Modulation of α1ß3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix.

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a ß-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABAA α1ß3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the ß+α- interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in ß3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [ß3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1ß3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the ß+α- interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to ß3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the ß+α- interface for the anesthetic propofol.

Cytotoxic effect of carbohydrate derivatives of digitoxigenin involves modulation of plasma membrane Ca2+ -ATPase.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na+ /K+ -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na+ /K+ -ATPase, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and plasma membrane Ca2+ -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na+ /K+ -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

Digitoxin analogues with improved anticytomegalovirus activity.

Cardiac glycosides are potent inhibitors of cancer cell growth and possess antiviral activities at nanomolar concentrations. In this study we evaluated the anticytomegalovirus (CMV) activity of digitoxin and several of its analogues. We show that sugar type and sugar length attached to the steroid core structure affects its anticytomegalovirus activity. Structure-activity relationship (SAR) studies identified the l-sugar containing cardiac glycosides as having improved anti-CMV activity and may lead to better understanding of how these compounds inhibit CMV replication.

C3'/C4'-Stereochemical Effects of Digitoxigenin α-L-/α-D-Glycoside in Cancer Cytotoxicity.

Sweet'n low in stereo: A Wharton reaction was employed along with a diastereoselective palladium-catalyzed glycosylation and other post-glycosylation transformations to synthesize digitoxin analogues. Cytotoxic evaluation against a panel of cancer cell lines uncovered the stereochemical and substitutional limits of the C3'/C4'-hydroxy functionality in digitoxin monosaccharide.