Prediction of Toxoplasma gondii virulence factor ROP18 competitive inhibitors by virtual screening

Abstract Background Rhoptry protein 18 (ROP18) is a key virulence factor of Toxoplasma gondii. The host’s immune responses mediated by immune-related GTPases (IRGs) could be blocked by ROP18’s kinase activity. ROP18 also interacts with various substrates, such as activating transcription factor 6 beta (ATF6β) and affects multiple physiological functions within host cells, thereby inducing intense virulence. In this study, competitive inhibitors targeted to ROP18 were subjected to virtual screening based on the principle of structure-based drug design (SBDD). Methods The preparation of the ROP18 structure was conducted using the “Structure Prepare” function of Molecular Operating Environment (MOE) software. The ATP-binding pocket was selected as the starting point for virtual screening. Construction of the pharmacophore model used Extended Hückel Theory (EHT) half-quantitative measurement and construction, as well as the characteristics of Type I kinase inhibitors. The pharmacophore model of ROP18 was imported into the Specs database for small molecule similarity screening using EHT pharmacophore measurement. Hit compounds were selected using the functions of London dG and generalized-born volume integral/weighted surface area (GBVI/WSA) scoring. The top 100 hits were analyzed by molecular docking and structure activity relationships (SAR) analysis. Results The final pharmacophore comprised three typical characteristics: three hydrogen bond acceptors/donors, two ring aromatic features occupying the hydrophobic core, and one cation group feature targeted to the terminus of ATP. A total of 1314 hit compounds analogous to ROP18 pharmacophore were passed through the Specs. After two rounds of docking, 25 out of 100 hits were identified as belonging to two main scaffold types: phthalimide ring structure, thiazole ring and styrene structure. Additionally, the screen also identified 13 inhibitors with distinct scaffold types. The docking models and SAR analysis demonstrated that these hits could engage in multiple hydrogen bonds, salt bridges halogen bonds, and hydrophobic interactions with ROP18, and para-position halo substituents on the benzene ring may enhance their affinity scoring. Conclusions A pharmacophore against the ROP18 ATP-binding pocket was successfully constructed, and 25 representative inhibitors from 15 scaffold clusters were screened using the Specs database. Our results provide useful scaffold types for the chemical inhibition of ROP18 or alternative conformations to develop new anti-toxoplasmosis drug leads

miR-29a/b enhances cell migration and invasion in nasopharyngeal carcinoma progression by regulating SPARC and COL3A1 gene expression.

Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with a genetic predisposition, Epstein-Barr virus infection and chromosomal abnormalities. Recently, several miRNAs have been shown to target specific mRNAs to regulate NPC development and progression. However, the involvement of miRNAs in processes leading to NPC migration and invasion remains to be elucidated. We predicted that miR-29a/b are associated with dysregulated genes controlling NPC through an integrated interaction network of miRNAs and genes. miR-29a/b over-expression in NPC cell lines had no significant effect on proliferation, whereas miR-29b mildly increased the percentage of cells in the G1 phase with a concomitant decrease in the percentage of cells in S phase. Furthermore, we demonstrated that miR-29a/b might be responsible for increasing S18 cell migration and invasion, and only COL3A1 was identified as a direct target of miR-29b despite the fact that both SPARC and COL3A1 were inhibited by miR-29a/b over-expression. Meanwhile, SPARC proteins were increased in metastatic NPC tissue and are involved in NPC progression. Unexpectedly, we identified that miRNA-29b expression was elevated in the serum of NPC patients with a high risk of metastasis. The 5-year actuarial overall survival rates in NPC patients with high serum miR-29b expression was significantly shorter than those with low serum miR-29b expression; therefore, serum miR-29b expression could be a promising prognostic marker

An analysis of 97 previously diagnosed de novo adult acute erythroid leukemia patients following the 2016 revision to World Health Organization classification

Abstract Background The incidence of acute erythroid leukemia subtype (AEL) is rare, accounting for 5% of cases of acute myeloid leukemia (AML), and the outcome is dismal. However, in 2016 revision to the WHO classification, the subcategory of AEL has been removed. Myeloblasts are redefined as the percentage of total marrow cells, not non-erythroid cells. Therefore, the previously diagnosed AEL cases are currently diagnosed as AML or myelodyspalstic syndrome (MDS) according to new criteria. Methods We respectively reviewed cases of 97 de novo previously diagnosed AEL and all the patients were diagnosed as AML or MDS according to the new classification scheme, and then the clinical characteristics of these two subtypes were compared. Statistical analyses were performed by SPSS software version 18.0. Results The median age was 37 years-old, the two-thirds of previous AEL cases were diagnosed as MDS, and there was no obvious difference between two subtypes except for male/female ratio and age. Cytogenetic, rather than MDS/AML subtypes, can better represent the prognostic factor of previously diagnosed AEL patients. When the cytogenetic risk of patients belonged to MRC intermediate category and age were below 40 years-old in previous AEL cases, the patients who received induction chemotherapy without transplantation had a similar survival compared with the patients who underwent transplantation (3-year OS: 67.2% vs 68.5%). Conclusions Cytogenetic, rather than MDS/AML subtypes, can better represent the prognostic factor of previously diagnosed AEL patients. Transplantation was a better choice for those whose cytogenetic category was unfavorable

