Discovery of N-benzylbenzamide-based allosteric inhibitors of Aurora kinase A.

Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6h) of N-benzylbenzamide backbone. Compound 6h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6h against AurkA (IC50 = 6.50 µM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure-activity relationship. In addition, 6h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors.

Cavomycins A-C, Linear Oligomer Depsipeptides from an Annelid-Associated Streptomyces cavourensis.

Three unique linear oligomeric depsipeptides, designated as cavomycins A-C (1-3), were identified from Streptomyces cavourensis, a gut bacterium associated with the annelid Paraleonnates uschakovi. The structures of these depsipeptides were determined through a combination of spectroscopic methods and chemical derivatization techniques, including methanolysis, the modified Mosher's method, advanced Marfey's methods, and phenylglycine methyl ester derivatization. The unique dipeptidyl residue arrangements in compounds 1-3 indicate that they are not degradation products of valinomycin. Compound 2 and its methylation derivative 2a exhibited antiproliferative activity against PANC-1 pancreatic cancer cells with IC50 values of 1.2 and 1.7 µM, respectively.

Hypothalamic astrocyte NAD+ salvage pathway mediates the coupling of dietary fat overconsumption in a mouse model of obesity.

Nicotinamide adenine dinucleotide (NAD)+ serves as a crucial coenzyme in numerous essential biological reactions, and its cellular availability relies on the activity of the nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed salvage pathway. Here we show that treatment with saturated fatty acids activates the NAD+ salvage pathway in hypothalamic astrocytes. Furthermore, inhibition of this pathway mitigates hypothalamic inflammation and attenuates the development of obesity in male mice fed a high-fat diet (HFD). Mechanistically, CD38 functions downstream of the NAD+ salvage pathway in hypothalamic astrocytes burdened with excess fat. The activation of the astrocytic NAMPT-NAD+-CD38 axis in response to fat overload induces proinflammatory responses in the hypothalamus. It also leads to aberrantly activated basal Ca2+ signals and compromised Ca2+ responses to metabolic hormones such as insulin, leptin, and glucagon-like peptide 1, ultimately resulting in dysfunctional hypothalamic astrocytes. Our findings highlight the significant contribution of the hypothalamic astrocytic NAD+ salvage pathway, along with its downstream CD38, to HFD-induced obesity.

Hypothalamic AMP-Activated Protein Kinase as a Whole-Body Energy Sensor and Regulator.

5´-Adenosine monophosphate (AMP)-activated protein kinase (AMPK), a cellular energy sensor, is an essential enzyme that helps cells maintain stable energy levels during metabolic stress. The hypothalamus is pivotal in regulating energy balance within the body. Certain neurons in the hypothalamus are sensitive to fluctuations in food availability and energy stores, triggering adaptive responses to preserve systemic energy equilibrium. AMPK, expressed in these hypothalamic neurons, is instrumental in these regulatory processes. Hypothalamic AMPK activity is modulated by key metabolic hormones. Anorexigenic hormones, including leptin, insulin, and glucagon-like peptide 1, suppress hypothalamic AMPK activity, whereas the hunger hormone ghrelin activates it. These hormonal influences on hypothalamic AMPK activity are central to their roles in controlling food consumption and energy expenditure. Additionally, hypothalamic AMPK activity responds to variations in glucose concentrations. It becomes active during hypoglycemia but is deactivated when glucose is introduced directly into the hypothalamus. These shifts in AMPK activity within hypothalamic neurons are critical for maintaining glucose balance. Considering the vital function of hypothalamic AMPK in the regulation of overall energy and glucose balance, developing chemical agents that target the hypothalamus to modulate AMPK activity presents a promising therapeutic approach for metabolic conditions such as obesity and type 2 diabetes mellitus.

Integrating computational methods guided the discovery of phytochemicals as potential Pin1 inhibitors for cancer: pharmacophore modeling, molecular docking, MM-GBSA calculations and molecular dynamics studies.

