The action of physiological and synthetic steroids on the calcium channel CatSper in human sperm

The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca2+ concentration ([Ca2+]i) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput screening (HTS) assay to measure changes in [Ca2+]i in human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca2+]i and reduced the P4-induced Ca2+ influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and P4-inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act on sperm acrosome reaction (AR) and penetration in viscous media. We found that these steroids show an inhibitory effect on P4 but not on prostaglandin E1-induced CatSper activation, suggesting that they compete for the same binding site as P4. Pregnenolone, dydrogesterone, epiandrosterone, nandrolone, and dehydroepiandrosterone acetate (DHEA) were found to activate CatSper at physiologically relevant concentrations within the nanomolar range. Like P4, most tested steroids did not significantly affect the AR while stanozolol and estropipate slightly increased sperm penetration into viscous medium. Furthermore, using a hybrid approach integrating pharmacophore analysis and statistical modelling, we were able to screen in silico for steroids that can activate the channel and define the physicochemical and structural properties required for a steroid to exhibit agonist activity against CatSper. Overall, our results indicate that not only physiological but also synthetic steroids can modulate the activity of CatSper with varying potency and if bound to CatSper prior to P4, could impair the timely CatSper activation necessary for proper fertilization to occur

Compounds from Toddalia asiatica: Immunosuppressant activity and absolute configurations

In a screening of an extract library from plants used in Traditional Chinese Medicine the MeOH extract of Toddalia asiatica inhibited proliferation of human primary T cells with an IC50 of 25.8 μg/mL. Activity in the extract was tracked by HPLC activity profiling, and a total of 15 compounds were characterized. Three compounds, toddalic acid (6) and both enantiomers (7a and 7b) of toddanolic acid (7), were new natural products, and two recently published compounds, (2′R)-toddalolactone 3′-O-β-d-glucopyranoside (10) and (2′S)-toddalolactone 2′-O-β-d-glucopyranoside (11), were described in detail for the first time. The absolute configurations of compounds 8, 9, 10, 12, 13, and 15 were determined by comparison of experimental and calculated ECD spectra. For glucosides 9 and 10, ECD data and chiral-phase HPLC of the aglycones after enzymatic hydrolysis confirmed the results. Nitidine chloride (4) inhibited proliferation of primary human T cells with an IC50 of 0.4 μM

Sesquiterpene Lactones from Artemisia argyi: Absolute Configuration and Immunosuppressant Activity

A library of extracts from plants used in Chinese Traditional Medicine was screened for inhibition of T lymphocyte proliferation. An ethyl acetate extract from aerial parts of Artemisia argyi showed promising activity and was submitted to HPLC-based activity profiling to track the active compounds. From the most active time window, three guaianolides (1, 2, and 5) and two seco-tanapartholides (3 and 4) were identified and, in a less active time window, five new sesquiterpene lactones (8–11, 17), along with six known sesquiterpene lactones and two known flavonoids. The absolute configurations of compounds 1, 2, 5–10, 13–15, 17, and 18 were established by comparison of experimental with calculated electronic circular dichroism (ECD) spectra. For seco-tanapartholides B (3) and A (4), ECD yielded ambiguous results, and their absolute configurations were determined by comparing experimental and calculated vibrational circular dichroism (VCD) spectra. Compounds 1–5 showed significant, noncytotoxic inhibition of T lymphocyte proliferation, with IC50 values between 1.0 and 3.7 μM

Microsecond MD simulations of human CYP2D6 wild-type and five allelic variants reveal mechanistic insights on the function.

Characterization of cytochrome P450 2D6 (CYP2D6) and the impact of the major identified allelic variants on the activity of one of the most dominating drug-metabolising enzymes is essential to increase drug safety and avoid adverse reactions. Microsecond molecular dynamics simulations have been performed to capture the dynamic signatures of this complex enzyme and five allelic variants with diverse enzymatic activity. In addition to the apo simulations, three substrates (bufuralol, veliparib and tamoxifen) and two inhibitors (prinomastat and quinidine) were included to explore their influence on the structure and dynamical features of the enzyme. Our results indicate that the altered enzyme activity can be attributed to changes in the hydrogen bonding network within the active site, and local structural differences in flexibility, position and shape of the binding pocket. In particular, the increased (CYP2D6*53) or the decreased (CYP2D6*17) activity seems to be related to a change in dynamics of mainly the BC loop due to a modified hydrogen bonding network around this region. In addition, the smallest active site volume was found for CYP2D6*4 (no activity). CYP2D6*2 (normal activity) showed no major differences in dynamic behaviour compared to the wild-type

DOLINA - Docking Based on a Local Induced-Fit Algorithm : Application toward Small-Molecule Binding to Nuclear Receptors

Docking algorithms allowing for ligand and - to various extent - also protein flexibility are nowadays replacing techniques based on rigid protocols. The algorithm implemented in the Dolina software relies on pharmacophore matching for generating potential ligand poses and treats associated local induced-fit changes by combinatorial rearrangement of side-chains lining the binding site. In Dolina, ligand flexibility is not treated internally, instead a pool of low-energy conformer identified in a conformational search screened for extended binding-pose candidates. Grouping rearranged residues in sterically independent families and side-chain conformer clustering are employed to achieve efficient use of the computational resources along with a good accuracy of e generated poses. Dolina was applied toward docking of small-molecule ligands to three different nuclear receptor ligand binding domains for which in total 18 high-resolution crystal structures were used as reference The selected nuclear receptors feature a deeply buried ligand-binding site where local induced-fit is to be expected, particularly for receptor antagonists. For each receptor, a crystal structure with a cocrystallized small steroid ligand (template) was chosen as a target system to which several synthetic ligands of different sizes were docked. Poses within an RMSD of 2.0 angstrom from the crystal reference pose were generated in 91% of the cases. In 28%, the pose with the lowest RMSD to the reference pose was ranked as the top one, and in 76% it was ranked among the top five poses. Detailed descriptions of the docking algorithm and observed results are included. Dolina is available free of charge for academic institutions