Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Bioactivity of Microencapsulated Cell-Free Supernatant of Streptococcus thermophilus in Combination with Thyme Extract on Food-Related Bacteria.

The bioactive properties of the combination of microencapsulated cell-free supernatant (CFS) from Streptococcus thermophilus and thyme extract on food-related bacteria (Photobacterium damselae, Proteus mirabilis, Vibrio vulnificus, Staphylococcus aureus ATCC29213, Enterococcus faecalis ATCC29212, and Salmonella Paratyphi A NCTC13) were investigated. The microencapsulated CFS of S. thermophilus, in combination with ethanolic thyme extract, had a particle size in the range of 1.11 to 11.39 µm. The microencapsulated CFS of S. thermophilus had a wrinkled, spherical form. In the supernatant, especially at 2% (v/w), the thyme extract additive caused a decrease in the wrinkled form and a completely spherical structure. A total of 11 compounds were determined in the cell-free supernatant of S. thermophilus, and acetic acid (39.64%) and methyl-d3 1-dideuterio-2-propenyl ether (10.87%) were the main components. Thyme extract contained seven components, the main component being carvacrol at 67.96% and 1,2,3-propanetriol at 25.77%. Significant differences (p < 0.05) were observed in the inhibition zones of the extracts on bacteria. The inhibitory effect of thyme extract on bacteria varied between 25.00 (P. damselae) and 41.67 mm (V. vulnificus). Less antibacterial activity was shown by the microencapsulated CFS from S. thermophilus compared to their pure form. (p < 0.05). As a result, it was found that microencapsulated forms of CFS from S. thermophilus, especially those prepared in combination with 2% (v/w) thyme extract, generally showed higher bioactive effects on bacteria.

Open resources for chemical probes and their implications for future drug discovery.

INTRODUCTION: The rational development of new therapeutics requires a thorough understanding of how aberrant signalling affects cellular homeostasis and causes human disease. Chemical probes are tool compounds with well-defined mechanism-of-action enabling modulation of, for example, domain-specific protein properties in a temporal manner, thereby complementing other target validation methods such as genetic gain- and loss-of-function approaches. AREAS COVERED: In this review, the authors summarize recent advances in chemical probe development for emerging target classes such as solute carriers and ubiquitin-related targets and highlight open resources to inform and facilitate chemical probe discovery as well as tool compound selection for target validation and phenotypic screening. EXPERT OPINION: Chemical probes are powerful tools for drug discovery that have led to fundamental insights into biological processes and have paved the way for the development of first-in-class drugs. Open resources can inform on various aspects of chemical probe development and provide access to data and recommendations on use of chemical probes to catalyse collaborative science and help accelerate drug target identification and validation.

Discovery of a Chemical Probe to Study Implications of BPTF Bromodomain Inhibition in Cellular and in†vivo Experiments.

The bromodomain and PHD-finger containing transcription factor (BPTF) is part of the nucleosome remodeling factor (NURF) complex and has been implicated in multiple cancer types. Here, we report the discovery of a potent and selective chemical probe targeting the bromodomain of BPTF with an attractive pharmacokinetic profile enabling cellular and in vivo experiments in mice. Microarray-based transcriptomics in presence of the probe in two lung cancer cell lines revealed only minor effects on the transcriptome. Profiling against a panel of cancer cell lines revealed that the antiproliferative effect does not correlate with BPTF dependency score in depletion screens. Both observations and the multi-domain architecture of BPTF suggest that depleting the protein by proteolysis targeting chimeras (PROTACs) could be a promising strategy to target cancer cell proliferation. We envision that the presented chemical probe and the related negative control will enable the research community to further explore scientific hypotheses with respect to BPTF bromodomain inhibition.

DNA double-strand break-derived RNA drives TIRR/53BP1 complex dissociation.

Tudor-interacting repair regulator (TIRR) is an RNA-binding protein and a negative regulator of the DNA-repair factor p53-binding protein 1 (53BP1). In non-damage conditions, TIRR is bound to 53BP1. After DNA damage, TIRR and 53BP1 dissociate, and 53BP1 binds the chromatin at the double-strand break (DSB) to promote non-homologous end joining (NHEJ)-mediated repair. However, the exact mechanistic details of this dissociation after damage are unknown. Increasing evidence has implicated RNA as a crucial factor in the DNA damage response (DDR). Here, we show that RNA can separate TIRR/53BP1. Specifically, RNA with a hairpin secondary structure, transcribed at the DSB by RNA polymerase II (RNAPII), promotes TIRR/53BP1 complex separation. This hairpin RNA binds to the same residues on TIRR as 53BP1. Our results uncover a role of DNA-damage-derived RNA in modulating a protein-protein interaction and contribute to our understanding of DSB repair.

The Impact of Thyme, Rosemary and Basil Extracts on the Chemical, Sensory and Microbiological Quality of Vacuumed Packed Mackerel Balls.

The effect of natural extracts (0.05%) and vacuum packaging on the sensory, chemical, and microbiological quality of mackerel balls were evaluated at refrigerated (4 ± 2 °C) storage. Natural extracts thyme (38.13 mg GAE/g), rosemary (81.85 mg GAE/g) and basil (21.08 mg GAE/g) were evaluated. Natural extracts imparted stability to lipids (TBA, FFA, and PV), and the ability was further improved by vacuum packaging. Biochemical changes (TVB-N, pH) and microbiological quality (total viable count) were also retained. Control samples packed under vacuum were found to cross over acceptable limits on day 28. Based on sensory quality evaluation, samples treated with rosemary and thyme extracts showed superior sensory quality over control, whilebasil-treated samples were not found acceptable at day 28. Consequently, the inclusion of thyme and rosemary extracts exhibits preservative quality when combined with vacuum packaging, retaining biochemical, microbial, and sensory quality.

