Design, Synthesis, and Biological Evaluation of 5-Amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide Derivatives as Novel and Potential MEK/RAF Complex Inhibitors Based on the "Clamp" Strategy.

Herein, we described the rational drug design and synthesis of a series of 5-amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide derivatives that inhibit MEK and RAF kinases. The detailed screening cascades revealed that 16b was a preferred compound, which might act like a "clamp" to stabilize the MEK/RAF complex, thereby effectively inhibiting MEK1, BRAF, and BRAFV600E with IC50 values of 28, 3, and 3 nM, respectively. 16b possessed an excellent selectivity over other 312 human-related kinases at 1 µM. In vitro, 16b showed potent antiproliferative activities against MIA PaCa-2 (G12C KRAS), HCT116 (G13D KRAS), and C26 (G12D KRAS) cells with IC50 values of 0.011, 0.079, and 0.096 µM, respectively. CoIP experiments demonstrated that 16b could induce MEK/RAF complex formation. Most importantly, in the C26 syngeneic colorectal and HCT116 mice xenograft tumor models, 16b demonstrated tumor growth inhibition of 70 and 93%, respectively, suggesting that 16b may be a promising MEK/RAF complex inhibitor and worthy of further development.

Discovery of Potent, Specific, and Orally Available NLRP3 Inflammasome Inhibitors Based on Pyridazine Scaffolds for the Treatment of Septic Shock and Peritonitis.

The NLRP3 inflammasome is a multiprotein complex that is a component of the innate immune system, involved in the production of pro-inflammatory cytokines. Its abnormal activation is associated with many inflammatory diseases. In this study, we designed and synthesized a series of NLRP3 inflammasome inhibitors based on pyridazine scaffolds. Among them, P33 exhibited significant inhibitory effects against nigericin-induced IL-1ß release in THP-1 cells, BMDMs, and PBMCs, with IC50 values of 2.7, 15.3, and 2.9 nM, respectively. Mechanism studies indicated that P33 directly binds to NLRP3 protein (KD = 17.5 nM), inhibiting NLRP3 inflammasome activation and pyroptosis by suppressing ASC oligomerization during NLRP3 assembly. Additionally, P33 displayed excellent pharmacokinetic properties, with an oral bioavailability of 62%. In vivo efficacy studies revealed that P33 significantly ameliorated LPS-induced septic shock and MSU crystal-induced peritonitis in mice. These results indicate that P33 has great potential for further development as a candidate for treating NLRP3 inflammasome-mediated diseases.

Distinct biological signature and modifiable risk factors underlie the comorbidity between major depressive disorder and cardiovascular disease.

Major depressive disorder (MDD) and cardiovascular disease (CVD) are often comorbid, resulting in excess morbidity and mortality. Here we show that CVDs share most of their genetic risk factors with MDD. Multivariate genome-wide association analysis of shared genetic liability between MDD and atherosclerotic CVD revealed seven loci and distinct patterns of tissue and brain cell-type enrichments, suggesting the involvement of the thalamus. Part of the genetic overlap was explained by shared inflammatory, metabolic and psychosocial or lifestyle risk factors. Our data indicated causal effects of genetic liability to MDD on CVD risk, but not from most CVDs to MDD, and showed that the causal effects were partly explained by metabolic and psychosocial or lifestyle factors. The distinct signature of MDD-atherosclerotic CVD comorbidity suggests an immunometabolic subtype of MDD that is more strongly associated with CVD than overall MDD. In summary, we identified biological mechanisms underlying MDD-CVD comorbidity and modifiable risk factors for prevention of CVD in individuals with MDD.

Single-cell RNA-sequencing provides new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass leaf blades.