Pin1 is a pivotal player in interactions with a diverse array of phosphorylated proteins closely linked to critical processes such as carcinogenesis and tumor suppression. Its axial role in cancer initiation and progression, coupled with its overexpression and activation in various cancers render it a potential candidate for the development of targeted therapeutics. While several known Pin1 inhibitors possess favorable enzymatic profiles, their cellular efficacy often falls short. Consequently, the pursuit of novel Pin1 inhibitors has gained considerable attention in the field of medicinal chemistry. In this study, we employed the Phase tool from Schrödinger to construct a structure-based pharmacophore model. Subsequently, 449,008 natural products (NPs) from the SN3 database underwent screening to identify compounds sharing pharmacophoric features with the native ligand. This resulted in 650 compounds, which then underwent molecular docking and binding free energy calculations. Among them, SN0021307, SN0449787 and SN0079231 showed better docking scores with values of -9.891, -7.579 and -7.097 kcal/mol, respectively than the reference compound (-6.064 kcal/mol). Also, SN0021307, SN0449787 and SN0079231 exhibited lower free binding energies (-57.12, -49.81 and -46.05 kcal/mol, respectively) than the reference ligand (-37.75 kcal/mol). Based on these studies, SN0021307, SN0449787, and SN0079231 showed better binding affinity that the reference compound. Further the validation of these findings, molecular dynamics simulations confirmed the stability of the ligand-receptor complex for 100 ns with RMSD ranging from 0.6 to 1.8 Å. Based on these promising results, these three phytochemicals emerge as promising lead compounds warranting comprehensive biological screening in future investigations. These compounds hold great potential for further exploration regarding their efficacy and safety as Pin1 inhibitors, which could usher in new avenues for combating cancer.

Multicenter comparison of analytical interferences of 25-OH vitamin D immunoassay and mass spectrometry methods by endogenous interferents and cross-reactivity with 3-epi-25-OH-vitamin D3.

Background: Vitamin D (vit-D) deficiency is highly prevalent in the Korean population, highlighting the need for accurate measurements. In this study, the interferences by endogenous and cross-reactive substances were compared between routine vit-D immunoassays and mass spectrometry (MS) methods. Methods: Two MS methods and 4 immunoassays from different manufacturers (Abbott, Beckman Coulter, Roche, Siemens) were compared. Residual samples that were icteric, lipemic, hemolyzed, high in rheumatoid factor, from myeloma patients, or patients undergoing hemodialysis were collected. Also, 4 levels of National Institute of Standards and Technology (NIST) Standard Reference Material 972a, and 12 samples serially spiked with 3-epi-25-OH-D3 were prepared. Results: Significant interferences were observed in hemolytic (Roche), icteric (Beckman and Siemens) and lipemic samples (all 4 immunoassays). Level 4 NIST material and 3-epi-25-OH-D3-spiked samples induced significant cross-reactivity, yielding higher total vit-D measurements in non-epimer-separating MS methods, and both the Beckman and Roche immunoassays. Conclusion: Most observed interferences were consistent with manufacturers' claims, but overall improvement of immunoassay bias limits is required. Awareness of potential interference is important to increase the accuracy of vit-D measurements. Moreover, care is due when interpreting vit-D results of newborns, infants and less commonly, pregnant women, who are known to have physiologically high levels of the highly cross-reactive 3-epi-25-OH-D3.

Design, Synthesis, In Vitro, and In Silico Studies of New N5-Substituted-pyrazolo[3,4-d]pyrimidinone Derivatives as Anticancer CDK2 Inhibitors.

CDK2 is a key player in cell cycle processes. It has a crucial role in the progression of various cancers. Hepatocellular carcinoma (HCC) and colorectal cancer (CRC) are two common cancers that affect humans worldwide. The available therapeutic options suffer from many drawbacks including high toxicity and decreased specificity. Therefore, there is a need for more effective and safer therapeutic agents. A series of new pyrazolo[3,4-d]pyrimidine analogs was designed, synthesized, and evaluated as anticancer agents against the CRC and HCC cells, HCT116, and HepG2, respectively. Pyrazolo[3,4-d]pyrimidinone derivatives bearing N5-2-(4-halophenyl) acetamide substituents were identified as the most potent amongst evaluated compounds. Further evaluation of CDK2 kinase inhibition of two potential cytotoxic compounds 4a and 4b confirmed their CDK2 inhibitory activity. Compound 4a was more potent than the reference roscovitine regarding the CDK2 inhibitory activity (IC50 values: 0.21 and 0.25 µM, respectively). In silico molecular docking provided insights into the molecular interactions of compounds 4a and 4b with important amino acids within the ATP-binding site of CDK2 (Ile10, Leu83, and Leu134). Overall, compounds 4a and 4b were identified as interesting CDK2 inhibitors eliciting antiproliferative activity against the CRC and HCC cells, HCT116 and HepG2, respectively, for future further investigations and development.

Diabetes mellitus: Classification, mediators, and complications; A gate to identify potential targets for the development of new effective treatments.

