Discovery and Proof of Concept of Potent Dual Polθ/PARP Inhibitors for Efficient Treatment of Homologous Recombination-Deficient Tumors.

DNA polymerase theta (Polθ) has recently emerged as a new attractive synthetic lethal target involved in DNA damage repair. Inactivating Polθ alone or in combination with PARP inhibitors has demonstrated substantial therapeutic potential against tumors with homologous recombination (HR) defects such as alternation of BRCA genes. Herein, we report the design and proof of concept of a highly potent dual Polθ/PARP inhibitor 25d, which exhibited low nanomolar inhibitory activities against both Polθ and PARP1. Compared to combination treatment, 25d demonstrated superior antitumor efficacy in both MDA-MB-436 cells and xenografts by inducing more DNA damage and apoptosis. Importantly, 25d retained sensitivity in PARP inhibitor-resistant MDA-MB-436 cells with 53BP1 defect. Altogether, these findings illustrate the potential advantages of 25d, a first-in-class dual Polθ/PARP inhibitor, over monotherapy in treating HR-deficient tumors, including those with acquired PARP inhibitor resistance.

Single-cell map of dynamic cellular microenvironment of radiation-induced intestinal injury.

Intestine is a highly radiation-sensitive organ that could be injured during the radiotherapy for pelvic, abdominal, and retroperitoneal tumors. However, the dynamic change of the intestinal microenvironment related to radiation-induced intestine injury (RIII) is still unclear. Using single-cell RNA sequencing, we pictured a dynamic landscape of the intestinal microenvironment during RIII and regeneration. We showed that the various cell types of intestine exhibited heterogeneous radiosensitivities. We revealed the distinct dynamic patterns of three subtypes of intestinal stem cells (ISCs), and the cellular trajectory analysis suggested a complex interconversion pattern among them. For the immune cells, we found that Ly6c+ monocytes can give rise to both pro-inflammatory macrophages and resident macrophages after RIII. Through cellular communication analysis, we identified a positive feedback loop between the macrophages and endothelial cells, which could amplify the inflammatory response induced by radiation. Besides, we identified different T cell subtypes and revealed their role in immunomodulation during the early stage of RIII through inflammation and defense response relevant signaling pathways. Overall, our study provides a valuable single-cell map of the multicellular dynamics during RIII and regeneration, which may facilitate the understanding of the mechanism of RIII.

Novel 6-amino-1,3,5-triazine derivatives as potent BTK inhibitors: structure-activity relationship (SAR) analysis and preliminary mechanism investigation.

Bruton's tyrosine kinase (BTK) is a promising drug target for the treatment of B-cell related malignancies. Irreversible inhibition of BTK by a covalent inhibitor has been proved to be a clinically effective therapy. However, most irreversible BTK inhibitors also inhibit other kinases including JAK3 and EGFR, leading to some adverse events. Herein, we reported the structure-based design and optimization of a series of irreversible BTK inhibitors bearing the 6-amino-1,3,5-triazine scaffold. Most of the synthesized compounds demonstrated considerable BTK inhibition and improved anti-proliferative activity against Raji and Ramos cells. Among them, compound C11 exhibited potent BTK inhibition (BTK IC50 = 17.0 nM) and a desirable selectivity profile especially over EGFR. Moreover, C11 effectively blocked activation of BTK and downstream signaling, arrested the cell cycle in G0/G1 phase and induced apoptosis in Raji cells. Its irreversible binding mode was further investigated by both molecular modeling and a washout experiment. Collectively, C11 is a novel selective irreversible BTK inhibitor worthy of further in-depth research.

RegVar: Tissue-specific Prioritization of Non-coding Regulatory Variants.

Non-coding genomic variants constitute the majority of trait-associated genome variations; however, the identification of functional non-coding variants is still a challenge in human genetics, and a method for systematically assessing the impact of regulatory variants on gene expression and linking these regulatory variants to potential target genes is still lacking. Here, we introduce a deep neural network (DNN)-based computational framework, RegVar, which can accurately predict the tissue-specific impact of non-coding regulatory variants on target genes. We show that by robustly learning the genomic characteristics of massive variant-gene expression associations in a variety of human tissues, RegVar vastly surpasses all current non-coding variant prioritization methods in predicting regulatory variants under different circumstances. The unique features of RegVar make it an excellent framework for assessing the regulatory impact of any variant on its putative target genes in a variety of tissues. RegVar is available as a web server at https://regvar.omic.tech/.

A Study on the Radiosensitivity of Radiation-Induced Lung Injury at the Acute Phase Based on Single-Cell Transcriptomics.

