Mass spectrometry and n-in-one analytics in early drug discovery: Combinatorial chemistry libraries, lipophilicity and absorption screening

This thesis describes current and past n-in-one methods and presents three early experimental studies using mass spectrometry and the triple quadrupole instrument on the application of n-in-one in drug discovery. N-in-one strategy pools and mix samples in drug discovery prior to measurement or analysis. This allows the most promising compounds to be rapidly identified and then analysed. Nowadays properties of drugs are characterised earlier and in parallel with pharmacological efficacy. Studies presented here use in vitro methods as caco-2 cells and immobilized artificial membrane chromatography for drug absorption and lipophilicity measurements. The high sensitivity and selectivity of liquid chromatography mass spectrometry are especially important for new analytical methods using n-in-one. In the first study, the fragmentation patterns of ten nitrophenoxy benzoate compounds, serial homology, were characterised and the presence of the compounds was determined in a combinatorial library. The influence of one or two nitro substituents and the alkyl chain length of methyl to pentyl on collision-induced fragmentation was studied, and interesting structurefragmentation relationships were detected. Two nitro group compounds increased fragmentation compared to one nitro group, whereas less fragmentation was noted in molecules with a longer alkyl chain. The most abundant product ions were nitrophenoxy ions, which were also tested in the precursor ion screening of the combinatorial library. In the second study, the immobilized artificial membrane chromatographic method was transferred from ultraviolet detection to mass spectrometric analysis and a new method was developed. Mass spectra were scanned and the chromatographic retention of compounds was analysed using extract ion chromatograms. When changing detectors and buffers and including n-in-one in the method, the results showed good correlation. Finally, the results demonstrated that mass spectrometric detection with gradient elution can provide a rapid and convenient n-in-one method for ranking the lipophilic properties of several structurally diverse compounds simultaneously. In the final study, a new method was developed for caco-2 samples. Compounds were separated by liquid chromatography and quantified by selected reaction monitoring using mass spectrometry. This method was used for caco-2 samples, where absorption of ten chemically and physiologically different compounds was screened using both single and nin- one approaches. These three studies used mass spectrometry for compound identification, method transfer and quantitation in the area of mixture analysis. Different mass spectrometric scanning modes for the triple quadrupole instrument were used in each method. Early drug discovery with n-in-one is area where mass spectrometric analysis, its possibilities and proper use, is especially important.

TOTEM early measurements

The status of the TOTEM experiment is described as well as the prospects for the measurements in the early LHC runs. The primary goal of TOTEM is the measurement of the total p-p cross section, using a method independent of the luminosity. A final accuracy of 1% is ex- pected with dedicated β∗ = 1540 m runs, while at the beginning a 5% resolution is achievable with a β∗ = 90 m optics. Accordingly to the running scenarios TOTEM will be able to measure the elastic scattering in a wide range of t and to study the cross-sections and the topologies of diffractive events. In a later stage, physics studies will be extended to low-x and forward physics collaborating with CMS as a whole experimental apparatus.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

Dynamic changes in mitogen-activated protein kinase (MAPK) activities in the corpus luteum of the bonnet monkey (Macaca radiata) during development, induced luteolysis, and simulated early pregnancy: A role for p38 MAPK in the regulation of luteal function

Changes in MAPK activities were examined in the corpus luteum (CL) during luteolysis and pregnancy, employing GnRH antagonist (Cetrorelix)-induced luteolysis, stages of CL, and hCG treatment to mimic early pregnancy as model systems in the bonnet monkey. We hypothesized that MAPKs could serve to phosphorylate critical phosphoproteins to regulate luteal function. Analysis of several indices for structural (caspase-3 activity and DNA fragmentation) and functional (progesterone and steroidogenic acute regulatory protein expression) changes in the CL revealed that the decreased luteal function observed during Cetrorelix treatment and late luteal phase was associated with increased caspase-3 activity and DNA fragmentation. As expected, human chorionic gonadotropin treatment dramatically increased luteal function, but the indices for structural changes were only partially attenuated. All three MAPKs appeared to be constitutively active in the mid-luteal-phase CL, and activities of ERK-1/2 and p38-MAPK (p38), but not Jun N-terminal kinase (JNK)-1/2, decreased significantly (P < 0.05) within 12 - 24 h after Cetrorelix treatment. During the late luteal phase, in contrast to decreased ERK-1/2 and p38 activities, JNK-1/2 activities increased significantly (P < 0.05). Although human chorionic gonadotropin treatment increased ERK-1/2 and p38 activities, it decreased JNK-1/2 activities. The activation status of p38 was correlated with the phosphorylation status of an upstream activator, MAPK kinase-3/6 and the expression of MAPK activated protein kinase-3, a downstream target. Intraluteal administration of p38 kinase inhibitor (SB203580), but not MAPK kinase-1/2 inhibitor (PD98059), decreased the luteal function. Together, these data suggest an important role for p38 in the regulation of CL function in primates.

