Imidazo[1,2-a]quinazolines as novel, potent EGFR-TK inhibitors: Design, synthesis, bioactivity evaluation, and in silico studies.

Tyrosine protein kinases (TKs) have been proved to play substantial roles on many cellular processes and their overexpression tend to be found in various types of cancers. Therefore, over recent decades, numerous tyrosine protein kinase inhibitors particularly epidermal growth factor receptor (EGFR) inhibitors have been introduced to treat cancer. Present study describes a novel series of imidazo[1,2-a]quinazolines 18 as potential -inhibitors. These imidazoquinazolines (18a and 18o, in particular) had great anti-proliferative activities with IC50 values in the micromolar (µM) range against PC3, HepG2, HeLa, and MDA-MB-231 comparing with Erlotinib as reference marketed drug. Further evaluations on some derivatives revealed their potential to induce apoptotic cell death and cell growth arrest at G0 phase of the cell cycle. Afterwards, the kinase assay on the most potent compounds 18a and 18o demonstrated their inhibitory potencies and selectivity toward EGFR (with EGFR-IC50 values of 82.0 µM and 12.3 µM, respectively). Additionally, western blot analysis on these compounds 18a and 18o exhibited that they inhibited the phosphorylation of EGFR and its downstream molecule extracellular signal-regulated kinase (ERK1/2). However, the level of B-Actin phosphorylation was not changed. Finally, density functional theory calculations, docking study, and independent gradient model (IGM) were performed to illustrate the structure-activity relationship (SAR) and to assess the interactions between proteins and ligands. The results of molecular docking studies had great agreement with the obtained EGFR inhibitory results through in vitro evaluations.

A review of 18O labelling Studies to probe the mechanism of aromatase (CYP191A).

Our previous studies, using precursors for two classes of estrogens, estrone and estriol, have highlighted the following facets of aromatase. The overall reaction, converting androgens into estrogens, occurs in three steps, each requiring NADPH and O2. In Step 1, a 19-hydroxy intermediate is produced, which in Step 2, is converted into a 19-oxo derivative via a gem -diol intermediate with the stereospecific loss of HRe. In Step 3, a scission of the C-10-C-19 bond occurs releasing C-19 as formic acid (HCOOH) and incorporating an atom of oxygen from O2, The other oxygen atom of formic acid is derived from the hydroxyl group introduced in Step 1. These experiments were performed using the classical placental microsomal system. Our findings were confirmed and extended by (the late) Caspi's group. However, incorporation of oxygen in Step 3, has been challenged in a subsequent study using a soluble reconstituted system. The latter authors have implied the superiority of their system over the microsomal preparation. However, several assumptions under pinning their own work were derived from the use of placental microsomes. Furthermore, the authors have not considered that when a previous work is challenged it needs to be repeated under the conditions described in the original publication.

[PECULIARITIES OF THE CELLULAR COMPOSITION OF SPLENIC LYMPHOID TISSUE IN MICE AFTER LONG-TERM USE OF LIGHT WATER AND IRRADIATION].

The changes of the cellular composition of splenic lymphoid tissue were studied 7, 15 and 30 days after irradiation with a dose of 50 rad, in BALB/c mice which received either distilled water or light (deuterium-depleted) water for a long time prior to and after irradiation. The irregular pattern of changes of splenic cellular composition was observed during the experiment. It was found that at day 7 after irradiation, the splenic structural zones in mice demonstrated a sharp decrease in the number of blast forms and mitotic cells, reflecting a lower level of lymphocytopoiesis, as well as an increased cellular destruction in mice consuming light water. By day 30 of the experiment, different responses of lymphoid structures were observed in the organ. In the periarteriolar lymphoid sheaths, the processes of cellular composition regeneration were more pronounced than in the germinal centers of lymphoid nodules, indicating the enhancement of body cell-mediated immunity and immunomodulating properties of light water in mice at later dates of post-irradiation period.

Adequacy of a compartment model for CMRO2 quantitation using ¹5O-labeled oxygen and PET: a clearance measurement of ¹5O-radioactivity following intracarotid bolus injection of ¹5O-labeled oxyhemoglobin on Macaca fascicularis.

We aimed at evaluating the adequacy of the commonly employed compartmental model for quantitation of cerebral metabolic rate of oxygen (CMRO2) using (15)O-labeled oxygen ((15)O2) and positron emission tomography (PET). Sequential PET imaging was carried out on monkeys following slow bolus injection of blood samples containing (15)O2-oxyhemoglobin ((15)O2-Hb), (15)O-labeled water (H2(15)O), and C(15)O-labeled hemoglobin (C(15)O-Hb) into the internal carotid artery (ICA). Clearance slopes were assessed in the middle cerebral artery territory of the injected hemisphere. The time-activity curves were bi-exponential for both (15)O2-Hb and H2(15)O. Single exponential fitting to the early (5 to 40 seconds) and late (80 to 240 seconds) periods after the peak was performed and the (15)O2-Hb and H2(15)O results were compared. It was found that a significant difference between the clearance rates of the (15)O2-Hb and H2(15)O injections is unlikely, which supports the mathematical model that is widely used to describe the kinetics of (15)O2-Hb and H2(15)O in cerebral tissues and is the basis of recent approaches to simultaneously assess CMRO2 and cerebral blood flow in a single PET session. However, it should be noted that more data are necessary to unequivocally confirm the result.

