Uncoupling Protein 2 Drives Myocardial Dysfunction in Murine Models of Septic Shock.

Cardiac dysfunction is a major component of sepsis-induced multiorgan failure in critical care units. Uncoupling protein 2 (UCP2) involves immune response, regulation of oxidative stress, and maintenance of mitochondrial membrane potential as well as energy production. However, whether and how UCP2 plays roles in the development of septic cardiac dysfunction are largely unknown. Here, intraperitoneal injection of LPS significantly activated UCP2 expression accompanied by a significant decrease of cardiac function and caused a significantly lower survival rate in mice. Of note, knockdown of UCP2 through a cardiotropic adenoassociated viral vector carrying a short hairpin RNA (shRNA) specifically targeting the UCP2 evoked resistance to LPS-triggered septic cardiac dysfunction and lethality in vivo. Moreover, UCP2 deficiency ameliorated the reduced levels of intracellular ATP in the LPS-challenged heart tissues and preserved mitochondrial membrane potential loss in primary adult mouse cardiomyocytes in LPS-challenged animals. Mechanistically, we confirmed that the inhibition of UCP2 promoted autophagy in response to LPS, as shown by an increase in LC3II and a decrease in p62. At last, the autophagy inhibitor 3-MA abolished UCP2 knockdown-afforded cardioprotective effects. Those results indicate that UCP2 drives septic cardiac dysfunction and that the targeted induction of UCP2-mediated autophagy may have important therapeutic potential.

Design and synthesis of isatin/triazole conjugates that induce apoptosis and inhibit migration of MGC-803 cells.

A series of new isatin/triazole conjugates were designed based on the hypothesis that the ester-linked compounds could be enzymatically hydrolyzed by cellular esterases inside the cells. These compounds showed moderate to good growth inhibition toward the tested cancer cells, exerted selective inhibition toward MGC-803 cells and were less toxic to normal cells HL-7702 and GES-1. Of these compounds, compound 5a showed the best inhibitory activity against MGC-803 cells (IC50 = 9.78 µM), induced apoptosis through multiple mechanisms, as well as inhibited migration of MGC-803 cells.

Structurally novel steroidal spirooxindole by241 potently inhibits tumor growth mainly through ROS-mediated mechanisms.

Cancer cells always have increased ROS levels, thus making them more vulnerable to persistent endogenous oxidative stress. The biochemical difference between cancer and normal cells could be exploited to achieve selective cancer cell killing by exogenous ROS-producing agents. Herein we described a structurally novel steroidal spirooxindole by241 and its anticancer efficacy. By241 exhibited potent inhibition against human cancer cells and less toxic to normal cells. By241 concentration-dependently induced apoptosis of MGC-803 and EC9706 cells, accompanied with the mitochondrial dysfunction and increased ROS levels. NAC can completely restore the decreased cell viability of MGC-803 cells caused by by241, suggesting ROS-mediated mechanisms. The expression levels of proteins involved in the mitochondrion-related pathways were detected, showing increased expression of proapoptotic proteins and decreased expression of anti-apoptotic proteins, and activation of caspases-9/-3, but without activating caspase-8 expression. Pretreatment with Z-VAD-FMK partially rescued by241-induced apoptosis of MGC-803 cells. Additionally, by241 inhibited mTOR, activated p53 and its downstream proteins, cleaved MDM2 and PI3K/AKT as well as NF-κB signaling pathway. In vivo experiments showed that by241 did not have significant acute oral toxicity and exerted good anticancer efficacy against MGC-803 bearing mice models. Therefore, by241 may serve as a lead for further development for cancer therapy.

Efficient synthesis of new antiproliferative steroidal hybrids using the molecular hybridization approach.

A series of steroidal hybrids with different terminal bioactive scaffolds were synthesized using the molecular hybridization approach and further evaluated for their antiproliferative activity against several cancer cell lines of different origins using the MTT assay. The preliminary results indicated that compounds 12a-h with the terminal isatin motif were remarkably sensitive to SH-SY5Y cells, thereby exerting potent growth inhibition in vitro. This selectivity is possibly attributed to LSD1 inactivation (IC50 = 3.18 µM). Besides, we also found that the chloro atom at the 7-position on the isatin core was beneficial for the activity through the SARs studies. Among this series, compound 12g showed the best inhibitory activity (IC50 = 4.06 µM) against SH-SY5Y cells, which was comparable to that of 5-FU. Compound 12g arrested cell cycle at G2/M phase, induced apoptosis accompanied with decrease of mitochondrial membrane potential, and inhibited LSD1 potently (IC50 = 3.18 µM). Docking studies showed that compound 12g formed interactions with surrounding amino acid residues and the steroid nucleus occupied the tubular hydrophobic cavity of the active site. Compounds 13-18 represented weak to moderate activity against the tested cancer cell lines. The steroidal dimer 20 and the structurally simplified non-steroidal dimer 21 were found to be devoid of the inhibitory activity.