Hot gaseous coronae of early-type galaxies and their radio luminosity function

Recent X-ray observations have revealed that early-type galaxies (which usually produce extended double radio sources) generally have hot gaseous haloes extending up to approx102kpc1,2. Moreover, much of the cosmic X-ray background radiation is probably due to a hotter, but extremely tenuous, intergalactic medium (IGM)3. We have presented4–7 an analytical model for the propagation of relativistic beams from galactic nuclei, in which the beams' crossing of the pressure-matched interface between the IGM and the gaseous halo, plays an important role. The hotspots at the ends of the beams fade quickly when their advance becomes subsonic with respect to the IGM. This model has successfully predicted (for typical double radio sources) the observed8 current mean linear-size (approx2Dsime350 kpc)4,5, the observed8–11 decrease in linear-size with cosmological redshift4–6 and the slope of the linear-size versus radio luminosity10,12–14 relation6. We have also been able to predict the redshift-dependence of observed numbers and radio luminosities of giant radio galaxies7,15. Here, we extend this model to include the propagation of somewhat weaker beams. We show that the observed flattening of the local radio luminosity function (LRLF)16–20 for radio luminosity Papproximately 1024 W Hz-1 at 1 GHz can be explained without invoking ad hoc a corresponding break in the beam power function Phi(Lb), because the heads of the beams with Lb < 1025 W Hz-1 are decelerated to sonic velocity within the halo itself, which leads to a rapid decay of radio luminosity and a reduced contribution of these intrinsically weaker sources to the observed LRLF.

Passive immunization with an antibody to the beta-subunit of ovine luteinizing hormone as a method of early abortion--a feasibility study in monkeys (Macaca radiata)

Antiserum to the beta-subunit of ovine luteinizing hormone (oLH-beta) raised in monkeys (Macaca radiata) has been tested by a variety of criteria both in vivo and in vitro to establish its ability to neutralize oLH, hLH, and human chorionic gonadotropin (hCG). Passive administration of this antiserum caused inhibition of ovulation and termination of pregnancy in recipient monkeys as indicated by premature vaginal bleeding and a significant reduction in serum progesterone and estrogen levels. The results suggest that antiserum raised in monkeys against oLH-beta can neutralize monkey LH as well as monkey CG.

Early biochemical events during adventitious root initiation in the hypocotyl of Phaseolus vulgaris

Indole butyric acid (IBA) initiates roots in the hypocotyl tissue of Phaseolus vulgaris (French bean). The response is dependent on the concentration of IBA and the duration of exposure to the hormone. IBA enhances the rate of total protein synthesis in ca 30 min after exposure of the hypocotyl segments to the hormone. There is no detectable change in total or poly(A)-containing RNA synthesis in this period although significant increases are seen 2 hr after hormone pre-treatment. The early IBA-mediated increase in protein synthesis (30 min) is not sensitive to Actinomycin D but the antibiotic blocks the increase manifested 2 hr after hormone pre-treatment. Inhibition of early protein synthesis by cycloheximide depresses and delays root initiation. Cytosol prepared from IBA-treated hypocotyl tissue stimulates protein synthesis in vitro to a greater extent than that of the control.

A monoclonal antibody recognizing the C-terminal region of chicken egg white riboflavin carrier protein terminates early pregnancy in mice

In order to identify the functionally relevant epitopes on chicken riboflavin carrier protein, we have raised monoclonal antibodies to the vitamin carrier. One of these, 6B2C12, was found to interact specifically with a synthetic oligopeptide corresponding to the C-terminal 17 amino acid residues of the chicken egg white riboflavin carrier protein, which is missing in part in the egg yolk riboflavin carrier protein. This epitope is conserved through evolution in mammals including humans. Administration of the ascites fluid of 6B2C12 to pregnant mice intraperitoneally, resulted in the termination of pregnancy indicating that this epitope is involved in or closely associated with the transplacental transport of the vitamin from the maternal circulation to the growing fetus.

Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.