No abstract

Only a small fraction of vitamin B 12 -requiring organisms are able to synthesize B 12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B 12 -binding enzymes and regulatory roles for B 12 . Here we report the development and use of a B 12 -based chemical probe to identify B 12 -binding proteins in a nonphototrophic B 12 -producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B 12 -binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B 12 likely modulates growth, and by limiting its availability to auxotrophs, B 12 -producing organisms may facilitate coordination of community metabolism.

Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography-mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.

The first ratiometric fluorescent reporter was designed for the detection of cardiac troponin I (cTnI), a key protein elicited during cardiac muscle cell death. In designing this abiotic fluorescent probe, docking simulation studies were performed to predict the probe/protein interactions along the solvent exposed regions of cTnI. Simple cuvette titration experiments in aqueous buffered solution indicate remarkable selectivity for cardiac troponin in the clinically relevant nM region versus skeletal troponin.

Testing small molecules for their ability to modify cysteine residues of proteins in the early stages of drug discovery is expected to accelerate our ability to develop more selective drugs with lesser side effects. In addition, this approach also enables the rapid evaluation of the mode of binding of new drug candidates in respect to thiol-reactivity and metabolism by glutathione. Herein, we describe the development of a fluorescence-based high throughput assay that allows the identification of thiol-reactive compounds. A thiol-containing fluorescent probe MSTI was synthesized and used to evaluate small molecules from the LOPAC collection of bioactive molecules. LOPAC compounds that are known to react with sulfur nucleophiles were identified with this assay, for example, irreversible protease inhibitors, nitric oxide releasing compounds, and proton-pump inhibitors. The results confirm that both electrophilic and redox reactive compounds can be quickly identified in a high throughput manner enabling the assessment of screening libraries in respect to thiol-reactive compounds.

A high throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes to modulate gene regulation mediated by VDR. The peroxisome proliferator-activated receptor δ (PPARδ) agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations higher than 12.1 µM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor (AR). Surprisingly, GW0742 behaved as PPAR agonist/antagonist activating transcription at lower concentration and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742+ increased fluorescence intensity and fluorescence polarization at an excitation wavelength of 595 nm and emission wavelength of 615 nm in a dose dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3 and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3.

A ten element matrix of 5- and 6-substituted-(2,3)-naphthalimides was prepared for the appropriate placement of substituents necessary to promote dual fluorescence (DF). As prescribed by our balanced seesaw photophysical model this matrix yielded nine new DF dyes out of a possible ten compounds. From this set of nine DF dyes, 4-fluoronaphthalic amide (37) was selected as a probe for ratiometric detection of DNA and demonstration of panchromatic emission.

The synthesis and photophysical characterization of a new white-light fluorophore is described. The optimization of excitation wavelengths allows the naphthalimide (NI) dyes to display blue, green or white light emission depending on the excitation wavelength.