As an important warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) flourishes in warm areas around the world due to the existence of the C4 photosynthetic pathway. However, how C4 photosynthesis operates in bermudagrass leaves is still poorly understood. In this study, we performed single-cell RNA-sequencing on 5296 cells from bermudagrass leaf blades. Eight cell clusters corresponding to mesophyll, bundle sheath, epidermis and vascular bundle cells were successfully identified using known cell marker genes. Expression profiling indicated that genes encoding NADP-dependent malic enzymes (NADP-MEs) were highly expressed in bundle sheath cells, whereas NAD-ME genes were weakly expressed in all cell types, suggesting C4 photosynthesis of bermudagrass leaf blades might be NADP-ME type rather than NAD-ME type. The results also indicated that starch synthesis-related genes showed preferential expression in bundle sheath cells, whereas starch degradation-related genes were highly expressed in mesophyll cells, which agrees with the observed accumulation of starch-filled chloroplasts in bundle sheath cells. Gene co-expression analysis further revealed that different families of transcription factors were co-expressed with multiple C4 photosynthesis-related genes, suggesting a complex transcription regulatory network of C4 photosynthesis might exist in bermudagrass leaf blades. These findings collectively provided new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass.

Distinct biological signature and modifiable risk factors underlie the comorbidity between major depressive disorder and cardiovascular disease.

Major depressive disorder (MDD) and cardiovascular disease (CVD) are often comorbid, resulting in excess morbidity and mortality. Here we show that CVDs share most of their genetic risk factors with MDD. Multivariate genome-wide association analysis of shared genetic liability between MDD and atherosclerotic CVD revealed seven loci and distinct patterns of tissue and brain cell-type enrichments, suggesting the involvement of the thalamus. Part of the genetic overlap was explained by shared inflammatory, metabolic and psychosocial or lifestyle risk factors. Our data indicated causal effects of genetic liability to MDD on CVD risk, but not from most CVDs to MDD, and showed that the causal effects were partly explained by metabolic and psychosocial or lifestyle factors. The distinct signature of MDD-atherosclerotic CVD comorbidity suggests an immunometabolic subtype of MDD that is more strongly associated with CVD than overall MDD. In summary, we identified biological mechanisms underlying MDD-CVD comorbidity and modifiable risk factors for prevention of CVD in individuals with MDD.

Fragment growth-based discovery of novel TNIK inhibitors for the treatment of colorectal cancer.

Traf2-and Nck-interacting protein kinase (TNIK) plays an important role in regulating signal transduction of the Wnt/ß-catenin pathway and is considered an important target for the treatment of colorectal cancer. Inhibiting TNIK has potential to block abnormal Wnt/ß-catenin signal transduction caused by colorectal cancer mutations. We discovered a series of 6-(1-methyl-1H-imidazole-5-yl) quinoline derivatives as TNIK inhibitors through Deep Fragment Growth and virtual screening. Among them, 35b exhibited excellent TNIK kinase and HCT116 cell inhibitory activity with IC50 values of 6 nM and 2.11 µM, respectively. 35b also shown excellent kinase selectivity, PK profiles, and oral bioavailability (84.64%). At a p. o. dosage of 50 mg/kg twice daily 35b suppressed tumor growth on the HCT116 xenograft model. Taken together, 35b is a promising lead compound of TNIK inhibitors, which merits further investigation.

Scleral remodeling during myopia development in mice eyes: a potential role of thrombospondin-1.

BACKGROUND: Scleral extracellular matrix (ECM) remodeling plays a crucial role in the development of myopia, particularly in ocular axial elongation. Thrombospondin-1 (THBS1), also known as TSP-1, is a significant cellular protein involved in matrix remodeling in various tissues. However, the specific role of THBS1 in myopia development remains unclear. METHOD: We employed the HumanNet database to predict genes related to myopic sclera remodeling, followed by screening and visualization of the predicted genes using bioinformatics tools. To investigate the potential target gene Thbs1, we utilized lens-induced myopia models in male C57BL/6J mice and performed Western blot analysis to detect the expression level of scleral THBS1 during myopia development. Additionally, we evaluated the effects of scleral THBS1 knockdown on myopia development through AAV sub-Tenon's injection. The refractive status and axial length were measured using a refractometer and SD-OCT system. RESULTS: During lens-induced myopia, THBS1 protein expression in the sclera was downregulated, particularly in the early stages of myopia induction. Moreover, the mice in the THBS1 knockdown group exhibited alterations in myopia development in both refraction and axial length changed compared to the control group. Western blotting analysis confirmed the effectiveness of AAV-mediated knockdown, demonstrating a decrease in COLA1 expression and an increase in MMP9 levels in the sclera. CONCLUSION: Our findings indicate that sclera THBS1 levels decreased during myopia development and subsequent THBS1 knockdown showed a decrease in scleral COLA1 expression. Taken together, these results suggest that THBS1 plays a role in maintaining the homeostasis of scleral extracellular matrix, and the reduction of THBS1 may promote the remodeling process and then affect ocular axial elongation during myopia progression.