Distinct genetic alteration profiles of acute myeloid leukemia between Caucasian and Eastern Asian population

Abstract Racial and ethnic disparities in malignancies attract extensive attention. To investigate whether there are racial and ethnic disparities in genetic alteration between Caucasian and Eastern Asian population, data from several prospective AML trials were retrospectively analyzed in this study. We found that there were more patients with core binding factor (CBF) leukemia in Eastern Asian cohorts and there were different CBF leukemia constitutions between them. The ratios of CBF leukemia are 27.7, 22.1, 21.1, and 23.4%, respectively, in our (ChiCTR-TRC-10001202), another Chinese, Korean, and Japanese Eastern Asian cohorts, which are significantly higher than those in ECOG1900, MRC AML15, UK NCRI AML17, HOVON/SAKK AML-42, and German AML2003 (15.5, 12.5, 9.3, 10.2, and 12%, respectively). And CBFbeta-MYH11 occurred more prevalently in HOVON/SAKK AML- 42 and ECOG1900 trials (50.0 and 54.3% of CBF leukemia, respectively) than in Chinese and Japanese trials (20.1 and 20.8%, respectively). The proportion of FLT3-ITD mutation is 11.2% in our cohort, which is lower than that in MRC AML15 and UK NCRI AML17 (24.6 and 17.9%, respectively). Even after excluding the age bias, there are still different incidence rates of mutation between Caucasian and Eastern Asian population. These data suggest that there are racial and ethnic disparities in genetic alteration between Caucasian and Eastern Asian population

Integrated microRNA-gene network.

<p>(A) The miRNA-gene network shows the relationships between 9 miRNAs and 51 dysregulated genes. Only genes reported in BioGrid are shown in this network. Interaction among genes and regulation of miRNAs are indicated by arrows. Red nodes represent up-regulated genes in NPC, and blue nodes represent down-regulated genes. miRNAs with more than 2 targets are shown in the figure. (B) The particular network for miR-29a/b and their targets stems from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120969#pone.0120969.g001" target="_blank">Fig. 1A</a>.</p

miR-29a/b target the SPARC/COL3A1 pathways in NPC cells.

<p>(A, B) S18 cells were transfected with 50 nmol of mimics-NC (control miRNA), mimics-miR-29a/b, anti-scramble (control anti-miRNA) and anti-miR-29a/b. The levels of miR-29a and miR-29b were assessed by qRT-PCR. Cell lysates were prepared for Western blotting with antibodies against SPARC and COL3A1, and GAPDH expression served as a loading control. Western blot figures are representative of at least three independent experiments. The value under each sample indicates the fold change of SPARC and COL3A1 protein levels relative to that of the control. (C) Western blot analysis of the expression level of SPARC and COL3A1 in NPC cells following treatment with vehicle, siRNA-SPARC and siRNA-COL3A1 for 24 h. The value under each sample indicates the fold changes of SPARC and COL3A1 protein levels relative to that of the control. Three independent experiments performed in triplicate. (D) A schematic model shows the function of miR-29a and miR-29b in NPC cell proliferation, migration and cell invasion. In response to miR-29a/b stimuli, the G1/S transition arrest triggers both a classical proliferative inhibition and an adapted metabolic switch. SPARC and COL3A1 protein levels inversely correlate with miR-29a and miR-29b expression in NPC cells, respectively. (i) The indirect effect of miR-29a to increase SPARC expression may be mediated by its unknown targets that affect cell survival, and subsequently SPARC could down-regulate the expression of COL3A1. (ii) COL3A1 is a direct target of miR-29b and affects the expression of the various ECM proteins, resulting in derepression of NPC cell migration and invasion.</p

Effects of miR-29a/b on NPC cell growth and cell cycle.

<p>The results of MTT assays following transfection of pre-miR-29a/b and their inhibitors into S18 cells for the indicated 24 h, 48 h and 72 h post-transfection times. The values are the mean and SD optical density (OD) units. (B) miR-29b increased the proportion of S18 cells at the G1/S transition, whereas miR-29a did not have a similar effect. Cells were treated with pEGFP-miR-29a/b or anti-miR-29a/b transfection and control vector pEGPF-C. Cell cycle distributions were detected 20 h later. A representative result of 3 independent experiments is shown. In all experiments, the negative control was pre-miRNA negative control.</p