Discovery of Potent and Selective Phosphatidylinositol 3-Phosphate 5-Kinase (PIKfyve) Inhibitors as Methuosis Inducers.

Cytoplasmic vacuolation-associated cell death, known as methuosis, offers a promising nonapoptotic approach for cancer treatment. In this study, we outline the synthesis and evaluation of potent methuosis-inducing compounds. These compounds selectively induce cell death, characterized by extensive cytoplasmic vacuolation in HeLa and MDA-MB-231 cells. Notably, compound L22 exhibited a remarkable interaction with PIKfyve kinase, boasting a Kd value of 0.47 nM, surpassing the positive controls D-13 and MOMIPP in potency. Furthermore, it is important to highlight that cell death induced by compound L22 is unequivocally attributed to methuosis as it differs from apoptosis, necrosis, or autophagy. Importantly, when administered orally, L22 effectively inhibited tumor growth in a HeLa xenograft model without any apparent signs of toxicity. These results underscore the potential of L22 as a valuable tool for in-depth investigations into the mechanisms of methuosis and as a promising lead compound to guide structural optimization.

Discovery of pyrimidine-5-carboxamide derivatives as novel salt-inducible kinases (SIKs) inhibitors for inflammatory bowel disease (IBD) treatment.

Salt-inducible kinases (SIKs) play a crucial role in inflammation process, acting as molecular switches that regulate the transformation of M1/M2 macrophages. HG-9-91-01 is a SIKs inhibitor with potent inhibitory activity against SIKs in the nanomolar range. However, its poor drug-like properties, including a rapid elimination rate, low in vivo exposure and high plasma protein binding rate, have hindered further research and clinical application. To improve the drug-like properties of HG-9-91-01, a series of pyrimidine-5-carboxamide derivatives were designed and synthesized through a molecular hybridization strategy. The most promising compound 8h was obtained with favorable activity and selectivity on SIK1/2, excellent metabolic stability in human liver microsome, enhanced in vivo exposure and suitable plasma protein binding rate. Mechanism research showed that compound 8h significantly up-regulated the expression of anti-inflammatory cytokine IL-10 and reduced the expression of pro-inflammatory cytokine IL-12 in bone marrow-derived macrophages. Furthermore, it significantly elevated expression of cAMP response element-binding protein (CREB) target genes IL-10, c-FOS and Nurr77. Compound 8h also induced the translocation of CREB-regulated transcriptional coactivator 3 (CRTC3) and elevated the expression of LIGHT, SPHK1 and Arginase 1. Additionally, compound 8h demonstrated excellent anti-inflammatory effects in a DSS-induced colitis model. Generally, this research indicated that compound 8h has the potential to be developed as an anti-inflammatory drug candidate.

Identification of abemaciclib derivatives targeting cyclin-dependent kinase 4 and 6 using molecular dynamics, binding free energy calculation, synthesis, and pharmacological evaluation.

CDK4/6 plays a crucial role in various cancers and is an effective anticancer drug target. However, the gap between clinical requirements and approved CDK4/6 drugs is unresolved. Thus, there is an urgent need to develop selective and oral CDK4/6 inhibitors, particularly for monotherapy. Here, we studied the interaction between abemaciclib and human CDK6 using molecular dynamics simulations, binding free energy calculations, and energy decomposition. V101 and H100 formed stable hydrogen bonds with the amine-pyrimidine group, and K43 interacted with the imidazole ring via an unstable hydrogen bond. Meanwhile, I19, V27, A41, and L152 interacted with abemaciclib through π-alkyl interactions. Based on the binding model, abemaciclib was divided into four regions. With one region modification, 43 compounds were designed and evaluated using molecular docking. From each region, three favorable groups were selected and combined with each other to obtain 81 compounds. Among them, C2231-A, which was obtained by removing the methylene group from C2231, showed better inhibition than C2231. Kinase profiling revealed that C2231-A showed inhibitory activity similar to that of abemaciclib; additionally, C2231-A inhibited the growth of MDA-MB-231 cells to a greater extent than did abemaciclib. Based on molecular dynamics simulation, C2231-A was identified as a promising candidate compound with considerable inhibitory effects on human breast cancer cell lines.

