Targeting cyclin-dependent kinases: From pocket specificity to drug selectivity.

The development of selective modulators of cyclin-dependent kinases (CDKs), a kinase family with numerous members and functional variations, is a significant preclinical challenge. Recent advancements in crystallography have revealed subtle differences in the highly conserved CDK pockets. Exploiting these differences has proven to be an effective strategy for achieving excellent drug selectivity. While previous reports briefly discussed the structural features that lead to selectivity in individual CDK members, attaining inhibitor selectivity requires consideration of not only the specific structures of the target CDK but also the features of off-target members. In this review, we summarize the structure-activity relationships (SARs) that influence selectivity in CDK drug development and analyze the pocket features that lead to selectivity using molecular-protein binding models. In addition, in recent years, novel CDK modulators have been developed, providing more avenues for achieving selectivity. These cases were also included. We hope that these efforts will assist in the development of novel CDK drugs.

Natural Prenylflavonoids from Sophora flavescens Root Bark against Multidrug-Resistant Methicillin-Sensitive Staphylococcus aureus Targeting the Membrane Permeability.

The overuse of antibiotics in animal farming and aquaculture has led to multidrug-resistant methicillin-sensitive Staphylococcus aureus (MR-MSSA) becoming a common pathogen in foodborne diseases. Sophora flavescens Ait. serves as a traditional plant antibacterial agent and functional food ingredient. A total of 30 compounds (1-30) were isolated from the root bark of S. flavescens, consisting of 20 new compounds (1-20). In the biological activity assay, compound 1 demonstrated a remarkable inhibitory effect on MR-MSSA, with an MIC of 2 µg/mL. Furthermore, 1 was found to rapidly eliminate bacteria, inhibit biofilm growth, and exhibit exceptionally low cytotoxicity. Mechanistic studies have revealed that 1 possesses an enhanced membrane-targeting ability, binding to the bacterial cell membrane components phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL). This disruption of bacterial cell membrane integrity increases intracellular reactive oxygen species, protein and DNA leakage, reduced bacterial metabolism, and ultimately bacterial death. In summary, these findings suggest that compound 1 holds promise as a lead compound against MR-MSSA.

The ADAM17 inhibitor ZLDI-8 sensitized hepatocellular carcinoma cells to sorafenib through Notch1-integrin ß-talk.

ZLDI-8 is an A disintegrin and metalloproteinase domain 17 (ADAM17) inhibitor that suppresses the shedding of Notch1 to the Notch1 intracellular domain (NICD). In previous studies, we found that ZLDI-8 was able to sensitize HCC to sorafenib, but the mechanism of action remains unclear. The sensitizing effects of ZLDI-8 were tested both in vitro and in vivo. EMT-related factors, sorafenib sensitivity-related proteins and ECM-related gene expression were assessed using immunohistochemistry, RTPCR and Western blotting. Knockdown assays were conducted to determine the relationship between the Notch and Integrin pathways. CoIP assays, nuclear and cytoplasmic fractionation and immunofluorescence colocalization were applied to explore the interaction between the Notch and Integrin pathways. Appropriate statistical analysis methods were used to assess the significance of the experimental results and to ensure the scientific validity and reliability of the experimental design. We found that ECM- and EMT-related proteins were downregulated after ZLDI-8 treatment (P<0.05). ZLDI-8 significantly downregulated Integrinß1 and Integrinß3 in HCC in vitro and in vivo (P<0.05), possibly through Foxc2-dependent regulation. Mechanistically, interfering with the expression of both Integrin-linked kinase (ILK) and the NICD may downregulate the expression of proteins targeted by sorafenib, thereby sensitizing cells to sorafenib. The retroregulation of Integrinß by ILK may occur through the interaction between the NICD and ILK and may be the result of the translocation of the complexus. Our study indicates that blocking the Notch pathway may affect Integrinß through crosstalk between the Notch1 and Integrinß/ILK signaling pathways, thus providing a potential therapeutic strategy for HCC.

Discovery of novel harmine derivatives as GSK-3ß/DYRK1A dual inhibitors for Alzheimer's disease treatment.