Nowadays, diabetes mellitus has emerged as a significant global public health concern with a remarkable increase in its prevalence. This review article focuses on the definition of diabetes mellitus and its classification into different types, including type 1 diabetes (idiopathic and fulminant), type 2 diabetes, gestational diabetes, hybrid forms, slowly evolving immune-mediated diabetes, ketosis-prone type 2 diabetes, and other special types. Diagnostic criteria for diabetes mellitus are also discussed. The role of inflammation in both type 1 and type 2 diabetes is explored, along with the mediators and potential anti-inflammatory treatments. Furthermore, the involvement of various organs in diabetes mellitus is highlighted, such as the role of adipose tissue and obesity, gut microbiota, and pancreatic ß-cells. The manifestation of pancreatic Langerhans ß-cell islet inflammation, oxidative stress, and impaired insulin production and secretion are addressed. Additionally, the impact of diabetes mellitus on liver cirrhosis, acute kidney injury, immune system complications, and other diabetic complications like retinopathy and neuropathy is examined. Therefore, further research is required to enhance diagnosis, prevent chronic complications, and identify potential therapeutic targets for the management of diabetes mellitus and its associated dysfunctions.

Discovery and optimized extraction of the anti-osteoclastic agent epicatechin-7-O-ß-D-apiofuranoside from Ulmus macrocarpa Hance bark.

Ulmus macrocarpa Hance bark (UmHb) has been used as a traditional herbal medicine in East Asia for bone concern diseases for a long time. To find a suitable solvent, we, in this study, compared the efficacy of UmHb water extract and ethanol extract which can inhibit osteoclast differentiation. Compared with two ethanol extracts (70% and 100% respectively), hydrothermal extracts of UmHb more effectively inhibited receptor activators of nuclear factor κB ligand-induced osteoclast differentiation in murine bone marrow-derived macrophages. We identified for the first time that (2R,3R)-epicatechin-7-O-ß-D-apiofuranoside (E7A) is a specific active compound in UmHb hydrothermal extracts through using LC/MS, HPLC, and NMR techniques. In addition, we confirmed through TRAP assay, pit assay, and PCR assay that E7A is a key compound in inhibiting osteoclast differentiation. The optimized condition to obtain E7A-rich UmHb extract was 100 mL/g, 90 °C, pH 5, and 97 min. At this condition, the content of E7A was 26.05 ± 0.96 mg/g extract. Based on TRAP assay, pit assay, PCR, and western blot, the optimized extract of E7A-rich UmHb demonstrated a greater inhibition of osteoclast differentiation compared to unoptimized. These results suggest that E7A would be a good candidate for the prevention and treatment of osteoporosis-related diseases.

Novel potent blockers for TWIK-1/TREK-1 heterodimers as potential antidepressants.

TREK-1 (TWIK-related potassium channel-1) is a subunit of the two-pore domain potassium (K2p) channel and is widely expressed in the brain. TREK-1 knockout mice were shown to have antidepressant-like effects, providing evidence for the channel's potential as a therapeutic target. However, currently there is no good pharmacological inhibitor specifically targeting TREK-1 containing K2p channels that also displays similar antidepressant-like effects. Here, we sought to find selective and potent inhibitors for TREK-1 related dimers both in vitro and in vivo. We synthesized and evaluated 2-hydroxy-3-phenoxypropyl piperidine derivatives yielding a library from which many TREK-1 targeting candidates emerged. Among these, hydroxyl-phenyl- (2a), piperidino- (2g), and pyrrolidino- (2h) piperidinyl substituted compounds showed high potencies to TREK-1 homodimers with significant antidepressant-like effects in forced swim test and tail suspension test. Interestingly, these compounds were found to have high potencies to TWIK-1/TREK-1 heterodimers. Contrastingly, difluoropiperidinyl-4-fluorophenoxy (3e) and 4-hydroxyphenyl-piperidinyl-4-fluorophenoxy (3j) compounds had high potencies to TREK-1 homodimer but lower potency to TWIK-1/TREK-1 heterodimers without significant antidepressant-like effects. We observed positive correlation between inhibition potency to TWIK-1/TREK-1 and immobility time, and no correlation between inhibition potency to TREK-1 homodimer and immobility time. This was consistent with molecular docking simulations of selected compounds to TREK-1 homodimeric and TWIK-1/TREK-1 heterodimeric models. Existing antidepressant fluoxetine was also found to potently inhibit TWIK-1/TREK-1 heterodimers. Our study reveals novel potent TWIK-1/TREK-1 inhibitors 2a, 2g, and 2h as potential antidepressants and suggest that the TWIK-1/TREK-1 heterodimer could be a potential novel molecular therapeutic target for antidepressants.