Study on tissue distribution, metabolite profiling, and excretion of [14C]-labeled flonoltinib maleate in rats.

Flonoltinib Maleate (FM) is a dual-target inhibitor that selectively suppresses Janus kinase 2/FMS-like tyrosine kinase 3 (JAK2/FLT3), which is currently in phase I/IIa clinical trial in China for the treatment of myeloproliferative neoplasms (MPNs). In this research, we used [14C]-labeled FM (14C-FM) to investigate the distribution, metabolism, and excretion of FM in rats using High-Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry/Radioactivity Monitoring (HPLC-HRMS/RAM) and liquid scintillation counter. The results revealed that FM displayed widespread distribution in rats. Furthermore, FM demonstrated rapid clearance without any observed risk of organ toxicity attributed to accumulation. Profiling of FM metabolites in rat plasma, feces, urine, and bile identified a total of 17 distinct metabolites, comprising 7 phase I metabolites and 10 phase II metabolites. The major metabolic reactions involved oxygenation, dealkylation, methylation, sulfation, glucuronidation and glutathione conjugation. Based on these findings, a putative metabolic pathway of FM in rats was proposed. The overall recovery rate in the excretion experiment ranged from 93.04 % to 94.74 %. The results indicated that FM undergoes extensive hepatic metabolism in SD rats, with the majority being excreted through bile as metabolites and ultimately eliminated via feces. A minor fraction of FM (<10 %) was excreted through renal excretion in the form of urine. Integration of the current results with previous pharmacokinetic investigations of FM in rats and dogs enables a comprehensive elucidation of the in vivo ADME processes and characteristics of FM, thereby establishing a solid foundation for subsequent clinical investigations of FM.

Local Scaffold Diversity-Contributed Generator for Discovering Potential NLRP3 Inhibitors.

Deep generative models have become crucial tools in de novo drug design. In current models for multiobjective optimization in molecular generation, the scaffold diversity is limited when multiple constraints are introduced. To enhance scaffold diversity, we herein propose a local scaffold diversity-contributed generator (LSDC), which can be utilized to generate diverse lead compounds capable of satisfying multiple constraints. Compared to the state-of-the-art methods, molecules generated by LSDC exhibit greater diversity when applied to the generation of inhibitors targeting the NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3). We present 12 molecules, some of which feature previously unreported scaffolds, and demonstrate their reasonable docking binding modes. Consequently, the modification of selected scaffolds and subsequent bioactivity evaluation lead to the discovery of two potent NLRP3 inhibitors, A22 and A14, with IC50 values of 38.1 nM and 44.43 nM, respectively. And the oral bioavailability of compound A14 is very high (F is 83.09% in mice). This work contributes to the discovery of novel NLRP3 inhibitors and provides a reference for integrating AI-based generation with wet experiments.