Multitarget-directed ligands (MTDLs) have recently attracted significant interest due to their superior effectiveness in multifactorial Alzheimer's disease (AD). Combined inhibition of two important AD targets, glycogen synthase kinase-3ß (GSK-3ß) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), may be a breakthrough in the treatment of AD. Based on our previous work, we have designed and synthesized a series of novel harmine derivatives, investigated their inhibition of GSK-3ß and DYRK1A, and evaluated a variety of biological activities. The results of the experiments showed that most of these compounds exhibited good activity against GSK-3ß and DYRK1A in vitro. ZLQH-5 was selected as the best compound due to the most potent inhibitory effect against GSK-3ß and DYRK1A. Molecular docking studies demonstrated that ZLQH-5 could form stable interactions with the ATP binding pocket of GSK-3ß and DYRK1A. In addition, ZLQH-5 showed low cytotoxicity against SH-SY5Y and HL-7702, good blood-brain barrier permeability, and favorable pharmacokinetic properties. More importantly, ZLQH-5 also attenuated the tau hyperphosphorylation in the okadaic acid SH-SY5Y cell model. These results indicated that ZLQH-5 could be a promising dual-target drug candidate for the treatment of AD.

Effects of FB1 and HFB1 on Autonomous Exploratory and Spatial Memory and Learning Abilities in Mice.

Fumonisin B1 (FB1) is a representative form of fumonisin and is widely present in food and feed. Hydrolyzed fumonisin B1 (HFB1) emerges as a breakdown product of FB1, which is accompanied by FB1 alterations. While previous studies have primarily focused on the liver or kidney toxicity of FB1, with limited studies existing on its neurotoxicity and even fewer on the toxicity of HFB1, this study focuses on the neurotoxicity of FB1 and HFB1 exposure in mice investigated by the open field test, Morris water maze test, histopathological analysis, and nontargeted metabolomics. Further, the levels of oxidative stress-related indices, neurotransmitters, and sphingolipids in the brain were measured to analyze their correlation with behavioral outcomes. The results showed that both FB1 (5 mg/kg) and HFB1 (2.8 mg/kg) reduced autonomous exploratory behavior in mice, impaired spatial learning and memory, and caused mild abnormalities in the brain structure. Quantitative analysis further indicated that exposure to FB1 and HFB1 disrupted neurotransmitter homeostasis, exacerbated oxidative stress, and significantly increased the sphinganine/sphingosine (Sa/So) ratio. Moreover, HFB1 exhibited neurotoxic effects similar to those of FB1, emphasizing the need to pay attention to the neurotoxicity effect of HFB1. These findings underscore the importance of understanding the risks and potential neurological damage associated with FB1 and HFB1 exposure, highlighting the necessity for further research in this crucial field.

An inhibitor with GSK3ß and DYRK1A dual inhibitory properties reduces Tau hyperphosphorylation and ameliorates disease in models of Alzheimer's disease.

Since Alzheimer's disease (AD) is a complex and multifactorial neuropathology, the discovery of multi-targeted inhibitors has gradually demonstrated greater therapeutic potential. Neurofibrillary tangles (NFTs), the main neuropathologic hallmarks of AD, are mainly associated with hyperphosphorylation of the microtubule-associated protein Tau. The overexpression of GSK3ß and DYRK1A has been recognized as an important contributor to hyperphosphorylation of Tau, leading to the strategy of using dual-targets inhibitors for the treatment of this disorder. ZDWX-12 and ZDWX-25, as harmine derivatives, were found good inhibition on dual targets in our previous study. Here, we firstly evaluated the inhibition effect of Tau hyperphosphorylation using two compounds by HEK293-Tau P301L cell-based model and okadaic acid (OKA)-induced mouse model. We found that ZDWX-25 was more effective than ZDWX-12. Then, based on comprehensively investigations on ZDWX-25 in vitro and in vivo, 1) the capability of ZDWX-25 to show a reduction in phosphorylation of multiple Tau epitopes in OKA-induced neurodegeneration cell models, and 2) the effect of reduction on NFTs by 3xTg-AD mouse model under administration of ZDWX-25, an orally bioavailable, brain-penetrant dual-targets inhibitor with low toxicity. Our data highlight that ZDWX-25 is a promising drug for treating AD.

Structural characterization and anti-inflammatory activity of a novel neutral polysaccharide isolated from Smilax glabra Roxb.

Crude polysaccharides isolated from Smilax glabra were screened for anti-inflammatory activity using mice ear swelling animal experiments, during which the neutral polysaccharide S1 was identified. The structural characteristics and anti-inflammatory effects of the anti-inflammatory S1 polysaccharide were then investigated. The results showed that S1 was mainly composed of rhamnose, arabinose, galactose, glucose, xylose, and mannose. The structure of the main chain consisted of →6)-α-Galp-(1 â†’ 6)-ß-Galp-(1 â†’ 4)-α-Xylp-(1 â†’ 6)-ß-Galp-(1→, with branched chains comprising α-Araf-(1 â†’ 4)-α â†’ Manp-(1 â†’ and ß-Rhap-(1 â†’ 4)-α-Glcp-(1 â†’ units. Furthermore, S1 did not have a triple helix conformation. S1 could inhibit NO secretion, reduce the levels of pro-inflammatory factors (IL-6 and TNF-α), and significantly reduce LPS-stimulated inflammatory damage in RAW 264.7 cells by inhibiting activation of the NF-κB (p65) pathway. These results shed light on the possibility of S1 to be developed as a novel anti-inflammatory drug for therapeutic purposes.

