David Sedlák

Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells

Antiangiogenic activity of the brassinosteroid plant hormones (BRs) and their derivative cholestanon was investigated in human umbilical vein endothelial cells (HUVEC) and in human microvascular endothelial cells (HMEC-1). 24-Epibrassinolide and 28-homocastasterone from group of 21 tested natural BRs inhibited migration of HUVEC cells. Seven tested BRs decreased the number of tubes significantly. Synthetic analogue cholestanon inhibited angiogenesis in vitro more effectively than natural BRs. Because of the similarity of BRs to human steroids, we have also studied interactions of BRs with human steroid receptors. Synthetic BRs cholestanon showed agonistic effects on estrogen-receptor-α, estrogen-receptor-β and androgen receptor. Of the natural BRs, 24-epibrassinolide was found to be a weak antagonist of estrogen-receptor-α (ERα). Our results provide the first evidence that large group of BRs can inhibit in vitro angiogenesis of primary endothelial cells. BRs constitute a novel group of human steroid receptor activators or inhibitors with capacity to inhibit angiogenesis.

Synthesis and biochemical characterization of a series of 17α-perfluoroalkylated estradiols as selective ligands for estrogen receptor α.

Despite intensive research efforts, the distinct biological roles of two closely related estrogen receptors, ERα and ERβ, are only partially understood. Therefore, ligands selective for either of two isotypes are useful research tools because they allow for exerting a desired subset of biological effects mediated by only one of the receptors. Here we report on the synthesis of a new class of potent and selective ligands for ERα represented by a series of 17α-substituted estradiols bearing lipophilic perfluoroalkyl chains. These 17α-perfluoroalkylated estradiols were synthesized by Ru-catalyzed cross metathesis reactions of 17α-allyl- or 17α-vinylestradiols with perfluoroalkylpropenes. Compounds were tested in both agonistic and antagonistic modes using a panel of stable steroid receptor reporter cell lines established in U2OS cells and consisting of ERα-LBD, ERβ-LBD, GR-LBD, and MR-LBD reporters. Some of the compounds are potent and selective agonists of ERα, exhibiting weak partial to no detectable agonistic activity on ERβ. Notably, 11c is the most ERα selective ligand of the prepared compounds because it activates ERα but inhibits ERβ. In addition, some compounds are pure agonists on ERα but show mixed agonistic/antagonistic profile on ERβ which is a typical pattern observed for selective estrogen receptor modulators (SERMs).

Silychristin: Skeletal Alterations and Biological Activities

Silychristin is the second most abundant flavonolignan (after silybin) present in the fruits of Silybum marianum. A group of compounds containing silychristin (3) and its derivatives such as 2,3-dehydrosilychristin (4), 2,3-dehydroanhydrosilychristin (5), anhydrosilychristin (6), silyhermin (7), and isosilychristin (8) were studied. Physicochemical data of these compounds acquired at high resolution were compared. The absolute configuration of silyhermin (7) was proposed to be identical to silychristin A (3a) in ring D (10R,11S). The preparation of 2,3-dehydrosilychristin (4) was optimized. The Folin-Ciocalteau reduction and DPPH and ABTS radical scavenging assays revealed silychristin and its analogues to be powerful antioxidants, which were found to be more potent than silybin and 2,3-dehydrosilybin. Compounds 4-6 exhibited inhibition of microsomal lipoperoxidation (IC50 4-6 μM). Moreover, compounds 4-8 were found to be almost noncytotoxic for 10 human cell lines of different histogenetic origins. On the basis of these results, compounds 3-6 are likely responsible for most of the antioxidant properties of silymarin attributed traditionally to silybin (silibinin).

Fluorescence-based high-throughput screening of dicer cleavage activity.

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

Synthesis of Ferrocenestrone: the First Metallocene Based Steroid Analogue

Ferrocenestrone, the first steroid derivative containing a metallocene moiety, was stereoselectively prepared. The key steps included the enantioselective functionalization of ferrocene, elongation of the side chain, intramolecular enyne metathesis, Diels-Alder reaction, heterogeneous hydrogenation of the sterically hindered double bond, and finally inversion of the configuration at C13.

Development of a high-throughput fluorescence polarization assay to identify novel ligands of glutamate carboxypeptidase II.

Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000–compound library. The novel assay presented here is robust, highly reproducible (Z′ = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.