Background and Aims: Radiation-induced lung injury (RILI) is the most common complication associated with chest tumors, such as lung and breast cancers, after radiotherapy; however, the pathogenic mechanisms are unclear. Single-cell RNA sequencing has laid the foundation for studying RILI at the cellular microenvironmental level. This study focused on changes during the acute pneumonitis stage of RILI at the cellular microenvironmental level and investigated the interactions between different cell types. Methods: An acute RILI model in mice and a single-cell transcriptional library were established. Intercellular communication networks were constructed to study the heterogeneity and intercellular interactions among different cell types. Results: A single-cell transcriptome map was established in a mouse model of acute lung injury. In total, 18,500 single-cell transcripts were generated, and 10 major cell types were identified. The heterogeneity and radiosensitivity of each cell type or subtype in the lung tissues during the acute stage were revealed. It was found that immune cells had higher radiosensitivity than stromal cells. Immune cells were highly heterogeneous in terms of radiosensitivity, while some immune cells had the characteristics of radiation resistance. Two groups of radiation-induced Cd8+Mki67+ T cells and Cd4+Cxcr6+ helper T cells were identified. The presence of these cells was verified using immunofluorescence. The ligand-receptor interactions were analyzed by constructing intercellular communication networks. These explained the origins of the cells and revealed that they had been recruited from endothelial cells to the inflammatory site. Conclusions: This study revealed the heterogeneity of in vivo radiosensitivity of different cell types in the lung at the initial stage post irradiation.

Development of novel tetrahydroisoquinoline-hydroxamate conjugates as potent dual SERDs/HDAC inhibitors for the treatment of breast cancer.

Concomitant inhibition of estrogen receptor alpha (ERα) and histone deacetylase (HDAC) signaling has been proven effective in endocrine-resistant ER+ breast cancers. Herein, a series of tetrahydroisoquinoline (THIQ)-hydroxamate conjugates were rationally designed and synthesized as dual SERDs/HDAC inhibitors by incorporating the hydroxamate, a known HDAC pharmacophore, into a privileged THIQ scaffold of selective ERα degraders (SERDs). Some of these THIQ-hydroxamate conjugates displayed remarkable HDAC6 inhibition and improved antiproliferative activity against MCF-7 cells. Particularly, the most potent HDAC inhibitor 19k also exhibits potent ERα binding affinity, good ERα degradation efficacy and the best antiproliferative activity. Besides, 19k displayed superior antitumor efficacy than the drug combination (Fulvestrant + SAHA) through promoting ERα degradation and histone acetylation in an MCF-7 xenograft model, without causing observable toxicity. Collectively, this study validates the therapeutic potential of a dual-acting compound with potent ERα degradation efficacy and HDAC6 inhibition in breast cancer.

Comparative study on the monosaccharide compositions, antioxidant and hypoglycemic activities in vitro of intracellular and extracellular polysaccharides of liquid fermented Coprinus comatus.

Polysaccharides were one of the important active ingredients of medical fungi, on account of its unique role caused the widespread concern of researchers at home and abroad. Currently, the structure and antioxidant activity of Coprinus comatus (C. comatus) fruit bodies polysaccharides have been reported. However, there is no article on the simultaneous study of intracellular and extracellular polysaccharides of liquid fermented C. comatus (IPs, EPs) so far. Hence, this paper innovatively investigated the monosaccharide compositions, antioxidative activities and inhibition of α-amylase activity in vitro of IPs and EPs. In this study, we adopted the PMP pre-column derivatization combined with HPLC method to determine the monosaccharide compositions of the IPs and EPs. Besides, the total antioxidant capacity, DPPH radical scavenging capacity, ·OH radical scavenging ability and inhibition effect on α-amylase in vitro of crude polysaccharides (ICPs, ECPs) and deproteinized polysaccharides (IDPs, EDPs) were severally compared. Only three kinds of monosaccharides were detected in IPs. Among the monosaccharides tested, the EPs contained five kinds, of which galactose and xylose accounted for a large proportion. ICPs showed better antioxidant activity and inhibition of α-amylase activity. This article innovatively studied the IPs and EPs, which could lay the foundation for the deep development of C. comatus.

A Purinergic P2 Receptor Family-Mediated Increase in Thrombospondin-1 Bolsters Synaptic Density and Epileptic Seizure Activity in the Amygdala-Kindling Rat Model.

Previous studies suggested that the thrombospondin-1/transforming growth factor-ß1 (TSP-1/TGF-ß1) pathway might be critical in synaptogenesis during development and that the purinergic P2 receptor family could regulate synaptogenesis by modulating TSP-1 signaling. However, it is unclear whether this pathway plays a role in synaptogenesis during epileptic progression. This study was designed to investigate this question by analyzing the dynamic changes and effects of TSP-1 levels on the density of synaptic markers that are related to epileptic seizure activity. In addition, we evaluated whether P2-type receptors could regulate these effects. We generated a rat seizure model via amygdala kindling and inhibited TSP-1 activity using small interfering RNA (siRNA) interference and pharmacological inhibition. We treated the rats with antagonists of P2 or P2Y receptors, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic (PPADS) or Reactive Blue 2. Following this, we quantified TSP-1 and TGF-ß1 immunoreactivity (IR), the density of synaptic markers, and seizure activity. There were significantly more synapses/excitatory synapses in several brain regions, such as the hippocampus, which were associated with progressing epileptic discharges after kindling. These were associated with increased TSP-1 and TGF-ß1-IR. Genetic or pharmacologic inhibition of TSP-1 significantly reduced the density of synaptic/excitatory synaptic markers and inhibited the generalization of focal epilepsy. The administration of PPADS or Reactive Blue 2 attenuated the increase in TSP-1-IR and the increase in the density of synaptic markers that follows kindling and abolished most of the epileptic seizure activity. Altogether, our results indicate that the TSP-1/TGF-ß1 pathway and its regulation by P2, particularly P2Y-type receptors, may be a critical promoter of synaptogenesis during the progression of epilepsy. Therefore, components of this pathway may be targets for novel antiepileptic drug development.

