Fit-for-purpose biomarker LC-MS/MS qualification for the quantitation of very long chain fatty acids in human cerebrospinal fluid.

Aim: Very long chain fatty acids (VLCFAs) have been identified as biomarkers for several peroxisomal disorders necessitating the need for reliable biomarker assays in particular C20, C22, C24, C26 in cerebrospinal fluid (CSF). Until now no absolute quantitation assay for total VLCFAs in CSF has been successfully developed and qualified for clinical use. Methodology: A quantitative LC-MS/MS assay for total VLCFA in human CSF was developed. Derivatization tag and coupling chemistry were optimized for sensitivity. CSF contamination by blood, non-specific binding of VLCFA to surfaces and exogenous VLCFA contamination was minimized. Discussion/conclusion: This fit for purpose biomarker assay was used to measure baseline healthy human VLCFA levels across multiple subjects to establish an understanding of concentration ranges and feasibility.

LC-MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human plasma.

DNA damaging chemotherapy and radiation are widely used standard-of-care modalities for the treatment of cancer. Nevertheless, the outcome for many patients remains poor and this may be attributed, at least in part, to highly effective DNA repair mechanisms. Ataxia-telangiectasia mutated and Rad3-related (ATR) is a key regulator of the DNA-damage response (DDR) that orchestrates the repair of damaged replication forks. ATR is a serine/threonine protein kinase and ATR kinase inhibitors potentiate chemotherapy and radiation. The ATR kinase inhibitor VX-970 (NSC 780162) is in clinical development in combination with primary cytotoxic agents and as a monotherapy for tumors harboring specific mutations. We have developed and validated an LC-MS/MS assay for the sensitive, accurate and precise quantitation of VX-970 in human plasma. A dilute-and-shoot method was used to precipitate proteins followed by chromatographic separation with a Phenomenex Polar-RP 80Å (4µm, 50×2mm) column and a gradient acetonitrile-water mobile phase containing 0.1% formic acid from a 50µL sample volume. Detection was achieved using an API 4000 mass spectrometer using electrospray positive ionization mode. The assay was linear from 3 to 5,000ng/mL, proved to be accurate (94.6-104.2%) and precise (<8.4% CV), and fulfilled criteria from the FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be a crucial tool in defining the clinical pharmacokinetics and pharmacology of VX-970 as it progresses through clinical development.

Recommendations on the Development of a Bioanalytical Assay for 4ß-Hydroxycholesterol, an Emerging Endogenous Biomarker of CYP3A Activity.

The availability of reliable assays for measuring 4ß-hydroxycholesterol (4ß-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4ß-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC). Plasma concentrations of 4ß-HC are low in humans (10-60 ng/ml), lower than many other cholesterol metabolites and far less than cholesterol itself. Stability of 4ß-HC has been established for at least 12 months at -20°C in plasma samples obtained with a typical clinical workflow. Oxidation of plasma cholesterol during storage produces both 4ß-HC and 4α-HC, and 4α-HC may be used as assessment of sample quality. As 4ß-HC concentrations over time in untreated individuals have low intra-individual variability, assay precision and reproducibility are the key assay attributes in assessing CYP3A4 induction, and potentially inhibition. Assessment of CYP3A4/5 activity with 4ß-HC relies on the differences between pre- and post-dose concentrations, in which each subject acts as their own control. To reduce analytical variability, samples from a single subject should be analyzed together to facilitate interpretation of study results. As an endogenous biomarker, 4ß-HC offers the opportunity for less invasive assessment of CYP3A induction potential of new drugs during clinical development.

Discovery of oxazolo[4,5-b]pyridines and related heterocyclic analogs as novel SIRT1 activators.

SIRT1 is an NAD(+)-dependent protein deacetylase that appears to produce beneficial effects on metabolic parameters such as glucose and insulin homeostasis. Activation of SIRT1 by resveratrol (1) has been shown to modulate insulin resistance, increase mitochondrial content and prolong survival in lower organisms and in mice on a high fat diet. Herein, we describe the identification and SAR of a series of oxazolo[4,5-b]pyridines as novel small molecule activators of SIRT1 which are structurally unrelated to and more potent than resveratrol.

Small molecule activators of SIRT1 replicate signaling pathways triggered by calorie restriction in vivo.

