Structure-Based Drug Design of ADRA2A Antagonists Derived from Yohimbine.

Yohimbine, a natural indole alkaloid and a nonselective adrenoceptor antagonist, possesses potential benefits in treating inflammatory disorders and sepsis. Nevertheless, its broader clinical use faces challenges due to its low receptor selectivity. A structure-activity relationship study of novel yohimbine analogues identified amino esters of yohimbic acid as potent and selective ADRA2A antagonists. Specifically, amino ester 4n, in comparison to yohimbine, showed a 6-fold higher ADRA1A/ADRA2A selectivity index (SI > 556 for 4n) and a 25-fold higher ADRA2B/ADRA2A selectivity index. Compound 4n also demonstrated high plasma and microsomal stability, moderate-to-low membrane permeability determining its limited ability to cross the blood-brain barrier, and negligible toxicity on nontumor normal human dermal fibroblasts. Compound 4n represents an important complementary pharmacological tool to study the involvement of adrenoceptor subtypes in pathophysiologic conditions such as inflammation and sepsis and a novel candidate for further preclinical development to treat ADRA2A-mediated pathologies.

Sulfonothioated meso-Methyl BODIPY Shows Enhanced Uncaging Efficiency and Releases H2Sn.

meso-Methyl BODIPY photocages stand out for their absorption properties and easy chromophore derivatization. However, their low uncaging efficiencies often hinder applications requiring release of protected substrates in high amounts. In this study, we demonstrate that the sulfonothioated BODIPY group photocleaves a sulfonylthio group from the meso-methyl position with a 10-fold higher quantum yield than the most efficient leaving groups studied to date. Photocleavage, observed in solution and in cells, is accompanied by the spatiotemporally controlled photorelease of H2Sn. For this reason, sulfonothioated BODIPY may be applied in cell signaling, redox homeostasis, and metabolic regulation studies.

Real-time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA-Incorporated Thiazole Orange-Modified Nucleotides.

A modified 2'-deoxycytidine triphosphate derivative (dCTO TP) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dCTO TP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dCTO TP. When dCTO TP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics.

Lipid-linked nucleoside triphosphates for enzymatic synthesis of hydrophobic oligonucleotides with enhanced membrane anchoring efficiency.

We designed and synthesized a series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing various lipid moieties. Fatty acid- and cholesterol-modified dNTPs proved to be substrates for KOD XL DNA polymerase in primer extension reactions. They were also mutually compatible for simultaneous multiple incorporations into the DNA strand. The methodology of enzymatic synthesis opened a pathway to diverse structurally unique lipid-ON probes containing one or more lipid units. We studied interactions of such probes with the plasma membranes of live cells. Employing a rational design, we found a series of lipid-ONs with enhanced membrane anchoring efficiency. The in-membrane stability of multiply modified ONs was superior to that of commonly studied ON analogues, in which a single cholesterol molecule is typically tethered to the thread end. Notably, some of the probes were detected at the cell surface even after 24 h upon removal of the probe solution. Such an effect was general to several studied cell lines.

trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging.

A series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 2- or 4-linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer were designed and synthesized. They were found to be good substrates for KOD XL DNA polymerase for primer extension enzymatic synthesis of modified oligonucleotides. We systematically tested and compared the reactivity of TCO- and BCN-modified nucleotides and DNA with several fluorophore-containing tetrazines in inverse electron-demand Diels-Alder (IEDDA) click reactions to show that the longer linker is crucial for efficient labeling. The modified dNTPs were transported into live cells using the synthetic transporter SNTT1, incubated for 1 h, and then treated with tetrazine conjugates. The PEG3-linked 4TCO and BCN nucleotides showed efficient incorporation into genomic DNA and good reactivity in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells within time periods as short as 15 min. The BCN-linked nucleotide in combination with TAMRA-linked (TAMRA = carboxytetramethylrhodamine) tetrazine was also efficiently used for staining of DNA for flow cytometry. This methodology is a new approach for in cellulo metabolic labeling and imaging of DNA synthesis which is shorter, operationally simple, and overcomes several problems of previously used methods.

Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA.

The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.

Structural revisions of small molecules reported to cross-link G-quadruplex DNA in vivo reveal a repetitive assignment error in the literature.

Two molecules of mistaken identity are addressed. Uncovering these assignment errors led us to formulate more general guidelines about additional misassignments in cases of published bis-imines derived from 1,2-phenylenediamine and hydroxybenzaldehydes having no substituent in ortho-positions. The main purpose of this article is to highlight this repetitive assignment error in the literature and thus increase the likelihood of correct assignments in future papers.

Endocrine relationships between rank - related behavior and antler growth in deer.

In this review, we analyze endocrine aspects of the relationships between antlerogenesis and rank-related behavior. The explanation of these relationships has been based on the presumption that the antler growth is regulated by hormones modulated by agonistic behavior. Originally, we assumed that these relationships are primarily testosterone dependent. In the eighties, it was reported that the insulin-like growth factor 1 (IGF-1) is the antler-stimulating hormone. This hypothesis was supposed to replace an earlier theory that the antler-stimulating hormones are either androgens or their derivatives. Here, we present historical and recent views on these issues. In particular, we analyze the arguments in favor and against the role of testosterone and IGF-1 in antler growth and present a comparison of the results obtained across some deer species. In this context, we review and discuss experiments with castration of various deer species and analyze data from papers dealing with in vivo studies. We conclude that testosterone and not IGF-1 is the main antler stimulating and regulating hormone, and that concentrations of testosterone may be modified by social behavior.

Effect of different factors on proliferation of antler cells, cultured in vitro.

Antlers as a potential model for bone growth and development have become an object of rising interest. To elucidate processes explaining how antler growth is regulated, in vitro cultures have been established. However, until now, there has been no standard method to cultivate antler cells and in vitro results are often opposite to those reported in vivo. In addition, many factors which are often not taken into account under in vitro conditions may play an important role in the development of antler cells. In this study we investigated the effects of the antler growth stage, the male individuality, passaged versus primary cultures and the effect of foetal calf serum concentrations on proliferative potential of mixed antler cell cultures in vitro, derived from regenerating antlers of red deer males (Cervus elaphus). The proliferation potential of antler cells was measured by incorporation of (3)H thymidine. Our results demonstrate that there is no significant effect of the antler growth stage, whereas male individuality and all other examined factors significantly affected antler cell proliferation. Furthermore, our results suggest that primary cultures may better represent in vivo conditions and processes occurring in regenerating antlers. In conclusion, before all main factors affecting antler cell proliferation in vitro will be satisfactorily investigated, results of in vitro studies focused on hormonal regulation of antler growth should be taken with extreme caution.