Interactions between curcumin and human salt-induced kinase 3 elucidated from computational tools and experimental methods.

Natural products are widely used for treating mitochondrial dysfunction-related diseases and cancers. Curcumin, a well-known natural product, can be potentially used to treat cancer. Human salt-induced kinase 3 (SIK3) is one of the target proteins for curcumin. However, the interactions between curcumin and human SIK3 have not yet been investigated in detail. In this study, we studied the binding models for the interactions between curcumin and human SIK3 using computational tools such as homology modeling, molecular docking, molecular dynamics simulations, and binding free energy calculations. The open activity loop conformation of SIK3 with the ketoenol form of curcumin was the optimal binding model. The I72, V80, A93, Y144, A145, and L195 residues played a key role for curcumin binding with human SIK3. The interactions between curcumin and human SIK3 were also investigated using the kinase assay. Moreover, curcumin exhibited an IC50 (half-maximal inhibitory concentration) value of 131 nM, and it showed significant antiproliferative activities of 9.62 ± 0.33 µM and 72.37 ± 0.37 µM against the MCF-7 and MDA-MB-23 cell lines, respectively. This study provides detailed information on the binding of curcumin with human SIK3 and may facilitate the design of novel salt-inducible kinases inhibitors.

Novel organoid construction strategy for non-involuting congenital hemangioma for drug validation.

BACKGROUND: Non-involuting congenital hemangiomas (NICHs) are fully formed vascular tumors at birth with distinctive clinical, radiologic, and histopathological profiles. In the literature, there is no effective therapy strategy for patients with NICH except surgery. Currently, no cell line or animal model exists for studying the mechanism of NICH and drug validation. We plan to construct a new strategy by constructing NICH organoids for further study. RESULT: Here, we report a novel NICH organoid system construction and optimization process. Both HE and immunohistological staining exactly matched NICH tissue. We further performed transcriptome analysis to elucidate the characteristics of NICH organoids. Both NICH tissue and NICH organoids manifested similar trends in download sites. NICH organoids display novel features to new cells derived from organoids and show spectacular multiplication capacity. In the preliminary verification, we found that cells splitting from NICH organoids were human endothelial cells. Drug validation demonstrated that trametinib, sirolimus, and propranolol showed no inhibitory effects on NICH organoids. CONCLUSION: Our data show that this new NICH-derived organoid faithfully captured the features of this rare vascular tumor. Our study will boost further research on the mechanism of NICH and drug filtering in the future.

Small Molecules Promote Selective Denaturation and Degradation of Tubulin Heterodimers through a Low-Barrier Hydrogen Bond.

Here, we report a novel mechanism to selectively degrade target proteins. 3-(3-Phenoxybenzyl)amino-ß-carboline (PAC), a tubulin inhibitor, promotes selective degradation of αß-tubulin heterodimers. Biochemical studies have revealed that PAC specifically denatures tubulin, making it prone to aggregation that predisposes it to ubiquitinylation and then degradation. The degradation is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and ßGlu198, which is identified as a low-barrier hydrogen bond (LBHB). In contrast, another two tubulin inhibitors that only form normal hydrogen bonds with ßGlu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. We then screened for compounds capable of forming an LBHB with ßGlu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimer degradation through the LBHB. Our study provided a unique example of LBHB function and identified a novel approach to obtain tubulin degraders.

The cancer angiogenesis co-culture assay: In vitro quantification of the angiogenic potential of tumoroids.

