Quantifying Target Occupancy of Small Molecules Within Living Cells.

The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.

Effects of acute pinitol supplementation on plasma pinitol concentration, whole body glucose tolerance, and activation of the skeletal muscle insulin receptor in older humans.

Limited research with rodents and humans suggests that oral ingestion of pinitol (3- O-methyl- D- CHIRO-inositol) might positively influence glucose tolerance. This double-blinded, placebo-controlled, and cross-over study assessed the effects of acute pinitol supplementation on plasma pinitol concentration, glucose tolerance, insulin sensitivity, and activation of the skeletal muscle insulin receptor. Fifteen older, nondiabetic subjects (62+/-1 years, mean+/-SEM) completed four, 1-day trials. Subjects consumed a non-nutritive beverage with nothing (placebo) or 1,000 mg pinitol. Sixty minutes later, the subjects consumed beverages that were either energy- and carbohydrate-free (Sham) or contained 75 g glucose (OGTT). Blood samples were collected frequently over the 240-min testing period. For the OGTT trials only, vastus lateralis samples were obtained before the placebo and pinitol supplementation and 60 min after consuming the 75 g glucose beverage. Plasma pinitol concentration increased and was maintained for 240 min. Pinitol did not influence the fasting state and 180-min area under the curves for plasma glucose and insulin during the Sham and OGTT trials or hepatic (placebo 0.83+/-0.08; pinitol 0.80+/-0.08) and whole-body (placebo 6.10+/-0.54; pinitol 6.22+/-0.52) insulin sensitivities. Activation of the muscle insulin receptor was increased by 140% with glucose ingestion (Pre 0.62+/-0.12; Post 1.49+/-0.35), but pinitol did not influence this response. These results show that the pinitol supplement was quickly absorbed, but did not acutely influence indices of whole-body glucose tolerance and insulin sensitivity, or the activation of the skeletal muscle insulin receptor in older, nondiabetic humans.

Anthocyanin quantification and radical scavenging capacity of Concord, Norton, and Marechal Foch grapes and wines.

The anthocyanin content and the radical scavenging capacity of three non-Vitis vinifera grapes (Marechal Foch, Norton, and Concord varieties) were determined. Analyses of anthocyanins in the skin (S) and wine (W) of these grape varieties were performed by spectrophotometry, HPLC with electrochemical detection, and matrix-assisted laser desorption ionization (MALDI). The total anthocyanin contents of S samples were 258 +/- 37 mg/100 g of wet weight for Foch, 888 +/- 78 mg/100 g for Norton, and 326 +/- 5.9 mg/100 g for Concord grapes. The malvidin 3,5-diglucoside content quantified by HPLC indicated that Norton S had the highest amount of the compound (327 +/- 110 mg/100 g). The MALDI mass spectrometric analysis indicated an abundance of malvidin glucosides in W of Foch grapes and in S and W of Norton grapes and of cyanidin aglycon in S and W of Concord grapes. S samples were subjected to a radical scavenging capacity test using the 2,2-diphenyl-1-picrylhydrazyl radical and compared to Trolox. The radical scavenging capacity for Foch S was 0.78 mM Trolox equiv, that of Concord S, 0.80 Trolox equiv, and that of Norton S was highest at 0.95 Trolox equiv. The higher concentrations of malvidin 3,5-diglucoside in S of grape varieties were associated with greater radical scavenging capacity.

Somaclonal variation: a morphogenetic and biochemical analysis of Mandevilla velutina cultured cells.

Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.

Somaclonal variation: a morphogenetic and biochemical analysis of Mandevilla velutina cultured cells

Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60 percent shorter and a metabolic rate 33.6 percent higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content

The effect of water on the ion trap analysis of trimethylsilyl derivatives of long-chain fatty acids and alcohols.

Gas chromatography/mass spectrometry (GC/MS), with an ion trap mass analyzer, was used to examine the very-long-chain cuticular acid and certain non-acid wax constituents on the leaf sheath surface of Sorghum bicolor before and during 36 hours of light exposure. The mass spectra of the trimethylsilylated acids and alcohols did not match any of those published in searchable mass spectral libraries. The observed differences can be related to the interaction between water and the trimethylsilylated acids and alcohols. Understanding the observed mass spectra of the very-long-chain plant waxes is critical for studies that employ GC/MS with the ion trap mass analyzer to elucidate cuticular wax compositions on plants.

Organization, management and operation of contemporary academic mass spectrometry service facilities.