The influences of fish infusion broth on the biogenic amines formation by lactic acid bacteria.

The influences of fish infusion decarboxylase broth (IDB) on biogenic amines (BA) formation by lactic acid bacteria (LAB) were investigated. BA productions by single LAB strains were tested in five different fish (anchovy, mackerel, white shark, sardine and gilthead seabream) IDB. The result of the study showed that significant differences in ammonia (AMN) and BA production were observed among the LAB strains in fish IDB (p < 0.05). The highest AMN and TMA production by LAB strains were observed for white shark IDB. The all tested bacteria had decarboxylation activity in fish IDB. The uppermost accumulated amines by LAB strains were tyramine (TYM), dopamine, serotonin and spermidine. The maximum histamine production was observed in sardine (101.69 mg/L) and mackerel (100.84 mg/L) IDB by Leuconostoc mesenteroides subsp. cremoris and Pediococcus acidophilus, respectively. Lactobacillus delbrueckii subsp. lactis and Pediococcus acidophilus had a high TYM producing capability (2943 mg/L and 1157 mg/L) in sardine IDB.

The influences of fish infusion broth on the biogenic amines formation by lactic acid bacteria

The influences of fish infusion decarboxylase broth (IDB) on biogenic amines (BA) formation by lactic acid bacteria (LAB) were investigated. BA productions by single LAB strains were tested in five different fish (anchovy, mackerel, white shark, sardine and gilthead seabream) IDB. The result of the study showed that significant differences in ammonia (AMN) and BA production were observed among the LAB strains in fish IDB (p < 0.05). The highest AMN and TMA production by LAB strains were observed for white shark IDB. The all tested bacteria had decarboxylation activity in fish IDB. The uppermost accumulated amines by LAB strains were tyramine (TYM), dopamine, serotonin and spermidine. The maximum histamine production was observed in sardine (101.69 mg/L) and mackerel (100.84 mg/L) IDB by Leuconostoc mesenteroides subsp. cremoris and Pediococcus acidophilus, respectively. Lactobacillus delbrueckii subsp. lactis and Pediococcus acidophilus had a high TYM producing capability (2943 mg/L and 1157 mg/L) in sardine IDB.

Stimulation of cadaverine production by foodborne pathogens in the presence of Lactobacillus, Lactococcus, and Streptococcus spp.

The effect of Lactobacillus plantarum (FI8595), Lactococcus lactis subsp. cremoris MG 1363), Lactococcus lactis subsp. lactis (IL 1403), and Streptococcus thermophilus on cadaverine and other biogenic amine production by foodborne pathogens was investigated lysine decarboxylase broth. Both of lactic acid bacteria and foodborne pathogens used (especially Staphylococcus aureus, E. coli, Lc. lactis subsp. lactis and Lb. plantarum) had an ability to convert aminoacids into biogenic amine. The conversion of lysine into cadaverine was the highest (167.11 mg/L) by Lactobacillus spp. Gram-positive bacteria generally had a greater ability to produce cadaverine with corresponding value of 46.26, 53.76, and 154.54 mg/L for Enterococcus faecalis, S. aureus, and Listeria monocytogenes, respectively. Significant variations on biogenic amine production were observed in the presence of lactic acid bacteria strains (P < 0.05). The role of lactic acid bacteria on biogenic amine production by foodborne pathogens varied depending on strains and specific amine. Cadaverine accumulation by Enterobactericeae was increased in the presence of lactic acid bacteria strains except for St. thermophilus, which induced 2-fold lower cadaverine production by S. Paratyphi A. Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris induced 10-fold higher increases in histamine for E. coli and K. pneumoniae, respectively. Lactic acid bacteria resulted in strong increases in cadaverine production by P. aeruginosa, although remarkable decreases were observed for histamine, spermidine, dopamine, agmatine, and TMA in the presence of lactic acid bacteria in lysine decarboxylase broth . The result of the study showed that amine positive lactic acid bacteria strains in fermented food led to significant amine accumulation by contaminant bacteria and their accumulation in food product may be controlled by the use of proper starters with amine-negative activity.

The effects of extraction methods on the contents of fatty acids, especially EPA and DHA in marine lipids.

This study aims to evaluate the efficiency of trans-methylation methods in fish oil obtained using Soxhlet extraction method and Bligh and Dyer method and also to observe the effects of extraction methods on the contents of polyunsaturated fatty acids [PUFAs, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in five marine species: sardine (Sardinella aurita), anchovy (Engraulis encrasicolus), sea bream (Sparus aurata), brushtooth lizardfish (Saurida undosquamis) and chub mackerel (Scomber japonicus). The results showed that Bligh and Dyer extraction method was more efficient in extracting polar and non-polar lipids than Soxhlet method and also prevented losses of PUFAs by a reduction in the oxidation. The level of EPA showed fluctuations for the two extraction methods. However, Soxhlet method showed significant decrease (p < 0.05) in the level of DHA for all fish species. The use of n-heptane proves to be superior for the recovery of unsaturated fatty acids, especially EPA and DHA.