The treatment response to anti-angiogenic agents varies among cancer patients and predictive biomarkers are needed to identify patients with resistant cancer or guide the choice of anti-angiogenic treatment. We present "the Cancer Angiogenesis Co-Culture (CACC) assay", an in vitro Functional Precision Medicine assay which enables the study of tumouroid induced angiogenesis. This assay can quantify the ability of a patient-derived tumouroid to induce vascularization by measuring the induction of tube formation in a co-culture of vascular cells and tumoroids established from the primary colorectal tumour or a metastasis. Furthermore, the assay can quantify the sensitivity of patient-derived tumoroids to anti-angiogenic therapies. We observed that tube formation increased in a dose-dependent manner upon treatment with the pro-angiogenic factor vascular endothelial growth factor A (VEGF-A). When investigating the angiogenic potential of tumoroids from 12 patients we found that 9 tumoroid cultures induced a significant increase in tube formation compared to controls without tumoroids. In these 9 angiogenic tumoroid cultures the tube formation could be abolished by treatment with one or more of the investigated anti-angiogenic agents. The 3 non-angiogenic tumoroid cultures secreted VEGF-A but we observed no correlation between the amount of tube formation and tumoroid-secreted VEGF-A. Our data suggests that the CACC assay recapitulates the complexity of tumour angiogenesis, and when clinically verified, could prove a valuable tool to quantify sensitivity towards different anti-angiogenic agents.

Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin with tubulin degradation effect.

Microtubules, composed of αß-tubulin heterodimers, have remained popular anticancer targets for decades. Six known binding sites on tubulin dimers have been identified thus far, with five sites on ß-tubulin and only one site on α-tubulin, hinting that compounds binding to α-tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our x-ray crystallography study reveals that, in addition to binding to the vinblastine site, cevipabulin also binds to a new site on α-tubulin. We find that cevipabulin at this site pushes the αT5 loop outward, making the nonexchangeable GTP exchangeable, which reduces the stability of tubulin, leading to its destabilization and degradation. Our results confirm the existence of a new agent binding site on α-tubulin and shed light on the development of tubulin degraders as a new generation of antimicrotubule drugs targeting this novel site.

In Vitro Coculture Assays of Angiogenesis.

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

Characterization of the polarized endothelial secretome.

Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin-α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.-Wei, H., Sundararaman, A., Dickson, E., Rennie-Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome.

Chlorophyllin Modulates Gut Microbiota and Inhibits Intestinal Inflammation to Ameliorate Hepatic Fibrosis in Mice.

Liver fibrosis is an abnormal wound healing response and a common consequence of chronic liver diseases from infection or alcohol/xenobiotic exposure. At the cellular level, liver fibrosis is mediated by trans-differentiation of hepatic stellate cells (HSCs), which is driven by persistent hepatic and systemic inflammation. However, impaired enterohepatic circulation and gut dysbiosis may indirectly contribute to the liver fibrogenesis. The composition of the gut microbiota depends on diet composition and host factors. In this study, we examined chlorophyllin, derived from green pigment chlorophyll, on gut microbiota, the intestinal mucosal barrier, and liver fibrosis. BALB/c mice received carbon tetrachloride through intraperitoneal injection to induce liver fibrosis and chlorophyllin was administrated in drinking water. The effects of chlorophyllin on liver fibrosis were evaluated for (1) survival rate, (2) hepatic morphologic analysis, (3) inflammatory factors in both the small intestine and liver, and (4) gut microbiota. Our results indicate that oral administration of chlorophyllin could attenuate intestinal and hepatic inflammation and ameliorate liver fibrosis. Importantly, oral administration of chlorophyllin promptly rebalanced the gut microbiota, exhibiting down-regulation of the phylum Firmicutes and up-regulation of the phylum Bacteroidetes. In vitro experiments on intestinal epithelial cells showed that chlorophyllin exposure could inhibit NF-κB pathway via IKK-phosphorylation suppression. In conclusion, this study demonstrates potential application of chlorophyllin to regulate the intestinal microbiota and ameliorate hepatic fibrosis.

Higher serum 25(OH)D level is associated with decreased risk of impairment of glucose homeostasis: data from Southwest China.