The rapid evolution of mass spectrometry in the past 15 years has moved mass spectrometry facilities from the traditional model in which instruments were located in and used for a single department's samples to a distributed model servicing entire universities. In this paper we describe two such shared instrument facilities that have evolved from a base in a single department to facilities that service a broad clientele. The Purdue University Campus-wide Mass Spectrometry Center (CWMSC) is a decentralized facility with multiple sites on campus. The CWMSC is a limited-access facility in which samples are run by service facility personnel in close cooperation with investigators. The Vanderbilt University Mass Spectrometry Research Center (VU-MSRC) is a centralized facility in the medical school that provides services to the university at large. The VU-MSRC is an open-access facility in which users are expected to prepare and analyze their own samples under the guidance of a trained operator. Perhaps the most significant benefit achieved by these models has been the minimization of academic barriers and the resultant intellectual cross-fertilization that has greatly enriched research at institutions where this approach has been adopted. The advantages and limitations of both models are discussed in terms of the traditional academic paradigm of service, research and education.

Three new adjacent bis-tetrahydrofuran acetogenins with four hydroxyl groups from Asimina triloba.

Three new adjacent bis-tetrahydrofuran ring Annonaceous acetogenins with four hydroxy groups, bullatetrocin (1), 10-hydroxyasimicin (2), and 10-hydroxytrilobacin (3), were isolated by activity-directed fractionation from the stem bark of Asimina triloba. Their structures were established on the basis of chemical and spectral evidence. The absolute stereochemistry at the C-10 hydroxy position was determined by converting 2 and 3 to their ketolactone isomers, 2,4-cis/trans 10-hydroxyasimicinones and 2,4-cis/trans 10-hydroxytrilobacinones, respectively. The bioactivities of the new compounds against brine shrimp larvae and six human solid-tumor cell lines are reported, and structure-activity relationships between trihydroxylated and tetrahydroxylated acetogenins are discussed. In addition to 1-3, gigantetrocin A, 2,4-cis/trans-gigantetrocin A-ones, annonacin, and annonacin A were also isolated for the first time from this species.

Applying Mosher's method to acetogenins bearing vicinal diols. The absolute configurations of muricatetrocin C and rollidecins A and B, new bioactive acetogenins from Rollinia mucosa.

Muricatetrocin C (1), rollidecin A (2), and rollidecin B (3), three new bioactive annonaceous acetogenins bearing vicinal diols, were isolated from the leaves of Rollinia mucosa (Annonaceae) using activity-directed fractionation. The total structural elucidations of 1-3, including the absolute stereochemistries of the vicinal diols, were achieved by analyzing their per-Mosher ester derivatives. All three compounds showed potent and selective inhibitory effects against several human cancer cell lines.

Novel features of drosophila period Transcription revealed by real-time luciferase reporting.

The rapid turnover of luciferase and the sensitive, non-invasive nature of its assay make this reporter gene uniquely situated for temporal gene expression studies. To determine the in vivo regulatory pattern of the Drosophila clock gene period (per), we generated transgenic strains carrying a luciferase cDNA fused to the promoter region of the per gene. This has allowed us to monitor circadian rhythms of bioluminescence from pacemaker cells within the head for several days in individual living adults. These high time-resolution experiments permitted neuronal per transcription and opens the door to vastly simplified experiments in general chronobiology and studies of temporally regulated transcription in a wide range of experimental systems.

Five novel mono-tetrahydrofuran ring acetogenins from the seeds of Annona muricata.

Bioactivity-directed fractionation of the seeds of Annona muricata L. (Annonaceae) resulted in the isolation of five new compounds: cis-annonacin (1), cis-annonacin-10-one (2), cis-goniothalamicin (3), arianacin (4), and javoricin (5). Three of these (1-3) are among the first cis mono-tetrahydrofuran ring acetogenins to be reported. NMR analyses of published model synthetic compounds, prepared cyclized formal acetals, and prepared Mosher ester derivatives permitted the determinations of absolute stereochemistries. Bioassays of the pure compounds, in the brine shrimp test, for the inhibition of crown gall tumors, and in a panel of human solid tumor cell lines for cytotoxicity, evaluated relative potencies. Compound 1 was selectively cytotoxic to colon adenocarcinoma cells (HT-29) in which it was 10,000 times the potency of adriamycin.

Transduction in microbial biosensors using multiplexed bioluminescence.

The enormous diversity of genetic responses in living microbes to their environment is an attractive resource on which to base biosensor designs. In particular, there is much interest in microbial sensors for environmental monitoring where toxicity can be ascertained directly by its action on cellular physiology. However, due to the complexities of living systems, the utility of genetic-based microbial sensors has been limited by the ability to accurately transduce the activities of specific genetic sensing systems into readily measurable signals. We present here a strategy for employing an additional signal in the sensor design, to provide an internal baseline control upon which to reliably interpret sensor responses. The strategy relies on using beetle luciferases capable of emitting optical signals of different wavelengths; the optical signals are a sensitive real-time indicator of genetic activity within the cells. The different wavelengths allow both a target and control signal to be incorporated into each cell, providing a means of differentiating between specific effects of a genetic sensing system and other non-specific interfering influences.

New bioactive adjacent bis-THF annonaceous acetogenins from Annona bullata.

