obtained through derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (Marfey's reagent). The assay was validated to determine concentrations over the range of 10-7500 ng/mL using electrospray ionization and multiple reaction monitoring (MRM). Both intra- and inter-day precision and accuracy were less than 16.5% (RE) and 7% (CV) for both analytes, respectively, and assay throughput was increased significantly relative to existing methodologies.

Quantification of heart fatty acid binding protein as a biomarker for drug-induced cardiac and musculoskeletal necroses.

Heart fatty acid binding protein (Fabp3) is a cytosolic protein expressed primarily in heart, and to a lesser extent in skeletal muscle, brain, and kidney. During myocardial injury, the Fabp3 level in serum is elevated rapidly, making it an ideal early marker for myocardial infarction. In this study, an MS-based selected reaction monitoring method (LC-SRM) was developed for quantifying Fabp3 in rat serum. Fabp3 was enriched first through an immobilized antibody, and the protein was digested on beads directly. A marker peptide of Fabp3 was quantified using LC-SRM with a stable isotope-labeled peptide standard. For six quality control samples with Fabp3 ranging from 0.256 to 25 ng, the average recovery following the procedure was about 73%, and the precision (%CV) between replicates was less than 7%. The Fabp3 concentrations in rat serum peaked 1 h after isoproterenol treatment, and returned to baseline levels 24 h after the dose. Elevated Fabp3 levels were also detected in rats administered with a PPAR α/δ agonist, which has shown to cause skeletal muscle necrosis. Fabp3 can be used as a biomarker for both cardiac and skeletal necroses. The cross-validation of the LC-SRM method with an existing ELISA method is described.

Quantitative analysis of amyloid beta peptides in cerebrospinal fluid of Alzheimer's disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry.

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.

Characterization of a novel endocannabinoid, virodhamine, with antagonist activity at the CB1 receptor.

The first endocannabinoid, anandamide, was discovered in 1992. Since then, two other endocannabinoid agonists have been identified, 2-arachidonyl glycerol and, more recently, noladin ether. Here, we report the identification and pharmacological characterization of a novel endocannabinoid, virodhamine, with antagonist properties at the CB1 cannabinoid receptor. Virodhamine is arachidonic acid and ethanolamine joined by an ester linkage. Concentrations of virodhamine measured by liquid chromatography atmospheric pressure chemical ionization-tandem mass spectrometry in rat brain and human hippocampus were similar to anandamide. In peripheral tissues that express the CB2 cannabinoid receptor, virodhamine concentrations were 2- to 9-fold higher than anandamide. In contrast to previously described endocannabinoids, virodhamine was a partial agonist with in vivo antagonist activity at the CB1 receptor. However, at the CB2 receptor, virodhamine acted as a full agonist. Transport of [(14)C]anandamide by RBL-2H3 cells was inhibited by virodhamine. Virodhamine produced hypothermia in the mouse and acted as an antagonist in the presence of anandamide both in vivo and in vitro. As a potential endogenous antagonist at the CB1 receptor, virodhamine adds a new form of regulation to the endocannabinoid system.

Recent advances in use of LC/MS/MS for quantitative high-throughput bioanalytical support of drug discovery.

LC/MS/MS based bioanalysis using atmospheric pressure ionization (API)-style interfaces has now been applied for over a decade. This technology, which initially found application for clinical bioanalysis, is now firmly established as the primary bioanalytical tool for ADME studies related to drug discovery and lead optimization (LO). This review focuses on recent advances in LC/MS/MS based bioanalysis in support of drug discovery and LO. The initial part of the article reviews the principal components of LC/MS/MS bioanalysis: sample preparation, chromatography, ionization and mass analysis. In each section, factors affecting high throughput bioanalysis are addressed. Because of the importance of on-line column switching methods to discovery bioanalysis, the section on sample preparation is divided into off-line and on-line approaches. In addition, the discussion of chromatography is limited to reversed phase liquid chromatography with emphasis given to the trend towards high-flow gradient elution techniques. The latter part of the review focuses on considerations for experimental design. In this section, pooling methods such as cassette dosing are discussed along with more highly integrated strategies linking bioanalysis with protocol generation and sample collection. The article concludes by briefly reviewing factors, which affect bioanalytical precision and accuracy, such as ion suppression, analyte stability and metabolite interference.

Effects of flow rate on high-throughput quantitative analysis of protein-precipitated plasma using liquid chromatography/tandem mass spectrometry.

The effects of flow rate and column length on analyte response (peak area and height), total cycle time, column backpressure, and elution volume are presented. Rapid chromatographic separations and tandem mass spectrometric (MS/MS) detection are applied to the supernatant of protein-precipitated plasma standards containing four compounds from a drug discovery screen. The plasma samples were injected onto three C-18 columns (2 x 10,2.1 x 30 and 2.1 x 50 mm) at flow rates of 0.25, 0.50, 1.00 and 1.50 mL/min. The plasma samples were detected using a Sciex API 3000 tandem mass spectrometer operated in the Turbo Ionspray mode. A post-column split was used to maintain a flow rate of 0.25 mL/min into the mass spectrometer source to avoid differences in nebulization efficiency. The data show that diluted protein-precipitated plasma supernatants show average matrix effects (i.e. suppression) of 60.0% (2 x 10 mm), 89.3% (2 x 30 mm), and 76.7% (2 x 50 mm) of expected response at 10 ng/mL. Average matrix effects of 70.2% (2 x 10 mm), 88.9% (2 x 30 mm), and 81.2% (2 x 50 mm) of expected response at 1000 ng/mL plasma. The data also show if peak widths remain relatively constant, analytes are less sensitive as flow rates are increased. These data are consistent with the concentration-dependent relationship of ionspray in the range of flow rates studied. The data show that, while analyte response decreased proportionately to increases in flow rate, the analysis cycle times did not decrease proportionately.