BACKGROUND: Recent epidemiological studies have suggested inverse associations between vitamin D status and metabolic diseases including type 2 diabetes (T2DM). The aim of this study was to examine whether a higher serum 25-hydroxyvitamin D (25(OH)D) was associated with a more favorable glucose homeostasis among adults without diabetes in Southwest China. METHODS: Serum 25(OH)D concentration was measured in a cross-sectional sample of 1514 adults without diabetes aged 25-65 years recruited from Southwest China. Indices describing glucose homeostasis included fasting plasma glucose (FPG), fasting insulin, glycated hemoglobin (HbA1c), the homeostatic model assessment 2-insulin resistance (HOMA2-IR) and odds of pre-diabetes. Data were analyzed by multivariable-adjusted regression models. RESULTS: The average serum 25(OH)D was 22.66 ng/ml, and percentages of vitamin D deficiency [25(OH)D < 20 ng/ml], insufficiency [20 ≤ 25(OH)D ≤ 30 ng/ml] were 47.6 and 32.2%, respectively. Serum 25(OH)D was inversely associated with fasting insulin (P = 0.0007), HbA1c (P = 0.0001) and HOMA2-IR (P = 0.0007), but not with FPG, after adjusting for age, gender, monthly personal income, smoking status, energy intake, moderate-to-vigorous physical activity (MVPA) and waist circumference (WC). Compared with the lowest 25(OH)D tertile, the odds ratio for pre-diabetes in the highest tertile was 0.68 (95%CI: 0.47-0.99) after adjustment for cofounders. In the following stratified analyses according to weight status, we only observed this inverse association between serum 25(OH)D and pre-diabetes in overweight or obese adults (n = 629, P = 0.047), but not in their counterparts with BMI < 24 kg/m2. CONCLUSIONS: Our results advocate that a higher serum 25(OH)D level is associated with decreased risk of impairment of glucose homeostasis among adults without diabetes in Southwest China. Further studies are warranted to determine the role of vitamin D in glucose homeostasis.

MicroRNA transcriptome analysis of porcine vital organ responses to immunosuppressive porcine cytomegalovirus infection.

BACKGROUND: Porcine cytomegalovirus (PCMV) is an immunosuppressive virus that mainly inhibits T-lymphocyte and macrophage immune functions; it has significantly damaged the farming industry. Although recent studies have shown that miRNAs play important roles in immune responses, the regulatory mechanisms of miRNAs during immunosuppressive virus infection remain unclear. METHODS: In this study, porcine small-RNA transcriptomes of PCMV-infected and uninfected vital organs were first characterised by high-throughput sequencing. miRDeep2 software was used to predict novel pig-encoded miRNAs. To verify the accuracy of the high-throughput sequencing results, stem-loop qRT-PCR was performed on 12 significantly DE miRNAs. The physical and functional interactions between the immune-related target genes of the DE miRNAs in PCMV-infected organs were analysed using the STRING database. RESULTS: In total, 306 annotated and 295 novel miRNAs were identified from PCMV-infected and uninfected porcine organs, respectively, through alignment with known Sus scrofa pre-miRNAs. Overall, 92, 107, 95, 77 and 111 miRNAs were significantly differentially expressed in lung, liver, spleen, kidney and thymus after PCMV infection, respectively. According to Gene Ontology enrichment analysis, target genes of the differentially expressed miRNAs associated with immune system processes, regulation of biological processes and metabolic processes were enriched in every sample. Integrated expression analysis of the differentially expressed miRNAs and their target mRNAs in PCMV-infected thymus showed that the significant differential expression of specific miRNAs under the pressure of PCMV infection in central immune organs interfered with the expression of genes involved in important immune-related signalling pathways, thus promoting the viral infection. CONCLUSIONS: This is the first comprehensive analysis of the responses of host small-RNA transcriptomes to PCMV infection in vital porcine organs. It provides new insights into the regulatory mechanisms of miRNAs during infection by immunosuppressive viruses.

Preparation of a small intestinal submucosa modified polypropylene hybrid mesh via a mussel-inspired polydopamine coating for pelvic reconstruction.