Discovery of 4-amino-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one derivatives as potential receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitors.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important regulatory factor in the necroptosis signaling pathway, and is considered an attractive therapeutic target for treating multiple inflammatory diseases. Herein, we describe the design, synthesis, and structure-activity relationships of 4-amino-1,6-dihydro-7H-pyrrolo [2,3-d]pyridazin-7-one derivatives as RIPK1 inhibitors. Among them, 13c showed favorable RIPK1 kinase inhibition activity with an IC50 value of 59.8 nM, and high RIPK1 binding affinity compared with other regulatory kinases of necroptosis (RIPK1 Kd = 3.5 nM, RIPK3 Kd = 1700 nM, and MLKL Kd > 30,000 nM). 13c efficiently blocked TNFα-induced necroptosis in both human and murine cells (EC50 = 1.06-4.58 nM), and inhibited TSZ-induced phosphorylation of the RIPK1/RIPK3/MLKL pathway. In liver microsomal assay studies, the clearance rate and half-life of 13c were 18.40 mL/min/g and 75.33 min, respectively. 13c displayed acceptable pharmacokinetic characteristics, with oral bioavailability of 59.55%. In TNFα-induced systemic inflammatory response syndrome, pretreatment with 13c could effectively protect mice from loss of body temperature and death. Overall, these compounds are promising candidates for future optimization studies.

Distinct genomic signatures and modifiable risk factors underly the comorbidity between major depressive disorder and cardiovascular disease.

Major depressive disorder (MDD) and cardiovascular disease (CVD) are often comorbid, resulting in excess morbidity and mortality. Using genomic data, this study elucidates biological mechanisms, key risk factors, and causal pathways underlying their comorbidity. We show that CVDs share a large proportion of their genetic risk factors with MDD. Multivariate genome-wide association analysis of the shared genetic liability between MDD and atherosclerotic CVD (ASCVD) revealed seven novel loci and distinct patterns of tissue and brain cell-type enrichments, suggesting a role for the thalamus. Part of the genetic overlap was explained by shared inflammatory, metabolic, and psychosocial/lifestyle risk factors. Finally, we found support for causal effects of genetic liability to MDD on CVD risk, but not from most CVDs to MDD, and demonstrated that the causal effects were partly explained by metabolic and psychosocial/lifestyle factors. The distinct signature of MDD-ASCVD comorbidity aligns with the idea of an immunometabolic sub-type of MDD more strongly associated with CVD than overall MDD. In summary, we identify plausible biological mechanisms underlying MDD-CVD comorbidity, as well as key modifiable risk factors for prevention of CVD in individuals with MDD.

Piper nigrum Extract Inhibits the Growth of Human Colorectal Cancer HT-29 Cells by Inducing p53-Mediated Apoptosis.

Colorectal cancer (CRC) is a prevalent malignancy of the digestive tract with the second highest mortality rate globally. Piper nigrum is a widely used traditional medicinal plant, exhibiting antitumor activity against various tumor cells. At present, research on the effect of Piper nigrum on CRC is limited to in vitro cytotoxicity, lacking comprehensive mechanism investigations. This study aimed to explore the inhibitory effect and mechanism of Piper nigrum extract (PNE) on HT-29 cells. Firstly, we identified the chemical components of PNE. Then, MTT assay, colony formation assay, JC-1 staining, and flow cytometry were used to analyze the effect of PNE on HT-29 cells in vitro. A xenograft model, histopathological examination, immunohistochemistry, and western blot were used to evaluate the tumor growth inhibitory activity and mechanism of PNE in vivo. The results indicated that PNE could inhibit cell proliferation and colony formation, reduce mitochondrial membrane potential, induce cell apoptosis in vitro, and inhibit tumor growth in vivo. Furthermore, PNE could regulate p53 and its downstream proteins, and subsequently activate the caspase-3 pathway. In summary, PNE probably induced apoptosis of HT-29 cells through the mitochondrial pathway mediated by p53. All these results suggested that PNE might be a potential natural-origin anti-CRC drug candidate.

NTRK gene alterations were enriched in hepatoid or enteroblastic differentiation type of gastric cancer.