Comparison of Four T-cell Assays and Two Binding Antibody Assays in SARS-CoV-2 Vaccinees With or Without Omicron Breakthrough Infection.

Background: Several T-cell response assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are available; however, their comparability and correlations with antibody responses remain unclear. We compared four SARS-CoV-2 T-cell response assays and two anti-SARS-CoV-2 spike antibody assays. Methods: We enrolled 89 participants who had received a booster dose of the BNT162b2 vaccine after two doses of the ChAdOx1 or BNT162b2 vaccine. Fifty-six participants without breakthrough infection (BI) (ChAdOx1/BNT162b2 group: N=27; BNT162b2 group: N=29) and 33 with BI were included. We evaluated two whole-blood interferon-gamma release assays (IGRAs) (QuantiFERON and Euroimmun), T-SPOT.COVID, an in-house enzyme-linked immunospot (ELISPOT) assay (targeting the spike and nucleocapsid peptides of wild-type and Omicron SARS-CoV-2), Abbott IgG II Quant, and Elecsys Anti-S, using Mann-Whitney U, Wilcoxon signed-rank, and Spearman's correlation tests. Results: The correlations between the IGRAs and between the ELISPOT assays (ρ=0.60-0.70) were stronger than those between the IGRAs and ELISPOT assays (ρ=0.33-0.57). T-SPOT.COVID showed a strong correlation with Omicron ELISPOT (ρ=0.70). The anti-spike antibody assays showed moderate correlations with T-SPOT.COVID, Euroimmun IGRA, and ELISPOT (ρ=0.43-0.62). Correlations tended to be higher in the BI than in the noninfected group, indicating that infection induces a stronger immune response. Conclusions: T-cell response assays show moderate to strong correlations, particularly when using the same platform. T-SPOT.COVID exhibits potential for estimating immune responses to the Omicron variant. To accurately define SARS-CoV-2 immune status, both T-cell and B-cell response measurements are necessary.

Identification of the novel HLA-DQA1*05:75 allele by next-generation sequencing in a Korean cord blood donor.

HLA-DQA1*05:75 differs from DQA1*05:05:01:01 by a single substitution at nucleotide 701 (G/A) in exon 4.

Novel topoisomerase II/EGFR dual inhibitors: design, synthesis and docking studies of naphtho[2',3':4,5]thiazolo[3,2-a]pyrimidine hybrids as potential anticancer agents with apoptosis inducing activity.

Topoisomerases II are ubiquitous enzymes with significant genotoxic effects in many critical DNA processes. Additionally, epidermal growth factor receptor (EGFR) plays pivotal role in tumour growth and angiogenesis. A novel series of naphtho[2',3':4,5]thiazolo[3,2-a]pyrimidine hybrids have been designed, synthesised and evaluated for their topo IIα/EGFR inhibitory and apoptotic inducer activities. Cytotoxicity of the synthesised hybrids was evaluated against MCF-7, A549 and HCT-116 cell lines. Of the synthesised hybrids, 6i, 6a and 6c experienced superior cytotoxic activity compared to doxorubicin and erlotinib against the tested cancer cells. The molecular mechanism of these hybrids revealed their ability to successfully inhibit topo IIα and EGFR activities in micromolar concentration and may serve as topo II catalytic inhibitor. Moreover, these hybrids significantly arrested cell cycle at G2/M phase together with increased p53, caspae-7, caspase-9 levels and Bax/Bcl-2 ratio. The synthesised hybrids showed efficient binding pattern in molecular docking study and have acceptable drug likeness characters.

The novel HLA-B allele, HLA-B*15:656, first identified in a Korean individual by next-generation sequencing.

HLA-B*15:656 differs from B*15:27:01:01 by two nucleotide substitutions in codon 163 (CTG > GAG).

Multimodal therapy strategy based on a bioactive hydrogel for repair of spinal cord injury.