Natural Product-Derived Spirooxindole Fragments Serve as Privileged Substructures for Discovery of New Anticancer Agents.

The utility of natural products for identifying anticancer agents has been highly pursued in the last decades and over 100 drug molecules in clinic are natural products or natural product-derived compounds. Natural products are believed to be able to cover unexplored chemical space that is normally not occupied by commercially available molecule libraries. However, the low abundance and synthetic intractability of natural products have limited their applications in drug discovery. Recently, the identification of biologically relevant fragments derived from biologically validated natural products has been recognized as a powerful strategy in searching new biological probes and drugs. The spirocyclic oxindoles, as privileged structural scaffolds, have shown their potential in designing new drugs. Several anticancer drug candidates such as SAR405838, RO8994, CFI-400945 and their bioisosteres are undergoing clinical trials or preclinical studies. To highlight the significant progress, we focus on illustrating the discovery of SAR405838, RO8994, CFI-400945 and their bioisosteres for cancer therapy using substructure-based strategies and discussing modes of action, binding models and preclinical data.

Efficient synthesis of novel antiproliferative steroidal spirooxindoles via the [3+2] cycloaddition reactions of azomethine ylides.

A series of novel steroidal spirooxindoles 3a-h were synthesized from pregnenolone in a high regioselective manner using the 1,3-dipolar cycloaddition as the key step. This protocol resulted in the formation of two C-C bonds, one C-N bond and the creation of one pyrrolidine ring and three contiguous stereocenters in a single operation. Biological evaluation showed that these synthesized steroidal spirooxindoles exhibited moderate to good antiproliferative activity against the tested cell lines and some of them were more potent than 5-FU. Among them, compounds 3e and 3f displayed the best antiproliferative activity against MCF-7 cells with the IC50 values of 4.0 and 3.9µM, respectively. Flow cytometry analysis demonstrated that compound 3d caused the cellular apoptosis and cell cycle arrest at G2/M phase in a concentration-dependent manner. Docking results indicated that compound 3d fitted well into the MDM2 active site 1RV1 by interacting with Lys94 and Thr101 residues.

Efficient three-component one-pot synthesis of steroidal polysubstituted anilines.

An efficient and practical base-promoted cascade reaction has been developed to access steroidal polysubstituted anilines from simple precursors. The protocol reported herein achieved the formation of a benzene ring as well as three continuous C-C bonds in a single operation. The reaction mechanism was proposed on the basis of the key intermediate obtained. Besides, this method could be potentially employed for the synthesis of biphenyl compounds. The adjacent amine and nitrile groups existed in the final products have the potential for late stage functionalization, which would provide efficient access to steroidal compound collections with structural diversity and complexity.

Drug discovery using spirooxindole cores: Success and Challenges [corrected].

The identification of novel anticancer agents with high efficacy and low toxicity has always been an intriguing topic in medicinal chemistry. The unique structural features of spirooxindoles together with diverse biological activities have made them promising structures in new drug discovery. "Among spirooxindoles, CFI-400945, recently discovered by Sampson et al., is a potent PLK4 inhibitor, which has entered phase I clinical trials for the treatment of solid tumors. However, questions remain as to whether PLK4 is the only relevant therapeutic target for CFI-400945. To highlight this significant progress of CFI-400945 in last two years, this review centers on the identification from a focused kinase library, structural optimizations and strategies involved, structure-activity relationships, modes of action, target validation, chemical synthesis and, more importantly, the kinase selectivity between PLK4 and other targets [corrected].

Discovery of novel steroidal pyran-oxindole hybrids as cytotoxic agents.

A series of novel steroidal pyran-oxindole hybrids were efficiently synthesized in a single operation through the vinylogous aldol reaction of vinyl malononitrile 3 with substituted isatins involving the construction of C-C and C-O bonds. Some compounds displayed moderate to good cytotoxicity against T24, SMMC-7721, MCF-7 and MGC-803 cells. Compounds 4f and 4i were more potent than 5-Fu against T24 and MGC-803 cells with the IC50 values of 4.43 and 8.45 µM, respectively. Further mechanism studies indicated that compound 4i induced G2/M arrest and early apoptosis in a concentration- and time-dependent manner.