Probes & Drugs portal: an interactive, open data resource for chemical biology

porated in commercial screening libraries and continue to be used by the community5,6. We developed the P&D portal (Fig. 1 and Supplementary Notes 1–3) to alleviate some of these problems. It is intended to serve as a central hub in chemical biology research, enabling users to easily answer questions both simple (e.g., “How many compounds are shared between these libraries?”) and complicated (e.g., “Which high-potent nuclear hormone receptor ligands, with molecular weight lower than 450, are similar to a given compound but have a different chemotype?”). The P&D portal offers an intuitive yet powerful filtering system (Fig. 1a and Supplementary Note 4), various visualization tools (Fig. 1b, Supplementary Note 5 and Supplementary Table 1) and an advanced chemical intelligence (structure-related queries and structural alerts; i.e., PAINS, aggregators and obsolete compounds) (Fig. 1c and Supplementary Note 6) supported by the implementation of several chemical and biological ontologies (Fig. 1d and Supplementary Note 7). The P&D portal is designed to reflect the current state of bioactive compound space. Its set of almost 30,000 compounds is assembled from 29 established public and commercial libraries, with high Probes & Drugs portal: an interactive, open data resource for chemical biology

Structure activity relationship studies on cytotoxicity and the effects on steroid receptors of AB-functionalized cholestanes.

Structure-activity relationship analysis and profiling of a library of AB-functionalized cholestane derivatives closely related to brassinosteroids (BRs) were performed to examine their antiproliferative activities and activities on steroid hormone receptors. Some of the compounds were found to have strong cytotoxic activity in several human normal and cancer cell lines. The presence of a 3-hydroxy or 3-oxo group and 2,3-vicinal diol or 3,4-vicinal diol moiety were found to be necessary for optimum biological activity, as well as a six-membered B ring. According to the profiling of all steroid receptors in both agonist and antagonist mode, the majority of the cholestanes were weakly active or inactive compared to the natural ligands. Estrogenic activity was detected for two compounds, two compounds possessed antagonistic properties on estrogen receptors and seven compounds showed agonistic activity. Two active cholestane derivatives were shown to strongly influence cell viability, proliferation, cell cycle distribution, apoptosis and molecular pathways responsible for these processes in hormone-sensitive/insensitive (MCF7/MDA-MB-468) breast cancer cell lines.

Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations

MicroRNAs (miRNAs) are small RNAs repressing gene expression. They contribute to many physiological processes and pathologies. Consequently, strategies for manipulation of the miRNA pathway are of interest as they could provide tools for experimental or therapeutic interventions. One of such tools could be small chemical compounds identified through high-throughput screening (HTS) with reporter assays. While a number of chemical compounds have been identified in such high-throughput screens, their application potential remains elusive. Here, we report our experience with cell-based HTS of a library of 12,816 chemical compounds to identify miRNA pathway modulators. We used human HeLa and mouse NIH 3T3 cell lines with stably integrated or transiently expressed luciferase reporters repressed by endogenous miR-30 and let-7 miRNAs and identified 163 putative miRNA inhibitors. We report that compounds relieving miRNA-mediated repression via stress induction are infrequent; we have found only two compounds that reproducibly induced stress granules and relieved miRNA-targeted reporter repression. However, we have found that this assay type readily yields non-specific (miRNA-independent) stimulators of luciferase reporter activity. Furthermore, our data provide partial support for previously published miRNA pathway modulators; the most notable intersections were found among anthracyclines, dopamine derivatives, flavones, and stilbenes. Altogether, our results underscore the importance of appropriate negative controls in development of small compound inhibitors of the miRNA pathway. This particularly concerns validation strategies, which would greatly profit from assays that fundamentally differ from the routinely employed miRNA-targeted reporter assays.

Trilobolide-steroid hybrids: Synthesis, cytotoxic and antimycobacterial activity

Graphical abstract Figure. No Caption available. HighlightsFive trilobolide‐steroid hybrids were synthesized using CuAAC approach.Cytotoxicity was tested on a 12 cancer and 3 non‐cancerous cell lines. The most cytotoxic compounds were tested for cell‐cycle analysis on CCRF‐CEM line.The potency on androgen (AR) and estrogen (&agr;,&bgr;‐ER) receptors was examined.Impact on cell morphology was studied by live‐cell microscopy.Compounds were tested against 8 sensitive and multiresistant bacterial and Candida strains. Abstract Sesquiterpene lactone trilobolide is a sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor, thus depleting the Ins(1,4,5)P3‐sensitive intracellular calcium stores. Here, we describe a synthesis of a series of 6 trilobolide‐steroids conjugates (estradiol, pregnene, dehydroepiandrosterone, and testosterone). We found that the newly synthesized Tb‐based compounds possess different remarkable biological activities. Cancer cell cytotoxicity and preferential selectivity is represented in our study by a Tb‐pregnene derivative. The most cytotoxic clickates of estradiol and pregnene were studied by FACS where impact on cell cycle and RNA synthesis was observed; live‐cell microscopy revealed the impact on cell organelle morphology particularly endoplasmic reticulum, mitochondria and nucleus. Further, we have studied the estrogenic and androgenic properties of the clickate molecules using cell‐based luciferase assays. Finally, antimycobacterial tests revealed that testosterone and estradiol derivatives potentiated the antimycobacterial activity up to IC50 of 10.6 &mgr;M.