Traumatic spinal cord injury results in permanent and serious neurological impairment, but there is no effective treatment yet. Tissue engineering approaches offer great potential for the treatment of SCI, but spinal cord complexity poses great challenges. In this study, the composite scaffold consists of a hyaluronic acid-based hydrogel, decellularized brain matrix (DBM), and bioactive compounds such as polydeoxyribonucleotide (PDRN), tumor necrosis factor-α/interferon-γ primed mesenchymal stem cell-derived extracellular vesicles (TI-EVs), and human embryonic stem cell-derived neural progenitor cells (NPC). The composite scaffold showed significant effects on regenerative prosses including angiogenesis, anti-inflammation, anti-apoptosis, and neural differentiation. In addition, the composite scaffold (DBM/PDRN/TI-EV/NPC@Gel) induced an effective spinal cord regeneration in a rat spinal cord transection model. Therefore, this multimodal approach using an integrated bioactive scaffold coupled with biochemical cues from PDRN and TI-EVs could be used as an advanced tissue engineering platform for spinal cord regeneration.

Circulating blood eNAMPT drives the circadian rhythms in locomotor activity and energy expenditure.

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor of critical enzymes including protein deacetylase sirtuins/SIRTs and its levels in mammalian cells rely on the nicotinamide phosphoribosyltransferase (NAMPT)-mediated salvage pathway. Intracellular NAMPT (iNAMPT) is secreted and found in the blood as extracellular NAMPT (eNAMPT). In the liver, the iNAMPT-NAD+ axis oscillates in a circadian manner and regulates the cellular clockwork. Here we show that the hypothalamic NAD+ levels show a distinct circadian fluctuation with a nocturnal rise in lean mice. This rhythm is in phase with that of plasma eNAMPT levels but not with that of hypothalamic iNAMPT levels. Chemical and genetic blockade of eNAMPT profoundly inhibit the nighttime elevations in hypothalamic NAD+ levels as well as those in locomotor activity (LMA) and energy expenditure (EE). Conversely, elevation of plasma eNAMPT by NAMPT administration increases hypothalamic NAD+ levels and stimulates LMA and EE via the hypothalamic NAD+-SIRT-FOXO1-melanocortin pathway. Notably, obese animals display a markedly blunted circadian oscillation in blood eNAMPT-hypothalamic NAD+-FOXO1 axis as well as LMA and EE. Our findings indicate that the eNAMPT regulation of hypothalamic NAD+ biosynthesis underlies circadian physiology and that this system can be significantly disrupted by obesity.

Development of a COX-2-Selective Fluorescent Probe for the Observation of Early Intervertebral Disc Degeneration.

Cyclooxygenase-2 (COX-2) is a biomolecule known to be overexpressed in inflammation. Therefore, it has been considered a diagnostically useful marker in numerous studies. In this study, we attempted to assess the correlation between COX-2 expression and the severity of intervertebral disc (IVD) degeneration using a COX-2-targeting fluorescent molecular compound that had not been extensively studied. This compound, indomethacin-adopted benzothiazole-pyranocarbazole (IBPC1), was synthesized by introducing indomethacin-a compound with known selectivity for COX-2-into a phosphor with a benzothiazole-pyranocarbazole structure. IBPC1 exhibited relatively high fluorescence intensity in cells pretreated with lipopolysaccharide, which induces inflammation. Furthermore, we observed significantly higher fluorescence in tissues with artificially damaged discs (modeling IVD degeneration) compared to normal disc tissues. These findings indicate that IBPC1 can meaningfully contribute to the study of the mechanism of IVD degeneration in living cells and tissues and to the development of therapeutic agents.

Association between Sleep Duration and Incident Diabetes Mellitus in Healthy Subjects: A 14-Year Longitudinal Cohort Study.

BACKGROUND: This study aimed to investigate whether sleep duration and/or quality are associated with incident diabetes mellitus (DM). METHODS: A total of 8816 of 10,030 healthy participants were enrolled in a prospective cohort study. Sleep duration and quality questionnaires were completed. Sleep quality was assessed using the Epworth Sleepiness Scale (ESS), which measures excessive daytime sleepiness in individuals. RESULTS: During the 14-year follow-up period, 18% (1630/8816) were diagnosed with DM. A U-shaped relationship was observed between sleep duration and incident DM, with the highest risk observed when sleep duration was ≥10 h/day (hazard ratios (HR) 1.65 [1.25-2.17]). This group exhibited decreased insulin glycogenic index, a marker of insulin secretory function, during the study period. Among study participants who slept less than 10 h/day, the risk of incident DM increased when the ESS score was >10. CONCLUSIONS: We found that the association between sleep duration and incident DM was U-shaped; both short (≤5 h) and long (≥10 h) sleep durations were associated with an increased risk for the occurrence of incident DM. When sleep duration was 10 h or longer per day, there was a tendency to develop DM due to decreased insulin secretory function.