AimsCurrently, the clinicopathological characteristics of gastric cancer (GC) with oncogenic NTRK alterations are not well known. Although NTRK fusion has been identified as prevalent in DNA mismatch repair protein deficient (dMMR) colorectal cancer (CRC), the relationship between NTRK alterations and dMMR protein expression in GC has not been previously explored. METHODS: Our study comprised 51 cases of EBV(Epstein-barr virus)-associated gastric carcinomas, 94 cases of dMMR GC, 90 cases of gastric adenocarcinoma with hepatoid or enteroblastic differentiation (GAHED) and 256 cases of conventional GC. Furthermore, to investigate the connection between NTRK fusion and dMMR proteins, we collected dMMR tumours of various types, including 21 cases of duodenal adenocarcinomas, 46 endometrioid carcinomas and 82 CRCs. NTRK fusion and amplification were screened in GC and various types of dMMR tumours using fluorescence in situ hybridisation (FISH), while cases positive for FISH translocation underwent next-generation sequencing testing. RESULTS: Our findings revealed the existence of two cases each of NTRK fusions and NTRK amplifications, which were all enriched in case of GAHED. Additionally, following an analysis of several types of cancers, we discovered that NTRK gene alterations were only present in dMMR CRC. CONCLUSIONS: Our results indicate that NTRK gene alterations are not enriched in GC with dMMR but are specifically enriched in cases of GAHED.

Enlargement of the knowledge of Cortinarius section Anomali (Agaricales, Basidiomycota): introducing three new species from China.

Cortinarius is a globally distributed agaricoid genus that has been well studied in Europe and America with over 1,000 described species. However, as part of an ongoing effort to investigate the diversity of Cortinarius section Anomali in China, the resource investigation and classification research are still limited, and the species diversity has not been clarified by far. During the re-examination of the Chinese Cortinarius specimens, C. cinnamomeolilacinus, C. subclackamasensis, and C. tropicus, belonging to the sect. Anomali, were described in China as new to science based on morphological examination and phylogenetic analysis. The three new species are described and illustrated in detail according to the Chinese materials. The phylogenetic analysis based on internal transcribed spacer sequences confirmed the placement of the three species in the Cortinarius sect. Anomali clade. Phylogenetically related and morphologically similar species to these three new species are discussed.

UPLC-MS/MS method development and application to pharmacokinetic study in rats and dogs of Flonoltinib Maleat.

Flonoltinib Maleate (FM) is a novel selective inhibitor of Janus kinase 2/FMS-like tyrosine kinase 3 (JAK2/FLT3). In this study, we developed an ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to measure the plasma concentrations of FM in rats and dogs for pharmacokinetic studies. For chromatographic separation, we used a BEH C18 column (2.1 × 50 mm, 1.7 µm particle size) in HPLC. The mobile phase A consisted of a water solution containing 0.1% formic acid (FA) and 2 mM NH4OAc, mixed with acetonitrile (ACN) (V:V = 95:5). The mobile phase B was a water solution containing 0.1% FA and 2 mM NH4OAc, mixed with ACN (V:V = 5:95), which was used for gradient elution. We used multiple reactive ion detection (MRM) mode and electrospray ionization positive (ESI+) mode for quantitative analysis. The standard curve was linear in the concentration range of 0.5 to 500 ng/ml in rat and dog plasma. The intra-batch and inter-batch precision (RSD%) of FM in rat and dog plasma was less than 15%. The intra-batch and inter-batch accuracy was 88.3-106.5% and 92.0-100.6% in rats, and 94.7-106.6% and 95.3-103.8% in dogs, respectively. The RSD (%) of matrix factors (MF) normalized to the internal standard (IS) of FM in rat and dog plasma was ≤5.6% and ≤3.0%, respectively. The extraction recovery and carryover were considered acceptable. When the sample concentration was higher than the upper limit of quantitation (ULOQ), the 10-fold dilution was reliable within the limits of acceptability. The UPLC-MS/MS method developed in this study was successfully applied in measuring the pharmacokinetic parameters of FM in rats and dogs after intravenous and oral administration, laying a foundation for the preclinical pharmacokinetic study of FM and providing a reference for clinical pharmacokinetic studies.

HIV Pre-Exposure Prophylaxis Use on a Global Scale Among Men Who Have Sex with Men: A Systematic Review and Meta-Analysis.