Metabolic Regulation Effect and Potential Metabolic Biomarkers of Pre-Treated Delphinidin on Oxidative Damage Induced by Paraquat in A549 Cells.

Delphinidin (Del) is an anthocyanin component with high in vitro antioxidant capacity. In this study, based on the screening of a cell model, gas chromatography-time of flight mass spectrometry (GC-TOF/MS) was used to evaluate the effect of Del pre-protection on the metabolite levels of intracellular oxidative stress induced by paraquat (PQ). According to the cytotoxicity and reactive oxygen species (ROS) responses of four lung cell lines to PQ induction, A549 cell was selected and treated with 100 µM PQ for 12 h to develop a cellular oxidative stress model. Compared with the PQ-induced group, the principal components of the Del pretreatment group had significant differences, but not significant with the control group, indicating that the antioxidant activity of Del can be correlated to the maintenance of metabolite levels. Del preconditioning protects lipid-related metabolic pathways from the disturbance induced by PQ. In addition, the levels of amino acid- and energy-related metabolites were significantly recovered. Del may also exert an antioxidant effect by regulating glucose metabolism. The optimal combinations of biomarkers in the PQ-treatment group and Del-pretreatment group were alanine-valine-urea and alanine-galactose-glucose. Cell metabolome data provided characteristic fingerprints associated with the antioxidant activity of Del.

Development and structure-activity relationship of tacrine derivatives as highly potent CDK2/9 inhibitors for the treatment of cancer.

CDK2/9 are members of the CDKs family, which play key roles in the occurrence and development of many cancers by regulating cell cycle and transcriptional prolongation, respectively. To further optimize and discuss the structure-activity relationships (SARs), a series of tacrine-based compounds were designed and synthesized from the compound ZLWT-37, which was studied by our group previously but no detailed SARs study was conducted on CDK2/9. Among this series, compounds ZLMT-12 (35) exhibited the most potent antiproliferative activity (GI50 = 0.006 µM for HCT116) and superior CDK2/9 inhibitory properties (CDK2: IC50 = 0.011 µM, CDK9: IC50 = 0.002 µM). Meanwhile, ZLMT-12 showed a weak inhibitory effect on acetylcholinesterase (AChE, IC50 = 19.023 µM) and butyrylcholinesterase (BuChE, IC50 = 2.768 µM). In addition, ZLMT-12 can suppress colony formation and migration in HCT116 cells, as well as induce the apoptosis and arrest the cell cycle in the S phase and G2/M phase. In vivo investigations revealed that ZLMT-12 inhibits tumor growth in the HCT116 xenograft tumor model at a low dose of 10 mg/kg without causing hepatotoxicity. The acute toxicity test showed low toxicity with a median lethal dosage (LD50) of 104.417 mg/kg. These findings showed that ZLMT-12 might be used as a drug candidate by targeting CDK2/9.

Discovery of novel α-carboline derivatives as glycogen synthase kinase-3ß inhibitors for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease, characterized by irreversible cognitive impairment, memory loss, and behavioral disturbances, ultimately resulting in death. The critical roles of glycogen synthase kinase-3ß (GSK-3ß) in tau pathology have also received considerable attention. Based on molecular docking studies, a series of novel α-carboline derivatives were designed, synthesized, and evaluated as GSK-3ß inhibitors for their various biological activities. Among them, compound ZCH-9 showed the most potent inhibitory activity against GSK-3ß, with an IC50 value of 1.71 ± 0.09 µM. The cytotoxicity assay showed that ZCH-9 had low cytotoxicity toward the cell lines SH-SY5Y, HepG2, and HL-7702. Moreover, Western blot analysis indicated that ZCH-9 effectively inhibited hyperphosphorylation of the tau protein in okadaic acid-treated SH-SY5Y cells. The binding mode between ZCH-9 and GSK-3ß was analyzed and further clarified throughout the molecular dynamics simulations. In general, these results suggested that the α-carboline-based small-molecule compounds could serve as potential candidates targeting GSK-3ß for the treatment of AD.

Discovery of novel benzofuro[3,2-b]quinoline derivatives as dual CDK2/Topo I inhibitors.

