Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer

Background

Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling.

Methods

Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots.

Results

Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~ 1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity.

Conclusion

In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies.

General significance

Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.     

Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.)

Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.     

Characterization of the interaction of diacylglycerol acyltransferase-2 with the endoplasmic reticulum and lipid droplets

Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.     

Synthesis,biological evaluation and molecular docking studies of benzyloxyacetohydroxamic acids as LpxC inhibitors

The inhibition of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represents a promising strategy to combat infections caused by multidrug-resistant Gram-negative bacteria. In order to elucidate the functional groups being important for the inhibition of LpxC, the structure of our previously reported hydroxamic acid 4 should be systematically varied. Therefore, a series of benzyloxyacetohydroxamic acids was prepared, of which the diphenylacetylene derivatives 28 (K_i = 95 nM) and 21 (K_i = 66 nM) were the most potent inhibitors of Escherichia coli LpxC. These compounds could be synthesized in a stereoselective manner employing a Sharpless asymmetric dihydroxylation and a Sonogashira coupling in the key steps. The obtained structure–activity relationships could be rationalized by molecular docking studies.     

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.     

Vitrification of in vitro produced bovine embryos: Effect of embryonic block and developmental kinetics

In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8–16 cell stage): I and II – early (day 2) and late (day 3) 5–8 cell, III and IV – early (day 3) and late (day 4) 8–16 cell, and V and VI – early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5–8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8–16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block.     

Acid sensing ion channels regulate neuronal excitability by inhibiting BK potassium channels

Acid sensing ion channels (ASICs), Ca²⁺ and voltage-activated potassium channels (BK) are widely present throughout the central nervous system. Previous studies have shown that when expressed together in heterologous cells, ASICs inhibit BK channels, and this inhibition is relieved by acidic extracellular pH. We hypothesized that ASIC and BK channels might interact in neurons, and that ASICs may regulate BK channel activity. We found that ASICs inhibited BK currents in cultured wild-type cortical neurons, but not in ASIC1a/2/3 triple knockout neurons. The inhibition in the wild-type was partially relieved by a drop in extracellular pH to 6. To test the consequences of ASIC-BK interaction for neuronal excitability, we compared action potential firing in cultured cortical neurons from wild-type and ASIC1a/2/3 null mice. We found that in the knockout, action potentials were narrow and exhibited increased after-hyperpolarization. Moreover, the excitability of these neurons was significantly increased. These findings are consistent with increased BK channel activity in the neurons from ASIC1a/2/3 null mice. Our data suggest that ASICs can act as endogenous pH-dependent inhibitors of BK channels, and thereby can reduce neuronal excitability.     

Toxicity of cryoprotectants to honey bee semen and queens

Given the threats to the intraspecific biodiversity of Apis mellifera and the pressure on bee breeding to come up with disease-tolerant lines, techniques to cryopreserve drone semen are of great interest. Freeze-thawed drone semen of high viability and/or motility has repeatedly been obtained, but fertility of such semen, when it was measured, was always low. The cryoprotective agent (CPA) most frequently used with drone semen is dimethyl sulfoxide (DMSO), although this substance has been suspected of causing genetic damage in sperm. No form of sperm washing is currently performed. Using a membrane permeability assay, we measured the short-term toxicity of four possible replacements for DMSO, 1,3-propane diol, 2,3-butane diol, ethylene glycol, and dimethyl formamide. We also tested whether the practice of inseminating queens with CPA-containing semen affects sperm numbers in the storage organs of queens, or sperm fertility. Finally, we tested whether CPA-toxicity in vivo can be reduced by using mixtures of two CPAs, DMSO, and ethylene glycol. Our results show that, although short-term toxicity of all CPAs tested was low, the presence of single CPAs in insemination mixtures at concentrations required for slow freezing greatly reduced the number of sperm reaching the spermatheca. Contrary to earlier reports, this was also true for DMSO. Ethylene glycol was additionally shown to reduce the viability of spermatozoa reaching the storage organ. Mixtures of DMSO and EthGly performed better than either substance used singly at the same concentration. We conclude that the toxicity of CPAs, including DMSO, on honey bee semen and/or queens has been underestimated in the past. This could partly explain the discrepancy between in vitro and in vivo quality of cryopreserved drone semen, described by others. Combinations of several CPAs and techniques to partly remove CPAs after thawing could help to solve this problem.     

Effects of propylene glycol drenching before and after luteolysis on blood glucose, ovarian steroids and follicular dynamics in heifers

The effect of propylene glycol drenching on ovarian and hormonal dynamics was studied in heifers. Five cycling heifers were used twice (as control and treatment) with crossover design. After the confirmation of ovulation (day 0), the heifers in the treatment group received propylene glycol on days 6, 7 and 8 as an oral drench (250 ml of 90% propylene glycol). On day 10, prostaglandin F2α (PGF2α), 15 mg per head of dinoprost, was administered intramuscularly to induce luteal regression followed by the follicular phase and then propylene glycol was again administered twice daily (500 ml/day) on days 10, 11 and 12. Palpation per rectum and ovarian ultrasonography were performed every other day from days 0 to 10, and daily after PGF2α administration until the subsequent ovulation (second ovulation) for analysis of follicular and luteal dynamics. Blood samples were also collected every other day from days 0 to 10, and then at 6 h intervals after PGF2α administration until the second ovulation. For the samples taken at 6-h intervals after PGF2α administration, the concentrations of glucose showed clear daily fluctuations in both groups. Changes in the plasma concentration of glucose in the treatment group were significantly (P < 0.05) higher than those of the control groups during the period between 0 and 72 h after PGF2α administration. No significant difference was detected in the growth of dominant follicles, maximum diameter of the ovulatory follicles and the changes in oestradiol and progesterone during the follicular phase between treatment and control groups. This study showed the clear daily fluctuations and stimulatory changes in the blood glucose concentrations at 24-h intervals during the short-term treatment of propylene glycol drenching in heifers. However, no significant changes in ovarian and hormonal dynamics were found under such metabolic conditions.