Rapid quantitative CBF and CMRO(2) measurements from a single PET scan with sequential administration of dual (15)O-labeled tracers.

Positron emission tomography (PET) with (15)O tracers provides essential information in patients with cerebral vascular disorders, such as cerebral blood flow (CBF), oxygen extraction fraction (OEF), and metabolic rate of oxygen (CMRO(2)). However, most of techniques require an additional C(15)O scan for compensating cerebral blood volume (CBV). We aimed to establish a technique to calculate all functional images only from a single dynamic PET scan, without losing accuracy or statistical certainties. The technique was an extension of previous dual-tracer autoradiography (DARG) approach, but based on the basis function method (DBFM), thus estimating all functional parametric images from a single session of dynamic scan acquired during the sequential administration of H(2)(15)O and (15)O(2). Validity was tested on six monkeys by comparing global OEF by PET with those by arteriovenous blood sampling, and tested feasibility on young healthy subjects. The mean DBFM-derived global OEF was 0.57±0.06 in monkeys, in an agreement with that by the arteriovenous method (0.54±0.06). Image quality was similar and no significant differences were seen from DARG; 3.57%±6.44% and 3.84%±3.42% for CBF, and -2.79%±11.2% and -6.68%±10.5% for CMRO(2). A simulation study demonstrated similar error propagation between DBFM and DARG. The DBFM method enables accurate assessment of CBF and CMRO(2) without additional CBV scan within significantly shortened examination period, in clinical settings.

3D multi-isotope imaging mass spectrometry reveals penetration of 18O-trehalose in mouse sperm nucleus.

The prevalence of genetically engineered mice in medical research has led to ever increasing storage costs. Trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing. Using multi-isotope imaging mass spectrometry, we are able to image and measure trehalose in individual spermatozoa. We provide the first evidence that trehalose penetrates the nucleus of a mammalian cell, permitting tolerance to desiccation. These results have broad implications for long-term storage of mammalian cells.

Correlation of physical parameters during radiochemical synthesis of (18)F positron emission tomography radiopharmaceuticals.

Positron emission tomography is a highly specialized imaging technique using short-lived radiolabel substances to produce extremely high resolution images of the body's biological function. The (18)F(-) ion is produced via the (18)O(p,n)(18)F reaction using a silver target cell filled with 1.4 mL of enriched [(18)O] water. On a typical run, the target is irradiated for 45 minutes with 16.5 MeV protons (on target) and an average beam current of 5-45 mA. When the same reaction takes place with [(16)O] water [(13)N] Ammonia is produced as the primary product by the abstraction of hydrogen from water. This study investigated the physical parameters of medical cyclotron during the radiochemical process with induced radioactivity flux and mutual correlation of physical parameters for 16.5 MeV medical cyclotron at the INMAS Delhi, India. It is observed that by getting farther from the target, the relative number of low-energy neutrons increases while the overall flux of neutrons decreases. This is due to multiple scattering of high-energy neutrons in the walls and eventually absorption of low-energy neutrons. The other parameters are also linked with each other which are correlatable.

18O labeling of chlorophyll d in Acaryochloris marina reveals that chlorophyll a and molecular oxygen are precursors.

The cyanobacterium Acaryochloris marina was cultured in the presence of either H(2)(18)O or (18)O(2), and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H(2)(18)O, newly synthesized Chl a and d, both incorporated up to four isotopic (18)O atoms. Time course H(2)(18)O labeling experiments showed incorporation of isotopic (18)O atoms originating from H(2)(18)O into Chl a, with over 90% of Chl a (18)O-labeled at 48 h. The incorporation of isotopic (18)O atoms into Chl d upon incubation in H(2)(18)O was slower compared with Chl a with approximately 50% (18)O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of (18)O(2) gas, one isotopic (18)O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H(2)(18)O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic (18)O atoms derived from molecular oxygen ((18)O(2)) was observed in the extracted Chl d, and the percentage of double isotopic (18)O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3(1)-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.

Perfluorooctanoic acid for shotgun proteomics.