Uncontrolled cell proliferation is a hallmark of cancer. The major regulator of the cell cycle, cyclin dependent kinase 2 (CDK2), has become a mature target for cancer treatment. Herein, we describe our efforts toward the discovery of a series of benzofuro[3,2-b]quinoline alkaloid derivatives as CDK2 inhibitors through a scaffold hopping strategy. Compound ZLHQ-5f has topoisomerase I (Topo I) inhibitory activity due to the unique structure of benzofurano[3,2-b]quinoline. Resultantly, ZLHQ-5f exhibited promising anti-proliferative and CDK2 inhibitory activities. It also arrests the cell cycle in S-phase, triggers apoptosis in HCT116 cells, and has a good safety profile in vivo. There has yet to be a report on dual CDK2/Topo I inhibitor, thus this will be a novel attempt.

Discovery of novel tacrine derivatives as potent antiproliferative agents with CDKs inhibitory property.

Tacrine was the first approved drug by the FDA for the treatment of Alzheimer's disease (AD) but was withdrawn from the market due to its dose-dependent hepatotoxicity. Herein, we describe our efforts toward the discovery of a novel series of tacrine derivatives for cancer therapeutics. Intensive structural modifications of tacrine led to the identification of N-(4-{9-[(3S)-3-aminopyrrolidin-1-yl]-5,6,7,8-tetrahydroacridin-2-yl}pyridin-2-yl)cyclopropanecarboxamide hydrochloride ((S)-45, ZLWT-37) as a potent antiproliferative agent (GI50 = 0.029 µM for HCT116). In addition, ZLWT-37 exhibited lower inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) compared to tacrine. The in vitro studies demonstrated that ZLWT-37 could significantly induce apoptosis and arrest the cell cycle in the G2/M phase in HCT116 cells. The in vivo studies revealed that compound ZLWT-37 showed excellent antitumor efficacy in HCT116 xenograft tumor model and favorable pharmacokinetics profiles (F% = 28.70%) as well as low toxicity in the acute toxicity test with a median lethal dose (LD50) of 380.3 mg/kg. Encouragingly, ZLWT-37 had no obvious hepatotoxicity, nephrotoxicity, and hematologic toxicity. Kinase assay suggested that ZLWT-37 possessed potent cyclin-dependent kinase 9 (CDK9) inhibitory activity (IC50 = 0.002 µM) and good selectivity over CDK2 (IC50 = 0.054 µM). Collectively, these findings indicate that compound ZLWT-37 is a promising anti-cancer agent that deserves further preclinical evaluation.

Discovery of novel ß-carboline derivatives as selective AChE inhibitors with GSK-3ß inhibitory property for the treatment of Alzheimer's disease.

The natural product harmine, a representative ß-carboline alkaloid from the seeds of Peganum harmala L. (Zygophyllaceae), possesses a broad spectrum of biological activities. In this study, a novel series of harmine derivatives containing N-benzylpiperidine moiety were identified for the treatment of Alzheimer's disease (AD). The results showed that all the derivatives possessed significant anti-acetylcholinesterase (AChE) activity and good selectivity over butyrylcholinesterase (BChE). In particular, compound ZLWH-23 exhibited potent anti-AChE activity (IC50 = 0.27 µM) and selective BChE inhibition (IC50 = 20.82 µM), as well as acceptable glycogen synthase kinase-3 (GSK-3ß) inhibition (IC50 = 6.78 µM). Molecular docking studies and molecular dynamics simulations indicated that ZLWH-23 could form stable interaction with AChE and GSK-3ß. Gratifyingly, ZLWH-23 exhibited good selectivity for GSK-3ß over multi-kinases and very low cytotoxicity towards SH-SY5Y, HEK-293T, HL-7702, and HepG2 cell lines. Importantly, ZLWH-23 displayed efficient reduction against tau hyperphosphorylation on Ser-396 site in Tau (P301L) 293T cell model. Collectively, harmine-based derivatives could be considered as possible drug leads for the development of AD therapies.

In Utero Exposure to Fine Particles Decreases Early Birth Weight of Rat Offspring and TLR4/NF-κB Expression in Lungs.