Design, synthesis and biological evaluation of novel steroidal spiro-oxindoles as potent antiproliferative agents.

Two series of novel steroidal spiro-pyrrolidinyl oxindoles 3a-t and 6a-c were designed and synthesized from dehydroepiandrosterone using the 1,3-dipolar cycloaddition as the key step and further evaluated for their antiproliferative activities for four human cancer cell lines (MGC-803, EC109, SMMC-7721 and MCF-7). This protocol achieved the formation of two CC bonds, one CN bond and the creation of one new five-membered pyrrolidine ring and three contiguous stereocenters in a single operation. Biological evaluation showed that these synthesized steroidal spiro-pyrrolidinyl oxindoles possessed moderate to good antiproliferative activities against the tested cell lines and some of them were more potent than 5-Fu. Particularly, compound 3g showed good antiproliferative activity against SMMC-7721 (IC50=0.71µM). Steroid dimer 6b showed improved antiproliferative activities against SMMC-7721 and MCF-7 with the IC50 values of 4.30 and 2.06µM, respectively. Flow cytometry analysis demonstrated that compound 3n caused the cellular early apoptosis and cell cycle arrest at G2/M phase in a concentration- and time-dependent manner. [Corrected]

Stereoselective synthesis of novel antiproliferative steroidal (E, E) dienamides through a cascade aldol/cyclization process.

The stereoselective and metal-free protocol involving a cascade aldol/cyclization process for the synthesis of steroidal (E, E) dienamides from steroidal α, α-dicyanoalkene was reported. This protocol efficiently achieved the construction of C=C bond and selective conversion of cyano group into carboxamide in one-pot procedure under mild condition. Further biological evaluation showed that some of these compounds had moderate to excellent cytotoxic activities against all the tested cancer cell lines and were more potent than well-known drug 5-fluorouracil. Particularly, compound 3c represented excellent inhibitory effect against MCF-7 (IC50=0.76 µM), which was about 10-fold more potent than 5-fluorouracil.

Efficient construction of novel D-ring modified steroidal dienamides and their cytotoxic activities.

Two series of steroidal dienamides 4a-q and 5a-f were designed, synthesized and evaluated for cytotoxic activities against five human cancer cell lines (MGC-803, EC109, PC-3, SMMC-7721 and MCF-7). The protocol developed efficiently achieved the construction of carbon-carbon double bond and selective conversion of nitrile group into carboxamide in one-pot procedure. Besides, compounds 4a-q and 5a-f showed moderate to excellent cytotoxic activities with the IC50 values ranging from 0.1 to 40 µM and most of them were more potent than 5-fluorouracil. Particularly, four compounds 4d, 4e, 4q and 5a showed excellent selectivity against MGC-803 with the IC50 values less than 1 µM. Flow cytometry analysis demonstrated that compound 4c caused the cellular early apoptosis and cell cycle arrest in G2/M phase in a concentration-independent manner.

[Eutrophication and pollution level of microcystin in Dianshan Lake].

A survey was conducted in Dianshan Lake to study the eutrophication indexes including total phosphorus (TP), total nitrogen (TN), pH, temperature, diaphaneity and chlorophyll-a level and dominant algae in seasons. The impacts of temperature, light, nitrogen and phosphorus on growth of and microcystin LR production by Microcystis aeraginosa strain under laboratory conditions were studied. Relationship between algal cell density and concentration of microcystin LR were studied. Results suggest that water in Dianshan Lake was eutrophicated. The suitable seasons for algae growth are the end of spring and summer. The annual average of TP and TN were 1.93 mg/L and 0.18 mg/L respectively. And 93.5 and 92.2 percent of TP and TN were higher than the criteria for the third class water body. Significant impact from agriculture was indicated since the peak of algae laged one month after the maxium use of fertilizer. The dominant algae in Dianshan Lake were cyanobacteria, bacillariophyta, cryptophyta and euglenophyta. Microcystis, anabaena and synedra, which excrete toxins and indicate water pollution, and are dominant algae species in summer. M. aeraginosa strain had a biggest growth rate at temperature of 25 degrees C and light intensity of 3 0001x, while microcystin LR production contents reached maximum at 20 degrees C and 5000lx respectively. The optimum TP and TN concentrations for growth of and toxin production by M. aeraginosa were found to be 650 micromol/L and 6.5 micromol/L respectively. TP is suspected to be the limiting factor for the growth of algae both in field and laboratory conditions. Positive correlations between total microcystin LR concentrations and algae cell density or M. aeraginosa cell densities are found. The algae cell density can be used to predict the level of algal toxins in water.