Here, we describe the novel use of a volatile surfactant, perfluorooctanoic acid (PFOA), for shotgun proteomics. PFOA was found to solubilize membrane proteins as effectively as sodium dodecyl sulfate (SDS). PFOA concentrations up to 0.5% (w/v) did not significantly inhibit trypsin activity. The unique features of PFOA allowed us to develop a single-tube shotgun proteomics method that used all volatile chemicals that could easily be removed by evaporation prior to mass spectrometry analysis. The experimental procedures involved: 1) extraction of proteins in 2% PFOA; 2) reduction of cystine residues with triethyl phosphine and their S-alkylation with iodoethanol; 3) trypsin digestion of proteins in 0.5% PFOA; 4) removal of PFOA by evaporation; and 5) LC-MS/MS analysis of the resulting peptides. The general applicability of the method was demonstrated with the membrane preparation of photoreceptor outer segments. We identified 75 proteins from 1 µg of the tryptic peptides in a single, 1-hour, LC-MS/MS run. About 67% of the proteins identified were classified as membrane proteins. We also demonstrate that a proteolytic (18)O labeling procedure can be incorporated after the PFOA removal step for quantitative proteomic experiments. The present method does not require sample clean-up devices such as solid-phase extractions and membrane filters, so no proteins/peptides are lost in any experimental steps. Thus, this single-tube shotgun proteomics method overcomes the major drawbacks of surfactant use in proteomic experiments.

Mapping protein abundance patterns in the brain using voxelation combined with liquid chromatography and mass spectrometry.

Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps.

Global quantitative proteomic profiling through 18O-labeling in combination with MS/MS spectra analysis.

Several stable-isotope-based peptide labeling methods have been developed to support large-scale relative quantitation, through mass spectrometry, of proteins present in two different biological samples. In one of these, trypsin-catalyzed 18O-based labeling, quantitation is typically performed at the full scan (MS) level by comparing the peak intensities of sister precursor ions corresponding to the labeled and unlabeled forms of an intact peptide as they co-elute during liquid chromatography (LC) separations. We show here that measuring relative abundance at the product ion (MS/MS) level after fragmentation provides excellent accuracy, sensitivity and signal-to-noise, while combining quantitation with global shotgun protein identification. To facilitate routine data analysis using this approach, we have developed two specialized software programs, ySelect and yRatios, which draw upon database search results for 18O-based data sets and combine fragmentation spectra peak lists to (1) accurately determine protein ratios between two samples while applying a correction for incomplete labeling and (2) tabulate these results in both intuitive summary reports and in formats amenable to systematic pathway level analysis. To validate our process, we subjected simple and complex test protein mixtures to single-step and multistep LC-MS/MS profiling experiments. Ratio distributions approached the expected means, allowing empirical derivation of confidence level cutoffs for determining statistically significant fold-changes in protein abundance. A set of stringent criteria for detecting spurious ratios based on consistency checking between unlabeled and labeled y-ion pairs was found to highlight putative false positive identifications. In summary, this toolkit facilitates comparative proteomic quantitation under conditions that are optimized for making reliable protein inferences.

A method to determine 18 O kinetic isotope effects in the hydrolysis of nucleotide triphosphates.

A method to determine 18 O kinetic isotope effects (KIEs) in the hydrolysis of GTP that is generally applicable to reactions involving other nucleotide triphosphates is described. Internal competition, where the substrate of the reaction is a mixture of 18 O-labeled and unlabeled nucleotides, is employed, and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18 O at sites of mechanistic interest also contains 13C at all carbon positions, whereas the 16 O-labeled nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by the use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink interface. Carbon isotope ratios can be determined with accuracy and precision greater than 0.04% and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1(333)-catalyzed hydrolysis of [beta18 O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (<0.1%). A single KIE measurement can be conducted in 25 min with less than 5 microg nucleotide reaction product.

Heme-dependent rubber oxygenase RoxA of Xanthomonas sp. cleaves the carbon backbone of poly(cis-1,4-Isoprene) by a dioxygenase mechanism.

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of (16)O(2), (18)O(2), H(2)(16)O, and H(2)(18)O. 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of (18)O-compounds. Incorporation of one (18)O atom in ODTD was found if the cleavage reaction was performed in the presence of (18)O(2) and H(2)(16)O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H(2)(18)O in the presence of (16)O(2) indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of (18)O(2), H(2)(16)O, and alcohol dehydrogenase/NADH, incorporation of two atoms of (18)O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.

An isotope coding strategy for proteomics involving both amine and carboxyl group labeling.

This paper describes a heavy isotope coding strategy for the analysis of all types of tryptic peptides, including those that are N-terminally blocked and from the C-terminus of proteins. The method exploits differential derivatization of amine and carboxyl groups generated during proteolysis as a means of coding. Carboxyl groups produced during proteolysis incorporate 18O from H218O. Peptides from the C-terminus of proteins were not labeled with 18O unless they contained a basic C-terminal amino acid. Primary amines from control and experimental samples were differentially acylated after proteolysis with either 1H3- or 2H3-N-acetoxysuccinamide. When these two types of labeling were combined, unique coding patterns were achieved for peptides arising from the C-termini and blocked N-termini of proteins. This method was used to (1) distinguish C-terminal peptides in model proteins, (2) recognize N-terminal peptides from proteins in which the amino terminus is acylated, and (3) identify primary structure variations between proteins from different sources.