Pelvic organ prolapse (POP) is a serious health issue that affects many adult women. Surgical treatments for POP patients comprise a common strategy in which scaffold materials are used to reconstruct the prolapsed pelvic. However, the existing materials for pelvic reconstruction cannot meet clinical requirements in terms of biocompatibility, mechanics and immunological rejection. To address these concerns, polypropylene (PP) mesh was selected because of its strong mechanical properties. Small intestinal submucosa (SIS) was used to modify the PP mesh via a mussel-inspired polydopamine coating to enhance its biocompatibility. The scanning electron microscopy (SEM) and atomic force microscopy (AFM) results demonstrated that SIS was successfully conjugated on the surface of the PP mesh. Moreover, the cytotoxicity results indicated that the PP mesh and SIS-modified PP mesh were safe to use. Furthermore, in vivo tests demonstrated that the fibroplasia around the implanted site in the SIS-modified PP mesh group was significantly less than the fibroplasia around the PP mesh group. In addition, the immunohistochemistry staining results indicated that the expression of pro-inflammatory macrophages (M1) was substantially lower and that the expression of pro-healing macrophages (M2) was higher in the SIS-modified PP mesh group. Furthermore, ELISA detection indicated that the expression of IL-1ß and IL-6 in the SIS-modified PP mesh group was reduced compared with the PP mesh group. These findings suggest that a SIS-modified polypropylene hybrid mesh via a mussel-inspired polydopamine coating is a promising approach in pelvic reconstruction.

Molecular epidemiology of Porcine torovirus (PToV) in Sichuan Province, China: 2011-2013.

BACKGROUND: Porcine torovirus (PToV) is a member of the genus Torovirus which is responsible for gastrointestinal disease in both human beings and animals with particular prevalence in youth. Torovirus infections are generally asymptomatic, however, their presence may worsen disease consequences in concurrent infections with other enteric pathogens. METHODS: A total of 872 diarrheic fecal samples from pigs of different ages were collected from 12 districts of Sichuan Province in the southwest of China. RT-PCR was done with PToV S gene specific primers to detect the presence of PToV positive samples. M gene specific primers were used with the PToV positive samples and the genes were sequenced. A phylogenetic tree was constructed based on the M gene nucleotide sequences from the 19 selected novel Sichuan strains and 21 PToV and BToV M gene sequences from GenBank. RESULTS: A total of 331 (37.96%, 331/872) samples were found to be positive for PToV and the highest prevalence was observed in piglets aged from 1 to 3 weeks old. Through phylogenetic inference the 40 PToV M gene containing sequences were placed into two genotypes (I & II). The 19 novel Sichuan strains of genotype I showed strong correlations to two Korean gene sequences (GU-07-56-11 and GU-07-56-22). Amino-acid sequence analysis of the 40 PToV M gene strains revealed that the M gene protein was highly conserved. CONCLUSIONS: This study uncovered the presence of PToV in Sichuan Province, and demonstrated the need for continuous surveillance PToV of epidemiology.

Molecular investigation of Torque teno sus virus in geographically distinct porcine breeding herds of Sichuan, China.

BACKGROUND: Torque teno sus virus (TTSuV), infecting domestic swine and wild boar, is a non-enveloped virus with a circular, single-stranded DNA genome. which has been classified into the genera Iotatorquevirus (TTSuV1) and Kappatorquevirus (TTSuV2) of the family Anelloviridae. A molecular study was conducted to detect evidence of a phylogenic relationship between these two porcine TTSuV genogroups from the sera of 244 infected pigs located in 21 subordinate prefectures and/or cities of Sichuan. RESULTS: Both genogroups of TTSuV were detected in pig sera collected from all 21 regions examined. Of the 244 samples, virus from either genogroup was detected in 203 (83.2%), while 44 animals (18.0%) were co-infected with viruses of both genogroups. Moreover, TTSuV2 (186/244, 76.2%) was more prevalent than TTSuV1 (61/244, 25%). There was statistically significant difference between the prevalence of genogroups 1 infection alone (9.4%, 23/244) and 2 alone (64.8%, 158/244), and between the prevalence of genogroups 2 (76.2%, 186/244) and both genogroups co-infection (18.0%, 44/244). The untranslated region of the swine TTSuV genome was found to be an adequate molecular marker of the virus for detection and surveillance. Phylogenetic analysis indicated that both genogroups 1 and 2 could be further divided into two subtypes, subtype a and b. TTSuV1 subtype b and the two TTSuV2 subtypes are more prevalent in Sichuan Province. CONCLUSIONS: Our study presents detailed geographical evidence of TTSuV infection in China.