BACKGROUND: Calorie restriction (CR) produces a number of health benefits and ameliorates diseases of aging such as type 2 diabetes. The components of the pathways downstream of CR may provide intervention points for developing therapeutics for treating diseases of aging. The NAD+-dependent protein deacetylase SIRT1 has been implicated as one of the key downstream regulators of CR in yeast, rodents, and humans. Small molecule activators of SIRT1 have been identified that exhibit efficacy in animal models of diseases typically associated with aging including type 2 diabetes. To identify molecular processes induced in the liver of mice treated with two structurally distinct SIRT1 activators, SIRT501 (formulated resveratrol) and SRT1720, for three days, we utilized a systems biology approach and applied Causal Network Modeling (CNM) on gene expression data to elucidate downstream effects of SIRT1 activation. RESULTS: Here we demonstrate that SIRT1 activators recapitulate many of the molecular events downstream of CR in vivo, such as enhancing mitochondrial biogenesis, improving metabolic signaling pathways, and blunting pro-inflammatory pathways in mice fed a high fat, high calorie diet. CONCLUSION: CNM of gene expression data from mice treated with SRT501 or SRT1720 in combination with supporting in vitro and in vivo data demonstrates that SRT501 and SRT1720 produce a signaling profile that mirrors CR, improves glucose and insulin homeostasis, and acts via SIRT1 activation in vivo. Taken together these results are encouraging regarding the use of small molecule activators of SIRT1 for therapeutic intervention into type 2 diabetes, a strategy which is currently being investigated in multiple clinical trials.

Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes.

Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme-peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

Fluorescent cardiac imaging: a novel intraoperative method for quantitative assessment of myocardial perfusion during graded coronary artery stenosis.

BACKGROUND: The purpose of the present study was to examine whether the effect of coronary stenoses of variable severity on myocardial perfusion can be quantitatively assessed in vivo by analysis of fluorescent cardiac imaging (FCI) compared with the gold standard, the fluorescent microsphere method. FCI is a novel technology to visualize coronary vessels and myocardial perfusion intraoperatively using the indocyanine green dye with an infrared-sensitive imaging device. METHODS AND RESULTS: Graded stenoses and total vessel occlusion of the left anterior descending coronary artery were created in 11 open-chest pigs. Stenoses were graded to reduce resting left anterior descending coronary artery flow by 25%, 50%, 75%, and 100% of baseline flow measured by transit-time flowmeter. FCI images were analyzed with a digital image processing system. The impairment of myocardial perfusion was quantified by background-subtracted peak fluorescence intensity and slope of fluorescence intensity obtained with FCI and compared with myocardial blood flow assessed by fluorescent microsphere. All stenoses resulted in an impairment of myocardial perfusion visualized by FCI. Occlusion of the left anterior descending coronary artery resulted in a total perfusion defect (no fluorescence intensity) of the corresponding anterior myocardial wall. During graded stenosis and total vessel occlusion, normalized background-subtracted peak fluorescence intensity and slope of fluorescence intensity decreased significantly (P<0.0001). Both background-subtracted peak fluorescence intensity (r=0.92, P<0.0001) and slope of fluorescence intensity (r=0.93, P<0.0001) analyzed by FCI demonstrated good linear correlation with fluorescent microsphere-derived myocardial blood flow. CONCLUSIONS: The impairment of myocardial perfusion in response to increased coronary stenosis severity and total vessel occlusion can be quantitatively assessed by FCI and correlates well with results obtained by fluorescent microsphere.

Structural determinants for inhibitor specificity and selectivity in PDE2A using the wheat germ in vitro translation system.