Five new adjacent bis-THF annonaceous acetogenins, 32-hydroxybullatacin, 31-hydroxybullatacin, 30-hydroxybullatacin, and (2,4-cis and trans)-28-hydroxybullatacinones, were isolated from the ethanolic extract of the bark of Annona bullata Rich. (Annonaceae). The absolute configurations of the above five compounds, as well as those of (2,4-cis and trans)-32-, 31-, and 30-hydroxybullatacinones and (2,4-cis and trans)-bulladecinones, previously isolated from the same extract, were defined by the application of the advanced Mosher ester [methoxy(trifluoromethyl)phenyl acetate or MTPA] methodology. The determination of the absolute configuration of C-20 of (2,4-cis and trans)-bulladecinones to be S supports our hypothesis that the cyclization of the THF rings of (2,4-cis and trans)-bulladecinones starts from C-12 (the right side). The first five compounds listed above showed potent bioactivities in the brine shrimp lethality test (BST) and among six human solid tumour cell lines.

Meliavolkenin, a new bioactive triterpenoid from Melia volkensii (Meliaceae).

Meliavolkenin, a new triterpene with an apotirucallane skeleton, has been isolated from the root bark of Melia volkensii (Meliaceae) by bioactivity-directed fractionation using the brine shrimp lethality test. The structure has been elucidated using spectral and chemical data. The relative stereochemistries were determined by reduction and acetonide derivations, and the ring conformations were analyzed using the results of NOESY experiments. Meliavolkenin was bioactive in the brine shrimp lethality test and gave moderate cytotoxicities against three human solid tumor lines.

Near-isogenic lines of maize differing for glycinebetaine.

A series of near-isogenic glycinebetaine-containing and -deficient F8 pairs of Zea mays L. (maize) lines were developed. The pairs of lines differ for alternative alleles of a single locus; the wild-type allele conferring glycinebetaine accumulation is designated Bet1 and the mutant (recessive) allele is designated bet1. The near-isogenic lines were used to investigate whether glycinebetaine deficiency affects the pool size of the glycinebetaine precursor, choline, using a new method for glycinebetaine and choline determination: stable isotope dilution plasma desorption mass spectrometry. Glycinebetaine deficiency in maize was associated with a significant expansion of the free choline pool, but the difference in choline pool size was not equal to the difference in glycinebetaine pool size, suggesting that choline must down-regulate its own synthesis. Consistent with this, glycinebetaine deficiency was also associated with the accumulation of the choline precursor, serine. A randomly amplified polymorphic DNA marker was identified that detects the bet1 allele. In 62 F8 families tested the 10-mer primer 5'-GTCCTCGTAG produced a 1.2-kb polymerase chain reaction product only when DNA from Bet1/bet1 or bet1/bet1 lines was used as template. All 26 homozygous Bet1/Bet1 F8 families tested were null for this marker.

Marker proteins for gene expression.

Reporter genes are widely used as a rapid and convenient means of measuring molecular genetic events. Their role in experimental strategies has expanded from analysis of the DNA sequences mediating RNA transcription to the broader ensemble of molecular events that define phenotype expression. The several genetic reporters available today impart a range of performance criteria to choose from, including assay convenience and reliability, sensitivity, linearity, simplicity and dynamics.

The volatiles of desert truffle: Tirmania nivea.

The volatile constituents of Tirmania nivea (white desert truffle) have been analysed, using gas chromatography/mass spectrometric technique. 11 compounds have been identified in the ascocarp volatiles. The major components were found to be unsaturated fatty acids; whereas hexadecanoic [correction of haxadecanoic] acid represented 49% of the volatiles isolate.

Gigantetronenin and gigantrionenin: novel cytotoxic acetogenins from Goniothalamus giganteus.

Gigantetronenin [1] and gigantrionenin [6], two new monotetrahydrofuran Annonaceous acetogenins each possessing a double bond along the hydrocarbon chain, have been isolated from the bark of Goniothalamus giganteus by the use of brine shrimp lethality for bioactivity-directed fractionation. The structures were elucidated based on spectroscopic and chemical methods. Compounds 1 and 6 both show selective and potent cytotoxicities to human tumor cells in culture as well as toxicity to brine shrimp. A known cytotoxic acetogenin, annomontacin [11], was also isolated from this plant. The biogenetic pathway of the acetogenins from G. giganteus is discussed.

Additional bioactive compounds and trilobacin, a novel highly cytotoxic acetogenin, from the bark of Asimina triloba.

Fractionation of the EtOH extract of the bark of Asimina triloba, monitoring by brine shrimp lethality, has led to the isolation and structural elucidation of a novel highly cytotoxic Annonaceous acetogenin, trilobacin [1], in addition to six known compounds: asimicin 2], bullatacin [3], bullatacinone [4], N-p-coumaroyltyramine [5], N-trans-feruloyltyramine [6], and (+)-syringaresinol [7]. Acetogenin 1 was identified as a diastereomer of asimicin [2] by spectral and chemical methods, and both 1 and 2 showed potent and selective cytotoxicities in the NCI human tumor cell line screen.