Knowledge of the proportion of Pre-Exposure Prophylaxis (PrEP) use among men who have sex with men (MSM) and the specific gaps in PrEP use can stimulate enhanced focus on HIV prevention policies and programs. To summarize the proportion of PrEP use and explore the temporal trend in the proportion of PrEP use and factors associated with PrEP use among MSM on a global scale, we searched PubMed, Embase, Web of Science Core Collection, and APA PsycINFO for studies reporting on the use of HIV PrEP among MSM before April 2022. Freeman-Tukey double arc-sine transformation and random-effects models were used to pool estimates. A total of 147 articles involving 395,218 MSM were included. The pooled proportions of PrEP use among MSM and PrEP-eligible MSM were 11.23% [95% confidence interval (CI): 9.71-12.84] and 16.04% (95% CI: 11.99-23.36), respectively. The proportion of PrEP use varied among countries with different support policies. ß regressions with the logit link showed that the proportion of PrEP use has increased in recent years. Interrupted time series analyses further supported that the approval of PrEP use would decrease the number of new HIV diagnoses among MSM. The main factors associated with PrEP use include health insurance, having a regular medical provider, prior HIV testing, past use of PrEP or Post-Exposure Prophylaxis, social networks, and stigma. Although the proportion of PrEP use among MSM has remained low, it has increased in recent years. More studies are needed to explore the factors associated with PrEP use, especially for PrEP-eligible MSM in low- and middle-income countries.

In vitro and in vivo metabolic profiling of PD105, a PI3Kδ inhibitor, using UHPLC-Q-Exactive plus-MS.

PD105, a PI3Kδ inhibitor, is a candidate for the treatment of rheumatoid arthritis. This study aims to identify the metabolic profiling in vitro and in vivo by UHPLC-Q-Exactive Plus-MS.The in vitro metabolism of PD105 was studied by mouse liver microsomes and hepatocytes, while the in vivo metabolic profiling was obtained from mouse plasma, urine, and faeces. A total of 20 metabolites were tentatively identified based on accurate mass, fragment pathways, and characteristic fragment ions, including 4 in vitro and 20 in vivo.The proposed metabolic pathways of PD105 showed that there were 18 phase I metabolites and 2 phase II metabolites. The phase I metabolic pathways included oxidation, hydration, desaturation and oxidative dechlorination, while the phase II metabolic reactions were mainly methylation and arginine conjugation. Among them, oxidation was the main metabolic pathway of PD105.The comprehensive metabolic profiling contributed to further elucidation of pharmacological action and mechanism of PD105.

Contralateral effect in progression and recovery of lens-induced myopia in mice.

PURPOSE: Apart from genetic factors, recent animal studies on myopia have focused on localised mechanisms. In this study, we aimed to examine the contralateral effects of monocular experimental myopia and recovery, which cannot be explained by a mere local mechanism. METHODS: One eye of 3-week-old C57BL/6 male mice was fitted with a -30 dioptre (D) lens. The mice were distributed into two groups based on different conditions in the contralateral eye: either no lens (NLC) (n = 10) or a Plano lens on the contralateral eye (PLC) group (n = 6). Mice receiving no treatment on either eye were set as a control group (n = 6). Lenses were removed after 3 weeks of myopia induction. All mice were allowed to recover for 1 week in the same environment. Refractive status, axial length (AL) and choroidal thickness were measured before myopia induction, after 1 and 3 weeks of lens wear and after 1 week of recovery. RESULTS: One week after removing the lenses, complete recovery was observed in the eyes that wore the -30 D lenses. In both the PLC and NLC groups, the refractive status showed a myopic shift after lens removal. Additionally, the choroid was significantly thinned in these eyes. The -30 D wearing eye showed a significant increase in AL after 3 weeks of lens wear. While the AL of the -30 D wearing eye ceased to grow after the lens was removed, the AL in the PLC and NLC contralateral eyes increased, and the binocular ALs gradually converged. CONCLUSIONS: Recovery of lens-induced myopia was observed in mouse models. In the fellow eyes, the effects, including thinning of the choroid and changes in refractive status, were triggered by contralateral visual cues.