Phosphodiesterases (PDEs) modulate signaling by cyclic nucleotides in diverse processes such as cardiac contractility, platelet aggregation, lipolysis, glycogenolysis, and smooth muscle contraction. Cyclic guanosine monophosphate (cGMP) stimulated human phosphodiesterase 2 (PDE2) is expressed mainly in brain and heart tissues. PDE2A is involved in the regulation of blood pressure and fluid homeostasis by the atrial natriuretic peptide (ANP), making PDE2-type enzymes important targets for drug discovery. The design of more potent and selective inhibitors of PDE2A for the treatment of heart disease would be greatly aided by the identification of active site residues in PDE2A that determine substrate and inhibitor selectivity. The identification of active site residues through traditional mutational studies involves the time-consuming and tedious purification of a large number of mutant proteins from overexpressing cells. Here we report an alternative approach to rapidly produce active site mutants of human PDE2A and identify their enzymatic properties using a wheat germ in vitro translation (IVT, also known as cell-free translation) system. We also present the crystal structure of the catalytic domain of human PDE2A determined at 1.7 A resolution, which provided a framework for the rational design of active site mutants. Using a rapid IVT approach for expression of human PDE2A mutants, we identified the roles of active site residues Asp811, Gln812, Ile826, and Tyr827 in inhibitor and substrate selectivity for PDE2A.

Examining reactivity and specificity of cytochrome c peroxidase by using combinatorial mutagenesis.

Combinatorial mutagenesis was used to investigate the role of three key residues in cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae, Arg48, Trp51, and Trp191, in control of the reactivity and selectivity of the heme-containing enzyme. Libraries were prepared by randomization of these residues and were subsequently screened for activity against the phenolic substrate guaiacol. Screening conditions were employed that favor either mutants with high activity or those with both high activity and stability of the reactive enzyme intermediates. The results obtained suggest a dual role for Arg48 of CCP: in addition to stabilizing reactive enzyme intermediates, the distal arginine residue plays a major role in restriction of access to the ferryl oxygen atom by small molecules and thereby controls reactivity and substrate specificity of the peroxidase. At position 51 of CCP, either a phenylalanine or a tryptophan residue is required both for catalytic and structural reasons. In contrast, either polar or positively charged residues are accepted at the position of Trp191, which is located inside the core of the protein. The variability at position 191 can be interpreted as a reflection of the mechanism of cytochrome c peroxidase, which transforms the nonpolar Trp191 into a transient cation radical.

Near-infrared fluorescence coronary angiography: a new noninvasive technology for intraoperative graft patency control.

BACKGROUND: Intraoperative graft patency verification is of major clinical importance for quality control after coronary artery bypass grafting (CABG), especially if surgery is performed on the beating heart. This is one of the first reports of fluorescence coronary angiography (FCA) using the dye indocyanine green (ICG), a noninvasive technology for direct visualization of coronary arteries, bypass grafts, and myocardial perfusion. METHODS: Twenty-three domestic pigs (weight, 45-72 kg) underwent FCA of the left anterior descending coronary artery (LAD). In the first group (n = 6 pigs), FCA was used to visualize the native coronary vessels and myocardial perfusion. In the second group (n = 8 pigs), 14 stenoses of various degrees and 4 total vessel occlusions were created by snares on different segments of the LAD, and FCA was used to visualize the effects of these obstructions. In the third group (n = 9 pigs), a coronary bypass procedure on the beating heart was performed by a left internal mammary artery or a human saphenous vein graft to the LAD, and FCA was used to visualize graft patency. Three pigs were removed from the study because of ventricular fibrillation. ICG was intravenously applied, and the heart was illuminated with near-infrared light emitted by laser diodes. The fluorescence emission was detected by an adapted charge-coupled device camera system. The images were displayed in real time on a high-resolution monitor. Subsequently, images obtained with FCA were compared to those obtained with coronary angiography (n = 10 pigs). RESULTS: In all cases, high-quality FCA images of coronary arteries and myocardial perfusion were obtained. All stenoses resulted in an impairment of the myocardial perfusion visualized by FCA. Occlusion of the LAD or the diagonal branch resulted in a total perfusion defect of the corresponding anterior myocardial wall with immediate reperfusion after releasing the snare. In 5 cases a patent bypass graft with an apparent homogenous perfusion of the corresponding myocardium was detectable. In one procedure, FCA images indicated total occlusion of the bypass graft and a total perfusion deficit in the distal LAD region. Correlation between FCA and coronary angiography in detection of stenoses and graft patency was excellent. CONCLUSION: With the fluorescence technique using ICG, visualization of blood flow in coronary vessels and bypass grafts, as well as of myocardial perfusion, is feasible. FCA is a highly sensitive and reproducible method and an excellent technique for intraoperative quality control in CABG.