Particulate matter (PM2.5) exposure is reported to have deleterious effects on health. Maternal PM2.5 exposure has been confirmed to damage the growth of somatic cells and enhance the incidence of chronic respiratory diseases in children. Here we aim to investigate the impact of in utero PM2.5 exposure on early birth weight and postnatal lung development. Pregnant Sprague-Dawley rats were administered PM2.5 (0.1, 0.5, 2.5, or 7.5 mg/kg) intraperitoneally every 3 days until birth. Maternal and birth outcomes and somatic growth were monitored. Lungs were collected on PND1 (where PND = postnatal day) and PND28; the lung wet-to-dry weight ratio (W/D) was analyzed, and reactive oxygen species (ROS) levels were measured. Expression of Toll-like receptor 4 (TLR4) and NF-κB were evaluated by Western blotting and quantitative RT-PCR. There were no significant intergroup differences for maternal outcomes; however, offspring exposed in utero to 2.5 and 7.5 mg/kg PM2.5 were significantly smaller in litter weight than the controls. In utero exposure to 2.5 and 7.5 mg/kg PM2.5 led to lower body weight after birth and disrupted lung development during infancy. ROS levels were significantly increased in the 7.5 mg/kg PM2.5 group. PM2.5-treated rats showed upregulated pulmonary expression of TLR4 and NF-κB. Maternal PM2.5 exposure enhances the risk of low birth weight and affects lung alveolar development. The underlying molecular mechanisms may involve TLR4/NF-κB signaling.

Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System.

Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC) represents one group of extracellular CISs that are present in both bacteria and archaea. Here, we report the cryo-EM structure of an intact PVC from P. asymbiotica. This over 10-MDa device resembles a simplified T4 phage tail, containing a hexagonal baseplate complex with six fibers and a capped 117-nanometer sheath-tube trunk. One distinct feature of the PVC is the presence of three variants for both tube and sheath proteins, indicating a functional specialization of them during evolution. The terminal hexameric cap docks onto the topmost layer of the inner tube and locks the outer sheath in pre-contraction state with six stretching arms. Our results on the PVC provide a framework for understanding the general mechanism of widespread CISs and pave the way for using them as delivery tools in biological or therapeutic applications.

Structural insights into chromosome attachment to the nuclear envelope by an inner nuclear membrane protein Bqt4 in fission yeast.

The dynamic association of chromosomes with the nuclear envelope (NE) is essential for chromosome maintenance. Schizosaccharomyces pombe inner nuclear membrane protein Bqt4 plays a critical role in connecting telomeres to the NE, mainly through a direct interaction with the telomeric protein Rap1. Bqt4 also interacts with Lem2 for pericentric heterochromatin maintenance. How Bqt4 coordinates the interactions with different proteins to exert their functions is unclear. Here, we report the crystal structures of the N-terminal domain of Bqt4 in complexes with Bqt4-binding motifs from Rap1, Lem2, and Sad1. The structural, biochemical and cellular analyses reveal that the N-terminal domain of Bqt4 is a protein-interaction module that recognizes a consensus motif and plays essential roles in telomere-NE association and meiosis progression. Phosphorylation of Bqt4-interacting proteins may act as a switch to regulate these interactions during cell cycles. Our studies provide structural insights into the identification and regulation of Bqt4-mediated interactions.

The Inner Nuclear Membrane Protein Bqt4 in Fission Yeast Contains a DNA-Binding Domain Essential for Telomere Association with the Nuclear Envelope.

Telomeres, the protective caps at the end of the chromosomes, are often associated with the nuclear envelope (NE). Telomere positioning to the NE is dynamically regulated during mitosis and meiosis. One inner nuclear membrane protein, Bqt4, in Schizosaccharomyces pombe plays essential roles in connecting telomeres to the NE. However, the structural basis of Bqt4 in mediating telomere-NE association is not clear. Here, we report the crystal structure of the N-terminal domain of Bqt4. The N-terminal domain of Bqt4 structurally resembles the APSES-family DNA-binding domain and has a moderate double-stranded DNA-binding activity. Disruption of Bqt4-DNA interaction results in telomere detachment from the NE. These data suggest that the DNA-binding activity of Bqt4 may function to prime the chromosome onto the NE and promote telomere-NE association.

Amorphous-MgGaO Film Combined with Graphene for Vacuum-Ultraviolet Photovoltaic Detector.

Vacuum-ultraviolet (VUV) detector equipped on satellites has extensive application in space exploration and cosmic science. For a VUV detector, a semiconductor material with a sufficiently wide band gap is eagerly desired. In this work, a wide-band gap amorphous-MgGaO (a-MGO) film was epitaxially grown on n-type GaN substrate by atomic layer deposition and a p-i-n-type heterojunction device for VUV detection was constructed with a-MGO film as a photosensitive layer and p-type graphene as a transparent conductive layer. The device exhibits a good spectral selectivity of VUV with photovoltaic response, a high responsivity (2 mA W-1) under zero bias, and an ultrafast response speed (rise and decay time of 0.76 µs and 0.56 ms, respectively) under nanosecond VUV pulse irradiation. This newly developed device shows great potential in VUV detection for space exploration.