[Advances in high-throughput transcriptome research of traditional Chinese medicines].

Traditional Chinese medicine is a treasure of Chinese culture, absorbing the wisdom of the Chinese people. Continuous application of new technologies makes traditional Chinese medicine research advance with the times. After several years of development, high-throughput transcriptome study has become a mature research tool in biology. This paper reviewed the advances in medicine transcriptome study, and compared two sequencing platforms, Roche's GS FLX platform and Illumina's HiSeq 2000 platform. Moreover, this paper introduced medicine transcriptome analysis process, with Panax quinquefolius and Lonicera japonica for examples, showing the characteristics of traditional Chinese medicine transcriptome studies. High-throughput transcriptome studies facilitate traditional Chinese medicine research with overall understand of functional genes, give clear elucidation of metabolic pathways, lay molecular foundation for the traditional Chinese medicine research and offer modern interpretation for traditional Chinese medicine theory. However, the current study faces several difficulties, including weak molecular basis, high sequencing cost and staff shortages in data anaysis. In the future, with the development in sequencing technology, the combination of transcriptome and other genomics, such as proteome and metabolome, will lay a solid foundation for the new high-throughput screening and developing model for the traditional Chinese medicine industry.

Proteomics analysis of UV-irradiated Lonicera japonica Thunb. with bioactive metabolites enhancement.

A previous study showed that the contents of caffeoylquinic acids and iridoids, the major bioactive components in the postharvest Lonicera japonica Thunb., were induced by enhanced ultraviolet (UV)-A or UV-B irradiation. To clarify the UV-responsive key enzymes in the bioactive metabolites biosynthetic pathway and the related plant defense mechanism in L. japonica, 2DE in combination with MALDI-TOF/TOF MS was employed. Seventy-five out of 196 differential proteins were positively identified. Based on the functions, these proteins were grouped into nine categories, covering a wide range of molecular processes including the secondary metabolites (caffeoylquinic acids and iridoids) biosynthetic-related proteins, photosynthesis, carbohydrate and energy metabolism, stress, DNA, transport-related proteins, lipid metabolism, amino acid metabolism, cell wall. Of note is the increasing expression of 1-deoxy-d-xylulose 5-phosphate reductoisomerase and 5-enol-pyruvylshikimate-phosphate synthase, which was crucial to supply more precursor for the secondary metabolites including caffeoylquinic acids and iridoids. Thus, this study provides both the clues at the protein level for the increase of the two bioactive components upon UV irradiation and the profile of UV-responsive proteins in L. japonica.

Genetic variation and metabolic pathway intricacy govern the active compound content and quality of the Chinese medicinal plant Lonicera japonica thunb.

BACKGROUND: Traditional Chinese medicine uses various herbs for the treatment of various diseases for thousands of years and it is now time to assess the characteristics and effectiveness of these medicinal plants based on modern genetic and molecular tools. The herb Flos Lonicerae Japonicae (FLJ or Lonicera japonica Thunb.) is used as an anti-inflammatory agent but the chemical quality of FLJ and its medicinal efficacy has not been consistent. Here, we analyzed the transcriptomes and metabolic pathways to evaluate the active medicinal compounds in FLJ and hope that this approach can be used for a variety of medicinal herbs in the future. RESULTS: We assess transcriptomic differences between FLJ and L. japonica Thunb. var. chinensis (Watts) (rFLJ), which may explain the variable medicinal effects. We acquired transcriptomic data (over 100 million reads) from the two herbs, using RNA-seq method and the Illumina GAII platform. The transcriptomic profiles contain over 6,000 expressed sequence tags (ESTs) for each of the three flower development stages from FLJ, as well as comparable amount of ESTs from the rFLJ flower bud. To elucidate enzymatic divergence on biosynthetic pathways between the two varieties, we correlated genes and their expression profiles to known metabolic activities involving the relevant active compounds, including phenolic acids, flavonoids, terpenoids, and fatty acids. We also analyzed the diversification of genes that process the active compounds to distinguish orthologs and paralogs together with the pathways concerning biosynthesis of phenolic acid and its connections with other related pathways. CONCLUSIONS: Our study provides both an initial description of gene expression profiles in flowers of FLJ and its counterfeit rFLJ and the enzyme pool that can be used to evaluate FLJ quality. Detailed molecular-level analyses allow us to decipher the relationship between metabolic pathways involved in processing active medicinal compounds and gene expressions of their processing enzymes. Our evolutionary analysis revealed specific functional divergence of orthologs and paralogs, which lead to variation in gene functions that govern the profile of active compounds.